Annual Review of Immunology Volume 21 2003

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Annual Review of Immunology Volume 21, 2003

Annu. Rev. Immunol. 2003.21:1-27. Downloaded from arjournals.annualreviews.org by HINARI on 09/01/07. For personal use only.

CONTENTS FRONTISPIECE, Thomas A. Waldmann THE MEANDERING 45-YEAR ODYSSEY OF A CLINICAL IMMUNOLOGIST, Thomas A. Waldmann

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CD8 T CELL RESPONSES TO INFECTIOUS PATHOGENS, Phillip Wong and Eric G. Pamer

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CONTROL OF APOPTOSIS IN THE IMMUNE SYSTEM: BCL-2, BH3-ONLY PROTEINS AND MORE, Vanessa S. Marsden and Andreas Strasser

CD45: A CRITICAL REGULATOR OF SIGNALING THRESHOLDS IN IMMUNE CELLS, Michelle L. Hermiston, Zheng Xu, and Arthur Weiss POSITIVE AND NEGATIVE SELECTION OF T CELLS, Timothy K. Starr, Stephen C. Jameson, and Kristin A. Hogquist

IGA FC RECEPTORS, Renato C. Monteiro and Jan G.J. van de Winkel REGULATORY MECHANISMS THAT DETERMINE THE DEVELOPMENT AND FUNCTION OF PLASMA CELLS, Kathryn L. Calame, Kuo-I Lin, and Chainarong Tunyaplin

71 107 139 177

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BAFF AND APRIL: A TUTORIAL ON B CELL SURVIVAL, Fabienne Mackay, Pascal Schneider, Paul Rennert, and Jeffrey Browning

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T CELL DYNAMICS IN HIV-1 INFECTION, Daniel C. Douek, Louis J. Picker, and Richard A. Koup

T CELL ANERGY, Ronald H. Schwartz TOLL-LIKE RECEPTORS, Kiyoshi Takeda, Tsuneyasu Kaisho, and Shizuo Akira

265 305 335

POXVIRUSES AND IMMUNE EVASION, Bruce T. Seet, J.B. Johnston, Craig R. Brunetti, John W. Barrett, Helen Everett, Cheryl Cameron, Joanna Sypula, Steven H. Nazarian, Alexandra Lucas, and Grant McFadden

IL-13 EFFECTOR FUNCTIONS, Thomas A. Wynn LOCATION IS EVERYTHING: LIPID RAFTS AND IMMUNE CELL SIGNALING, Michelle Dykstra, Anu Cherukuri, Hae Won Sohn, Shiang-Jong Tzeng, and Susan K. Pierce

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THE REGULATORY ROLE OF Vα14 NKT CELLS IN INNATE AND ACQUIRED IMMUNE RESPONSE, Masaru Taniguchi, Michishige Harada, Satoshi Kojo, Toshinori Nakayama, and Hiroshi Wakao

ON NATURAL AND ARTIFICIAL VACCINATIONS, Rolf M. Zinkernagel COLLECTINS AND FICOLINS: HUMORAL LECTINS OF THE INNATE IMMUNE DEFENSE, Uffe Holmskov, Steffen Thiel, and Jens C. Jensenius THE BIOLOGY OF IGE AND THE BASIS OF ALLERGIC DISEASE,


Hannah J. Gould, Brian J. Sutton, Andrew J. Beavil, Rebecca L. Beavil, Natalie McCloskey, Heather A. Coker, David Fear, and Lyn Smurthwaite

483 515 547

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GENOMIC ORGANIZATION OF THE MAMMALIAN MHC, Attila Kumánovics, Toyoyuki Takada, and Kirsten Fischer Lindahl

MOLECULAR INTERACTIONS MEDIATING T CELL ANTIGEN RECOGNITION, P. Anton van der Merwe and Simon J. Davis TOLEROGENIC DENDRITIC CELLS, Ralph M. Steinman, Daniel Hawiger, and Michel C. Nussenzweig

629 659 685

MOLECULAR MECHANISMS REGULATING TH1 IMMUNE RESPONSES, Susanne J. Szabo, Brandon M. Sullivan, Stanford L. Peng, and Laurie H. Glimcher

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BIOLOGY OF HEMATOPOIETIC STEM CELLS AND PROGENITORS: IMPLICATIONS FOR CLINICAL APPLICATION, Motonari Kondo, Amy J. Wagers, Markus G. Manz, Susan S. Prohaska, David C. Scherer, Georg F. Beilhack, Judith A. Shizuru, and Irving L. Weissman

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DOES THE IMMUNE SYSTEM SEE TUMORS AS FOREIGN OR SELF? Drew Pardoll

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B CELL CHRONIC LYMPHOCYTIC LEUKEMIA: LESSONS LEARNED FROM STUDIES OF THE B CELL ANTIGEN RECEPTOR, Nicholas Chiorazzi and Manlio Ferrarini

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INDEXES Subject Index Cumulative Index of Contributing Authors, Volumes 11–21 Cumulative Index of Chapter Titles, Volumes 11–21

ERRATA An online log of corrections to Annual Review of Immunology chapters may be found at http://immunol.annualreviews.org/errata.shtml

895 925 932


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Annu. Rev. Immunol. 2003. 21:1–27 doi: 10.1146/annurev.immunol.21.120601.140933 First published online as a Review in Advance on September 17, 2002

THE MEANDERING 45-YEAR ODYSSEY OF ∗ A CLINICAL IMMUNOLOGIST


Thomas A. Waldmann Metabolism Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 10 Center Drive, MSC 1374, Building 10, Room 4N115, Bethesda, Maryland 20892-1374; email: [email protected]

Key Words interleukin-2 (IL-2), interleukin-15 (IL-15), monoclonal antibody, suppressor T cell, immunoglobulin metabolism ■ Abstract My work on basic and clinical immunology has focused on the regulation of the human immune response and how its dysregulation can lead to immunodeficiency, autoimmune, and malignant disorders. The early focus in our laboratory was on pathogenic mechanisms underlying hypogammaglobulinemia. Our demonstration of active suppression by human suppressor T cells changed thinking about the pathogenesis of certain immunodeficiency disorders. Recently we have focused on the cytokines interleukin-2 (IL-2) and IL-15, which have competitive functions in adaptive immune responses. IL-2 is necessary to destroy self-reactive lymphocytes and thus favors peripheral tolerance to self-antigens, whereas IL-15 favors the persistence of lymphocytes involved in the memory and effector responses to invading pathogens but risks the development of inflammatory autoimmune diseases. Our murine anti-Tac monoclonal antibody exploits these differences, as does a humanized form (daclizumab) now approved for the prevention of renal allograft rejection. New forms of therapy directed at IL-2 and IL-15 receptors may be effective against certain neoplastic diseases and autoimmune disorders and in the prevention of allograft rejection.

MY INTRODUCTION TO RESEARCH At 3 AM on a cold February night in 1954, Sherman Weissman, my friend and classmate from the University of Chicago and Harvard Medical School, opened the curtains around my hospital bed on a ward at the Peter Brent Brigham Hospital in Boston, where I was a patient with a minor condition. He handed me my huge blue winter coat that I had used on the Chicago Midway and we silently sneaked out of the hospital to a small laboratory in the Harvard Dental School. We had received a $50 grant and permission to use a niche in a lab to try to extend the observations made by Allan Erslev, who had just reported the discovery of a ∗ The US Government has the right to retain a nonexclusive, royalty-free license in and to any copyright covering this paper.

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novel erythropoiesis-stimulating factor he called erythropoietin. Our investigations involved phenylhydrazine-induced anemia in rabbits. While I had been in the hospital, a friend substituting for me had held one of the rabbits inadequately and it had died. Sherm and I decided we could not afford to lose another rabbit, so each night I sneaked out of the hospital to continue the studies until morning light. This represented the dawn of my research career. The 1950s were also the time of awakening of research into non-endocrine growth factors, as well as that of modern immunology in general. During this era Byron Waksman, a professor in the medical school, was allotted only one hour to summarize all that was important and then known in immunology. Our brief laboratory experience was a critical one for both Sherm and me. For the 45th Harvard Medical School (HMS) reunion report, he was asked to comment on a teacher, incident, or situation at HMS that had a telling influence on his life. He chose as pivotal to the initiation of his research career “someone unknown who left supplies out so Tom Waldmann and I could sneak into the lab at night to try to reproduce early erythropoietin experiments.” This experience exposed me to the excitement of biomedical research and also provided me with an entrée into the National Institutes of Health (NIH) in the era of the doctor’s draft, when only a small proportion of applicants were being accepted.

THE BEGINNINGS I was born in New York City on September 21, 1930, the only child of Elizabeth Sipös and Charles Waldmann. My father and mother had emigrated from Nitra, Czechoslovakia, in 1920 and from Budapest, Hungary, in 1927, respectively. My mother was a teacher with her own elementary school in Budapest. Later in the United States she founded and ran her own nursery school. My father was an engineer educated at the Royal University for Engineers in Budapest, graduating with degrees in civil, mechanical, and electrical engineering. His career in the United States and my early life were dominated by the Great Depression and by World War II. Although he had been the chief engineer of the Comstock Industrial Construction Company, with the crash in the stock market and the onset of the depression the company declared bankruptcy, and my father was out of work. In 1936 he became an engineer in housing development for the National Housing Authority of the Resettlement Administration in Washington, D.C., where he was involved in the construction of such communities as The Greenbelt Towns in Maryland. After the war, in 1946 my father became chief engineer and member of the board of directors of American Community Builders, a company involved in the generation from scratch of Park Forest, Illinois, a community of over 30,000 individuals that has been the subject of many sociological studies including The Organization Man. From this home base I attended the University of Chicago during the Robert Maynard Hutchins era. It was a magnificent, intellectually challenging experience that forced all the students to become de facto philosophy majors who concentrated

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on the “great books” and who were challenged to address questions concerning fundamental values and broad life issues in small, interactive classes. One of my interests that persisted from an early age through my initial decades at the NIH was photography. At age five I was given a 15-cent camera. My father, impressed with some of my results, trotted one of my first photographs to the local camera store, where in exchange for permission to put it in the store window I was given a new camera worth a dollar and a quarter. This mini-triumph started a pleasurable avocation. Through the years my photographic and scientific efforts have often interconnected. In each arena I have tried to seek simplicity for impact and to approach challenges in maverick ways by seeking odd interconnections between diverse elements, a process that has proved pivotal in expressing originality in both photography and research. For example, in my four years at Harvard Medical School I not only learned to appreciate clinical medicine, but also had my first encounter, albeit minimal, with research on growth factors. In 1955–1956 I was a medical intern at the Massachusetts General Hospital, where I met Katharine Spreng, my medical resident. On March 29, 1958, we were married. The subsequent years marked the birth of our children: Richard Allen, now a neonatologist in New Bedford, Massachusetts; Robert James, now an economics professor in Rome who received his Ph.D. degree from Harvard mentored by Lawrence Summers (now the president of Harvard), and our daughter Carol Ann, a physician and internist who is working with Health Care for the Homeless in Boston. We have three grandchildren. My wife and children, each in different ways, have been caregivers who focus on people in need including the Dinka in the southwest of Sudan, the Masai in Kenya, those in poverty at Health Care for the Homeless in Boston, and individuals with HIV at the Dennis Avenue Public Health Clinic in Montgomery County, Maryland, where my wife works. My daughter and wife especially focus on those people that my Hungarian mother would have called Vizes Vereb, the “wet sparrows” of the world.

I JOIN THE NATIONAL CANCER INSTITUTE While I was completing my medical internship at the Massachusetts General Hospital, the doctor’s draft was in force and many of us looked for alternatives to a two-year appointment in the army. Although I had not seriously considered a research career, I joined the National Cancer Institute (NCI) at the NIH in 1956 following what was essentially a sojourn as a philosophy major at the University of Chicago, medical training at Harvard Medical School, and a single year of medical internship. Other than the effort supported by the $50 grant, I had no research experience. Thus, it is my associates at NIH who have been my teachers. These include my mentor, Nathaniel Berlin, my collaborators, and especially my postdoctoral fellows, students, and technicians who have so often inverted the conventional mentor-student relationship by providing me with ideas, by teaching me by example how to think in innovative ways, and by converting my vague ideas into experimental realities. I believe that more than the other writers of prefatory

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chapters I owe any success I discuss here to my associates and peers within the corridors of the NIH. A pivotal factor in my research efforts has been an intimate association with the Clinical Center of the NIH that opened in 1953, shortly before I joined the NCI. This research hospital provides a close proximity between the laboratory and the clinical care units, facilitating the development of renaissance groups that can be productive in a full scientific range extending from fundamental laboratory-based efforts to patient-oriented clinical research. Many major challenges can be addressed only by patient-oriented clinical research that involves a real-time relationship between the patient and the physician/scientist. Patient-oriented research is necessary for the translation of fundamental laboratory insights into new approaches for the prevention, diagnosis, and treatment of human disease. In comparison with the extramural academic community, clinical research at NIH increased in relative importance in recent decades as sources of funding for patient-oriented clinical research outside of the intramural NIH research community became limited, and the research community in general moved toward the application of molecular-biological approaches to fundamental scientific questions, and away from patient-oriented clinical research. Although basic research efforts became widely distributed throughout the nation, approximately 50% of all NIH-supported clinical research beds are within the NIH Clinical Center. As a result, intramural NIH has made some of its most unique contributions through translational patientoriented research. It is a major success story that provides a model for the renewed enthusiasm for such research in the extramural research community as well. During my first year at the NCI, I rotated through each of its clinical branches. Subsequently I was assigned to the Metabolism Service (now the Metabolism Branch), then directed by Nathaniel Berlin. The Metabolism Branch has through its 45 years of existence been an exemplar of the type of research requiring the presence of the patient. The research efforts on the Branch have led to the discovery of new diseases, the definition of new infectious agents, and through serendipitous observations the development of novel insights that have had both fundamental and clinical implications. This type of clinical research was necessary to test hypotheses concerning the pathogenesis of human diseases, to develop therapeutic agents on the basis of our fundamental laboratory observations, and finally to evaluate them in clinical trials. My own research has especially benefited from the analyses of certain patient-based observations that were initially paradoxical and that could not be understood without a challenge to the prevailing paradigms. The dominant arena of my research, clinical immunology, has undergone a revolution, changing from a largely phenomenological endeavor into a deeply analytical and technical field. Movement in this field, like the growth of our children, may not appear impressive when viewed from one day to the next; however, its progress has been dramatic when viewed using the portfolio of images taken over four to five decades. Questions that could not even be asked 45 years ago have been definitively answered. To give a sense of the dramatic progress in the field of immunology in my own scientific lifetime, I quote from the textbook Clinical Hematology by Maxwell Wintrobe that appeared in 1956, the year I joined the NIH:

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The function of the lymphocyte is still obscure. Because of their strategic position in lymph nodes and because they are rich in adenosinase which splits adenosine it has been suggested that the lymphocyte is instrumental in the destruction of toxic products of protein metabolism . . . a role in transference of fat from intestinal epithelium to the lacteals has been denied. Of course, this view did not do justice to the contributions of Landsteiner & Chase, who had demonstrated the cellular transfer of certain forms of immunity. However, it did reflect the primitive understanding we had concerning the immune system, especially cellular immunity, at that time. The role of the thymus and of T cells in immunity had not been defined. One could not even consider the nature of the T cell receptor for antigen nor meaningfully discuss how antibody and T cell diversity were generated when neither the T cell receptor nor the multichain structure of an immunoglobulin molecule had been demonstrated. By 1951 none of the primary immunodeficiency diseases had been shown to represent a defined molecular defect of an element of the immune system. Moreover, we did not have anything approaching our present understanding of a disease such as AIDS caused by a retrovirus, a form of pathogen that had not been defined, that infects CD4-expressing target T cells that had not been discovered. In this chapter, I discuss scientific issues and experiments that have been of interest to me over the decades. To complete the discussion of a specific issue I fast-forward to the present to report what has been accomplished in that particular scientific arena. In short, I consider what scientific fruits have developed from the seeds provided by early fundamental laboratory and clinical observations.

ERYTHROPOIETIN AND THE CONTROL OF ERYTHROPOIESIS After joining the NIH in 1956, I continued my interest in erythropoietin and erythropoiesis on the NCI Metabolism Service with Nathaniel Berlin. I was assigned to a co-mentor, Jessie Steinfeld, who focused on the field of serum protein metabolism. Jessie left the NIH shortly after I joined his laboratory, and despite my lack of research training, I became de facto a senior investigator with my own laboratory. For a short period I followed two scientific paths: the study of erythropoietin and erythropoiesis and the metabolism of serum proteins. In terms of erythropoietin and erythropoiesis I was lucky to have Nathaniel Berlin as my mentor and to have Wendell Rosse join me as the first medical staff fellow that I chose myself. As has often been true with my associates, Wendell brought with him an interesting scientific challenge and his own novel approach to answering it. His question was, Are extrarenal sites involved in the production of erythropoietin in response to anoxia? To address this issue, we generated parabiotic rats and held them in a device that permitted the introduction of room air or hypoxic air to either one of the two joined animals whose circulations shared only a capillary connection. We nephrectomized

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one of the partners of the parabiotic pairs. When only the nephrectomized partner was exposed to hypoxic air, increased erythropoiesis of the pair was observed, which confirmed an extrarenal site of erythropoietin production. This observation became of special relevance when we examined patients who had erythrocytosis associated with nonrenal tumors. We demonstrated that in patients with erythrocytosis their extrarenal tumors produced erythropoietin (1). In particular, we focused on a patient, JGH, who came to the NIH complaining of a staggering gait. He had been picked up by the police, not for the first time, on the assumption that he was drunk. This recurrent humiliation discouraged him and in his depressed state, he told the police that he would go home by jumping off the Calvert Street Bridge (now the Duke Ellington Bridge) if a solution to his medical dilemma was not sought. The police delivered him to the recently established Clinical Center of the NIH. We were called and admitted him to our service, where he participated in a study concerning the metabolism of serum proteins. However, we soon recognized that he had marked erythrocytosis not associated with an elevation of the other formed elements of the blood. He had von Hippel-Lindau disease with a cerebellar hemangioblastoma, which caused his ataxia as well as the associated polycythemia. Wendell and I chemically extracted from this patient’s cerebellar hemangioblastoma tumor, which had been surgically removed as required for his clinical care, the tissue that we demonstrated contained an erythropoiesis-stimulating molecule that shared the physical characteristics of erythropoietin. In the four decades since these early studies, there have been dramatic advances in the field involving erythropoietin. These include the identification and molecular cloning of the gene encoding erythropoietin. This normal hormone has become a major therapeutic agent for patients with anemia associated with renal disease, those with AIDS, and those receiving cancer chemotherapy. In addition, in the field of von Hippel-Lindau (VHL) disease, other investigators at the NCI including Berton Zbar, Marston Linehan, and Richard Klausner demonstrated that a disordered VHL gene is responsible for the disease as well as for select forms of renal cancer, and they have defined its role as a suppressor oncogene (2). Furthermore, the von Hippel-Lindau tumor suppressor protein was shown to play a role in regulating the degradation of the hypoxia-inducible factor Ia, providing a linkage between the genetic error in the VHL disease and the excessive erythropoietin production that led to the erythrocytosis observed in our patient.

´ INTO IMMUNOLOGY THROUGH ENTREE THE STUDY OF NORMAL AND DISORDERED IMMUNOGLOBULIN METABOLISM The dominant focus of my research over the past four decades has been part of the fabric of immunology dedicated to understanding the complex of interacting cells and antibodies that protect us from infection. My own entry into this field was through the back door, by the study of serum protein metabolism and turnover.

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Initially the focus of our studies was on the metabolism of albumin in normal individuals and those with hypoalbuminemia due to either reduced synthesis or a short protein survival that we showed to be associated, in many cases, with proteinlosing enteropathy. We soon turned from albumin metabolism to the metabolism of the five major classes of immunoglobulins. We demonstrated that they have different rates of synthesis, patterns of distribution, and rates of catabolism in normal human individuals (3). Furthermore, we found that naturally occurring and synthetically produced Fc immunoglobulin fragments have metabolic characteristics comparable to those of the parent molecule, whereas light chains and Fab fragments are far more rapidly catabolized and appear to be handled by renal metabolic pathways that are different from those of the whole molecule. We indicated that the metabolism of such small immunoglobulin fragments involves passage through the glomerulus and then catabolism in the proximal convoluted tubule of the kidney. The studies of the pharmacokinetics of the five immunoglobulin classes and their subunits provided the scientific basis for the dosing schedules that are now used in the development of rational regimens that employ monoclonal antibodies and their fragments in the treatment of human disease. One of the physiological factors that controls the immunoglobulin G (IgG) catabolic rate, as well as its specific saturable transport across the neonatal gut of the mouse and the placenta of humans, was shown by Brambell to involve the IgG concentration (4). We also demonstrated a direct relationship between the serum IgG concentration and its fractional catabolic rate in humans. A high serum IgG concentration was associated with a short IgG survival (3). In particular, in humans the fraction of the intravascular pool of IgG catabolized daily rose from 2% in patients with hypogammaglobulinemia associated with decreased IgG synthesis to an upper asymptomatic limit of 16–18% in patients with multiple myeloma and associated serum IgG concentrations over 30 mg/ml (3). Brambell suggested that this concentration-catabolism effect could be explained by postulating a saturable carrier-mediated protection system specific for IgG molecules (4). Similarly, the transport of immunoglobulins from the mother to the fetus or newborn appeared to utilize a saturable IgG-specific process that could be competitively inhibited by the addition of homologous or heterologous IgG or its Fc fragment. In the early 1970s we reexamined this issue and demonstrated that receptors could be extracted from the rat neonatal intestine and from the adult carcass. These receptors bound IgG molecules and had the characteristics of an IgG-specific receptor involved in the normal perinatal transport, as well as the concentration-catabolism effect of this immunoglobulin class (5). In 1989, using biochemical and molecular techniques, Simister & Mostov (6) demonstrated that this IgG-specific transport and catabolism receptor was a heterodimer involving the β2-microglobulin light chain that was associated with an IgG Fc–specific receptor structurally related to major histocompatibility complex (MHC) class I molecules. In subsequent years we applied our analyses of immunoglobulin metabolism to the study of the pathogenesis of the reduced serum immunoglobulin concentrations in patients with different forms of hypogammaglobulinemia. We demonstrated that

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such disorders of immunoglobulin concentration may occur secondary to a variety of pathophysiological mechanisms that include decreased synthesis of all classes of immunoglobulin or reduced synthesis of only one or two immunoglobulin classes. In one of our early contributions we defined new categories of immunoglobulin deficiency diseases caused by defects of endogenous protein catabolic pathways or loss rather than by abnormalities of immunoglobulin synthesis (3, 7–9). For example, we demonstrated that hypogammaglobulinemia may be the result of hypercatabolism affecting a single immunoglobulin class such as we observed in patients with myotonic dystrophy and a selective decrease in IgG survival (7). In other patients the short survival of a particular immunoglobulin class was due to the development of circulating antibodies to that class, as observed in select patients with IgA deficiency who had developed an anti-IgA antibody. Alternatively, we showed that endogenous hypercatabolism may affect all immunoglobulin classes and albumin, as we reported in patients with what we called familial hypercatabolic hypoproteinemia, a previously unreported syndrome that involved reduced protein serum concentrations associated with reduced survival of diverse immunoglobulin classes and albumin (8). Our group in collaboration with Robert Gordon discovered yet another form of disordered protein metabolism leading to hypogammaglobulinemia, in this case, associated with the excessive bulk loss of serum proteins into the gastrointestinal tract by what we called protein-losing gastroenteropathy (9). By using radiolabeled proteins, we demonstrated that diverse patients previously diagnosed as having idiopathic hypoproteinemia had protein-losing gastroenteropathy as the pathophysiological mechanism leading to their hypoproteinemia (9). We developed two of the techniques that permitted the identification and quantification of such gastrointestinal protein loss, the 67Cu-ceruloplasmin and 51Cr labeled albumin clearance tests (10). Protein-losing enteropathy was shown by our group and by others to occur as an associated feature of over one hundred disorders that affect the gastrointestinal tract (9). Through the analysis of these disorders we were able to define a number of new clinical syndromes that included allergic gastroenteropathy, which involves an abnormal response to milk, and the syndrome intestinal lymphangiectasia (11). Intestinal lymphangiectasia, which we defined four decades ago, was shown by our group to represent a generalized disorder of the development of lymphatic channels including those of the gastrointestinal tract (termed Waldmann’s disease by the National Organization for Rare Disorders). In conjunction with my associate Warren Strober, we demonstrated that intestinal lymphangiectasia involves the gastrointestinal loss of both serum proteins and lymphocytes, which represents the pathological equivalent of a thoracic duct fistula and causes a novel form of immunodeficiency characterized by profound hypogammaglobulinemia, lymphocytopenia, skin anergy, and impaired homograft rejection (12). Both sporadic and familial cases of intestinal lymphangiectasia were defined. By way of an update on this syndrome, the VEGF R-3, a receptor tyrosine kinase known to be pivotal for normal lymphatic development, was shown to be defective in some patients with a familial, generalized lymphatic abnormality (13).

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IMMUNOREGULATORY DISORDERS STUDIED VIA GENETIC IMMUNODEFICIENCY DISEASES As noted above, we used an analysis of disorders of immunoglobulin metabolism as our entrée into the field of immunology, but we soon enlarged our focus to involve the pathogenesis of the immunological disorders of patients with hereditary abnormalities in their levels of immunoglobulins as well as cellular elements of the immune system. We initially focused on these rare but instructive patients to help define the stages in the sequential development of bone marrow stem cells into mature effectors of the immune system, and to obtain insights into the pathogenic role played by disorders in the network of interacting regulatory T cells that normally control the immune response. During the late 1950s and early 1960s, the understanding of immunology expanded rapidly as the result of experimental work in whole animals and the study of human patients who had either a lymphocytic leukemia or one of the hereditary primary immunodeficiency diseases. The analysis of these latter patients, who essentially had experiments of nature in which one or another essential component of body defenses was missing or aberrant, led to the recognition that disordered immune responses could reflect either intrinsic defects in the development and function of B lymphocytes or abnormalities of the regulatory network involving either helper or suppressor T cells. In the course of our studies of immunoglobulin metabolism, we identified patients with an array of disorders associated with defective immunoglobulin synthesis. In an effort to define the pathogenic mechanisms underlying these synthetic defects we and others developed techniques to analyze the terminal differentiation of human B lymphocytes examined ex vivo. The B cells were stimulated with lectins such as pokeweed mitogen to induce them, in the presence of T cells, to become immunoglobulin-synthesizing and -secreting cells. In addition, we developed co-culture techniques to assist in identifying disorders of the helper and suppressor T cells that normally regulate B cell maturation by facilitating or inhibiting this process (14). In the majority of patients, the hereditary immunoglobulin deficiency was associated with an intrinsic B cell defect. Within this broad group we were able to identify new immunodeficiency syndromes including X-linked hypogammaglobulinemia associated with isolated growth hormone deficiency, a syndrome that could be distinguished from Bruton’s X-linked agammaglobulinemia, which is associated with a Btk tyrosine kinase abnormality (15). In our co-culture studies the mononuclear cells of some groups of patients with immunodeficiency but with normal B cells were shown to manifest a helper T cell defect. In particular a helper T cell abnormality was the major abnormality in patients with X-linked hyper-IgM. The B cells of such patients could produce IgM molecules but could not accomplish a normal immunoglobulin class switch. The addition of irradiated normal T cells ex vivo to the peripheral blood B cells of such patients allowed them to produce additional classes of immunoglobulin molecules in vitro in the biosynthesis co-culture system stimulated by pokeweed mitogen. In a collaborative effort, Lloyd Mayer demonstrated that the

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lymphocytes of such patients with the X-linked form of hyper-IgM would synthesize the other immunoglobulin classes when the patients’ cells were co-cultured with the “switch” T cells from a patient with the helper T cell leukemia—the Sézary syndrome (16). Subsequently a number of other groups demonstrated that patients with the Xlinked hyper-IgM syndrome have a defect in their expression of the CD40 ligand (CD40L) on activated helper T cells that normally are involved in the immunoglobulin class switch manifested by B lymphocytes. Helper T cell disorders along with B cell defects also contributed to the dysgammaglobulinemia that characterizes the ataxia telangiectasia syndrome. Such patients have an immature thymus gland that lacks Hassall’s corpuscles. It had been proposed that this failure of thymus development was secondary to an abnormality of the interactions between the mesodermal and entodermal anlage that are necessary for the maturation of this organ. I reasoned that a similar failure of organ development might lead to immaturity of the liver, an organ that also requires these interactions. To address this hypothesis we established a radioimmunoassay for alpha fetoprotein and reported an elevation of this fetal protein in all of the patients with ataxia telangiectasia we studied (17). The establishment of this radioimmunoassay along with one for human chorionic gonadotropin (HCG) permitted us to segue into tumor immunology by introducing these assays into the evaluation of patients undergoing therapy for germ cell tumors of the testis. In a series of studies in collaboration with different clinical trial groups we showed that the two radioimmunoassays taken together provided valuable surrogate markers of disease activity (18). Persistence of an elevated alpha fetoprotein or HCG level indicated the presence of residual tumor and predicted a recurrence in those patients who do not receive further chemotherapy (18). The application of these tests was of critical value in the subsequent development of curative chemotherapeutic regimens for germ cell tumors.

Antigen-Nonspecific Suppressor T Cells Stimulated by Richard Gershon’s discovery of antigen-specific suppressor T cells in mice, I considered the possibility that a subset of patients with common variable hypogammaglobulinemia might have aberrant antigen-nonspecific suppressor T cell activity as a pathogenic factor underlying their broad immunodeficiency. When the immunoglobulin biosynthesis in vitro co-culture assays were applied to the peripheral blood mononuclear cells of patients with common variable immunodeficiency, the majority of patients with this disorder were shown to have intrinsic B cell defects. However, in another subset involving 10–15% of the patients, excessive numbers of circulating activated suppressor T cells inhibited the B cell maturation and immunoglobulin synthesis of co-cultured normal peripheral blood mononuclear cells (14). The B cells of the patients in this latter subset were able to synthesize IgM normally when freed ex vivo of their suppressor T cells but could not do so when their own T cells were returned to the mononuclear cell cultures. These studies represented the first demonstration of aberrant activated

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T cell–mediated suppression of an immune response in humans, thus changing the way investigators thought about the pathogenesis of certain immunological disorders. During these early efforts to understand immunodeficiency diseases, the diseases were viewed as disorders of the pathways of B cell differentation and maturation and their interactions with regulatory T cells. In the years since, over 75 molecular genetic defects have been defined that not only provide major insights concerning the pathogenesis of immunodeficiency diseases but also define the role of different immunological cells and their interactions in the normal immune response. These more recent discoveries by others have also provided new approaches to the diagnosis, including intrauterine diagnosis of immunodeficiency diseases. Furthermore, the definition of the underlying genetic defects provided the scientific basis for rational therapeutic strategies, including the first successful approach to gene therapy for one of these genetic disorders, the X-linked severe combined immunodeficiency disease (SCID), which is associated with a disorder of the common gamma chain (γ c) (19).

HUMAN LEUKEMIAS WITH RETAINED FUNCTIONS PROVIDED INSIGHTS INTO THE REGULATORY NETWORK OF T CELLS Although the studies of genetic primary human immunodeficiency diseases were of great heuristic value, our understanding of the immune system was hindered by the fact that unseparated lymphocytes represent mixtures of complex populations of cells with different and at times opposing functions. They presented a confusing Tower of Babel sending diverse and often conflicting signals that were difficult to analyze. Monoclonal antibodies and the techniques to clone lymphocytes had not been developed. To circumvent this problem, my associate Samuel Broder and I took advantage of the fact that in a given patient human lymphoid leukemic cells represent homogeneous populations of T cells that could theoretically retain a single function. Using the ex vivo immunoglobulin biosynthesis co-culture assay we were able to show that some leukemic T cells, in particular those of patients with the Sézary syndrome, can act as helper T cells when co-cultured with normal B cells. In contrast, the T cells of patients with human T cell lymphotropic virus I (HTLV-I)-associated adult T cell leukemia (ATL) profoundly inhibited immune responses by functioning as immune suppressors in such co-cultures (20, 21). Our patients with this latter leukemia were profoundly anergic and were unable to make either antibody responses or skin test responses to recall antigens. Effective therapy of these patients with ATL was associated with a return to normal immune function in vivo. In the period following the publications of our studies on suppressor T cell abnormalities in common variable hypogammaglobulinemia and ATL, concern was expressed about our focus on such suppressor T cells as pathogenetic factors underlying the immunodeficiency state. This in part reflected the difference in the

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scientific interest of investigators focusing on the immune responses of mice and our interest focusing on those of humans. The scientists focusing on mice in their analysis of immune responses were especially interested in antigen-specific, genetically restricted immune responses, whereas our studies focused on genetically nonrestricted, antigen-nonspecific events mediated by suppressor elements. With the decline in acceptance of antigen-specific suppressor cells in mice there was a parallel loss of interest in human suppressor T cells. However, recently there has been a major reawakening in the acceptance of antigen-nonspecific suppressor T cells with the definition of the importance, in mice, of a population of CD4+, CD25+ (IL-2Rα) suppressor or negative regulatory T cells (22). It should be noted that in 1984 we reported that the HTLV-I-associated ATL cells of our patients functioned as profound suppressor cells and had the phenotype CD4+, CD25+, the phenotype that has now been identified as that of the negative immunoregulatory cell (21). More recently, in conjunction with Thomas Fleisher of the NIH Clinical Center, we demonstrated that these ATL cells have the full phenotype characteristic of the suppressor cells that have recently been the focus of this reawakened interest. In particular, ATL T cells manifest the CD4+, CD25+, CD62L, high CD45RO+, and CTLA4+ phenotype. It is our hypothesis that the HTLV-I-associated ATL cells that we reported functioned as suppressor cells in our ex vivo co-culture studies in the early 1980s represent a human leukemic T cell expansion of the recently reaccepted CD4+, CD25+ negative immunoregulatory T cell. Taken as a whole, these studies of human immunodeficiency diseases and T cell leukemias have provided valuable insights into the complex regulatory network of cells that controls the human immune response.

MOLECULAR GENETIC ANALYSIS OF IMMUNOGLOBULIN AND T CELL RECEPTOR GENES IN HUMAN LYMPHOID NEOPLASMS Until the mid-1970s, the cardinal question regarding the immune system remained unanswered: How does our body with its limited amount of genetic material generate a diversity of antibodies and T cells that can recognize a myriad of foreign configurations in our environment? The solution to this paradox emerged from the brilliant studies of Tonegawa, Leder, and Hood, who used recombinant DNA technology to show that the genes encoding antibodies and those for T cell receptors utilize discontinuous bits of genetic material. Like letters of the alphabet, these bits can be shuffled and rearranged into many combinations to yield diverse sequences, a mechanism underlying one component of the observed immunoglobulin and T cell receptor diversity. Phillip Leder, aware of our interest in the hereditary immunodeficiency diseases, suggested a collaboration to examine the immunoglobulin genes of such patients. However, the disordered genes associated with the majority of primary immunodeficiency diseases and those encoding the immunoglobulin molecules were present on different chromosomes. As an alternative subject

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for study we correctly reasoned that Southern blot analysis of immunoglobulin gene rearrangements could serve as tumor-specific clonal markers capable of detecting even minority populations of monoclonal cells in patients with different forms of lymphocytic leukemia. Our initial studies applying immunoglobulin gene rearrangement analysis to human cells have had wide-ranging implications for the diagnosis and the monitoring of therapy of B and T cell neoplasms. Stanley Korsmeyer, Phillip Leder, and I used this analysis of receptor gene rearrangements to define the lineage (T or B cell) of leukemic cells lacking conventional markers, to establish whether the abnormal lymphocytic populations were polyclonal, oligoclonal, or monoclonal, to determine the state of maturation of leukemic B and T cell precursors, and to broaden the scientific basis for the diagnosis and monitoring of the therapy of lymphoid neoplasms (23). For example, we established that the majority of what had been referred to as the non-T/non-B form of acute lymphoblastic leukemia represented a developmental series of B cell precursors. Furthermore, this analysis was the first to establish a hierarchical order of immunoglobulin gene rearrangements in humans wherein heavy chain gene rearrangements precede those of light chains and kappa gene rearrangements precede lambda gene rearrangements.

INTERLEUKIN-2 AND ITS RECEPTOR The latest adventures in my odyssey in immunology have involved the critical roles played by the cytokines IL-2 and IL-15 and their receptors on the growth and differentiation of normal and neoplastic T cells. The basic insights concerning the IL-2 and IL-15 systems are being translated into receptor-directed, monoclonal antibody–mediated strategies for the treatment of patients with leukemias and lymphomas and those with autoimmune diseases, as well as for the prevention of allograft rejection. Our work on the IL-2 receptor emerged during a halcyon period for our group, a moment two decades ago when within our laboratory we were joined by Takashi Uchiyama, Stanley Korsmeyer, Warner Greene, Warren Leonard, and Andrew Arnold, and by Carolyn Goldman, who has been my indispensable coworker for over 25 years. In the early 1980s, when these studies were initiated, the HIV virus had not been identified, the T cell antigen receptor had not been cloned, and our understanding of the regulation of the immune system and its function was quite different from that of today. Our efforts became focused on the question, How do T cells grow and develop effector functions following activation? Takashi Uchiyama joined the laboratory shortly after Köhler & Milstein’s development of hybridoma technology, and their initial production of monoclonal antibodies captured the imagination of biomedical scientists. Uchiyama’s production of the anti-Tac monoclonal antibody directed toward the IL-2 receptor alpha subunit was a classic example of serendipity (24). He used a T cell line we developed from a patient, CR, who carried the diagnosis of Sézary T cell leukemia, but who in retrospect had HTLV-I-associated adult T cell leukemia, as a target in our efforts

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to produce an anti-CD4 antibody during a period when such antibodies were being embargoed and were not made available to the scientific community. However, the antibody we produced, anti-Tac (T cell activation antigen), did not target the CD4 antigen but reacted with what we now recognize as the alpha subunit of the receptor for interleukin-2 (then termed T cell growth factor) that had just been discovered by Morgan, Ruscetti, and Gallo (25). At that time the mode of action of IL-2 was undefined; that is, which cells expressed its receptor and under what conditions. Warren Leonard and Warner Greene joined our laboratory and determined that anti-Tac blocked the ability of IL-2 to stimulate T cells to divide (26). This was an important finding since anti-Tac was one of the first, if not the first, monoclonal antibody to define a receptor for one of the cytokines that immune cells use to communicate with one another. We biochemically characterized the receptor peptide identified by anti-Tac as a densely glycosylated, sulfated, integral membrane protein with an apparent Mr of 55,000. Along with two other groups, using the anti-Tac monoclonal antibody to purify the receptor peptide, Warren Leonard and other members of our group succeeded in cloning, sequencing, and expressing cDNAs encoding the 33-kDa polypeptide backbone of the 55-kDa IL-2 receptor protein (27). Based on this DNA sequence, the primary structure of this IL-2Rα receptor peptide was shown to be composed of 272 amino acids including a 21-residue signal peptide and a short 13-amino-acid intracytoplasmic domain that was too short to function in signaling. This raised the issue of how this receptor’s signals were transduced to the nucleus. Furthermore, questions were posed concerning the IL-2 receptor that were difficult to answer when only the 55-kDa IL-2Rα peptide was considered. These questions included: What is the structural explanation for the great difference in affinity between high (10−11 M) and low (10−8 M) affinity receptors? How could certain non-Tac-expressing cells including resting natural killer cells respond to IL-2? In association with Mitsuru Tsudo, who had joined our laboratory, we resolved these issues in parallel with investigators in the Leonard laboratory by codiscovering a novel non-Tac IL-2 binding protein, IL-2Rβ, with an Mr of 75,000 (28). We proposed a multichain model for the high affinity IL-2 receptor. Subsequently, Sugamura and coworkers discovered the IL-2Rγ chain, or γ c, that is required for high affinity IL-2 binding and signaling (29). In subsequent studies, we demonstrated that the alpha chain of the IL-2 receptor identified by the anti-Tac monoclonal antibody is not expressed by the majority of normal resting cells but is expressed by the abnormal T cells of patients with a wide range of diseases including different forms of human leukemia, the T cells involved in the pathogenesis of autoimmune disorders, and the T cells participating in organ allograft rejection. This discovery represented a major turning point for our clinical efforts because it suggested that we could exploit the difference in IL-2 receptor expression between normal cells and T cells by designing IL-2R-directed agents to eliminate unwanted CD25-expressing T cells and thereby treat human disease. Our developments of IL-2Rα- and IL-2Rβ-directed monoclonal antibody strategies that dominate our clinical trials are considered below.

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INTERLEUKIN-15 The most recent scientific pathway in the immunological odyssey of our lab was initiated with our codiscovery of the cytokine interleukin-15 (30). This discovery emerged from a paradoxical observation that was difficult to explain with the paradigms of the time. The observation was made during our IL-2 receptor– directed clinical trials that involved patients with HTLV-I-associated adult T cell leukemia (ATL). In the early phases of ATL, the leukemic cell proliferation is linked to an autocrine process involving the coordinate expression of IL-2 and IL-2Rα. However, in late phase ATL, the leukemic cells no longer produce IL-2 yet continue to express increasing numbers of cell surface IL-2 receptors. As part of our studies of this IL-2-independent phase of the ATL leukemic cell proliferation, we focused on HuT-102, an ATL cell line that was derived from patient CR and that manifested over 100,000 IL-2Rs per cell yet did not express mRNA encoding IL-2. We showed that the supernatants from cultures of this cell line stimulated the proliferation of an indicator cytokine-dependent cell line, CTLL-2. Furthermore, this stimulatory action was not inhibited by the addition of an antibody to IL-2 but was associated with a previously undefined 14- to 15-kDa lymphokine that required expression of the IL-2Rβ subunit for its stimulation of T cell proliferation and for its induction of NK cell activation (30). Grabstein and coworkers (31) simultaneously reported the recognition of this cytokine, now known as IL-15, which they isolated from supernatants of a simian kidney epithelial cell line, CV1/EBNA. With the use of an antibody to IL-15 we demonstrated that our factor and that of Grabstein’s group were identical. The cDNA defining IL-15 encodes a 162-amino-acid peptide with a 48-amino-acid leader sequence yielding a 114amino-acid mature protein. Our codiscovery of IL-15 emphasizes the important role played by translational research wherein observations made using material derived directly from patients can open new basic science arenas. It is of note that the HuT-102 cell line used in these studies to identify IL-15 was from the patient CR discussed above, who had adult T cell leukemia. Cell lines from this same patient were utilized by Poiesz and coworkers (32) to discover the first pathogenic human retrovirus, HTLV-I, and were the target cells that we used in the generation of the anti-Tac monoclonal antibody that first defined an IL-2 receptor subunit (24) and that, as discussed below, has been used in our IL-2 receptor–directed clinical trials. We helped to define two distinct receptor and signaling pathways that are used by IL-15 in diverse cells. As predicted from the ability of IL-15 to stimulate the proliferation of the putatively IL-2-specific, cytokine-dependent CTLL-2 cell line, we demonstrated that IL-15 uses a multisubunit receptor in T and NK cells that involves the IL-2Rβ chain shared with IL-2, as well as the common γ c subunit shared with IL-2, IL-4, IL-7, IL-9, and IL-21 (30). Giri and coworkers (34) demonstrated that the high affinity IL-15 receptor also includes a private IL-15Rα receptor element. In an effort to define functions mediated by IL-15 and not shared with IL-2, Yutaka Tagaya of our group focused on mast cells that do not respond to

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IL-2 since they lack the required IL-2Rβ chain (33). He postulated that if mast cells responded to IL-15, it would indicate that IL-15 utilizes non-IL-2R components in these cells. In accord with his prediction, we demonstrated that IL-15 is a mast cell growth factor and that its signaling in such cells involves a receptor system that does not share any subunits with the IL-2/IL-15 receptor system of T cells. Rather this mast cell receptor uses a 60- to 65-kDa novel receptor, IL-15RX. Furthermore, this receptor employs a signal transduction pathway involving Jak-2 and STAT-5, in contrast to the Jak-1/Jak-3 and STAT-3/STAT-5 pathway used by the IL-2/IL-15R system in T cells.

The Contrasting Roles of IL-2 and IL-15 in the Life and Death of Lymphocytes We compared and contrasted IL-2- and IL-15-mediated functions in terms of the fundamental goals of the immune system. In a simplified form these include (a) the generation of rapid innate (e.g., NK cell) and adaptive (e.g., antibody and T cell) immune responses to invading pathogens, (b) the maintenance of a specific memory response to these pathogens, and (c) the elimination of autoreactive T cells to yield tolerance to self. As might be anticipated from their shared use of the IL-2Rβ and γ c subunits in T and NK cells, IL-15 and IL-2 share a number of functions including the stimulation of T cell proliferation, activation of NK cells, and the induction of immunoglobulin synthesis by human B cells costimulated with anti-IgM or anti-CD40. Furthermore, a special role for IL-15 has been demonstrated in the development of NK cells, as well as in the development and persistence of memory phenotype CD8+ cells. Yutaka Tagaya in our laboratory prepared transgenic mice expressing human IL-15. These mice manifested a major increase in the number of their NK cells, NK-T cells, and CD44hi, CD8+ memory phenotype T cells (35). A major advance emerging from our laboratory and those of others is the finding that although IL-2 and IL-15 share two receptor subunits and some functions, their contributions in some aspects of the life and death of lymphocytes are distinct and at times competing (35–38). Although IL-2 is an important growth and survival factor, it also plays a pivotal role in FasL-mediated activation induced cell death (AICD) of CD4 T cells. Receptor-mediated stimulation of CD4 T cells by antigen at high concentration induces the expression of both IL-2 and the IL-2 receptor, which in turn interact to yield T cell activation and cell cycling. Antigen restimulation of the cycling T cells at this stage through the T cell antigen receptor increases the transcription and expression of the death effector molecule FasL. We demonstrated that IL-15, in contrast to IL-2, acts to extend the survival of lymphocytes both by acting as a growth factor and by inhibiting CD4+ T cell AICD mediated by IL-2 (35, 37). In particular, in ex vivo studies, CD4 T cells from the IL-15 transgenic mice that Tagaya developed did not manifest IL-2-mediated AICD (35). In addition to their distinct actions in AICD, IL-2 and IL-15 play opposing roles in the control of the homeostasis of CD8+ memory phenotype T cells (35–38). Again IL-15 is involved in the maintenance of T lymphocyte

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survival whereas IL-2 has the opposite effect, leading to their loss. Ku and coworkers (36) reported that the division of CD8+ T cells of memory phenotype is stimulated by IL-15, but is inhibited by IL-2. We, in turn, demonstrated that IL-15 transgenic mice have abnormally elevated numbers of CD8+ memory phenotype T cells (35). Furthermore, we defined a role for IL-15 and its beta receptor in HTLVI-associated ATL and the neurological disease tropical spastic paraparesis (TSP) (38). Using tetramer technology, Nazli Azimi of our group and Steven Jacobson showed that patients with TSP have a marked increase in circulating MHC class I (A 201) restricted, antigen-specific (amino acids 11–19 of the HTLV-I-encoded tax protein) reactive CD8+ cells, suggested to be involved in the pathogenesis of TSP (38). Azimi monitored the survival of such CD8 antigen-specific T cells ex vivo in the presence or absence of antibodies to the cytokines IL-2 and IL-15 or to their receptors and thereby demonstrated that the addition of antibodies to IL-15 or to its receptor beta subunit in such ex vivo cultures led to a rapid (within six days) reduction in the number and function of antigen-specific memory phenotype CD8+ cells. In contrast, the addition of antibodies to IL-2 or to its private IL-2Rα receptor did not have this effect. Taken together, the studies support the view that in their special adaptive immune functions, IL-2 and IL-15 favor opposing actions that emphasize one or the other of the two competing major goals of the immune response. IL-2, through its contribution to AICD for CD4+cells and its interference with the persistence of memory CD8+ phenotype T cells, favors the elimination of lymphocytes directed toward self-antigens and thus plays a critical role in the maintenance of peripheral self-tolerance. In contrast, IL-15, through its inhibition of IL-2-mediated AICD and its positive role in the maintenance of CD8+ memory phenotype T cells, favors the maintenance and survival of T cells that are of value in providing a long-term specific memory immune response to foreign pathogens. In accord with this role for IL-15, the elimination of vaccinia virus was more effective in our IL-15 transgenic mice than in wild-type mice (35). An analysis of IL-2 and IL-2Rα as well as of IL-15 and IL-15Rα knockout mice support these conclusions concerning the competing roles for IL-2 and IL-15 in AICD and in the expression of memory phenotype CD8+ cells (37).

Regulatory Controls Affecting IL-15 Expression The uncontrolled expression of IL-15 carries the risk to the organism of the survival of autoreactive T cells that could lead to the development of autoimmune diseases. As just noted, IL-15 is a potentially dangerous inflammatory cytokine in that it inhibits self-tolerance mediated by AICD, facilitates the persistence of CD8+ memory T cells, and induces the expressions of TNFα, IL-1β, and inflammatory chemokines. In terms of the regulation of cytokine expression, IL-2 is produced by activated T cells, and its synthesis is controlled at the levels of mRNA transcription and stabilization. In contrast, we have defined a complex multifaceted regulation of IL-15 expression (39–42).

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IL-15 is not produced by T cells, but there is widespread expression of IL-15 mRNA in a wide variety of tissues and cells. Using interferon regulatory factor-1 (IRF-1) knockout mice, we in conjunction with the laboratory of Tadamitu Taniguchi showed IRF-1 to be a critical element in the induction of IL-15 transcription (42). Furthermore, we demonstrated that the HTLV-I tax protein transactivates IL-15 transcription through the action of NF-κB. Nevertheless, the regulation of IL-15 expression is predominantly posttranscriptional and occurs at the levels of IL-15 mRNA translation and protein trafficking and translocation within the cell. In particular, although IL-15 message (that is, mRNA encoding IL-15) is widely expressed constitutively, it has been difficult to demonstrate the IL-15 cytokine in the supernatants of many cells that express such message (39–41). In particular, we observed that although monocytes activated with LPS and IFN-γ express high levels of mRNA encoding IL-15, culture supernatants from these cells did not contain sufficient IL-15 to be identified by either an IL-15-specific assay (enzyme-linked immunosorbent assay, ELISA) or by the CTLL-2 proliferation assay (39). This demonstration of a discordance between IL-15 message expression and IL-15 protein secretion led us to examine normal IL-15 mRNA for posttranscriptional controls, particularly for features that would impede IL15 production at the level of mRNA translation. We demonstrated that the IL-15 message includes a number of elements that are impediments to its translation. In particular, the 50 UTR of the normal human IL-15 message is burdened with 13 upstream AUGs that interfere with efficient IL-15 translation. Furthermore, the unusually long 48-amino-acid signal peptide sequence and a cis-acting element in the 30 mature protein-coding region interfere with this process. Taken together, these observations suggest that IL-15 mRNA, unlike IL-2 mRNA, may exist in translationally inactive pools. As a hypothesis, we propose that by maintaining a pool of translationally inactive IL-15 mRNA, mononuclear cells may respond to an intracellular infectious agent by unburdening the IL-15 message, thereby transforming it into one that can be efficiently translated and yielding IL-15 that would facilitate the activation of NK and T cells that could clear the pathogen. Our current efforts are directed toward defining the putative molecular events involved in unburdening the IL-15 message to facilitate its translation.

Disorders of IL-15 Expression in Patients with Autoimmune Diseases and Disorders Associated with the Retrovirus HTLV-I Despite the complex regulation of IL-15, abnormalities of IL-15 expression have been described in patients with rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, autoimmune chronic liver disease, T cell–mediated alveolitis, and diseases associated with the retroviruses HIV and HTLV-I. Our group has had a special interest in the study of HTLV-I-associated tropical spastic paraparesis (TSP). In particular, T cells from patients with TSP manifest spontaneous proliferation in ex vivo culture in the absence of exogenously added cytokines. We

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demonstrated that this proliferation is partly a result of the fact that the HTLV-Iencoded tax protein transactivates the expression of IL-2 and IL-2Rα; addition of antibodies to IL-2 or its private receptor subunit partially inhibited the proliferation (43). This coordinate expression of IL-2 and its receptor establish an autocrine loop, which participates in the abnormal proliferation of HTLV-I-infected T cells. However, this observation left open the question of why the blockade of the interaction of IL-2 with its receptor in TSP did not completely abrogate the ex vivo mononuclear cell proliferation. In studies to address this question, we demonstrated that the HTLV-I-encoded tax protein also transactivates the transcription of both the IL-15 and IL-15Rα genes through a mechanism involving an NF-kB site. The addition of antibodies to IL-15 or directed toward the IL-2Rβ receptor shared by IL-15 and IL-2 partially inhibited the spontaneous proliferation of peripheral blood mononuclear cells from patients with TSP, in a fashion similar to the parallel observation with antibodies to IL-2 or to its private receptor. When a combination of both anti-IL-2 and anti-IL-15 antibodies was used, a dramatic inhibition of the ex vivo proliferation was observed, suggesting that both cytokines contribute to the spontaneous proliferation observed in TSP (44). We proposed a model in which at least two autocrine loops involving IL-2 and IL-15 and their receptors are active in HTLV-I-infected T cells and contribute to their abnormal proliferation. Furthermore, as noted above, disordered IL-15 expression in TSP plays a role in the persistence of MHC-I restricted, antigen-specific CD8+ memory and effector T cells that are thought to play a role in the pathogenesis of the disease. As discussed below, we plan to use the monoclonal antibody HuMikβ1 that blocks IL-15 interaction with IL-2/15Rβ, thereby inhibiting IL-15 function, to exploit these observations concerning its role in the pathogenesis of this autoimmune, neurological disease.

THE IL-2/IL-15 RECEPTOR SYSTEMS: TARGETS FOR IMMUNOTHERAPY The FDA on December 10, 1997, approved daclizumab (Zenapax), the humanized version of our anti-Tac monoclonal antibody, for use in humans to prevent acute kidney transplant rejection. This was the first humanized monoclonal antibody approved for use in transplantation. Furthermore, it was the first antibody directed to a cytokine or interleukin receptor to be approved. The FDA decision marked the culmination of a 16-year odyssey in our laboratory which was responsible for a series of discoveries that laid the scientific foundation for the randomized clinical trials and ultimately led to the approval of daclizumab. As noted above, one of our group’s most critical contributions was the recognition that the IL-2 receptor represents an extraordinarily useful therapeutic target for monoclonal antibody action (45–47). Although Köhler & Millstein’s development of hybridoma technology had rekindled interest in the use of antibodies to treat patients, the initial use of monoclonal antibodies as therapeutic agents was

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relatively disappointing. The magic bullet of antibody therapy that had been the dream of immunotherapists since the time of Paul Ehrlich proved to be elusive. Nevertheless, monoclonal antibody–mediated therapy has recently been revolutionized by advances such as genetic engineering to create less immunogenic, more effective agents with better pharmacokinetics by the arming of such antibodies with toxins or radionuclides to enhance their effector functions, and most critically by the definition of new surface structures on cancer and other cells such as growth factor and death pathway receptors as targets for effective monoclonal antibody action. Since 1981, we have focused our therapeutic efforts on the use of monoclonal antibodies directed toward the receptors for the lymphokines IL-2 and IL-15 (45–51). The scientific basis for this approach emerged from our application of the anti-Tac monoclonal antibody to the analysis of IL-2Rα expression by normal and abnormal cells. We demonstrated that the majority of resting T cells and monocytes do not display the alpha subunit of IL-2 receptor identified by anti-Tac. In contrast to this lack of IL-2Rα expression by most normal resting cells, this receptor subunit is constitutively expressed by the abnormal cells in certain forms of lymphoid neoplasia including HTLV-I-associated ATL, cutaneous T cell lymphoma, anaplastic large cell lymphoma, hairy cell B cell leukemia, and Hodgkin’s disease, as well as by the activated T cells in an array of autoimmune diseases such as T cell–mediated uveitis and HTLV-I-associated tropical spastic paraparesis (TSP) and by the T cells of individuals undergoing allograft rejection (48, 49). We took advantage of this difference in IL-2Rα expression between most normal resting cells and T cells in our strategies to eliminate abnormal, activated T cells. In the next phase of these efforts, we translated the fundamental insights from the laboratory and validated them in preclinical animal studies. In particular, in collaboration with Robert Kirkman of Harvard Medical School, we demonstrated the efficacy of therapy with anti-IL-2Rα-directed monoclonal antibodies in the prevention of renal allograft rejection in cynomolgus monkeys. In our own laboratory, we showed that the murine version of the anti-Tac monoclonal antibody delayed cardiac allograft rejection in cynomolgus monkeys (50). The first clinical trials of murine anti-Tac were directed toward the treatment of patients with adult T cell leukemia, in which the malignancy of mature CD4+, CD25+ lymphocytes is caused by the retrovirus HTLV-I (45). No chemotherapeutic regimen appeared successful in altering the survival of these patients, who have a median survival duration of only 9 months. The retrovirus encodes a transactivating protein, tax, that indirectly stimulates the transcription of numerous host genes including those of IL-2 and IL-2Rα. The malignant ATL cells constitutively express approximately 10,000 IL-2Rα subunits identified by the anti-Tac monoclonal antibody, whereas most of the patients’ normal resting cells do not express this receptor. These observations stimulated us to perform therapeutic trials with the unmodified murine version of the anti-Tac monoclonal antibody. Six of the 19 patients treated developed a partial (4 cases) or complete (2 cases) remission;

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in one case remission persists more than 12 years after initiation of therapy (45). Using the murine model of human ATL we developed, we demonstrated that the effector action of anti-Tac appears to result partly from a form of antibody dependent cellular cytotoxicity (ADCC) involving Fcγ R III receptors (46). In patients in the early phase of ATL the effective action of anti-Tac involves both ADCC and the interruption of the interaction of IL-2 with its growth factor receptor, which leads to cytokine deprivation–mediated apoptosis. Despite the encouraging results in these studies, there were three problems inherent in the use of the murine anti-Tac monoclonal antibody: its immunogenicity, its ineffectiveness in recruiting host-effector functions, and its short survival in vivo. To address these issues, I joined with Cary Queen, then of the Biochemistry Branch of the National Cancer Institute and subsequently of Protein Design Labs, to humanize anti-Tac (51). Humanized anti-Tac retains the complementarity determining regions (CDR) from the mouse, whereas virtually all the rest of the molecule is derived from human IgG1κ. The humanized version of anti-Tac is virtually nonimmunogenic, has improved pharmacokinetics (a survival half-life of 40 h for the murine as compared to 20 days for the humanized version), and functions in antibody-dependent cellular cytotoxicity with human mononuclear cells, in contrast to the absence of this effector function for its parent murine version (51). After our encouraging phase I/II trials, Hoffmann-LaRoche, Inc. conducted two double-masked, placebo-controlled randomized trials involving 535 evaluated patients to determine the value of humanized anti-Tac (daclizumab) in preventing renal allograft rejection. In each trial, the patients received a standard immunosuppressive regimen. The parallel treatment groups also received either intravenous placebo or daclizumab prior to the transplant and on four subsequent occasions. No drug-specific adverse events or increased morbidity were observed. Acute rejection episodes were reduced by 40% in the patients treated with daclizumab ( p < 0.01); 98% of the patients receiving triple immunosuppression and daclizumab retained their renal allograft for at least 6 months whereas only 92% of the patients in the placebo-controlled group retained their grafts (p = 0.02) (52). As noted above, on the basis of these phase III clinical trials, daclizumab has received approval for use in the prevention of acute renal rejection episodes in patients undergoing kidney transplantation. In addition to its use in the prevention of organ allograft rejection, our collaborators have shown that humanized anti-Tac is of value in the therapy of T cell–mediated autoimmune disorders including uveitis, multiple sclerosis, and tropical spastic paraparesis. For example, in a clinical trial, patients with noninfectious uveitis who were receiving multiple immunosuppressive agents were weaned off their systemic immunosuppressive medications and in parallel received daclizumab infusions every 4 weeks (53). In 9 of 10 patients treated with daclizumab over a 4-year period, improvement was noted in visual acuity without the use of the previously required immunosuppressive agents. On the basis of these encouraging findings, a phase III controlled trial is being initiated involving humanized anti-Tac therapy for patients with active noninfectious uveitis.

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Systemic Radioimmunotherapy Directed to IL-2Rα One limitation in the use of unmodified monoclonal antibodies to treat leukemia and lymphoma is that they are relatively ineffective as cytocidal agents. This is especially true with late stage HTLV-I-associated ATL, wherein the leukemic cells continue to express IL-2Rα but no longer produce nor require IL-2 for their proliferation. This limited efficacy of unmodified monoclonal antibodies in cancer therapy has led to an alternative approach that involves the use of agents such as anti-Tac as carriers of cytotoxic substances including toxins or radionuclides. In one series of studies, we have collaborated with Ira Pastan and Robert Kreitman of the NCI in the evaluation of their IL-2Rα-directed immunotoxin, LMB-2, that includes a truncated version of Pseudomonas exotoxin A linked with anti-Tac fv to yield a single antibody toxin fusion protein that is used in the therapy of patients with IL-2Rα-expressing leukemias and lymphomas. Our dominant efforts, however, have been to develop a generalized reproducible approach for the systemic radioimmunotherapy of IL-2Rα-expressing malignancy. We augmented the efficacy of murine and humanized anti-Tac by arming the monoclonal antibody with the β-emitting radionuclide 90Y and observed a partial or complete remission in over 50% of patients with ATL treated in clinical trials (47). In sum, the progressive increase in our understanding of the IL-2 receptor and its involvement in disease has opened the possibility for more specific immune intervention. The clinical application of IL-2R-directed therapy has represented a novel approach for the treatment of certain neoplastic diseases and select autoimmune disorders, and for the prevention of organ allograft rejection.

FUTURE THERAPEUTIC DIRECTIONS: IL-15 AS A THERAPEUTIC AGENT AND IL-2/15Rβ-DIRECTED THERAPY FOR AUTOIMMUNE DISEASES In our translational clinical trials program we wish to exploit our expanding understanding of the IL-15/IL-15 receptor system in the normal immune response and its disorders in disease. The opposing effects of IL-2 and IL-15 discussed above have implications for the use of these cytokines as elements in cancer therapy strategies and as components of vaccines. IL-2 has been approved for use in metastatic renal carcinoma and malignant melanoma; however, in the presence of IL-2, the tumor-specific T cells generated may interpret the tumor cells as self and by the activation induced cell death (AICD) process may undergo apoptotic cell death. Furthermore, the inhibition mediated by IL-2 on the survival of those memory CD8+ T cells that are directed toward cancer-associated antigens is not desirable. In contrast, IL-15 with its activation of T cells, its inhibitory action on AICD, and its facilitation of the persistence of memory phenotype CD8+ T cells may be superior to IL-2 in the treatment of cancer and as a component of vaccines. Thus in the future we wish to evaluate IL-15 as a therapeutic agent. We are encouraged

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by the observations of Yutaka Tagaya that syngeneic tumor cells injected intravenously into IL-15 transgenic mice did not develop metastatic foci in the lungs, whereas in wild-type mice, pulmonary tumor masses developed within 2 to 3 weeks. Following preclinical evaluation of IL-15 in murine tumor models, we hope to evaluate IL-15 as a replacement for IL-2 therapy of malignant melanoma and renal cell carcinomas and as a component of vaccines for cancer and AIDS. Our previous studies using intact unmodified monoclonal antibodies have targeted IL-2Rα as the private receptor for IL-2. Although such IL-2Rα-directed therapy has met with considerable success, culminating in the approval by the FDA of the humanized version of the anti-Tac antibody (daclizumab), such approaches with this subunit have their limitations. In particular, antibodies directed to IL-2Rα do not inhibit the actions of IL-15, a cytokine that does not bind to this receptor subunit. An antibody, humanized Mikβ1 (HuMikβ1), that acts on the IL-2/IL-15Rβ receptor subunit shared by IL-2 and IL-15 blocks all actions of IL-15 but not those of IL-2 on T and NK cells. We have shown that the administration of HuMikβ1 as a single agent leads to prolonged cardiac allograft survival in cynomolgus monkeys. We plan to initiate clinical trials of this agent for patients with diseases in which a disorder of the inflammatory cytokine IL-15 has been observed and is proposed to play a role in the disease pathogenesis. In particular, we plan clinical trials of HuMikβ1 in groups of patients with T cell large granular lymphocytic leukemia associated with hematocytopenia, tropical spastic paraparesis, rheumatoid arthritis, and multiple sclerosis. These studies may help us to test the hypothesis that IL-15 plays a pathogenic role in these disorders and to determine whether disrupting IL-15 interaction with its beta receptor subunit will provide clinical benefit to patients with such disorders. As I look to the future, the task of preventing and curing cancer is a difficult one. The road ahead seems long and daunting. I am, however, encouraged about cancer research in general and in particular the use of immune approaches in preventing, diagnosing, and treating cancer. Recent advances in the knowledge of disordered expression of cytokine and growth factor receptors by neoplastic cells in conjunction with progress in linking toxins and radionuclides to monoclonal antibodies or their fragments, as well as the genetic engineering of these antibodies to produce humanized versions of the monoclonal antibodies with reduced immunogenicity and improved pharmacokinetics and functions, provide new hope for the treatment of neoplastic disease. We have come close to fulfilling the vision of Paul Ehrlich, who stated in his Croonian lecture, “On Immunity with Special Reference to Cell Life,” to the Royal Society of London in 1900: It is hoped that immunizations such as these which are of great theoretic interest may also come to be available for clinical application . . . attacking epithelial new formations, particularly carcinoma, by means of specific antiepithelial sera. . . . I trust my lords and gentlemen that we no longer find ourselves lost on a boundless sea but that we have already caught a distinct glimpse

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of the land where we hope, nay, which we expect, will yield rich treasures for biology and therapeutics. It must be emphasized that the outcome of the efforts to prevent and cure cancer is not solely in the hands of scientists. Cancer research including that involving immunological approaches is a public endeavor requiring the support of our entire society. What we have is the hope that all of us together will ennoble humankind by seeing this great and hopeful adventure through to its end.


The Annual Review of Immunology is online at http://immunol.annualreviews.org

LITERATURE CITED 1. Waldmann TA, Levin EH, Baldwin M. 1961. The association of polycythemia with a cerebellar hemangioblastoma: the production of an eythropoiesis stimulating factor by the tumor. Am. J. Med. 31:318–24 2. Latif F, Tory K, Gnarra J, Yao M, Duh FM, Orcutt ML, Stackhouse T, Kuzmin I, Modi W, Geil L, Schmidt L, Zhou F, Li H, Wei MH, Chen F, Glenn G, Choyke P, Walther MM, Weng YK, Dah-Shuhn R, Duan DSR, Dean M, Glavac D, Richards FM, Crossey PA, Ferguson-Smith MA, Le Paslier D, Chumakov I, Cohen D, Chinault AC, Maher ER, Linehan WM, Zbar B, Lerman MI. 1993. Identification of the von HippelLindau disease tumor-suppressor gene. Science 260:1317–20 3. Waldmann TA, Strober W. 1969. Metabolism of immunoglobulins. Prog. Allerg. 13:1–110 4. Brambell FWR. 1966. The transmission of immunity from the mother to young and the catabolism of immunoglobulins. Lancet 1087–93 5. Jones EA, Waldmann TA. 1972. The mechanism of intestinal uptake and transcellular transport of IgG in the neonatal rat. J. Clin. Invest. 51:2916–27 6. Simister NE, Mostov KE. 1989. An Fc receptor structurally related to MHC Class I antigens. Nature 337:184–87 7. Wochner RD, Drews G, Strober W, Waldmann TA. 1966. Accelerated breakdown of immunoglobulin G (IgG) in myotonic dys-

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trophy: a hereditary error of immunoglobulin catabolism. J. Clin. Invest. 45:321–29 Waldmann TA, Terry WD. 1990. Familial hypercatabolic hypoproteinemia: a disorder of the endogenous catabolism of albumin and immunoglobulin. J. Clin. Invest. 86:2093–98 Waldmann TA. 1975. Protein losing gastroenteropathies. In Gastroenterology, ed. HL Bockus, pp. 361–85. Philadelphia: Saunders. 3rd ed. Waldmann TA. 1961. Gastrointestinal protein loss demonstrated by 51Cr-labelled albumin. Lancet 2:121–23 Waldmann TA, Steinfeld JL, Dutcher TF, Davidson JD, Gordon RS. 1961. The role of the gastrointestinal system in “idiopathic” hypoproteinemia. Gastroenterology 41:197–207 Strober W, Wochner RD, Carbone PP, Waldmann TA. 1967. Intestinal lymphangiectasia: a protein-losing enteropathy with hypogammaglobulinemia, lymphocytopenia and impaired homograft rejection. J. Clin. Invest. 46:1643–56 Karkkainen MJ, Saaristo A, Jussila L, Karila KA, Lawrence EC, Pajusola K, Bueler H, Eichmann A, Kauppinen R, Kettunen MI, Yla-Herttuala S, Finegold DN, Ferrell RE, Alitalo K. 2001. A model for gene therapy of human hereditary lymphedema. Proc. Natl Acad. Sci USA 98:12677–82 Waldmann TA, Durm M, Broder S, Blackman M, Blaese RM, Strober W. 1974.

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Role of suppressor T cells in pathogenesis of common variable hypogammaglobulinema. Lancet 2:609–13 Fleisher TA, White RM, Broder S, Nissley SP, Blaese RM, Mulvihill JJ, Olive G, Waldmann TA. 1980. X-linked hypogammaglobulinemia and isolated growth hormone deficiency. N. Engl. J. Med. 302: 1429–34 Mayer L, Kwan SP, Thompson C, Ko HS, Chiorazzi N, Waldmann T, Rosen F. 1986. Evidence for a defect in “switch”: T-cells in patients with immunodeficiency and hyperimmunoglobulinemia M. N. Engl. J. Med. 314:409–13 Waldmann TA, McIntire KR. 1972. Serum alpha-fetoprotein levels in patients with ataxia-telangiectasia. Lancet 2:1112–15 Perlin E, Engeler JE, Edson M, Karp D, McIntire KR, Waldmann TA. 2002. The value of serial measurement of both human chorionic gonadotropin and alphafetoprotein for monitoring germinal cell tumors. (Reprinted from Cancer 37:215–19, 1976) J. Urol. 167:934–37 Hacein-Bey-Abina S, Le Deist F, Carlier F, Bouneaud C, Hue C, De Villartay J, Thrasher AJ, Wulffraat N, Sorensen R, Dupuis-Girod S, Fischer A, CavazzanaCalvo M, Davies EG, Kuis W, Lundlaan WHK, Leiva L. 2002. Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy. N. Engl. J. Med. 346:1185–93 Broder S, Waldmann TA. 1978. The suppressor-cell network in cancer (Pt 1). N. Engl. J. Med. 199:1281–84 Waldmann TA, Greene WC, Sarin PS, Saxinger C, Blayney DW, Blattner WA, Goldman CK, Bongiovanni K, Sharrow S, Depper JM, Leonard W, Uchiyama T, Gallo RC. 1984. Functional and phenotypic comparison of human T-cell leukemia/lymphoma virus positive adult T cell leukemia with human T-cell leukemia/lymphoma virus negative Sézary leukemia, and their distinction using anti-Tac: monoclonal antibody identifying the human receptor for T

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cell growth factor. J. Clin. Invest. 73:1711– 18 Shevach EM. 2001. Certified professionals: CD4(+)CD25(+) suppressor T cells. J. Exp. Med. 193:F41–F45 Korsmeyer SJ, Arnold A, Bakshi A, Ravetch JV, Siebenlist U, Hieter PA, Sharrow SO, LeBien TW, Kersey JH, Poplack DG, Leder P, Waldmann TA. 1983. Immunoglobulin gene rearrangement and cell surface antigen expression of acute lymphocyte leukemias of T-cell and B-cell precursor origins. J. Clin. Invest. 71:301–13 Uchiyama T, Broder S, Waldmann TA. 1981. A monoclonal antibody (anti-Tac) reactive with activated and functionally mature human T cells. I. Production of antiTac monoclonal antibody and distribution of Tac+ cells. J. Immunol. 126:1393–97 Morgan DA, Ruscetti FW, Gallo R. 1976. Selective in vitro growth of T lymphocytes from normal human bone marrows. Science 193:1007–8 Leonard WJ, Depper JM, Uchiyama T, Smith KA, Waldmann TA, Greene WC. 1982. Monoclonal antibody that appears to recognize the receptor for human T-cell growth factor: partial characterization of the receptor. Nature 300:267–69 Leonard WJ, Depper JM, Crabtree GR, Rudikoff S, Pumphrey J, Robb RJ, Kronke M, Svetlik PB, Peffer NJ, Waldmann TA, Greene WC. 1984. Molecular cloning and expression of cDNAs for the human interleukin-2 receptor. Nature 311:626–31 Tsudo M, Kozak RW, Goldman CK, Waldmann TA. 1986. Demonstration of a nonTac peptide that binds interleukin 2: a potential participant in a multichain interleukin 2 receptor complex. Proc. Natl. Acad. Sci. USA 83:9694–98 Takeshita T, Asao H, Ohtani K, Ishii N, Kumaki S, Tanaka N, Munakata H, Nakamura M, Sugamura K. 1992. Cloning of the γ chain of the human IL-2 receptor. Science 257:379–82 Bamford RN, Grant AJ, Burton JD,

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WALDMANN Peters C, Kurys G, Goldman CK, Brennan J, Roessler E, Waldmann TA. 1994. The interleukin (IL) 2 receptor β chain is shared by IL-2 and a cytokine, provisionally designated IL-T, that stimulates T-cell proliferation and the induction of lymphokineactivated killer cells. Proc. Natl. Acad. Sci. USA 91:4940–44 Grabstein KH, Eisenman J, Shanebeck K, Rauch C, Srinivasan S, Fung V, Beers C, Richardson J, Schoenborn MA, Ahdieh M, Johnson L, Alderson MR, Watson JD, Anderson DM, Giri JG. 1994. Cloning of a T cell growth factor that interacts with the β chain of the interleukin-2 receptor. Science 264:965–68 Poiesz BJ, Ruscetti FW, Gazdar AF, Bunn PA, Minna JD, Gallo RC. 1980. Detection and isolation of type-C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma. Proc. Natl. Acad. Sci. USA 77:7415–19 Tagaya Y, Burton JD, Miyamoto Y, Waldmann TA. 1996. Identification of a novel receptor/signal transduction pathway for IL-15/T in mast cells. EMBO J. 15:4928– 39 Giri JG, Kumaki S, Ahdieh M, Friend DJ, Loomis A, Shanebeck K, Dubose R, Cosman D, Park LS, Anderson DM. 1995. Identification and cloning of a novel IL-15 binding protein that is structurally related to the α chain of the IL-2 receptor. EMBO J. 14:3654–63 Marks-Konczalik J, Dubois S, Losi JM, Sabzevari H, Yamada N, Feigenbaum L, Waldmann TA, Tagaya Y. 2000. IL-2 induced activation-induced cell death is inhibited in IL-15 transgenic mice. Proc. Natl. Acad. Sci. USA 97:11445–50 Ku CC, Murakami M, Sakamato A, Kappler J, Marrack P. 2000. Control of homeostasis of CD8(+) memory T cells by opposing cytokines. Science 288:675–78 Waldmann TA, Dubois S. Tagaya Y. 2001. Contrasting roles of IL-2 and IL-15 in the life and death of lymphocytes; implications for immunotherapy. Immunity 14:105–10

38. Azimi N, Nagai M, Jacobson S, Waldmann TA. 2001. IL-15 plays a major role in the persistence of tax-specific CD8 cells in HAM/TSP patients. Proc. Natl. Acad. Sci. USA 98:14559–64 39. Bamford RN, Battiata AP, Burton JD, Sharma Hann TA. 1996. Interleukin (IL) 15/IL-T production by the adult T-cell leukemia cell line HuT-102 is associated with a human T-cell lymphotrophic virus type I R region/IL-15 fusion message that lacks many upstream AUGs that normally attenuate IL-15 mRNA translation. Proc. Natl. Acad. Sci. USA 93:2897–902 40. Tagaya Y, Bamford R, DeFilippis A, Waldmann TA. 1996. IL-15: a pleiotropic cytokine with diverse receptor/signaling pathways whose expression is controlled at multiple levels. Immunity 4:329–36 41. Waldmann TA, Tagaya Y, Bamford R. 1998. Interleukin-2, interleukin-15, and their receptors. Intern. Rev. Immunol. 16: 205–26 42. Ogasawara K, Hida S, Azimi N, Tagaya Y, Sato T, Yokochi-Fukuda Y, Waldmann TA, Taniguchi T, Taki S. 1998. Requirement of IRF-1 for the microenvironment supporting natural killer cell development. Nature 391:700–3 43. Tendler CL, Greenberg SJ, Blattner WA, Manns A, Murphy E, Fleisher T, Hanchard B, Morgan O, Burton JD, Nelson DL, Waldmann TA. 1990. Transactivation of interleukin-2 and its receptor induces immune activation in HTLV-I associated myelopathy: pathogenic implications and a rationale for immunotherapy. Proc. Natl. Acad. Sci. USA 87:5218–22 44. Azimi N, Jacobson S, Leist T, Waldmann TA. 1999. Involvement of IL-15 in the pathogenesis of human T lymphotropic virus type I–associated myelopathy/tropical spastic pareparesis: implications for therapy with a monoclonal antibody directed to the IL-2/15Rβ receptor. J. Immunol. 163:4064–72 45. Waldmann TA, White JD, Goldman CK, Top L, Grant A, Bamford R, Roessler E,

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Horak ID, Zaknoen S, Kasten-Sportes C, England R, Horak E, Mishra B, Dipre M, Hale P, Fleisher TA, Junghans RP, Jaffe ES, Nelson DL. 1993. The interleukin-2 receptor: a target for monoclonal antibody treatment of human T-cell lymphotrophic virus I-induced adult T-cell leukemia. Blood 82:1701–12 Phillips KE, Herring B, Wilson LA, Rickford MS, Zhang M, Goldman CK, Tso JY, Waldmann TA. 2000. IL-2Rα-Directed monoclonal antibodies provide effective therapy in a murine model of adult Tcell leukemia by a mechanism other than blockade of IL-2/IL-2Rα interaction. Cancer Res. 60:6977–84 Waldmann TA, White JD, Carrasquillo JA, Reynolds JC, Paik CH, Gansow OA, Brechbiel MW, Jaffe ES, Fleisher TA, Goldman CK, Top LE, Bamford R, Zaknoen S, Roessler E, Katen-Sportes C, England R, Litou H, Johnson JA, Jackson-White T, Manns A, Hanchard B, Junghans RP, Nelson DL. 1995. Radioimmunotherapy of IL-2Rα-expressing adult T-cell leukemia with yttrium-90 labeled anti-Tac. Blood 86:4063–75 Waldmann TA. 1992. Immune Receptors: targets for therapy of leukemia/lymphoma, autoimmune diseases and for the prevention of allograft rejection. Annu. Rev. Immunol. 10:675–704 Waldmann TA. 1988. The multichain interleukin-2 receptor: from the gene to the

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bedside. In: Sabatini D, ed. The Harvey Lectures, Series 82. New York. Alan R. Liss. 1–17 Brown PS JR, Parenteau GL, Dirbas FM, Garsia RJ, Goldman CK, Bukowski MA, Junghans RP, Queen C, Hakimi J, Benjamin W, Clark RE, Waldmann TA. Anti-Tac-H, a humanized antibody to the interleukin-2 receptor prolongs primate cardiac allograft survival. Proc. Natl. Acad. Sci. USA 88:2663–67 Junghans RP, Waldmann TA, Landolfi NF, Avdalovic NM, Schneider WP, Queen C. 1990. Anti-Tac-H, a humanized antibody to the interleukin-2 receptor with new features for immunotherapy in malignant and immune disorders. Cancer Res. 50:1495– 502 Vincenti F, Kirkman R, Light S, Bumgardner G, Pescovitz M, Halloran P. Neylan J, Wilkinson A, Ekberg H, Gaston R, Backman L, Burdick J. 1998. Interleukin2-receptor blockade with daclizumab to prevent acute rejection in renal transplantation. N Engl J. Med. 338:161–65 Nussenblatt RB, Fortin E, Schiffman R, Rizzo L, Smith J. vanVeldhuisen P, Sran P, Yaffe A, Goldman CK, Waldmann TA, Whitcup SM. 1999. Treatment of non-infectious intermediate and posterior uveitis with the humanized anti-Tac monoclonal antibody: a phase I/II clinical trial. Proc. Natl. Acad. Sci. USA 96:7462– 66

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Annu. Rev. Immunol. 2003. 21:29–70 doi: 10.1146/annurev.immunol.21.120601.141114 c 2003 by Annual Reviews. All rights reserved Copyright ° First published online as a Review in Advance on October 16, 2002

CD8 T CELL RESPONSES TO INFECTIOUS PATHOGENS


Phillip Wong and Eric G. Pamer Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021; email: [email protected], [email protected]

Key Words cytotoxic T lymphocytes, microbial immunity, bacteria, viruses, protozoans ■ Abstract CD8 T cells respond to viral infections but also participate in defense against bacterial and protozoal infections. In the last few years, as new methods to accurately quantify and characterize pathogen-specific CD8 T cells have become available, our understanding of in vivo T cell responses has increased dramatically. Pathogenspecific T cells, once thought to be quite rare following infection, are now known to be present at very high frequencies, particularly in peripheral, nonlymphoid tissues. With the ability to visualize in vivo CD8 T cell responses has come the recognition that T cell expansion is programmed and, to a great extent, independent of antigen concentrations. Comparison of CD8 T cell responses to different pathogens also highlights the intricate relationship between microbially induced innate inflammatory responses and the kinetics, magnitude, and character of long-term T cell responses. This review describes recent progress in some of the major murine models of CD8 T cell–mediated immunity to viral, bacterial, and protozoal infection.

INTRODUCTION CD8 T cells recognize pathogen-derived peptides complexed with major histocompatibility complex (MHC) class I molecules on the surface of infected cells. As a rule, peptides generated by proteasome-mediated protein degradation are transported into the endoplasmic reticulum, bound by newly synthesized MHC class I molecules and transported to the cell surface (1). Because most peptides bound by MHC class I molecules derive from cytosolic proteins, CD8 T cells principally defend against microbes that introduce antigens into the cytosol of infected cells. Our understanding of MHC class I antigen presentation as it relates to priming of naive CD8 T cells, however, has evolved over the last decade. Examples of effective priming of CD8 T cells by exogenous proteins abound, particularly if antigens are administered with anti-CD40 (2), heat shock proteins (HSPs) (3), or in complex with cells or other particulates (4). There is increasing evidence that cross-priming, as this form of MHC class I antigen presentation is referred to (5), involves uptake of antigen by specialized dendritic cells (DC). These recent 0732-0582/03/0407-0029$14.00

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advances in our understanding of CD8 T cell priming are of particular relevance to antimicrobial immunity. How important are CD8 T cells in antimicrobial defense? The answer to this question is not simple, since many arms of the adaptive and innate immune system functionally overlap with CD8 T cells during immune defense. While adoptively transferred CD8 T cells can protect immunologically na¨ıve individuals from lethal infection, or control viral infection in immunocompromised recipients, individuals with defects in the MHC class I antigen processing pathway are remarkably resistant (with some important exceptions) to pathogens that induce CD8 T cell responses. Thus, for many of the pathogens discussed in this review, CD8 T cell responses can and do confer protective immunity, but in their absence other arms of the immune system provide protective cover. CD8 T cells express a range of effector molecules that mediate defense against pathogens. Direct cytolysis of target cells, mediated by perforin release and Fas, tops the list. CD8 T cells also secrete cytokines such as tumor necrosis factor (TNF) and interferon-γ (IFN-γ ), which play important roles in antimicrobial defense. In addition, pathogen-specific CD8 T cells express chemokines that attract inflammatory cells to sites of infection. The effector functions of CD8 T cells during microbial infections have been comprehensively reviewed recently (6). Viruses, bacteria, fungi, and protozoa cause acute and chronic infections in mammalian hosts. Some viruses, such as influenza virus, cause acute infections and are eliminated. In contrast, herpes viruses cause latent infections and remain with the host for life. CD8 T cells play distinct roles in these two circumstances. Following influenza virus infection, CD8 T cells eliminate the pathogen and provide longterm immunity from reinfection. In contrast, herpes family viruses such as EpsteinBarr virus (EBV) and cytomegalovirus (CMV) are perpetually held in check by CD8 T cells. Bacterial and protozoal pathogens also cause acute and chronic infections, with similar challenges for CD8 T cell–mediated immune defense. CD8 T cell responses to infection occur in the setting of inflammation. The site of initial infection, the anatomic and cellular localization of pathogens, the in vivo tempo of pathogen growth, and the transduction of signals by receptors that recognize pathogen-derived molecules are factors that, in aggregate, create the inflammatory milieu. The inflammatory environment at the time of CD8 T cell priming plays a critical role in the proliferation, differentiation, and survival of pathogenspecific T cells. It is increasingly clear that Toll-like receptor (TLR)-mediated pathways and chemokine/cytokine cascades provide the context and stage for the generation of pathogen-specific CD8 T cell populations, and the components of the complex immune network initiated by infection are only beginning to be defined. In this review we summarize our knowledge of CD8 T cell immunity to pathogens, as learned from the major mouse models of infectious disease. This is an ambitious endeavor, as there are many exciting and informative animal models of infection, and the literature on this topic is vast. We review viral, bacterial, and protozoal pathogens that have been used in mouse models to characterize CD8

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T cell responses and discuss new concepts of CD8 T cell–mediated antimicrobial immunity that have emerged from these studies.

VIRAL PATHOGENS


Lymphocytic Choriomeningitis Virus Murine infection with lymphocytic choriomeningitis virus (LCMV) is a widely used model to study CD8 T cell responses. LCMV primarily infects macrophages, lymphocytes, dendritic cells, and glial cells (7, 8) and stimulates a powerful cytotoxic T lymphocyte (CTL) response (9, 10). Acute infection of mice with LCMV results in rapid viral growth that causes little host damage since LCMV is noncytopathic. Viral growth induces an LCMV-specific CTL response that leads to viral elimination within 8 to 10 days (11). In some circumstances, such as high dose infection, the virus can persist in the host (12, 13). CD8 T cells are essential for viral clearance (14, 15), and perforin is a critical CTL effector component (16). Remarkably, infection with large doses or with rapidly replicating strains of LCMV, provides an overwhelming stimulus that decreases the number of virusspecific CD8 T cells (17) and results in persistent viral infection. CTL exhaustion is decreased in perforin-deficient mice, suggesting that this effector molecule is involved in the deletion of antigen-specific T cells (18). The CD8 T cell response to LCMV infection focuses on a few MHC class I–restricted epitopes derived from the viral glycoprotein (gp) and nucleoprotein (np). In C57BL/6 mice, three H-2Db-restricted epitopes, gp33 (gp33-41), gp276 (gp276-286), and np396 (np396-404) are the major targets of the CTL response, while in BALB/c mice, the single immunodominant epitope is np118 (np118-126) presented by H-2Ld (19). Subdominant epitopes have also been defined, and immunization with these peptides confers protection against LCMV infection (20, 21). In C57BL/6 mice, the three dominant peptides are bound by H-2Db with high affinity and with long half-lives, but they are present at different densities on the cell surface, with gp33 > np396 > gp276 (22). Their abundance correlates with the magnitude of CTL responses to these epitopes following LCMV infection. However, the magnitude of a particular response does not necessarily correlate with its protective antiviral capacity because subsequent adoptive transfer experiments showed that when equal numbers of CTLs specific for the gp33, np396, or gp276 peptides were injected into infected mice, np396-specific CTLs conferred optimum protection, followed by gp276- and finally gp33-specific T cells. Protective capacity correlated with the sensitivities of each CTL line for its cognate peptide. The CD8 T cell response to LCMV infection is massive. ELISPOT, intracellular cytokine staining, and MHC/peptide tetramer staining demonstrated that nearly 70% of CD8 T cells at the peak of the response were virus-specific (11, 23–25). In C57BL/6 mice, the majority of CD8 T cells are specific for the gp33 and np396 epitopes, and a smaller percentage recognize the gp276 peptide. These studies showed that bystander activation of nonspecific CD8 T cells is probably negligible.

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The CTL response to LCMV infection does not depend upon CD4 T cells or B cells, although neutralizing antibodies are necessary to prevent viral reemergence (26–28). Whereas CD4 T cells are not necessary for LCMV clearance, their paucity has a detrimental effect on long-term memory CD8 T cell populations (29, 30). Priming of LCMV-specific CTL most likely involves bone marrow– derived antigen-presenting cells (APC), but CTL responses are also primed by nonprofessional APC (31, 32). The absence of B7-CD28 signals dampens the CTL response to LCMV but does not prevent the development of protective CD8 T cell memory (33). CD40 signaling is not required for primary CD8 T cell responses to LCMV (34), but its absence reduces the ability of CD8 T cells to provide long-term immunity (35). The size of antigen-specific memory populations correlates with the burst size of the primary response (36). TCR diversity is conserved between primary and memory CD8 T cell populations following LCMV infection. These findings suggest that selection of cells into the memory population is stochastic (37, 38). Recent studies demonstrate that LCMV-specific CD8 T cells undergo functional avidity maturation during the expansion phase, a process that involves optimization of TCR signaling (39). LCMV-specific memory CD8 T cells persist in mice in the absence of viral antigens (36, 40). Whereas na¨ıve T cells require constant low levels of TCR stimulation by self-MHC molecules to survive (41), memory cells persist without such a requirement (42). It has been shown, however, that memory cells surviving in the absence of MHC molecules have functional deficits (43). The longevity of memory CD8 T cells may be related to increased expression of anti-apoptotic genes such as bcl-2 (44, 45). Memory CD8 T cells require IL-15 for in vivo maintenance; in the absence of this cytokine, LCMV-specific memory CD8 T cells are generated, but their frequency declines (46). Recent studies show that the size of memory CD8 T cell populations is influenced by infection with unrelated pathogens (47–49). Using sequential infection with LCMV and several other viruses, memory CD8 T cell populations specific for prior infections were found to incrementally decrease with each subsequent infection. In contrast, memory CD4 T cell populations are stable after multiple heterologous infections (50). Occasionally, memory T cells specific for a prior infection are cross-stimulated by antigens present in a more recent infection, resulting in their expansion and a shift in the immunodominance hierarchy (51). Indeed, LCMV-specific memory CTLs cross-reactive against heterologous antigens may enhance and alter the course of the immune response to subsequent infection with such unrelated agents (52, 53).

Influenza Virus Influenza viruses cause infections of the respiratory tract and can reinfect individuals because two viral surface proteins, hemagglutinin and neuraminidase (HN), rapidly evolve and evade previously established humoral immunity. While

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neutralizing antibodies play an essential role in immunity to influenza virus infection, T lymphocytes can, in the absence of humoral immunity, provide significant protection. Mice lacking B cells develop CD8 and, to a lesser extent, CD4 T cell– mediated protective immunity (54). Influenza-specific T cells mediate pulmonary viral clearance by perforin or Fas-mediated lysis (55). Staining with MHC tetramers showed that primary and memory T cell responses to respiratory infection of mice with influenza virus are substantial (56), and memory responses to reinfection show rapid in vivo proliferation of influenza-specific T cells (57). While the magnitude of T cell responses to different viral epitopes is hierarchical (58), measurements of T cell population sizes in parent versus F1 mouse strains demonstrated that the presence of other MHC class I alleles can diminish some peptide-specific CD8 T cell responses. Also, thymic selection and na¨ıve T cell precursor frequencies can determine immunodominance hierarchies (59). Understanding immunodominance at a mechanistic level has been a vexing problem. Some evidence suggests that antigen presentation determines immunodominance, while other studies suggest that T cell repertoire is more important. In a comprehensive study of this issue, CD8 T cell responses to five different influenza virus–derived peptides were characterized. The results were gratifying because TCR repertoire, antigen processing efficiency, and T cell competition all contribute to the T cell response hierarchy (60). Analysis of influenza virus–specific T cell responses in mice lacking the LMP2 proteasome subunit demonstrated that antigen processing influences immunodominance at two levels (61). First, LMP2 influences the repertoire of na¨ıve T cells that respond to specific viral peptides by altering thymic selection and, second, LMP2 affects peripheral presentation of viral peptides during influenza virus infection. CD8 T cell responses to different influenza epitopes can have disparate kinetics. For example, an epitope derived from influenza virus polymerase 2 protein (PA 224-233) primes T cells that undergo prolonged in vivo expansion when compared to other influenza-specific T cell responses (62). With respect to T cell differentiation, influenza virus–specific T cells that differed in peptide specificity also differed in terms of cytokine production (63). Along similar lines, studies of an influenza virus–specific CD8 T cell population specific for a dominant and a subdominant epitope derived from HA also differed in terms of cytolytic activity (64). Phenotypic analysis of memory T cells following infection demonstrated diversity in surface expression of CD62L, cytolytic activity, and the capacity to undergo proliferation upon stimulation (65). These studies demonstrated that T cells differing in peptide specificity can have disparate cytokine secretion profiles and that T cells sharing antigen specificity can differ phenotypically, providing additional complexity to the antiviral response. Costimulatory molecules contribute to expansion and differentiation of CD8 T cells following influenza virus infection. Absence of CD27, a tumor necrosis factor family member, diminishes primary and memory T cell responses to influenza virus (66). Blocking CD28 reduces the number of cytotoxic T cells and the amount of IFN-γ produced in infected lungs, but mice clear influenza virus (67).

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On the other hand, mice lacking 4-1BB ligand (4-1BBL) exhibit normal primary CD8 T cell expansion but decreased numbers of antigen-specific T cells that remain late in the primary response as well as reduced memory responses (68). This suggests that CD28 is critical for initial CD8 expansion during influenza infection, whereas 4-1BBL is needed to maintain T cell numbers later in the response that give rise to the memory pool. Not surprisingly, mice lacking both CD28 and 4-1BBL have markedly diminished T cell responses to influenza virus infection (69). CD4 T cells significantly impact influenza virus–specific CD8 T cell responses. Depletion of CD4 T cells decreases recruitment of virus-specific CD8 T cells to infected lungs (70), worsening viral infection and delaying viral clearance. CD4 T cells, induced by peptide immunization, enhance viral clearance and antibody formation but decrease priming of antiviral CD8 T cells (71). Trafficking of virus-specific CD8 T cells to sites of infection is essential for viral clearance. Influenza virus infection is a particularly useful model for studies of trafficking because infection is restricted to the respiratory system. During acute infection, influenza-specific T cells in the lungs are highly cytolytic, and the majority produce IFN-γ upon peptide stimulation (72). This contrasts with CD8 T cells of similar antigen specificity in the liver, a site that is not infected by influenza virus. In liver, most influenza-specific T cells do not produce IFN-γ , and apoptosis is markedly increased (72). The kinetics of influenza virus–specific CD8 T cell expansion, differentiation, and contraction are distinct in different tissues (73). Virus-specific CD8 T cells persist at very high frequencies in airways and lung parenchyma after infection and, in contrast to splenic and lymph node memory T cells, rapidly express effector functions upon re-exposure to antigen (74). Rapidly responsive, virus-specific T cells also persist in the nasal mucosa (75). While adoptive transfer of na¨ıve, influenza virus–specific T cells does not protect from viral infection, transfer of memory or activated T cell does. Activated and memory T cells are superior at trafficking to influenza virus–infected lungs (76), a process that is not driven by antigen because even non–virus specific, activated T cells traffic to infected lungs (77). Whereas antigen presentation activates virus-specific T cells, little is known about the kinetics and localization of in vivo antigen presentation. In an interesting study, adoptively transferred reporter T cells specific for nucleoprotein (NP) 324-332 and HN 421-436 demonstrated in vivo antigen presentation in mediastinal lymph nodes for 9 days following viral challenge, while live virus was cleared within 6 days (78).

Respiratory Syncytial Virus Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract viral infections in infants, and RSV causes respiratory disease in elderly adults and bone-marrow transplant patients (79–81). Reinfection with this single-stranded RNA virus is common throughout life, suggesting that protective immunity to RSV is limited (82).

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T cells play a major role in viral clearance during RSV infection, but also enhance lung immunopathology (83–85). In particular, RSV-specific T helper type 2 (Th2)-phenotype CD4 T cells infiltrating the lung contribute to pulmonary eosinophilia and inflammation (83, 86, 87). CD8 T cells can either augment or suppress the Th2 response, depending on the model system studied. During primary murine infection with RSV, CD8 T cells have been implicated in airway eosinophilia and bronchial hyperresponsiveness (88, 89), but when mice are first immunized with recombinant vaccinia virus expressing RSV antigens and subsequently challenged with RSV, CD8 T cells downregulate Th2 cytokine production and prevent pulmonary eosinophilia (90, 91). Recent studies visualizing T cell responses in mice have uncovered a suppressive effect of RSV on CD8 T cell effector activity in the lung. In BALB/c mice, the majority of antiviral CTLs are directed toward a single immunodominant H-2Kd-restricted epitope in the matrix 2 protein (M282-90) of RSV (92, 93). A second subdominant peptide targeted by RSV-specific CTLs in BALB/c mice is derived from the fusion glycoprotein (F85-93/Kd). At the peak of the primary response following intranasal RSV infection, M282-90-specific CD8 T cells constitute ∼50% of the pulmonary CD8 T cell population, while F85-93-specific CTLs make up ∼4.8% (94). Interestingly, intracellular cytokine staining showed lower numbers of IFN-γ -producing antigen-specific CD8 T cells in the lung when compared to tetramer staining for M2-specific and F-specific CD8 T cells (94, 95). The discrepancy between tetramer staining and intracellular IFN-γ production occurred in primary and secondary CTL responses to RSV, but it was not found when mice were infected with a different pulmonary virus (influenza A). This discordance occurred only in the lung, as splenic RSV-specific CD8 T cells are not deficient in IFN-γ synthesis. In addition, RSV-specific CD8 T cells in the lung have reduced ex vivo cytolytic activity on a per cell basis compared to spleen T cells (95). Lung RSV-specific CD8 T cells also fail to upregulate perforin expression and do not downmodulate surface TCR in response to antigen. Thus, RSV infection does not reduce the initial expansion of primary and secondary CD8 T cells, but it selectively interferes with TCR signaling in the lung during primary and memory responses. Impaired CTL responses during RSV infection may explain earlier findings that the protection afforded by RSV-specific CD8 T cells is transient, waning after about 2 months (85). Although expression of perforin is not absolutely required for CD8 T cell–mediated clearance of RSV, because Fas-FasL interactions can substitute, its absence does delay viral clearance and results in increased cellular infiltrates in the lung (96). It is tempting to speculate that Th2 cytokines induced during RSV infection may dampen CTL activity in the lung because IL-4 can reduce perforin-mediated cytotoxicity by RSV-specific CD8 T cells in vivo (97, 98).

Herpes Simplex Virus Infection by the lytic herpes simplex virus (HSV) causes a variety of diseases ranging from mild cutaneous lesions to life-threatening encephalitis (99). HSV type 1 (HSV-1) is typically associated with orofacial infections and encephalitis,

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while HSV type 2 (HSV-2) generally infects the genital region. A fundamental characteristic of these viruses, however, is the establishment of latent infection in sensory neurons and periodic reactivation, resulting in lesions at or near the site of latent infection. Studies of murine HSV infection have provided a picture of immune responses to localized viral infection, because this virus does not replicate systemically, but rather targets skin and neurons. CD4 T cells are critical for clearing primary HSV infection (100–102), which may be related to elaborate mechanisms HSV uses to evade MHC class I antigen presentation, such as expression of ICP47, which blocks the transporter associated with antigen-processing molecule (TAP) (103–105). Nevertheless, macrophages, γ δ T cells, and CD8 T cells also control HSV spread in the nervous system (106–109). In particular, CD8 T cells maintain HSV-1 in a latent state in sensory neurons through noncytolytic mechanisms (110), possibly by producing antiviral cytokines or granzyme A, a noncytolytic serine protease (111). Although CD8 T cell responses to HSV are unimpaired in the absence of CD40/CD154 interactions, costimulation through CD28 is essential (112). Additionally, lymphotoxin participates in CTL differentiation following HSV infection (113). Footpad infection of B6 mice with HSV-1 results in CD8 T cell accumulation in the draining popliteal lymph node, with antigen-specific CTLs peaking 5 days after inoculation (114–116), at which time, approximately 6% of CD8 T cells are HSV-1 specific (117, 118). The CD8 T cell response is focused on an H-2Kb-restricted immunodominant peptide derived from HSV glycoprotein B (gB498-505) (118), while smaller CD8 T cell populations recognize subdominant epitopes from the viral ribonucleotide reductase (RR822-829) and the immediate-early protein ICP27 (ICP27445-452) (119, 120). Between 70% and 90% of HSV-1-specific CD8 T cells are directed against the gB498-505 epitope as measured by intracellular IFN-γ staining of draining lymph node cells (118). Mice immunized with a viral variant that lacks the dominant gB epitope have reduced HSV-1-specific T cell responses. While responses to the known subdominant epitopes were not increased in the absence of the gB498-505 determinant, some unidentified cryptic specificities became apparent (118). The tightly focused HSV-1 gB-specific CTL response has exemplified how VDJ recombinational diversity within the na¨ıve TCR repertoire influences immunodominance. CD8 T cells specific for HSV-1 gB498-505 express highly restricted TCRβ variable regions. Approximately 60% of CTLs specific for gB498-505 express Vβ10, while another 20% express Vβ8.1 (121). Within the Vβ10 subset there is almost complete conservation of a tryptophan-glycine doublet within the junctional CDR3 regions (121, 122). These amino acids are encoded not by N or J nucleotides, but by the germline Dβ2 element. When mice naturally lacking the Dβ2-Jβ2 cluster were immunized with HSV-1, a significant reduction of the dominant gB-specific Vβ10+ T cell subset was observed (121, 122), demonstrating that germline-encoded elements and rearrangement biases have significant effects on the magnitude and makeup of T cell responses.

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Kinetic analysis of CD8 T cell priming after HSV-1 infection demonstrates the rapidity of antigen transport by professional APCs into draining lymph nodes. Lymph node CD8 T cells specific for the dominant gB epitope are activated within 6–8 h after footpad injection, and they begin dividing approximately 24 h later (123). This is consistent with in vivo antigen presentation in the draining lymph nodes occurring within 4–6 h after cutaneous infection. Proliferation of CTLs occurs without any evidence of virus in the lymph node, indicating that HSV-1 antigen is rapidly transported by APC from peripheral tissues to draining lymph nodes. Acquisition of effector functions by gB-specific CTLs occurs rapidly, with significant cytolytic activity detectable within 30 h of infection (123). T cells migrate to the spleen where they continue to proliferate in an antigen-independent fashion, resulting in a secondary peak of CTL expansion 2 days following peak expansion in draining lymph nodes (114).

Hepatitis B Virus Hepatitis B virus (HBV) is an enveloped DNA virus that infects hepatocytes. HBV does not infect mice, precluding conventional studies of immunity and viral pathogenesis. To circumvent this problem, a mouse strain that is transgenic for the whole HBV genome was generated (124). Remarkably, this mouse strain expresses infectious HBV in the liver but does not develop hepatitis. Thus, HBV infection is not directly toxic to hepatocytes, but the inflammatory response to viral antigens induces hepatic inflammation. Transfer of HBV-specific CD8 T cells into HBV transgenic mice results in transient, mild hepatitis and a dramatic drop in the HBV RNA transcripts in hepatocytes (125). CTLs do not inhibit transcription of HBV genes but do enhance degradation of HBV transcripts. On the basis of serologic studies, it is estimated that roughly 10% of hepatocytes are lysed by CTL in a perforin-dependent manner (126). Remarkably, HBV-specific, perforin-deficient CD8 T cell lines do not induce hepatitis in HBV transgenic mice but do mediate clearance of viral transcripts. How do CD8 T cells mediate viral clearance from hepatocytes? One product of activated CD8 T cells, IFN-γ , has been implicated in this process (127). HBVspecific CD8 T cells derived from mice deficient for IFN-γ do not mediate HBV clearance. This result is consistent with previous results showing that blocking antibodies against TNF and IFN-γ prevent the downregulation of HBV transcripts following CD8 T cell transfer (126). These investigators calculate that direct interactions between CTLs and each infected (in this case transgenic) hepatocyte is a physical and numeric impossibility. A recent study demonstrated that HBV-specific T cells induce the production of Mig and IP10, two CXC chemokines that recruit a range of mononuclear cells to sites of inflammation (128). Induction of these chemokines depends upon IFN-γ production by HBV-specific CD8 T cells, and results in the recruitment of NK cells, NK T cells, CD8 and CD4 T cells, and lymphoid and myeloid DCs (128).

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Blocking recruitment of these cells to the liver ameliorates hepatocyte damage, and HBV transcripts are still cleared from hepatocytes. Thus, the salutary effect of HBV-specific CD8 T cells in this system does not depend upon recruitment of inflammatory cells by Mig or IP10. The finding that CD8 T cell–mediated downregulation of HBV transcripts does not require contact with “infected” cells suggests that inflammatory cytokines clear viral infection. Heterologous infection (e.g., with malaria or LCMV) dramatically downregulates HBV transcripts in HBV transgenic mice (129), and even administration of IL-12 or TNF results in viral clearance (130). The ability of CD8 T cells to clear HBV without direct contact with infected cells is not limited to mice: Confirmatory results were obtained with a chimpanzee model, which very closely reflects human infection (131).

Murine γ -Herpesvirus γ -herpesviruses (γ HV) cause acute infections at mucosal sites but then persist by establishing latency in B cells and other cell types. Epstein-Barr virus (EBV) and Kaposi’s sarcoma herpesvirus (KSHV or human herpesvirus 8) represent two important human γ HV associated with tumors in immunocompromised individuals (132–134). The mouse model of γ HV infection is used to study γ HV pathogenesis and immune mechanisms that regulate persistent viral infections (135). Murine γ -herpesvirus 68 (MHV-68 or γ HV68), a type 2 γ -herpesvirus, occurs naturally in rodents and shares biological and genetic features with EBV and KSHV (136, 137). Intranasal administration of γ HV68 results in acute infection of alveolar epithelial cells and latent infection of B cells, macrophages, DCs, and epithelial cells (138–141), whereas intraperitoneal inoculation causes acute infection of splenic B cells (142). T cells clear infectious virus from the lungs within 10–13 days after intranasal infection and 15–20 days after intraperitoneal infection. CD8 T cells are particularly important for viral clearance during the acute phase of γ HV68 infection and for immune surveillance during the persistent latent phase; their depletion increases viral titers in lung and spleen after intranasal inoculation (143). Similarly, β 2m-deficient mice have increased viral titers in spleen following intraperitoneal infection (142). Perforin and Fas-mediated cytotoxicity by CD8 T cells limits spread of γ HV68 infection. Mice deficient in either molecule clear lytic virus from lung, but animals with perforin-deficient T cells and Fas-null lung epithelial cells fail to clear infectious virus (144, 145). CD8 T cells, however, are not sufficient to control γ HV68 infection. MHC class II–deficient mice succumb to a chronic wasting disease associated with increasing virus titers in the lung beginning 25–35 days after infection despite initial control of the acute infection (146). This does not result from CTL exhaustion, as seen following high-dose LCMV infection; indeed, normal expansion of virus-specific CD8 T cells is observed (147). Although mice can recover from acute infection in the absence of IFN-γ (148), clearance of γ HV68 infection by CD4 T cells is

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mediated in part by IFN-γ (149). Boosting γ HV68-specific CD8 T cell responses in the absence of CD4 T cells does not increase long-term survival (150). CD8 T cells that expand in the absence of CD4 T cells have decreased CTL function and decreased expression of IFN-γ and TNF (151). CD4 T cells may optimize CD8 T cell priming by conditioning APCs via CD40-CD154 ligand interactions (152–154). Agonistic anti-CD40 antibody treatment substitutes for CD4 T cells by preventing latent γ HV68 reactivation in the lungs of MHC class II–deficient mice (155). CD8 T cells in γ HV68-infected C57BL/6 mice recognize peptides derived from single-stranded DNA-binding protein (p56) and ribonucleotide reductase (p79) presented by H-2Db and H-2Kb (156, 157). Several minor epitopes have also been identified in early lytic phase genes of γ HV68. Intracellular IFN-γ and tetramer staining show that CTL responses in lung, lymph nodes, and spleen peak 10–20 days after infection and then contract for 30–40 days (157). The expansion kinetics of T cells responding to the two major epitopes differ, with peak expansion of p79-specific CTL occurring later than for p56-specific T cells. This may reflect disparate epitope presentation in spleen and mediastinal lymph nodes during the course of infection (158). γ HV68 has evolved strategies to avoid CTL detection. For example, the K3 gene encodes a zinc-finger-containing protein that diminishes antigen presentation by decreasing the half-life of newly synthesized MHC class I molecules, leading to reduced surface class I expression (159). The γ HV68 M3 gene encodes a protein that binds to a wide range of chemokines (160), potentially interfering with migration of CTL and other cells to sites of infection. These evasion mechanisms may explain the inability of CTL responses to completely control γ HV68 infection. γ HV68 infection induces dramatic expansion of an oligoclonal population of activated Vβ4+CD8+ T cells after clearance of lytic virus from the lung (161). A syndrome of splenomegaly and lymphocytosis develops 2–3 weeks after intranasal infection, with increased circulating CD8 T cells bearing Vβ4+ TCR (162, 163). These CD8 T cells are not responsive to any known H-2b γ HV68 peptides, do not depend on the presence of MHC class I, and are prevalent when the frequency of peptide-specific CTL has declined (157, 162, 164). CD4 T cells, however, are necessary for the expansion of Vβ4+CD8+ T cells (162, 165). The stimulatory ligand for these T cells remains unknown but is expressed on latently infected B cells (138), which are activated during γ HV68 infection (166).

BACTERIAL PATHOGENS Whereas CD8 T cells are principally associated with defense against viral infections, they also combat intracellular bacterial infections (167). Intracellular bacterial pathogens can be categorized on the basis of the subcellular compartment they enter. Mycobacteria and Salmonella species remain in vacuoles, while Listeria monocytogenes, Shigella, and some Rickettsia species enter the cytosol. While bacterial entry into cytosol provides direct access to the MHC class I

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antigen-processing pathway, offering a ready explanation for the priming of CD8 T cells, vacuolar pathogens such as mycobacteria and Salmonella also induce CD8 T cell responses that play a role in protective immunity. Salmonella causes infection via the gastrointestinal route, while mycobacterial infections are usually acquired by inhalation. Nevertheless, both pathogens survive and replicate in macrophages, and immunity is highly dependent upon T lymphocytes. While CD4 T cells are essential for protective immunity against these two pathogens (168, 169), the contribution of MHC class I–restricted CD8 T cells is increasingly appreciated. In the following sections we review murine CD8 T cell responses to Listeria monocytogenes, Mycobacterium tuberculosis, and Salmonella typhimurium.

Listeria monocytogenes Listeria monocytogenes is a gram-positive bacterium that infects many mammalian species, including mice and humans. The prominent role of CD8 T cells in defense against Listeria infection directly relates to this pathogen’s virulence strategy: L. monocytogenes rapidly escapes the vacuolar compartment of infected macrophages by secreting the membranolytic protein Listeriolysin O (170). By entering the cytosol of infected cells, Listeria more closely resembles viral than typical bacterial infections. L. monocytogenes–specific CD8 T cells fall into two groups: One recognizes peptides generated by cytosolic degradation of secreted bacterial proteins; the other recognizes short, hydrophobic bacterial peptides that contain N-formyl methionine at the amino-terminus. A number of distinct peptide antigens derived from the degradation of secreted proteins have been identified (171–174), and their processing and presentation have been characterized. These studies, which have been performed predominantly in vitro with L. monocytogenes–infected cells, have provided some interesting insights into the presentation of bacterial antigens by MHC class I molecules. First, because the rate of intracellular protein synthesis and degradation could be determined in bacterially infected cells, it was possible to estimate the efficiency of presentation of epitopes from two dominant Listeria antigens (175–177). These studies demonstrated that, depending on the specific epitope, between 3 and 35 protein molecules were degraded to generate peptides bound by MHC class I molecules. Bacterially secreted proteins in the cytosol of infected macrophages are rapidly degraded by proteasomes (176, 177). Some secreted L. monocytogenes proteins such as p60 are rapidly degraded because their amino termini contain destabilizing residues (175), as defined by the N-end rule (178). On the other hand, another Listeria protein, ActA, has enhanced stability in the cytosol because it contains a stabilizing amino acid at the amino-terminus (179). Interestingly, LLO, the membranolytic virulence factor that is potentially highly toxic to the host cell, is rapidly degraded in a proteasome-dependent fashion (177) because it contains a PEST-like sequence (180). Investigation of bacterial antigen presentation is much more difficult in vivo than in vitro. Many cell types may be infected with Listeria in vivo, some of

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which may prime CD8 T cells while others may only be targets of activated CD8 T cells. Indeed, while epithelial cells and hepatocytes are infected with Listeria, only bone marrow–derived cells are capable of priming na¨ıve cells in vivo (31). Some attempts have been made to measure antigen presentation in vivo (181), but we remain constrained by the limitations of available methods. The antigenicity of secreted versus nonsecreted (somatic) proteins has been investigated. Hess and colleagues studied immunity induced by Salmonella strains expressing Listeria antigens in secreted or somatic forms. They showed that secreted antigens were superior to somatic antigens at inducing protective T cell responses (182). Priming of CD8 T cells by Salmonella secreting a Listeria protein is TAP dependent, indicating that, despite initial vacuolar localization, secreted antigens access the APC cytosol (183). Expression of Listeria antigens in secreted versus somatic form in the Mycobacterium bacille Calmette-Guerin (BCG) strain yielded similar results (184). In a more recent study, Listeria strains expressing a nonsecreted antigen were shown to effectively prime CD8 T lymphocytes (185). Importantly, antigen-specific CD8 T cells could not protect mice infected with Listeria expressing antigen in a nonsecreted form, indicating that the antigen processing pathways involved with T cell priming draw on a broader antigen pool than the pathways involved in effector mediated bacterial clearance. In vivo processing and presentation of nonsecreted Listeria antigens to CD8 T lymphocytes occurs in the absence of IFN-γ (186). ActA, the Listeria virulence factor that allows intracellular bacteria to move by actin polymerization, can also prime CD8 T cells. Interestingly, ActA is not an important antigen during natural infection with Listeria because it is tethered to the bacterial cell surface, limiting access to the MHC class I antigen-processing pathway (187, 188). CD8 T cells are primed when mice are immunized with bacteria that secrete ActA. The slow rate of ActA degradation in the cytosol may also limit its antigenicity (179). Quantitative studies of in vitro antigen presentation by Listeria-infected cells demonstrated that some epitopes are prevalent on the cell surface, while others are sparse. Using ELISPOT assays to quantify epitope-specific T cell frequencies, it was found that the largest T cell response was elicited by one of the less prevalent epitopes, while the most prevalent and most efficiently presented epitope elicited only a minor, subdominant T cell response (189). Altering the efficiency of antigen presentation had a negligible impact on CD8 T cell response magnitudes until the efficiency of antigen presentation fell below a threshold level (190). CD8 T cell responses to primary Listeria infection are modest, with peak frequencies to dominant MHC class I–restricted epitopes in the range of 2% to 3% (189). Deletion of one or two dominant L. monocytogenes epitopes has no detectable impact on the magnitude of responses to subdominant epitopes (191). Thus, immunodomination, as described by Yewdell and colleagues (192), does not apply to the primary CD8 T cell response to L. monocytogenes infection. Kinetic analysis of CD8 T cell responses to L. monocytogenes infection, measured by MHC class I tetramer staining (193), demonstrated synchronous

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expansion and contraction of T cells that differed in antigen specificity. This result was surprising because the cognate antigens differ in stability and quantity. Subsequent studies demonstrated that the kinetics of CD8 T cell expansion and contraction are similar in mice undergoing prolonged infection, or infection curtailed by antibiotic administration (194). These studies, and in vitro studies of CD8 T cell activation, demonstrate that transient exposure of na¨ıve T cells to antigen and costimulatory signals activates a program of prolonged cell division and differentiation into effector and memory T cells (195–197). Recent studies demonstrate that CD8 T cell contraction is also programmed (198). The factors determining immunodominance hierarchies are complex, and MHCpeptide stability, T cell repertoire and competition have been implicated in different systems. A new concept of immunodominance came from studies of CD8 T cell responses in mice lacking IFN-γ , perforin, or both molecules. Surprisingly, the magnitude of CD8 T cell responses was greater in mice lacking both IFN-γ and perforin, and the hierarchy was also shifted (199, 200). These studies demonstrate that in the absence of perforin, CD8 T cell responses to different epitopes are uniformly increased, while the absence of IFN-γ has more selective effects on individual epitopes, thereby altering the immunodominance hierarchy. Presentation of Listeria antigens in mice lacking IFN-γ suggests that the abundance of different epitopes is affected (201). The relative roles of antigen and inflammation in the expansion and differentiation of CD8 T cells remain undefined. Increased inflammation 5 days after priming modestly enhances T cell expansion, but whether this reflects increased proliferation or decreased apoptosis is unclear (202). Enhanced expansion by inflammation, however, does not translate into larger memory T cell populations. In contrast, Listeria infection of IL-15 transgenic mice results in increased populations of memory CD8 T cells and enhanced immunity (203, 204). L. monocytogenes is effective at inducing an inflammatory response that optimizes CD8 T cell priming and memory formation, bypassing the requirement for CD40-CD154 activation (205). In contrast, CD28 is critical for optimal CD8 T cell expansion but not effector differentiation during Listeria infection (206). By mechanisms that remain undefined, γ δ T cells also influence the magnitude of CD8 T cell responses to Listeria infection (207). CD8 T cells responding to a dominant L. monocytogenes epitope express a broad array of TCR Vβ chains (208). Reinfection of mice induces a prominent and accelerated CD8 T cell response resulting in a T cell population that expresses a narrower range of T cell receptors that is of higher avidity for the cognate epitope (209). While CD8 T cells confer protection against L. monocytogenes infection, the mechanism of microbial clearance remains undefined. IFN-γ expression by Listeria-specific CD8 T cells is dispensable for protection (210). Remarkably, cytolytic function of CD8 T cells is also not required, as Listeria-specific CD8 T cells lacking perforin provide protection in mice deficient in Fas (CD95) (211). Finally, even Listeria-specific CD8 T cells lacking TNF conferred protection (212).

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Interestingly, CD8 T cells lacking both perforin and TNF could provide protection against Listeria infection in the liver, but not the spleen (212). Only one CD8 T cell product has been implicated in Listeria-specific protective immunity: MIP-1α. Listeria-specific CD8 T cells from MIP-1α-deficient mice are incapable of conferring protective immunity (213). This suggests that CD8 T cells may recruit cells to sites of L. monocytogenes infection, thereby indirectly leading to bacterial clearance. Immunization with heat-killed Listeria (HKLM), or with Listeria lacking LLO, does not induce protective immunity. Immune responses to killed L. monocytogenes can be enhanced, however, by concurrent activation of the innate immune system. For example, immunization with HKLM and IL-12 enhances CD4 T cell responses and provides long-lived protective immunity (214). Co-administration of anti-CD40 antibody with HKLM also induces protective immunity (215). AntiCD40 treatment induces IL-12 production by antigen-presenting cells, thereby enhancing T cell priming. A recent study examined in vivo CD8 T cell responses to HKLM immunization. HKLM immunization is remarkably effective at priming CD8 T cells, but primed CD8 T cells do not differentiate into effector cells (216). HKLM-primed CD8 T cells, upon re-exposure to live infection, divide and differentiate into effector T cells. Concurrent immunization with live Listeria and HKLM induces distinct responder T cell populations, suggesting that priming by live and killed bacteria occurs in distinct compartments. Recent studies have highlighted the prevalence of effector T cells in nonlymphoid tissues following infection (217). Because L. monocytogenes causes disseminated infection, it is not surprising to find Listeria-specific CD8 T cells in a range of tissues. In fact, CD8 T cells mediate immunity to Listeria infection by direct recognition of peripheral cells such as hepatocytes in the absence of syngeneic bone marrow–derived antigen presenting cells (218). Thus, trafficking of CD8 T cells to nonlymphoid tissues is essential for protective immunity. The lamina propria (LP) of the small intestine contains a high frequency of L. monocytogenes–specific memory T cells (219, 220). Although the LP and secondary lymphoid T cells share antigen specificity, subtle differences in their TCR repertoires suggest that these compartments are not entirely overlapping (219). Although the explanation for the high frequency of Listeria-specific memory T cells in the LP remains unclear, several facts are known. In contrast to T cell responses in the spleen, those in the lamina propria are dependent upon CD4 T cells and CD40-mediated signals (220). Interestingly, Listeria infection of intestinal epithelial cells was demonstrated to induce IL-15 production (221), a cytokine that plays a major role in memory T cell homeostasis (222). The role of mucosal T cells in protective immunity remains unresolved. Mice lacking T, B, and NK cells are not more susceptible to early intestinal infection with Listeria than wild-type mice, suggesting that mucosal T cells, at least in na¨ıve mice, do not play a major role in defense (223). Careful characterization of CD8 T cell clones derived from Listeria-infected mice demonstrated that a substantial fraction were not MHC restricted, i.e., they recognized Listeria-infected macrophages from diverse mouse strains (224).

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Subsequently, the MHC class Ib molecule, H2-M3, was found to present L. monocytogenes–derived N-formyl methionine peptides to CD8 T cells (225, 226). Most laboratory mouse strains (BALB/c, C57BL/6, C3H, SJL) express identical alleles of H2-M3, explaining the apparent lack of MHC restriction. Three Listeriaderived peptides recognized by H2-M3-restricted clones have been identified, all of which are short, hydrophobic, and contain N-formyl methionine at the amino terminus (227–229). CD8 T cell responses to these peptides following primary and secondary Listeria infection are distinct from MHC class Ia–restricted responses. Specifically, H2-M3-restricted responses during primary infection are often larger and more rapid than conventional T cell responses (230, 231). During secondary infection, H2-M3-restricted T cells undergo limited expansion, generally achieving peak frequencies that are smaller than those seen during the primary response (231–233). In contrast, memory responses to MHC class Ia–restricted peptides are far more robust, generally peaking at 7 to 10 times the frequency seen during the primary response (231). Listeria infection of mice lacking MHC class Ia molecules induces CD8 T cell– mediated immunity almost equivalent to that seen in normal mice (234). H2-M3-restricted CD8 T cells are cytolytic and produce IFN-γ and TNF (230) and can mediate protective immunity (235). Transfer of H2-M3-restricted CTL into TAP-deficient mice confers partial protection, indicating that TAP-dependent and -independent antigen processing pathways are operative (235). Processing and presentation of L. monocytogenes N-formyl-methionine peptides by infected cells is poorly defined. In uninfected cells, most H2-M3 molecules remain in the ER because endogenous N-formyl methionine peptides are scarce (236). Some L. monocytogenes–derived N-formyl methionine peptides are bound by gp96 prior to association with H2-M3 (237). The flanking region of the LemA-derived, H2-M3-associated epitope is critical for efficient presentation and T cell priming (238). Remarkably, immunization with the 27 amino acid flanking region of LemA fused to known L. monocytogenes epitopes primes protective CD8 T cell responses.

Salmonella typhimurium Salmonella cause disease by traversing an epithelial layer and surviving in macrophage vacuoles. Immunization with Salmonella strains expressing malaria circumsporozoite protein primes CD8 T cells that mediate protection against malaria (239, 240), and oral immunization with Salmonella expressing simian immunodeficiency virus (SIV) capsid induces SIV-specific CTL (241). In early studies of Salmonella-induced CD8 T cells, antigens were synthesized but not secreted by bacteria. A novel approach for targeting the cytosol of cells exposed to Salmonella exploited the type III secretion system of gram-negative pathogens. This multiprotein virulence determinant mechanically injects bacterial proteins into target cell cytosol, providing direct access to the class I MHC antigen-processing pathway. The approach was successful (242) and effectively induced long-term protective immunity against a viral pathogen (243). This strategy was used to generate

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Salmonella secreting L. monocytogenes epitopes that induced protective CD8 T cell responses (244). Although type III secretion of antigen may enhance priming of CD8 T cells, whether this route serves as the major pathway into the MHC class I antigen-processing pathway during Salmonella infection remains unclear. Whereas some evidence indicates that Salmonella-derived antigens are degraded and bound by MHC class I molecules in vacuolar compartments (245), more recent attention has focused on dendritic cell presentation of bacterial antigens (246). Salmonella infection induces dramatic redistribution of DC populations, with a marked increase in the number of CD8+, CD11c+ DCs in the red pulp of infected spleens (247). Infection of mice with green fluorescent protein (GFP)–expressing Salmonella demonstrated their localization within CD11c-expressing DCs (248). Immunization with DCs that had phagocytosed live or killed Salmonella primed CD4 and CD8 T cells (248). Salmonella infection induces remarkable T cell activation, with 20% to 30% of CD4 and CD8 T cells producing IFN-γ 3 to 4 weeks after infection (249). Activation of T cells during Salmonella infection requires CD28 costimulation (250). While the specificity of CD8 T cells activated by Salmonella infection is largely unknown, at least some CD8 T cells are specific for a peptide derived from the bacterial heat-shock protein GroEL in the context of the Qa-1 MHC class Ib molecule (251, 252). This finding is remarkable because the bacterial GroEL peptide is conserved among diverse gram-negative bacterial species and is so similar to mammalian HSP-60 that GroEL-specific CD8 T cells crossreact with Qa-1/self HSP-60 peptides (252).

Mycobacterium tuberculosis Mycobacteria are phagocytosed by pulmonary macrophages and can survive within phagosomes. A key feature of infection with Mycobacterium tuberculosis is longterm latency: Viable organisms survive in the host for decades. T lymphocytes play distinct roles during defense against primary infection and control of latent infection. Selective depletion of T cell subsets demonstrated that CD4 T cells play a predominant role in mycobacterial clearance, but CD8 T cells also contribute (253). Indeed, CD8 T cells isolated from infected mice lyse infected macrophages and produce IFN-γ (254). Mycobacterium BCG, an attenuated cousin of M. tuberculosis, also induces specific CD8 T cells (255). The importance of CD8 T cells in anti-mycobacterial defense received further confirmation when β 2m−/− mice were found to be more susceptible to intravenous infection with M. tuberculosis (256). Mycobacterial infection of mice lacking TAP-1 also demonstrated a role for MHC class I–restricted CD8 T cells (257, 258). Whereas CD8 T cells play a significant role in defense against intravenously inoculated mycobacteria, their contribution to defense against primary aerosol infection is less impressive. Thus, while mice lacking either all T cells or only CD4 T cells are more susceptible to aerosol infection with M. tuberculosis, mice lacking

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CD8 T cells survive almost as long as wild-type mice and have only a tenfold increase in mycobacterial burden (259). Analysis of pulmonary T cell infiltration following aerosol infection revealed that both CD4 and CD8 T cells expand, peaking approximately 8 weeks following infection (260). IFN-γ is essential for clearance of mycobacterial infection, with CD4 and CD8 T cells producing IFN-γ in vivo following lung infection with M. tuberculosis (261, 262). Indeed, transferred T cells only provide protection if they produce IFN-γ (263). Interestingly, CD4 T cells prominently produce IFN-γ during the first few weeks of infection, while CD8 T cell production of IFN-γ occurs later. Thus, CD4 T cells play a greater role in early defense against M. tuberculosis infection, and both CD4 and CD8 T cells control latent mycobacteria (169, 262). Recruitment of CD4 and CD8 T cells to the lung occurs simultaneously, with CD4 T cells predominating in frequency and IFN-γ production (264). CD8 T cells that enter the lung are cytolytic, express perforin, and lyse M. tuberculosis–infected macrophages (265). Interestingly, while recruitment, expansion, and IFN-γ production by CD8 T cells in the lung of M. tuberculosis–infected mice does not depend upon the presence of CD4 T cells, the acquisition of cytolytic activity does (266). Determining the specificity of CD8 T cells following M. tuberculosis infection is challenging. While the murine CD1d molecule does not appear to play a significant role in the presentation of mycobacterial antigens to CD8 T cells (257, 258), the H2-M3 MHC class Ib molecule presents several M. tuberculosis peptides to CD8 T cells (267). Thus, H2-M3 presentation of N-formyl peptides, which occurs following L. monocytogenes infection, may also contribute to defense against M. tuberculosis infection. Other antigens have also been identified, including an epitope identified in the mycobacterial protein MPT64 (268).

PROTOZOAL PATHOGENS Murine models of parasitic diseases have been instrumental in providing a better understanding of how the immune system handles infection by protozoan pathogens, which are characterized by complex and usually antigenically distinct life cycle stages in their host.

Plasmodium Malaria, caused by Plasmodium species, is one of the most important parasitic diseases in humans and a leading cause of mortality worldwide. A bite from an infected mosquito transmits the malaria sporozoites into the bloodstream and eventually to the liver where they invade hepatocytes and undergo a series of rapid divisions to give rise to the merozoite stage form of the parasite. The merozoites reenter the bloodstream and invade erythrocytes, where they multiply to produce either more merozoites or gametes, which can be taken up by a feeding mosquito.

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The complex life cycle of the pathogen involving invasion of two distinct host cells types, one of which lacks MHC class I expression, implies that different arms of the immune system are utilized to combat the distinct life cycle forms of the parasite. Studies using the rodent parasites Plasmodium berghei and Plasmodium yoelli have shown that CD8 T cells are critical for protective immunity specifically against liver stages of infection. Immunization with radiation-attenuated sporozoites induces long-lasting protective immunity against challenge with sporozoites, but not with infected erythrocytes. In vivo depletion of CD8 T cells reduces sporozoitespecific immunity (269, 270), and transfer of cytotoxic T cell clones specific for circumsporozoite protein or sporozoite surface protein 2 confers protection (271– 273). Protective immune responses are restricted to the liver stage because transfer of CD8 T cells does not confer immunity to blood stage P. yoelli infection, and β 2m-deficient mice resolve blood stage malarial infections with kinetics of normal mice (274, 275). CD4 T cell production of IL-4 helps maintain liver-stage specific CD8 T cell responses by either sustaining the proliferative response or preventing activation-induced death of CD8 T cells (276). Thus, while CD8 T cells play a central role in protective immunity to malaria, CD4 T cell help is necessary for optimal protective responses (277). CD8 T cells protect against the pre-erythrocytic stage of malaria by producing IFN-γ upon recognition of MHC class I–bound parasite-derived peptides on infected hepatocytes (278, 279). Although efficient CTL contact with infected hepatocytes is necessary for protective immunity (280), direct cytotoxicity via perforin, granzyme B, or Fas-FasL interactions is not crucial (281, 282). On the other hand, IFN-γ -deficient mice fail to develop protective immunity after P. yoelli irradiated sporozoite immunization, despite effective induction of CD8 CTL (269, 283). CD8 T cell–derived IFN-γ may induce hepatocyte production of nitric oxide (NO), thereby destroying infected hepatocytes or inactivating intracellular parasites (284–287). CD8 T cell secretion of IFN-γ may also stimulate DCs and macrophages to produce IL-12, which is essential for protective immunity (283). BALB/c mice lacking IL-12 produce less IFN-γ upon sporozoite challenge and do not develop immunity (283). On the other hand, injection of IL-12 into BALB/c mice prior to sporozoite challenge confers IFN-γ independent protection (288). Since NK cells also contribute to protective immunity conferred by irradiated sporozoite immunization (283), it is possible that IL-12 acts on NK cells to augment IFN-γ production. Transfer of na¨ıve T cells specific for P. yoelli circumsporozoite protein (CS252-260/H-2Kd) into na¨ıve mice followed by sporozoite challenge demonstrated that antigen-specific T cells expanded for 4–5 days postinfection, decreasing in frequency rapidly thereafter (123). IFN-γ and perforin expression and CD44 upregulation on antigen-specific T cells was detected 24 h postinfection and prior to T cell proliferation. Cytolytic function was acquired 48 h after infection. T cells could inhibit parasite development in a dose-dependent fashion within the first 24 h following infection, consistent with findings from other model systems, such as HSV infection.

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Toxoplasma gondii Toxoplasma gondii infects a wide variety of vertebrate hosts, living within a broad range of nucleated cell types. The acute stage of infection is characterized by rapid multiplication and widespread dissemination of tachyzoites, followed by formation of quiescent tissue cysts in the central nervous system and muscle during the latent phase. Infection with this parasite is clinically important during pregnancy and in highly immunocompromised individuals, such as AIDS patients (289, 290). Several murine experimental systems have shed light on the host response to acute and chronic stages of T. gondii infection. One uses an attenuated temperaturesensitive mutant strain of the parasite, ts-4, which infects multiple tissues but does not persist in mice (291). ME49 is another low virulence strain that forms longlasting cysts within brains of infected animals. Immunization of mice with ts-4 induces protective immunity against challenge with a lethal dose of the virulent RH strain of Toxoplasma, mediated primarily by T cells (292, 293). Although neutrophils and NK cells play critical roles in containing the early stages of infection (294, 295), CD8 T cells, by producing IFN-γ , are the major effector cells providing resistance to T. gondii infection (292, 296–301). Similar to malaria infection, effective CD8 T cell immunity to T. gondii requires CD4 T cell help. Although CD8 T cell responses are induced normally by primary oral immunization with T. gondii, immunity wanes without CD4 T cells, and mice become susceptible to T. gondii (302). Interestingly, in MHC class II–deficient mice, which lack CD4 T cells, CD4+NK1.1+ T cells may provide help to CD8 T cells during T. gondii infection (303). Antigen-specific CD8 T cells from infected mice express MHC-restricted cytolytic activity (304–306), but the importance of cytolysis in vivo appears to be limited to chronic infection. Perforin-deficient mice previously immunized with the ts-4 mutant are completely resistant to lethal T. gondii challenge but are more susceptible to the persistent ME49 strain (307). CD8 T cell production of IFN-γ is believed to activate macrophages to produce nitric oxide, which inhibits the growth of T. gondii (308, 309). However, mice deficient in inducible nitric oxide synthase (iNOS) survive ts-4 parasite immunization and develop protective immunity. On the other hand, control of persistent T. gondii infection appears to be NO-dependent (310, 311). IL-15 plays a role in the generation and maintenance of CD8 T cell memory to T. gondii. Exogenous IL-15 administration enhances the CD8 T cell response to T. gondii infection and prolongs the duration of CD8 T cell immunity (312, 313). Conversely, blocking endogenous IL-15 in vivo with a soluble IL-15 receptor fragment reduces the memory CD8 T cell response to T. gondii infection (314).

Trypanosoma cruzi Trypanosoma cruzi, the causative agent of Chagas’ disease, is an intracellular protozoan parasite endemic to Central and South America. Transmitted by the bite of a hematophagous insect, it causes chronic, probably life-long infection

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(315). A number of mammalian hosts exist for T. cruzi, and the parasite can invade many different nucleated cell types. Once intracellular, trypomastigotes enter the cytoplasm where they multiply as amastigotes (316, 317). Entry of T. cruzi into the host cell cytosol suggests that CD8 T cells might respond to this parasite, and several studies confirm this suspicion. Mice depleted of CD8 T cells or deficient in CD8 T cell development exhibit increased susceptibility to T. cruzi infection and do not develop protective immunity upon vaccination with avirulent strains of the parasite (318–324). Moreover, adoptive transfer of antigen-specific CTLs confers protection to challenge infection (325). CD8 T cell epitopes have been identified within trypomastigote sialidase/trans-sialidase surface antigens and in two amastigote surface proteins of T. cruzi (325, 326). Although CD8 T cell responses play a major role in immunity to this parasite, MHC class II–deficient mice or CD4-depleted mice exhibit defective responses to T. cruzi infection (322, 327). CD8 T cells exhibit cytolytic activity during T. cruzi infection (328). Whereas mice deficient in perforin or granzyme B are capable of controlling low dose T. cruzi infection (318), infection with higher doses of virulent strains of the parasite can be lethal in perforin knockout, but not wild-type mice (329). Production of IFN-γ by CD8 T cells appears to be most important for protection (330, 331). The mechanism of IFN-γ -mediated resistance most likely involves induction of NO-dependent microbicidal activity (332).

CONCLUDING REMARKS Comparing CD8 T cell responses to different pathogens has revealed differences but also some interesting similarities. The kinetics of T cell expansion and contraction in response to infection by LCMV, L. monocytogenes and influenza virus are remarkably similar, for example, with peak expansion occurring 8 days following the initiation of these infections. In addition, the finding by several groups that transiently stimulated T cells undergo prolonged proliferation in the absence of antigen (195–197) demonstrates that T cells can be programmed. The new concept, that T cell expansion is “programmed” at the time of T cell priming, is replacing the more conventional idea that expansion is driven by antigen, terminating when antigen is depleted in vivo. However, some infections induce more prolonged expansion of CD8 T cells, such as in γ -herpesvirus infection, and the kinetics of T cell expansion and contraction can differ in different tissues. This suggests that T cells can be programmed differently, depending upon the nature of the initial exposure to antigen or the tissue to which activated T cells are targeted. It is interesting to compare the requirements for costimulation and CD4 help in the priming of CD8 T cell responses following infection by different pathogens. Infection by L. monocytogenes, for example, primes CD8 T cells in the absence of CD4 T cell help, CD40 stimulation, and even CD28 ligation (205, 206). On the other hand, priming of CD8 T cells specific for protozoal pathogens is CD4

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T cell–dependent (276, 302, 327). As the in vivo mechanisms of CD8 T cell priming are further delineated for different classes of pathogens, it is likely that patterns of costimulatory requirements will emerge that will reflect the intensity and nature of the inflammatory response induced by the specific pathogen. Thus, infection by L. monocytogenes, which is known to elicit an exuberant inflammatory response, bypasses some of the costimulatory pathways for CD8 T cell priming required for immune responses to less inflammatory pathogens. Our understanding of in vivo trafficking of CD8 T cells has increased dramatically in the last several years. The concept of central and effector memory T cells, first introduced by Sallusto and Lanzavecchia (333), has gained additional support by the findings that activated memory T cells are remarkably prevalent in peripheral tissues of mice infected with a variety of pathogens. Interestingly, the localization of memory CD8 T cells to peripheral tissues is not restricted to the original site of infection. Rather, memory T cells congregate throughout immune animal tissues. While the frequency of antigen-specific memory T cells in lymphoid tissues detected by MHC tetramer staining is surprising, the frequency of memory T cells in tissues such as lung following influenza virus infection is truly astounding. How these high frequencies of pathogen-specific T cells are maintained long after infection has resolved is starting to be deciphered and appears to relate, at least in part, to localized IL-15 production. The coming decade will provide unprecedented opportunities to dissect CD8 T cell responses to a range of pathogens. The complete genome sequences of most of the important microbial pathogens are already available, greatly enabling antigen and epitope identification. The ever-increasing number of mouse strains with deletions of genes involved in immune recognition, signaling, and effector functions provides a myriad of opportunities to uncover mechanisms of antimicrobial defense. Indeed, we are only beginning to decipher the complex but beautifully orchestrated circuitry that defends us from the universe of invasive pathogens. The Annual Review of Immunology is online at http://immunol.annualreviews.org

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305. Kasper LH, Khan IA, Ely KH, Buelow R, Boothroyd JC. 1992. Antigen-specific (p30) mouse CD8+ T cells are cytotoxic against Toxoplasma gondiiinfected peritoneal macrophages. J. Immunol. 148:1493–98 306. Subauste CS, Koniaris AH, Remington JS. 1991. Murine CD8+ cytotoxic T lymphocytes lyse Toxoplasma gondiiinfected cells. J. Immunol. 147:3955– 59 307. Denkers EY, Yap G, Scharton-Kersten T, Charest H, Butcher BA, Caspar P, Heiny S, Sher A. 1997. Perforin-mediated cytolysis plays a limited role in host resistance to Toxoplasma gondii. J. Immunol. 159:1903–8 308. Adams LB, Hibbs JB Jr, Taintor RR, Krahenbuhl JL. 1990. Microbiostatic effect of murine-activated macrophages for Toxoplasma gondii. Role for synthesis of inorganic nitrogen oxides from Larginine. J. Immunol. 144:2725–29 309. Deckert-Schluter M, Rang A, Weiner D, Huang S, Wiestler OD, Hof H, Schluter D. 1996. Interferon-γ receptordeficiency renders mice highly susceptible to toxoplasmosis by decreased macrophage activation. Lab. Invest. 75:827–41 310. Khan IA, Matsuura T, Kasper LH. 1998. Inducible nitric oxide synthase is not required for long-term vaccine-based immunity against Toxoplasma gondii. J. Immunol. 161:2994–3000 311. Scharton-Kersten TM, Yap G, Magram J, Sher A. 1997. Inducible nitric oxide is essential for host control of persistent but not acute infection with the intracellular pathogen Toxoplasma gondii. J. Exp. Med. 185:1261–73 312. Khan IA, Casciotti L. 1999. IL-15 prolongs the duration of CD8+ T cellmediated immunity in mice infected with a vaccine strain of Toxoplasma gondii. J. Immunol. 163:4503–9 313. Khan IA, Kasper LH. 1996. IL-15 augments CD8+ T cell-mediated immunity

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nitrogen oxide-mediated mechanism inhibitable by interleukin-10 and transforming growth factor-β. Eur. J. Immunol. 22:2501–6 333. Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A. 1999. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401:708–12

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ON NATURAL AND ARTIFICIAL VACCINATIONS, Rolf M. Zinkernagel COLLECTINS AND FICOLINS: HUMORAL LECTINS OF THE INNATE IMMUNE DEFENSE, Uffe Holmskov, Steffen Thiel, and Jens C. Jensenius THE BIOLOGY OF IGE AND THE BASIS OF ALLERGIC DISEASE,



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CONTROL OF APOPTOSIS IN THE IMMUNE SYSTEM: Bcl-2, BH3-Only Proteins and More Annu. Rev. Immunol. 2003.21:71-105. Downloaded from arjournals.annualreviews.org by HINARI on 09/01/07. For personal use only.

Vanessa S. Marsden and Andreas Strasser Molecular Genetics of Cancer Division, Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia; email: [email protected], [email protected]

Key Words lymphocyte development, myeloid development, Fas, cell survival, repertoire selection ■ Abstract Apoptotic cell death plays a critical role in the development and functioning of the immune system. During differentiation, apoptosis weeds out lymphocytes lacking useful antigen receptors and those expressing dangerous ones. Lymphocyte death is also involved in limiting the magnitude and duration of immune responses to infection. In this review, we describe the role of the Bcl-2 protein family, and to a lesser extent that of death receptors (members of the tumor necrosis factor receptor family with a death domain), in the control of lymphoid and myeloid cell survival. We also consider the pathogenic consequences of failure of apoptosis in the immune system.

INTRODUCTION TO APOPTOSIS The removal of superfluous, defective, damaged, or dangerous cells is critical for normal development and tissue homeostasis in multicellular organisms (1). The death of these unwanted cells occurs by a process called apoptosis (2), which is characterized morphologically and biochemically by blebbing of the plasma membrane, surface exposure of phosphatidylserine, condensation of the nucleus, internucleosomal DNA fragmentation, and phagocytosis of the corpse by neighboring cells (3–5).

Molecular Control of Apoptosis The molecular control of this genetically programmed cell death process has been evolutionarily conserved in metazoans (1). The death effector machinery is driven by a family of aspartate-specific cysteine proteases, known as caspases, which cleave many vital cellular proteins (e.g., lamins) and proteolytically activate enzymes that contribute to cell destruction, such as the DNAse DFF40/CAD (6). Many caspases are present in healthy cells as catalytically dormant pro-enzymes (zymogens), which are activated in response to developmentally programmed cues and stress stimuli that trigger apoptosis. 0732-0582/03/0407-0071$14.00

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Two mechanisms exist for caspase activation. (a) Certain zymogens (caspases 1, 2, 4, 5, 8, 9, 10, 11, and 12) have long N-terminal pro-domains that undergo homotypic interaction with specific adaptor proteins, such as FADD/MORT1 or Apaf-1. This promotes aggregation of zymogens that allows their low enzymatic activity to bring about autocatalytic processing to generate polypeptides of ∼20 kDa and ∼10 kDa that assemble into the fully active caspase (7). (b) Caspases with short pro-domains (caspases 3, 6, and 7) are activated predominantly through proteolysis by already active caspases (or by granzymes: aspartate-specific serine proteases). Thus, caspases form a self-amplifying avalanche of proteolytic activity, and it appears that the adaptor protein-regulated (long pro-domain) caspases kick-start the death effector machinery (“initiator caspases”), whereas those with short prodomains perform most of the proteolysis of vital substrates (“effector caspases”) (6). Caspase activity in the cell can be controlled at two levels (Figure 1). First, as discussed below, caspase activity is induced by upstream apoptosis signaling pathways that are highly regulated. Second, certain caspases can be antagonized by the inhibitor of apoptosis proteins (IAPs). These IAPs bind to the active site of caspases, acting as competitive inhibitors (8). The anti-apoptotic effect of IAPs is neutralized upon release of certain mitochondrial proteins, such as Smac/DIABLO and HTRA2, which can sequester the IAPs (8).

TWO DISTINCT APOPTOSIS SIGNALING PATHWAYS IN MAMMALS Death Receptor-Induced Apoptosis Mammals have two distinct apoptosis signaling pathways that require different initiator caspases but converge at the level of effector caspase activation (9). Ligation of death receptors (e.g., Fas/APO-1/CD95, TNF-R1) causes formation of a death-inducing signaling complex (DISC) (10) in which the adaptor proteins FADD and TRADD bind with their death domain (DD) to a DD in the cytoplasmic region of the receptors (11, 12). Through homotypic interaction of death effector domains (DED), FADD causes recruitment and activation of caspase-8 (and in humans also caspase-10) (13, 14). Experiments with knockout mice and transgenic mice have shown that FADD (15–17) and caspase-8 (18, 19) are essential for death receptor–induced apoptosis. In contrast, these signal transducers, and death receptors per se, appear to be dispensable for apoptosis triggered by cytokine withdrawal or cytotoxic stress, such as that imposed by γ -radiation or chemotherapeutic drugs. Signaling by death receptors can be inhibited by cellular and viral FLICE-inhibitory proteins (FLIPs), which consist either of two DED domains or of an entire caspase-8-like molecule in which the active site (and other critical residues) are missing (20). FLIPs are recruited to the DISC, preventing the activation and release of caspase-8 in cells stimulated with ligands for death receptors (21, 22). FLIPs have no effect on apoptosis induced by cytokine withdrawal

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Figure 1 Pathways to apoptosis in hematopoietic cells. Mammalian cells may die either following ligation and multimerization of cell surface death receptors or via a pathway regulated by the Bcl-2 protein family that is initiated in response to many stress stimuli. These pathways converge to activate effector caspases that function to demolish the cell. While mitochondrial damage is thought by many to be essential for Bcl-2-inhibitable apoptosis, some evidence suggests a pathway that may act upstream and/or in parallel to the mitochondrial events (grey arrows). The precise mediators of this are not yet elucidated.

or cytotoxic drugs (23), consistent with the notion that death receptor signaling is not involved in those cell death signaling pathways. At least in lymphocytes and myeloid cells, death receptor–induced apoptosis is not controlled by the Bcl-2 protein family (24–26). In contrast, pro- and anti-apoptotic members of the Bcl-2 family are critical regulators of cytokine withdrawal- and stress-induced apoptosis (27–31). The death receptor signaling pathway and the Bcl-2 regulated pathway can be connected through

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caspase-mediated activation of the pro-apoptotic Bcl-2 family member Bid (32, 33), but it is presently not clear in which physiological cell death program this plays a critical role.


The Bcl-2-Regulated Cell Death Signaling Pathway The mammalian Bcl-2 family consists of both pro- and anti-apoptotic proteins, which show sequence and structural similarity in the Bcl-2 homology (BH) regions (34). In addition to Bcl-2, the anti-apoptotic faction includes A1/Bfl-1 (35, 36), Bclw (37), Bcl-xL(38), Boo/Diva/Bcl-B (39–41), and Mcl-1 (42). These proteins all share three or four BH regions, and they localize to the cytoplasmic faces of intracellular membranes, such as the outer mitochondrial membrane, the endoplasmic reticulum, and the nuclear envelope (43). A subgroup of pro-apoptotic Bcl-2 family members, including Bax (44), Bak (45–47), Bok/Mtd (48, 49), Bcl-xs (a splice variant of the bcl-x gene) (38), and Bcl-GL (50), have two or three BH regions and appear to be structurally very similar to their prosurvival relatives (51). A divergent subgroup of apoptosis-inducing Bcl-2-related proteins share with the family only the short BH3 region (52). These “BH3-only” proteins include Bad (53), Bcl-Gs (a splice variant of the bcl-g gene) (50), Bid (54), Bik/Nbk (55), Bim/Bod (56), Blk (57), Bmf (58), Hrk/DP5 (59, 60), Noxa (61), and PUMA/Bbc3 (62–64). How the Bcl-2 family regulates stress-induced apoptosis and which initiator caspase is essential for this process remain hotly debated issues (9, 65–67). Genetic studies in Caenorhabditus elegans have provided important insight. In this organism three proteins are required for programmed cell death: the pro-apoptotic BH3-only Bcl-2 family member EGL-1, the caspase CED-3 and its adaptor CED4, whereas the anti-apoptotic Bcl-2 ortholog CED-9 is essential for cell survival (4, 68). Biochemical experiments and studies in the yeast two-hybrid system have indicated that CED-9 promotes survival by direct binding to CED-4, thereby preventing this adaptor protein from activating the caspase CED-3 (4). Programmed cell death is initiated by transcriptional induction of EGL-1, which binds to CED-9 and thereby blocks its ability to keep CED-4 in check (4). Bcl-2 and CED-9 share significant sequence similarity and human Bcl-2 can (at least in part) substitute for CED-9 function in C. elegans (4). It has therefore been proposed that Bcl-2 regulates apoptosis in mammalian cells by a biochemical mechanism analogous to the one regulated by CED-9 in C. elegans (1, 9). In agreement with this notion, initiation of cell death in mammals requires EGL-1-related BH3-only proteins, such as Bim (56), which is essential for the killing of lymphocytes deprived of growth factors (30) of those expressing autoreactive antigen receptors (69). A mammalian CED-4-related caspase adaptor, called Apaf-1, was found (70), but unlike CED-4, which binds to CED-9, Apaf-1 did not bind to Bcl-2 (or its homologs) (71). Moreover, at its C-terminus Apaf-1 has multiple WD40 repeats that have autoinhibitory activity, and it therefore requires cytochrome c to activate caspase-9. In contrast, there is no evidence for a role of cytochrome c in CED4 activation and programmed cell death in C. elegans (4). In mammalian cells, cytochrome c is released from mitochondria during apoptosis, and this can be

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prevented by Bcl-2 (4). It is therefore currently widely believed that Bcl-2 functions to preserve mitochondrial membrane integrity and that stress-induced apoptosis signaling relies entirely on the cytochrome c/Apaf-1/caspase-9 apoptosome (65–67). BH3-only proteins act as sensors for apoptotic stimuli and initiate the apoptotic cascade (52), but pro-apoptotic Bax and Bak are also essential for cell killing in response to death stimuli that can be antagonized by Bcl-2 (31). Bax/Bak-like proteins appear to function downstream of BH3-only proteins because bax−/−bak−/− fibroblasts are resistant to enforced overexpression of Bim, Bad, or Noxa (72, 73). Upon induction of apoptosis, Bax moves from the cytosol to the mitochondria to form clusters with Bak, facilitating cytochrome c release (67). It has been suggested that BH3-only proteins induce apoptosis by directly influencing Bax/ Bak-like proteins and that Bcl-2 promotes cell survival by sequestering BH3-only proteins (73). However, for most BH3-only proteins, binding to Bax or Bak with significant affinity has not been demonstrated, and it is therefore equally likely that BH3-only proteins induce apoptosis by activating Bax/Bak-like proteins indirectly (9). It might appear that C. elegans does not have a Bax/Bak-like protein. However, CED-9 is not only essential for cell survival, but it can also promote apoptosis under certain conditions (74). Because Bax and Bcl-xL are very similar in structure (51), the postulate has therefore been put forward that CED-9 may be able to switch conformation from an anti-apoptotic (Bcl-2-like) to a pro-apoptotic (Bax-like) state. The events that lead to mitochondrial membrane disruption and release of proapoptotic molecules (e.g., cytochrome c, Smac/DIABLO) are still a matter of much debate (9, 65–67). According to one theory, Bax/Bak-like proteins (directly or indirectly) form mitochondrial pores, and all initiation of the caspase cascade occurs downstream of mitochondria in the cytochrome c/Apaf-1/caspase-9 apoptosome (65–67). However, there are some indications that the order of events may be in the opposite orientation—i.e., caspases are activated upstream of the mitochondrial pathway and are essential for cytochrome c release. For example, active caspase-2 can cause the release of cytochrome c from isolated mitochondria (75), and novel caspase inhibitors can prevent mitochondrial release of cytochrome c in lymphoid and fibroblastoid cells exposed to apoptotic stimuli (253, 254). Moreover, hematopoietic cells that lack either Apaf-1 or caspase-9 can undergo classical caspase-mediated apoptosis despite the absence of the requisite molecules for cytochrome c–induced apoptosis (254). In a similar way, release of cytochrome c does not appear to be a prerequisite for caspase-mediated apoptosis in Drosophila (76, 77). We therefore postulate that apoptosis is initiated upstream of mitochondria by caspases activated by adaptors that are directly regulated by Bcl-2 (9). Accordingly, the mitochondrial pathway is not essential for initiation of cell death but may instead function as a machinery for amplification of caspase activity. MECHANISMS FOR CONTROLLING BH3-ONLY PROTEINS Death stimuli antagonized by Bcl-2 initiate apoptosis through the BH3-only proteins (52). In C. elegans, EGL-1 is transcriptionally regulated in response to developmental cues (78).

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Similarly, in mammals some death stimuli can induce death through increasing the transcription of certain BH3-only genes (79). For example, both Noxa (61) and PUMA/Bbc3 (62–64) are targets of the tumor suppressor p53 and are induced in response to DNA damage. As described below, prosurvival Bcl-2 family members are also transcriptionally regulated in the hematopoietic compartment, with certain stimuli (for example, cytokine signaling) upregulating their transcripts, thereby promoting cell survival. Posttranslational control of BH3-only proteins is also important for apoptosis regulation (79). For example, in healthy cells both Bim and Bmf are held on cytoskeletal structures, the microtubular dynein motor or the actin-based myosin V motor complex. Release of Bim or Bmf occurs in response to specific apoptotic stimuli and allows the induction of apoptosis (58, 80). Another BH3-only protein, Bad, is a target for phosphorylation by protein kinase A and Akt. Cytokines that activate these protein kinases promote phosphorylation and inactivation of Bad by allowing its sequestration by the phosphoserine-binding 14-3-3 protein. Withdrawal of cytokine signals may thus induce apoptosis by permitting the dephosphorylation and release of Bad (81–85). However, bad−/− mice have no detectable abnormality, and their neurons are normally sensitive to NGF deprivation in culture (86). It therefore appears that Bad is not essential for cytokine withdrawal–induced apoptosis, but it may contribute to the function of Bim, which is clearly essential for this pathway to cell death (30, 87).

Caspase Independent Apoptosis While the C. elegans caspase CED-3 has been proven to be indispensable for apoptosis (88), some have suggested that mammalian apoptosis can occur in the absence of caspase activation. These studies have mainly relied upon the use of chemical caspase inhibitors, and the induction of apoptosis in their presence may reflect the inefficiency of the compounds, particularly in vivo (89). Nonetheless, two factors have been identified as potential mediators of some apoptotic events without a need for caspase activation. These are apoptosis-inducing factor (AIF, Ref. 90) and endonuclease G (91). Both have been suggested to be capable of inducing apoptotic changes in the nucleus (i.e., DNA fragmentation), but neither have been demonstrated to be responsible for other stereotypical changes that occur in apoptosis (e.g., exposure of phosphatidylserine and proteolysis of vital substrates), and their importance to programmed cell death is not clear.

APOPTOSIS IN THE DEVELOPMENT AND FUNCTIONING OF THE IMMUNE SYSTEM The regulation of cell survival and death is critical for the functioning of the mammalian immune system. Experiments with transgenic mice and knockout mice have revealed the importance of apoptosis in the maintenance of cell number, the deletion of both useless or autoreactive cells, as well as the removal of expanded

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lymphocyte clones that are generated during an immune response. The lpr and gld mice, which lack Fas (92) and FasL function (93), respectively, show excess lymphocytes, proving the importance of Fas-mediated signals in removing leukocytes in some circumstances. Similarly, abnormalities in the Bcl-2-regulated signaling pathway, such as expression of a bcl-2 transgene (94–96), loss of the BH3-only protein Bim (30), or a combined deletion of Bax and Bak (31), all cause extensive lymphoid and myeloid cell accumulation. Conversely, loss of Bcl-2 causes reduced survival of lymphoid (97–99) and myeloid (A. Villunger, C. Scott, P. Bouillet, and A. Strasser, manuscript submitted) cells and thereby elicits profound immunodeficiency. Similarly, absence of Bcl-x causes massive death of developing erythroid cells in fetal liver (100). Bim appears to be an important physiological regulator of Bcl-2 in lymphocytes, as deletion of both Bim and Bcl-2 prevents the lymphopoenia seen in bcl-2−/− mice (87). The distinct nature of the death receptor–induced and the Bcl-2-regulated pathways to apoptosis is highlighted by the observation that expression of a bcl-2 transgene in lpr or gld mice enhances lymphadenopathy (24, 101). While overexpression or loss of Bcl-2 family members can lead to hyperplasia or hypoplasia of hematopoietic cell populations, to date none of the knockout mutations of any of the caspases have had such dramatic effects in the hematopoietic system. Mice lacking caspases-1 (102), 2 (103), 3(104), 6 (quoted in Ref. 105), 9 (106, 107, 254), 11 (108), and 12 (109) all have hematopoietic systems of normal size and composition. This indicates that there is some redundancy in the use of these caspases in the developmentally programmed death of leukocytes. As yet, no information for the roles of caspases-7 (quoted in Ref. 105) and 8 (19) in the immune system is available from knockout mice, but interestingly, caspase-8−/− embryos have abnormally low numbers of blood cells. As discussed further below, detailed studies have elucidated the specific roles of individual molecules or signaling pathways in the developmentally programmed deaths that occur at the different checkpoints during lymphocyte and myeloid cell development.

Death During B Cell Development and Function DEVELOPMENT OF IMMATURE B CELLS B cells develop in the fetal liver and postnatal bone marrow from a common lymphoid progenitor (Figure 2). Pro-B cells (CD45R/B220+c-Kit+sIg-) are the earliest recognizable stage of cells committed to the B lineage. Pro-B cells have their immunoglobulin (Ig) genes in germline configuration. For development to a mature B cell, both the genes encoding the Ig heavy (H) and light (L) chains must be productively rearranged to produce a functional B cell receptor (BCR). Moreover, selection occurring both in the bone marrow and the peripheral lymphoid organs (particularly the spleen) ensure that autoreactive B cells are deleted (110, 111). Rearrangement of the Ig heavy chain genes is the first step of development of pro-B cells. Productive recombination of the VH, DH, and JH gene segments allows

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Figure 2 Role of apoptosis in the development of B cells. During development in the bone marrow, B cells that express either a nonfunctional B cell receptor (BCR) or an autoreactive BCR are deleted. Within peripheral lymphoid organs, apoptosis serves as a mechanism to regulate the activation of B cells.

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expression of a pre-BCR composed of the IgH chain with the λ5 and Vpre-B surrogate light chains and the Igα and Igβ signal transduction complex (112). Signaling from the pre-BCR permits both the survival of pro-B cells and their differentiation to the pre-B (B220+c-Kit−sIg−) stage (110, 111). B cells do not progress beyond the pro-B stage in rag-1−/−, rag-2−/− and scid mutant mice, which cannot express a pre-BCR due to failure to productively rearrange Ig gene segments (113–115). The survival signal from the BCR is likely to involve upregulation of prosurvival Bcl-2 family members, as overexpression of Bcl-2 or Bcl-xL can prevent the deletion of pro-B cells in scid mutant (116) and Rag-deficient mice (117–119). Differentiation to the pre-B stage is not merely a consequence of the survival signal provided by the pre-BCR, because although Bcl-2 or Bcl-xL overexpression allow the accumulation of pro-B cells in rag-1−/− and rag-2−/− mice, these cells do not show the features of more mature B cells (117–119). Instead, the pre-BCR and BCR must also provide specific differentiation signals. In addition to pre-BCR signaling, development of pre-B cells from pro-B cells also requires signaling from the interleukin-7 receptor (IL-7R) (120, 121). Although Bcl-2 overexpression can restore normal T cell development and function in mice lacking IL-7R (122, 123), it has only a relatively small effect on B cell production (124, 125). This may indicate that the IL-7R activates different prosurvival signaling pathways in different cell types. Alternatively, and in our opinion more likely, the IL-7R may be essential for proliferation in developing B cells but not in pro-T cells. A further role for signaling through the pre-BCR is to promote proliferation to expand the population of developing B cells that have productively rearranged IgH genes (110). After dividing several times, pre-B cells start to rearrange their Ig light chain genes. Productive V-J recombination permits the expression of cell surface IgM heavy chains with Igκ or Igλ light chains, forming the BCR and marking the conversion to the immature (virgin) B cell stage (110, 111). Failure to produce a light chain results in apoptosis. Generation of bcl-2 transgenic rag-2−/− mice that express a functional IgH transgene (and hence can express a pre-BCR and progress to the pre-B stage, but are unable to rearrange their IgL genes) has shown that Bcl-2 can inhibit the death that occurs in the absence of a BCR (119). Differentiation of short-lived virgin B cells to the long-lived mature B cell stage requires signaling by the TNF-related cytokine BAFF (BLys, THANK, TALL1), most likely through BAFF receptor 3 (reviewed in Refs. 126, 127; see also 127a). The function of the two other BAFF-binding receptors, BCMA and TACI, is presently not clear. It appears that BAFF promotes B cell differentiation and survival by upregulating expression of Bcl-2 (128). This signal probably requires NF-κB activation because B cells lacking BAFF (129) or BAFF-R3 (130) or those with combined loss of c-Rel and RelA transcription factors (131) all have a similar defect in differentiation and survival. At least in the c-rel−/−relA−/− B cells, this defect can be rescued by expression of a bcl-2 transgene (131). Sustained survival of mature B cells also requires continuous BCR expression. Induced deletion of the expressed IgH gene in mature B cells of gene targeted mice

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results in their rapid deletion in vivo, and this death is due to a Bcl-2-inhibitable apoptotic pathway (132). NEGATIVE SELECTION OF B CELLS Removal of self-reactive B cells is critical for the prevention of autoimmunity. This negative selection is enforced in immature B cells in the bone marrow and the spleen, in which BCR ligation can result in three possible outcomes: apoptosis, anergy (unresponsiveness), or receptor editing (133). Use of transgenic mouse models in which expression of an Ig specific for an endogenous self-antigen or a pseudo-self-antigen has allowed the investigation of the pathways that silence autoreactive cells. Deletion of immature autoreactive cells does not require Fas (134, 135) but utilizes a pathway that can be inhibited by Bcl-2 or Bcl-xL (136–139). The BH3-only protein Bim is a critical inducer of death induced by BCR ligation in immature B cells, as Bim deficiency prevents the elimination of autoreactive B cells (A. Enders, D. Tarlinton, P. Bouillet, and A. Strasser, in preparation), causing accumulation of autoantibody-secreting plasma cells and systemic lupus erythematosus–like autoimmunity (30). Although overexpression of Bcl-2 can prevent apoptosis induced by BCR ligation, it does not always permit autoreactive B cells to differentiate further. Instead, development of surviving self-reactive B cells is often arrested or the cells are rendered unresponsive to antigenic stimulation by a process known as anergy (138). Anergic B cells have a reduced lifespan compared to normal, mature B cells (140), and this deletion may occur when anergized, autoreactive B cells present antigen to T cells. Studies using mutant gld and lpr mice have shown that in this situation the death signal from the T cell is provided through ligation of Fas on the B cell (141). A possible outcome of BCR ligation in immature B cells is the re-initiation of Rag-induced Ig light-chain gene rearrangement, potentially to yield a new, nonautoreactive BCR, a process known as receptor editing (142). Receptor editing occurs normally in B cells (143), but extension of the lifespan of autoreactive cells by enforced Bcl-2 or Bcl-xL expression increases receptor editing (136, 139), perhaps by allowing the B cells more time to produce a suitable BCR when normally death would have been induced. The decision of an immature B cell to die, inactivate, or alter specificity may be a consequence of the strength or duration of BCR ligation (144). It has also been suggested that early stage immature B cells (IgMloIgD−) may be more likely to undergo receptor editing than later stage cells (IgMhiIgD−), either because they are less susceptible to BCR-induced ligation or because they are more efficient at initiating receptor editing (145). The latter may be more likely, as enforced Bcl-2 expression does not enhance receptor editing in later stage IgMhi cells, despite blocking their death (145). SURVIVAL OF ACTIVATED B CELLS Productive activation of B cells, leading to proliferation and differentiation to antibody-secreting plasma cells, requires signals from the BCR plus signals from co-stimulatory receptors. Presentation of antigen to T helper cells results in the activation of B cells because activated T helper cells produce the costimulatory signals that enhance B cell survival.

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The TNF family members CD40L (146), BAFF (BLys/THANK/TALL-1) (147, 148) and APRIL (149) are important T cell– and macrophage-derived signals for B cell survival. CD40L signals to B cell CD40 (146). Both BAFF and APRIL bind the receptors BCMA and TACI (150, 151), but the binding of BAFF to BAFF-R3 appears to be the physiologically relevant interaction for B cell survival (129, 130, 152). CD40 (153), TACI, BCMA (150, 151) and BAFF-R3 (127, 255) all signal through the Rel/NF-κB family of transcription factors. This allows the upregulation of genes involved in B cell activation as well as of prosurvival genes, which include bcl-2, bcl-xL (154), and A1 (155). The synergy between BCR ligation and costimulatory signals in promoting B cell survival is highlighted by the finding that CD40 ligation causes an upregulation of Fas (156). Without BCR stimulation, this heightens the B cells’ sensitivity to FasL-induced apoptosis, but concomitant BCR stimulation protects cells (156), presumably through upregulation of FLIP (157). Anergic B cells have abnormal BCR signaling, which probably explains why they are deleted by a FasL-Fas-induced mechanism upon presentation of antigen to T cells (141). Studies with mutant mice in which expression of an autoreactive BCR is induced only in mature B cells have shown that these cells are rapidly deleted in vivo (158). In this system, autoreactive B cells presumably receive no T cell help, and consequently the absence of CD40/BAFF/APRIL signaling prevents the required upregulation of Bcl-2, which counters BCR ligation-induced death. Accordingly, these cells are rescued through overexpression of Bcl-2 (158). SURVIVAL OF B CELLS DURING THE HUMORAL IMMUNE RESPONSE Once activated, B cells proliferate and many differentiate into antibody-secreting plasma cells. Antibodies produced early in the immune response are of low affinity and originate from foci of plasma cells in the spleen. As the immune response progresses, activated B cells are recruited to the germinal centers and undergo hypermutation of their Ig genes, followed by selection for higher affinity antibody (159). The high-affinity antibody-forming cells of the germinal center supercede the antibody-forming cells of the foci as the important antibody producers, and the latter undergo apoptosis. This death is probably a consequence of limited access to essential cytokines (e.g., IL-6) and can be inhibited by Bcl-2 overexpression (160). Selection of B cells producing high-affinity antibody is thought to involve competition of B cells for limiting T cell or follicular DC interaction (159). B cells producing the highest affinity antibody are recruited to form antibody-forming plasma cells, while those of intermediate affinity become memory B cells (161). Cells in which hypermutation has produced Ig of low affinity undergo apoptosis, presumably through neglect, and this is inhibited by Bcl-2 (162) or Bcl-xL (163). Despite Fas being highly expressed in germinal center B cells, experiments with mutant lpr mice have demonstrated that this death receptor is not required for germinal center selection (164). At the conclusion of an immune response, expanded clones of antibody forming cells must be reduced to a size that is suited for production of antibodies that

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provide long-lasting protection against infection but not too large to cause immunecomplex mediated disorders. This process relies on the BH3-only protein Bim, and is inhibited by Bcl-2, as both bcl-2 transgenic and Bim-deficient mice have accumulation of antibody-forming cells, and consequently abnormally high serum Ig levels, which can be pathogenic (see below) (30, 95, 160).


Death During T Cell Development and Function THYMIC DEVELOPMENT AND SELECTION Cells of the T lineage are derived from immature progenitors that migrate from the fetal liver or postnatal bone marrow to the thymus (Figure 3). Early T cell progenitors, here referred to as pro-T cells, express neither the CD4 nor CD8 co-receptors, and have their T cell receptor (TCR) genes in germline configuration. Expression of CD25 and CD44 delineates four populations of pro-T cells: CD25−CD44− pro-T1, CD25+CD44+ pro-T2, CD25+CD44− pro-T3 and CD25−CD44− pro-T4, with the developmental sequence being in this order (Figure 3). IL-7R signaling is required for cell survival during the pro-T1 to pro-T3 stages (120, 121). Bcl-2 overexpression restores normal production and function of T cells in IL-7R-deficient mice (122, 123). This indicates that the predominant role of IL-7R signaling in T cells is to block the Bcl-2-regulated apoptosis pathway and other receptors can promote cell proliferation. In contrast, in developing B lymphocytes IL-7R signaling appears to be critical for cell proliferation (see above). As for B lymphopoiesis, T lymphocyte development involves the testing of a cell’s antigen receptor for functionality and autoreactivity. TCR gene rearrangement begins as T cells develop from the pro-T2 to the pro-T3 stage (165), with the TCRβ gene initially being rearranged and its protein expressed in combination with the pTα chain and the CD3 signaling molecules as the pre-TCR (166). Expression of the pre-TCR allows differentiation of pro-T3 cells (CD25+44−) to the pro-T4 (CD25−44−) stage and then into CD4+8+ immature, cortical thymocytes. A failure of pre-TCR signaling results in developmental arrest at the pro-T3 stage, and apoptosis as can be seen in scid, rag-1−/− or rag-2−/− mice, which cannot rearrange their antigen receptor genes (114, 167–169), and in CD3ε−/− mice, in which pro-T3 cells cannot signal following pre-TCR ligation (170). The prosurvival signals transmitted by the pre-TCR do not appear to involve the Bcl-2-protein family, as Bcl-2 overexpression does not cause accumulation of pro-T cells in scid (116) or rag-1−/− mice (171). Apoptosis caused by the absence of a pre-TCR signal requires the tumor suppressor p53, as Rag-deficient or scid mice lacking p53 contain some cells progressing past the pro-T3 stage (172–174). Death receptor signaling also appears to be involved in the deletion of pro-T cells lacking a pre-TCR, as inhibition of this apoptotic pathway by deletion of FADD or expression of a dominant interfering FADD mutant permits the transition to the pro-T4 and CD4+8+stages in Rag-deficient mice (175, 176). The death receptor that is used to kill pre-TCRdeficient cells has not been identified but is unlikely to be Fas because it is not expressed by pro-T cells (177, 178) and because Fas deficiency (lpr mutation)

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Figure 3 Role of apoptosis in the development of T cells. Within the thymus, developing T cells are subject to apoptosis if they fail to produce a T cell receptor (TCR) or if they produce an autoreactive TCR. Within peripheral lymphoid organs, interactions of the TCR with MHC are required for T cell survival. Upon activation, T cells become sensitive both to Fas-mediated activation induced cell death (AICD) and to cytokine deprivation.

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does not promote the survival and differentiation of pro-T3 cells in rag-1−/− mice (176). TCRα gene rearrangement occurs at the CD4+8+stage (179), and expression of the TCR α and β chains plus CD3 enables selection of T cells on the basis of their antigen receptor specificity. A requirement for T cells is that their TCRs bind with low affinity to self major histocompatibility complex (MHC) molecules bearing peptides (180). TCR-MHC interactions promote the differentiation of CD4+8+ thymocytes, including loss of pTα and Rag expression, which leads to cessation of further TCRα gene rearrangement (181–183). In addition, TCR-ligation during positive selection promotes thymocyte survival through upregulation of Bcl-2 expression (183). Bcl-2 overexpression prevents the death of thymocytes bearing TCRs that cannot bind to MHC molecules in their environment, but their differentiation is arrested at the CD4+8+ stage (184, 185). This indicates that TCR-ligation activates signals for cell survival and for differentiation. While thymocytes are positively selected on the basis of recognition of selfMHC, a high avidity of binding to self-peptides presented by MHC (or a high affinity for self-MHC alone) confers potential autoreactivity, and such T cells are deleted in a process termed negative selection (180). The death receptor pathway is not required for thymocyte negative selection. This has been shown by crossing transgenic mice expressing either a dominant interfering mutant of FADD (15) or the cowpox virus serpin CrmA (18), which inhibits caspase-8, with mice expressing autoreactive TCRs. In contrast, the pro-apoptotic BH3-only Bcl-2 family member Bim is essential for negative selection, since Bim-deficient thymocytes are resistant to death in a number of models for negative selection (69). Furthermore, Bcl-2 overexpression can also antagonize negative selection (29, 69, 184). In thymocytes TCR-ligation causes accumulation of Bim protein and its association with Bcl-xL (69), but the signaling pathways regulating this process are not yet understood. Experiments with transgenic mice expressing a dominant inhibitor of Nur77, which also blocks the closely related proteins Nurr1 and Nor-1, demonstrated that these transcription factors are critical for thymocyte negative selection (186, 187). The genes that these factors regulate to induce cell death are presently not identified, but bim does not appear to be a direct target (P. Bouillet and A. Strasser, unpublished observations). Nur77/Nurr1/Nor1 transcription factors might therefore regulate a pathway to apoptosis that acts in negative selection in parallel to the one activated by Bim, or they may control upstream regulators of Bim. Targeted deletion from the T lineage of the gene encoding the phosphatase PTEN revealed the importance of this molecule in deletion of autoreactive thymocytes (188). The apoptosisinducing mechanism of PTEN is unclear, but through its ability to attenuate PI3 kinase signaling, it may act by negatively regulating the expression of Bcl-xL or its homologs (189). T CELL APOPTOSIS IN PERIPHERAL LYMPHOID ORGANS Bcl-2 expression is required for the survival of mature T cells, as bcl-2−/− T cells are progressively lost from the blood and peripheral lymphoid organs (97–99). The abnormal death

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of bcl-2−/− T cells can be prevented by concomitant loss of the BH3-only protein Bim (87). The cytokines IL-4, 6, and 7 promote the survival of mature, resting T cells (190, 191) and do so probably by promoting the expression of Bcl-2 and Bcl-xL (191). Interaction of the TCR with MHC is also essential for long-term survival of mature T cells in vivo (192–195). Interestingly, while na¨ıve T cells require interaction with MHC molecules for which they were positively selected in the thymus, survival of memory T cells can be mediated by interaction with MHC molecules of any haplotype (194). The mechanism by which TCR-MHC interactions promote survival of resting T cells is presently not understood, but we speculate that it may involve regulation of the expression of Bcl-2 or its homologs. The antigen-mediated stimulation of T lymphocytes results in a change in their requirements for survival. Activated T cells produce IL-2 and are dependent on IL2 and related cytokines for their survival (196, 197) and proliferation (198–200). The prosurvival function of cytokines that signal through γ c-containing receptors appears to be mediated through maintenance of Bcl-2 and Bcl-xL expression (196, 200), and accordingly, activated bcl-2 transgenic T cells are able to survive but do not proliferate in the absence of IL-2 (29). bim−/− and bcl-2 transgenic T lymphocytes behave similarly, indicating that Bim must be essential for IL-2withdrawal induced death (30). Repeated TCR-stimulation sensitizes activated T cells to apoptosis, a process known as activation-induced cell death (AICD). In these cells apoptosis occurs through upregulation of both Fas and FasL, which then causes paracrine cell killing (201–205). Upon activation, T cells are initially resistant to FasL-induced death, but after several days gain sensitivity (206, 207). This change in propensity to undergo AICD has been attributed to an initially high level of FLIP expression, which diminishes with time (206). IL-2 has been reported to be required for the reduction in FLIP expression (208). When the infection has subsided, most activated T lymphocytes are killed to prevent immunopathology caused by the many cytotoxic molecules produced by these. Culling of T cells may occur by two mechanisms: (a) AICD by repeated TCR-stimulation from antigen presenting cells or (b) by reduction in cytokine levels (due to decreased inflammation). The second mechanism requires the proapoptotic BH3-only protein Bim (30, 30a) and can be inhibited by Bcl-2 overexpression (29). Antigen-stimulated T lymphocytes persist longer in bcl-2 transgenic lpr mice than in mice expressing only a bcl-2 transgene or those bearing only the lpr mutation, indicating that the two killing mechanisms cooperate in terminating immune responses (24, 101).

Regulation of Myeloid Cell Survival Despite having a critical contribution to both the innate and acquired immune responses, compared to lymphocytes relatively little is understood about the regulation of myeloid cell survival and death. Granulocytes, monocytes, and dendritic

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cells are formed from myeloid precursors under the influence of cytokines (209). They play critical roles in immune defense, mediating inflammation, phagocytosis of pathogens, and uptake of antigen for presentation and activation of T cells. Modulation of survival is one means by which the function of myeloid cells is regulated (Figure 4).

Figure 4 Regulation of apoptosis in the myeloid compartment. The survival of granulocytes, mast cells, monocyte/macrophages, and dendritic cells is tightly regulated. In all these cells inflammatory signals tend to upregulate anti-apoptotic molecules, reducing cell death by both the death receptor–mediated and the Bcl-2-inhibitable pathways. The Bcl-2 homologs A1 and Mcl-1 both appear to play physiologically important roles in regulating myeloid cell survival.

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Myeloid cells tend to have a much shorter lifespan than lymphocytes, but their survival can be extended in periods of infection or inflammation. For example, peripheral blood granulocytes have a half-life of less than a day. Upon exposure to pro-inflammatory cytokines, which in vivo corresponds to migration into an infected tissue, granulocyte lifespan is prolonged (209, 210). Bcl-2 overexpression (96) or Bim deficiency (30) both promote accumulation of granulocytes in vivo and prolonged survival in culture (26). This indicates that the spontaneous death of granulocytes is mediated (at least in part) by the Bcl-2-regulated pathway. A1 and Mcl-1 appear to be the major prosurvival proteins involved in the response to cytokine stimulation because both are upregulated by G-CSF (211–213). Moreover, granulocytes lacking A1a (one of four A1 genes existing in mice) show an enhanced susceptibility to cytokine withdrawal-induced apoptosis in culture (214). Although Fas-deficient lpr mice have normal numbers of granulocytes (215), overexpression of Bcl-2 synergizes with the lpr mutation to cause an acute myeloblastic leukemia–like disease (216). This demonstrates that, as in activated T cells, Bcl2-regulated apoptosis signaling and the death receptor pathway cooperate to limit granulocyte production and accumulation in vivo.


GRANULOCYTES

The Bcl-2 homolog A1 is also important for the survival of mast cells. These cells bind IgE via their FcεR1, and in response release inflammatory mediators (217). FcεR1 cross-linking also provides an anti-apoptotic signal by upregulating A1 expression (218). Mast cells lacking the A1a gene do not show enhanced survival in response to FcεR1 ligation, and accordingly, A1a−/− mice have abnormally reduced allergic responses (218). However, since A1a-deficiency does not cause a depletion of mast cells under normal (non-allergic) conditions, other Bcl-2-like prosurvival proteins are probably also important for mast cell survival in vivo (218). MAST CELLS

MACROPHAGES Both the death receptor–mediated and the Bcl-2 inhibitable pathways to apoptosis seem important in control of macrophage numbers. Like granulocytes, macrophages upregulate A1 and Mcl-1 under inflammatory conditions (219), and Bcl-2 overexpression (96) or Bim-deficiency (30) cause accumulation of both monocytes and macrophages in vivo. T cells can kill macrophages via Fas ligation (220), and this may explain why Fas-deficient lpr mice show increased numbers of macrophages (221). Under conditions of inflammation, activated macrophages may protect themselves from death receptor killing through upregulation of FLIP (222). Thus, also in the macrophage lineage Bcl-2-regulated apoptotic pathways and death receptor signaling probably cooperate to limit cell production. DENDRITIC CELLS The term dendritic cell (DC) encompasses a range of antigen presenting cells that are generated through various developmental pathways. The best studied DCs are those of the myeloid lineage; they serve as sentinels, sampling their environment prior to maturation and carrying antigenic particles to lymphoid

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organs for presentation to T cells (223). DC survival is enhanced both by inflammatory signals, such as those that signal through TOLL-like receptors (e.g., CpG motifs) (224) and by T cell–derived factors, including the TNF-related cytokines CD40L (146) and TRANCE (225). TRANCE prolongs DC survival through upregulation of Bcl-xL (225), while CD40 ligation upregulates Bcl-2 (226) and TOLLlike receptor signaling upregulates Bcl-2 and Bcl-xL as well as IAPs (227). CD40 ligation has also been found to reduce DC sensitivity to death receptor–mediated killing (226), and this may be mediated through upregulation of FLIP (228). This combined role of inflammatory and T cell–derived signals, to both activate and enhance the survival of dendritic cells, may ensure their persistence during an immune response.

PATHOGENIC CONSEQUENCES OF DEFECTS IN APOPTOSIS SIGNALING IN THE IMMUNE SYSTEM Autoimmunity Mice genetically modified to lack or overexpress cell death regulators have indicated that autoimmunity can be caused by a failure of lymphocyte apoptosis. Inhibiting apoptosis by expression of a bcl-2 transgene or loss of Bim prevents negative selection of both B cells (136, 138, 139) (A. Enders, D. Tarlinton, P. Bouillet, and A. Strasser, manuscript in preparation) and T cells (29, 30a, 69), and so allows the survival of potentially autoreactive cells. Accordingly, both Bcl-2-overexpressing (95) and Bim-deficient (30) mice show a systemic lupus erythematosus–like autoimmune syndrome on certain genetic backgrounds. Failure of Fas-mediated apoptosis can also cause autoimmune disease. The human autoimmune lymphoproliferative syndrome (ALPS) is due to mutations in Fas (229). Similarly, on some genetic backgrounds Fas- and FasL-deficient mice (lpr and gld, respectively) show autoimmune glomerulonephritis and vasculitis in addition to lymphoaccumulation (230).

Leukemogenesis The initial identification of upregulation of Bcl-2 expression due to an oncogenic chromosomal translocation in human follicular lymphoma (231), and its subsequent identification as an antagonist of cell death (27) highlighted the importance of abrogation of apoptosis in tumorigenesis. Tumor cells are subjected to many signals which would normally induce death. Such apoptotic stimuli include DNA damage, anoxia, overexpression of oncogenes, absence of growth factors and loss of attachment to a substratum. Failure to undergo cell death is thus essential for development and progression of tumors, although clearly other transforming events (e.g., upregulation of oncogenes) are also required. Within the hematopoietic system, overexpression of Bcl-2 facilitates B cell transformation (232). Bcl-2 potently synergizes with the myc (233) and pim-1 (234) oncogenes in lymphomagenesis. Bcl-2 overexpression has also been shown to cooperate with the pro-myelocytic

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leukemia (PML) retinoic acid receptor α to hasten the development of acute PML (235). Similarly, expression of the Bcl-2 relative A1 is required for leukemogenesis mediated by BCR/ABL (236). A possible role for Bim as a tumor suppressor has been identified by showing that Bim-deficiency accelerates myc lymphomagenesis (A. Egle, A. Harris, and S. Cory, unpublished results). The death receptor pathway also appears be important for tumor suppression. Defects in Fas have been identified in some T cell leukemias (237, 238), and Fas-deficient mice are susceptible to plasmacytoma development (239, 240). Inhibition of death receptor signaling by expression of a dominant interfering mutant of FADD can also cause thymic lymphomas when expressed on a rag-1−/− background (176). The importance for leukemogenesis of both the death receptor and Bcl-2-regulated paths to apoptosis is highlighted by the development of myeloid leukemia in Fas-deficient lpr mice expressing a bcl-2 transgene in myeloid cells (216).

Infection Viruses have evolved many mechanisms to promote the survival of host cells, evading both intrinsic pathways activated in cells in response to infection, as well as the host immune system. The lymphotrophic viruses Epstein Barr virus (EBV) and human herpes virus 8 (HHV8) both carry genes for Bcl-2 homologs, BHRF-1 (241) and KSbcl-2 (242, 243), respectively. These enable inhibition of a host cell’s intrinsic apoptotic response to viral infection, as well as potentiating the generation of lymphomas by both viruses (244). Similarly, the HIV tat protein upregulates cellular Bcl-2 (245), possibly allowing the short-term survival of infected T cells. As protection from death receptor–induced apoptosis, HHV8 also encodes a viral FLIP (246), while EBV may upregulate cellular FLIP (247).

Sepsis Apoptosis of lymphocytes has been observed in both septic animals (248) and humans (249). This appears to be a causal event in sepsis-induced death, as mortality can be reduced by inhibition of this apoptosis, either by expression of a bcl-2 transgene (250, 251) or through drug-mediated inhibition of caspases (251, 252). The death of lymphocytes during sepsis may be detrimental to the infected host because it removes a portion of the immune defense against the causative infection.

CONCLUSIONS Homeostasis and normal functioning of the immune system require regulation of the survival of leukocytes. Apoptosis can be induced by death receptor ligation or via Bcl-2-inhibitable pathways, and both these pathways are critical to many developmental events. Modulation of apoptotic pathways allows an animal to delete autoreactive cells, but it promotes the survival of certain cell populations during an immune response. The importance of apoptosis in the immune system

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is emphasized by the many observations that defects in the cell death program contribute to autoimmunity, leukemogenesis, and infection.


ACKNOWLEDGMENTS We thank our many colleagues, especially J. Adams, S. Cory, A. Harris, C. Scott, L. O’Reilly, H. Puthalakath, A. Villunger, P. Bouillet, D. Huang, L. Coultas, M. Pellegrini, D. Vaux, A. Egle, Y. Laâbi, S. Gerondakis, and D. Tarlinton for illuminating discussions and critical appraisal of this manuscript. Work in our laboratories is supported by grants and fellowships from the National Health and Medical Research Council (Canberra; Reg. Key 973002), the U.S. National Cancer Institute (CA80188), the Leukemia and Lymphoma Society of America, the Dr. Josef Steiner Cancer Foundation (Bern, Switzerland), the Cancer Research Institute (New York), the Anti-Cancer Council of Victoria (Melbourne), and the Commonwealth Department of Employment, Education, Training and Youth Affairs (Canberra). The Annual Review of Immunology is online at http://immunol.annualreviews.org

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ON NATURAL AND ARTIFICIAL VACCINATIONS, Rolf M. Zinkernagel COLLECTINS AND FICOLINS: HUMORAL LECTINS OF THE INNATE IMMUNE DEFENSE, Uffe Holmskov, Steffen Thiel, and Jens C. Jensenius THE BIOLOGY OF IGE AND THE BASIS OF ALLERGIC DISEASE, Annu. Rev. Immunol. 2003.21:71-105. Downloaded from arjournals.annualreviews.org by HINARI on 09/01/07. For personal use only.


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CD45: A Critical Regulator of Signaling Thresholds in Immune Cells Michelle L. Hermiston1,2,3, Zheng Xu2,3, and Arthur Weiss2 Annu. Rev. Immunol. 2003.21:107-137. Downloaded from arjournals.annualreviews.org by HINARI on 09/01/07. For personal use only.

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Department of Pediatrics, 2Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California 94143; email: [email protected], [email protected], [email protected] 3 These authors contributed equally to this review.

Key Words protein tyrosine phosphatase, alternative splicing, dimerization, autoimmunity ■ Abstract Regulation of tyrosine phosphorylation is a critical control point for integration of environmental signals into cellular responses. This regulation is mediated by the reciprocal actions of protein tyrosine kinases and phosphatases. CD45, the first and prototypic receptor-like protein tyrosine phosphatase, is expressed on all nucleated hematopoietic cells and plays a central role in this process. Studies of CD45 mutant cell lines, CD45-deficient mice, and CD45-deficient humans initially demonstrated the essential role of CD45 in antigen receptor signal transduction and lymphocyte development. It is now known that CD45 also modulates signals emanating from integrin and cytokine receptors. Recent work has focused on regulation of CD45 expression and alternative splicing, isoform-specific differences in signal transduction, and regulation of phosphatase activity. From these studies, a model is emerging in which CD45 affects cellular responses by controlling the relative threshold of sensitivity to external stimuli. Perturbation of this function may contribute to autoimmunity, immunodeficiency, and malignancy. Moreover, recent advances suggest that modulation of CD45 function can have therapeutic benefit in many disease states.

INTRODUCTION CD45 was first reviewed in this journal by the late Matt Thomas in 1989 and again by Trowbridge & Thomas in 1994 (1, 2). At that time, it was clear that CD45 was the prototypic receptor-like protein tyrosine phosphatase (PTP) and an essential regulator of signal transduction pathways in immune cells. Our knowledge concerning CD45 has grown significantly during the past eight years. A Medline search for CD45 now generates a list of over 5000 references. In this review, we highlight progress made in four key areas of CD45 biology: structure, regulation of gene expression and alternative splicing, CD45 function in immune cells, and regulation of phosphatase activity. We conclude by discussing the roles of CD45 in disease, its potential as a therapeutic target, and the areas of focus for the next decade. 0732-0582/03/0407-0107$14.00

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STRUCTURE


CD45 is one of the most abundant cell surface glycoproteins, comprising up to 10% of the cell surface area (1). Its structural features are summarized in Figure 1. The cytoplasmic region shares a remarkable 95% homology among all mammalian species analyzed. In contrast, the extracellular domain is only 35% homologous (1).

Figure 1 Structure of CD45. CD45 exists as multiple isoforms due to alternative splicing of three exons (4, 5, and 6, designated A, B, and C) in the extracellular domain. The largest isoform RABC (including all three exons) and the smallest isoform RO (lacking all three exons) are shown. The three exons encode multiple sites of O-linked glycosylation and are variably modified by sialic acid. As a result, various isoforms with molecular weight ranging from 180 kDa of RO to 240 kDa of RABC differ substantially in size, shape, and negative charge. The remaining extracellular domain is heavily N-glycosylated and contains a cysteine-rich region followed by three fibronectin type III repeats (3). CD45 has a single transmembrane domain and a large cytoplasmic tail containing two tandemly duplicated PTPase homology domains, D1 and D2. Only D1 has enzymatic activity and is necessary to rescue TCR signaling in a CD45deficient cell line (10). In addition, molecular modeling indicates the juxtamembrane region may form a structural wedge (124).

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However, analysis of CD45 from divergent species indicates a conserved overall organization of the extracellular domain (3). The heavy glycosylation of CD45 is attributable to N-glycosylation throughout the extracellular domain and O-glycosylation of the variable exons. The Nglycoconjugates of CD45 are mainly tetra- and triantennary complex-type sugar chains containing poly(N-acetyllactosamine) groups and exclusively α-2,6-linked sialic acid residues (4). The O-glycoconjugates contain mostly core 1 and 2 oligosaccharides (5). The glycosylation pattern of CD45 depends not only on differential usage of variable exons but also on the cell type, developmental stage, and activation state of the cell [reviewed in (1)], suggesting functional importance. For example, the interaction of the α-2,6-linked sialic acid residues on N-linked sugar chains of CD45 expressed in T cells with CD22, a sialic acid–binding lectin expressed in B cells, may contribute to cell adhesion (6). Moreover, the high mannose or hybrid-type N-linked oligosaccharides of CD45 expressed on CD4+CD8+CD3lo immature thymocytes interact specifically with the serum mannan-binding protein, which may modulate the development and maturation of thymocytes (7). Finally, CD45 also binds to specific isoforms of the resident endoplasmic reticulum lectin, glucosidase II (8). This association is developmentally regulated and may change the carbohydrate content of CD45, potentially influencing thymocyte development (9). The cytoplasmic domain contains two tandemly duplicated protein tyrosine phosphatase homology (PTPase) domains (D1 and D2). Only D1 has phosphatase activity and is necessary to rescue TCR signaling in a CD45-deficient cell line (10). The function of the D2 domain is unclear. It may contribute to the stability and optimal activity of CD45 via an intramolecular interaction with D1 and/or the spacer region between D1 and D2 (11–13). D2 may also regulate CD45 function via a unique 19 amino acid insert that is rich in serine and acidic residues and conserved between human, murine, and rat CD45. This insert physically associates with and is phosphorylated by casein kinase II (CK2) (14, 15).

REGULATION OF CD45 GENE EXPRESSION AND ALTERNATIVE SPLICING Although the extracellular domain is dispensable for reconstitution of antigen receptor signaling in several cell culture systems (10, 16, 17), it is subject to exquisite regulation. The CD45 extracellular domain is expressed as multiple isoforms in a cell type, developmental stage, and activation state dependent manner [summarized in Figure 2, see (2, 18, 19) for further review]. This pattern of isoform expression is highly conserved across species, suggesting functional importance in vivo. Alternative splicing of exons 4, 5, and 6 can generate at least eight different isoforms (1, 2). Evidence for five of these has been detected at the protein level in humans (2). Recent mRNA studies have also provided evidence for alternative splicing of exons 7, 8, and 10 in several cell lines (20). Corresponding protein expression for these isoforms has yet to be demonstrated.

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Figure 2 Cell surface expression patterns of CD45 isoforms. Only the predominant isoforms are depicted. Less abundant isoforms are denoted by smaller font size. The triangles represent increasing expression of the protein during development of T, B, and myeloid lineages and decreasing expression during erythroid differentiation. An exception is plasma cells, which have decreased total CD45 expression.

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Most studies evaluating isoform-specific differences in CD45 function have been complicated by the inability to obtain endogenous levels of CD45 expression in either cell culture or transgenic mouse systems. The importance of adequate levels of CD45 expression is highlighted by studies of mice heterozygous for a null CD45 allele. Whereas a 50% reduction in cell surface levels of CD45 had no effect on in vitro responses, in vivo studies employing a TCR transgenic system revealed marked enhancement of both positive and negative selection relative to wild-type controls (21). The recent progress regarding the regulation of CD45 by cis- and trans-activating factors may aid in design of constructs that recapitulate physiologic expression levels.

Cis- and Trans-Activating Factors Regulating CD45 Gene Expression The genomic organization of CD45 has been previously reviewed (1). Briefly, murine CD45 consists of 34 exons extending over more than 120 kilobases (kb). Both the size and organization of introns is similar in humans, suggesting functional importance (1, 22). Exon 1, which encodes 50 untranslated sequence, is split into two alternatively used exons 1a and 1b. Exon 1a is preferentially expressed. Transcription can be initiated at three positions (P1a, P1b, and P2) (23). Interestingly, both human and murine CD45 genes lack traditional TATA boxes or initiator-like sequences within 2.5 kb of exon 1 (24). A strong, but non-tissuespecific, promoter has been identified within intron 1 in a region that is highly conserved among mouse, chicken, and human (22, 25). Minigene constructs have been used to identify the sequences required for high level expression of CD45 (26). Inclusion of the 30 -untranslated region or up to 19 kb of sequence 50 of the translation start is unable to support adequate quantity or tissue-specific expression of CD45. However, inclusion of intronic sequences between exons 3 and 9 results in reproducible, tissue-specific expression of CD45. Notably, protein expression in this minigene is still substantially below that of endogenous levels, suggesting additional elements are necessary. One attractive candidate for additional control elements is the 50-kb intron between exons 2 and 3 because its unusual size and location are highly conserved between species (1). The presence of important regulatory elements in the vicinity of the alternatively spliced exons may explain why mice containing a targeted mutation of exon 6 (27), designed to disrupt only the expression of larger isoforms, express minimal protein of any isoform. Analysis of mice deficient for the Ets family transcription factor PU.1 has led to identification of a potential trans-acting factor governing tissue-specific expression of CD45 (28). PU.1 is expressed in both lymphoid and myeloid cells. While PU.1−/− mice develop significant developmental and functional defects in both lineages, a complete lack of CD45 mRNA or protein expression is seen only in the myeloid lineage (28). Importantly, a PU.1 binding site was identified 50 of the P1b and P2 CD45 transcriptional start sites. While T and B cells are capable of

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efficient transcriptional initiation from all three potential start sites, myeloid cells preferentially use the P2 site (28). Moreover, ablation of P2, but not P1, specifically disrupts CD45 expression in myeloid cells. In addition, retroviral-mediated expression of PU.1 is sufficient to restore normal surface expression of CD45 in PU.1-deficient myeloid cells. It is tempting to speculate that many of the functional deficits in PU.1-deficient myeloid cells, including abnormal adherence, chemotaxis, phagocytosis, and killing of microbes, could be attributed to the absence of CD45 (29).

Regulation of Isoform Expression by Alternative Splicing Several studies have begun to identify the potential cis- and trans-acting factors that govern alternative splicing of the CD45 extracellular domain. Initial studies focused on identification of cis-acting elements required for tissue-specific alternative splicing. Linker scanning studies have demonstrated that sequences within and flanking exons 4 and 6 are both necessary and sufficient for cell type–specific alternative splicing [reviewed in (2)]. Although exons 4, 5, and 6 are variably included in CD45 mRNA, only the inclusion of exons 4 and 6 is tightly regulated. The inclusion of exon 5 appears stochastic. These same cis-acting regulatory sites have also been shown to govern the isoform switch that occurs during T cell activation (30). Protein expression of mixed larger isoforms (RA+) transiently increases during the first day post stimulation and then slowly decreases as it is replaced by the smallest isoform (RO) over the ensuing three days (31, 32). This change in protein expression reflects alterations in the mRNA level. RA+ mRNA increases during the first four hours post activation. It then rapidly decreases and is undetectable by 24 hours. At the same time, RO mRNA begins to accumulate. In addition, studies in a model T cell system employing a minigene approach have demonstrated that activation of protein kinase C and Ras, as well as de novo protein synthesis, are essential for the RA+ to RO switch (30). Approximately 1% of the human population express aberrantly high levels of exon 4–containing isoforms in all cell lineages and reduced levels of smaller isoforms (33). The genetic basis for this misregulation correlates with the autosomal dominant inheritance of a C to G polymorphism at nucleotide 77 (C77G) of exon 4 (34, 35). While the mutation is translationally silent, CD45 minigene constructs carrying this mutation clearly demonstrate that the C77G polymorphism is sufficient to confer abnormally high expression of exon 4 in mature CD45 mRNA and protein (30). Further analyses have shown that this polymorphism disrupts a strong exonic splicing silencer site (ESS1) that normally associates with a distinct complex in nuclear extracts and represses the exon 4 50 splice site (36). The functional consequences of the C77G polymorphism remain controversial. Initial studies demonstrated a link to the development of multiple sclerosis (MS) in three German kindreds (37). A fourth family in this cohort lacks the C77G polymorphism but expresses a distinct polymorphism, C59G in exon 4 (38). Interestingly,

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C59G also maps to ESS1. Recent analyses of American and Swedish cohorts, as well as a larger German cohort, failed to find a similar association (39–41). Links to other autoimmune diseases, such as Grave’s disease or type 1 diabetes mellitus, have not been identified either (42). One possible explanation for these disparate results is that genetic modifiers act directly or indirectly to alter CD45 function and to modify these disease-associated susceptibilities. Supporting this notion, the phenotypes of CD45-deficient (43) and CD45E613R mice (44; M.L. Hermiston and A. Weiss, unpublished observations) appear to be modulated by genetic background. Isoform expression in rats also appears subject to genetic modification (45). Lewis rats primarily express the high molecular weight isoforms CD45RC+ and have increased susceptibility to autoimmune disease, while Brown Norway rats predominantly express low molecular weight isoforms and are resistant to autoimmune disease. Bone marrow chimera experiments show that strain-specific isoform expression is cell autonomous and genetically controlled. Linkage analyses further suggest that the difference may be due to the presence or absence of trans-acting factors located outside the CD45 locus (45). Trans-acting factors that regulate cell-type and activation-state specific alternative splicing are largely unknown. Evidence for both positive and negative regulatory factors exists [reviewed in (2)]. The SR family of proteins is one attractive candidate for factors that mediate tissue-specific splicing of CD45. SR proteins, essential components of the splicosome machinery, are expressed in a tissue-specific manner and regulated by phosphorylation. For example, expression of the SR protein SRp20 is altered in T cells during activation (46). Moreover, use of a heterologous system employing a CD45 minigene and overexpression of various SR proteins has demonstrated that the SR proteins SRp20 and 9G8 can facilitate exon inclusion whereas SF2/ASF, SC35, SRp30c, SRp40, and SRp75 can promote exon exclusion (46–48). Interestingly, thymus-specific deletion of the SR protein SC35 results in impaired thymocyte development with a partial block at the double negative (DN) to double positive (DP) transition (49). This correlates with inappropriate downregulation of larger CD45 isoforms. While intriguing, it is unclear if the thymic defect is the direct result of altered CD45 expression or secondary to alterations in other proteins affected by the deletion of SC35.

CD45 FUNCTION CD45 in T Cell Development, Signaling, and Function Studies employing CD45-deficient cell lines initially identified CD45 as an obligate positive regulator of antigen receptor signaling (10, 16, 17, 50, 51). This is verified by the subsequent observations that CD45-deficient humans (52, 53) and mice (27, 43, 54) develop a severe-combined immunodeficiency (SCID) phenotype. CD45-deficient mice, independently generated by three groups through the targeting of exons 6, 9, or 12, have profound defects in thymic development due to enhanced basal apoptosis and dysfunctional signaling through the pre-TCR and

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TCR (27, 43, 54). As a result, the absolute number of DP thymocytes is reduced twofold, and the number of single positive (SP) thymocytes is reduced five-fold. In addition to these defects in beta- and positive selection, studies employing TCR transgenic mice (43, 55, 56) and fetal thymic organ cultures (FTOC) (57) have revealed an additional defect in negative selection that is dependent upon the strength of antigenic stimuli. Further supporting the signal strength model of thymic selection, generation of hybridomas between the few CD45−/− T cells that do survive to the periphery and a control thymoma line clearly demonstrates that the majority of CD45−/− peripheral T cells are self-reactive (58). These T cells are hyporesponsive to antigenic stimulation, providing an explanation for the lack of stigmata of autoimmune disease in the CD45−/− mice. Insight into the molecular basis for these findings is provided by biochemical studies of CD45-deficient cell lines and mice. BRIEF OVERVIEW OF TCR SIGNAL TRANSDUCTION Ligation of the T cell receptor (TCR) by peptide antigen presented on major histocompatibility complex (MHC) molecules initiates a signal transduction cascade that ultimately leads to T cell activation (reviewed in 59, 60). The earliest event is activation of the Src family protein tyrosine kinases (SFKs) Lck and Fyn. These SFKs subsequently phosphorylate the immunoreceptor tyrosine-based activation motifs (ITAMs) present in the ζ and CD3 ε, δ, and γ subunits of the TCR. Doubly phosphorylated ITAMs promote recruitment and subsequent activation of ZAP-70 protein tyrosine kinase (PTK). The resultant increase in PTK activity leads to phosphorylation of adapter proteins and enzymes that facilitate the stimulation of downstream signaling pathways, culminating in T cell activation with new gene transcription, cytoskeletal reorganization, cytokine production, and proliferation. MODULATION OF SFK BY CD45 SFKs are a primary substrate for CD45 (reviewed in 61–63). SFKs are responsible for initiation of immune responses in both T and B cells. They may also modulate signals emanating from growth factor, cytokine, and integrin receptors. In addition to these positive roles, recent evidence suggests that SFKs also function as negative regulators by phosphorylating immunoreceptor tyrosine-based inhibitory motifs (ITIMs) on inhibitory receptors, which then leads to recruitment and activation of inhibitory molecules such as the PTPs SHP-1 and SHP-2 and the SH2-containing 50 inositol phosphatase SHIP-1. Because of their proximal position in TCR signal transduction and rapid activation upon TCR cross-linking, modulation of SFK activity represents a critical regulatory node in T cells. SFK activity is controlled, in part, by the phosphorylation of two key tyrosine residues (59, 63, 64 and references within). Autophosphorylation of a site in the activation loop of the catalytic domain potentiates kinase activity. Conversely, the ubiquitous PTK, C-terminal Src kinase (Csk), negatively regulates kinase activity by phosphorylating the carboxy-terminal inhibitory tyrosine of SFKs. An intramolecular interaction between this phosphotyrosine and the SFK’s own SH2 domain blocks the substrate binding site, thereby maintaining the SFK in an inactive “closed” conformation. Csk activity, in turn, is also highly regulated by the PEP family phosphatases and by interactions with the adapter Csk

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binding protein/phosphoprotein associated with glycosphingolipid-enriched microdomains (cbp or PAG), which determines its subcellular localization and access to substrate (59, 64). Molecular dynamic simulations indicate that the connector region between the SH2 and SH3 domains is also crucial for stabilizing the kinase in the inactive closed conformation. This connector appears to function as a snap lock that couples the SH2 and SH3 domains in a configuration that constrains the conformation of the catalytic domain. Dephosphorylation of the C-terminal tail or the binding of ligands to the SH2 and/or the SH3 domains restores flexibility to the connector region, thus enabling the kinase to adopt a more open “primed” conformation (65). Subsequent clustering of SFKs on the cell surface may allow for transphosphorylation of the catalytic domain and full activation of the kinase. Adding to the complexity of regulation, SFKs can be found in distinct membraneassociated and intracellular pools [reviewed in (62, 63)]. It is possible that the activation status, as well as access to substrate, may vary between different subcellular locations. Importantly, current assays to assess SFK activity generally involve immunoprecipitation from whole cell lysates and thus report the average SFK activity or phosphorylation status for the cell rather than for individual molecules or intracellular pools. Lck and Fyn are the predominant SFK members in T cells. Lck is unique in its noncovalent association with the CD4 and CD8 coreceptors. Several lines of evidence highlight a role for CD45 as a positive regulator of SFKs. In most CD45deficient cell lines and CD45−/− thymocytes, Lck and Fyn are hyperphosphorylated at their respective negative regulatory tyrosines and the TCR is completely uncoupled from intracellular signals (50, 51, 57, 66–68). Moreover, expression of a constitutively active Lck Y505F mutant rescues the block in thymic development in CD45−/− mice, providing genetic evidence that the negative regulatory tyrosine is a physiologically relevant CD45 substrate in vivo (68, 69). These observations led to a model proposing that a primary function of CD45 is to counteract the effect of Csk on SFKs by dephosphorylating their negative regulatory tyrosine. This results in a signal-competent pool of Lck that can phosphorylate the TCR ITAMs if foreign antigen is encountered. Complicating this model is the observation that the average cellular SFK activity is paradoxically increased in CD45−/− thymocytes and in some CD45-deficient cell lines, despite hyperphosphorylation of the negative regulatory tyrosine (61, 70, 71). This led to the suggestion that the autocatalytic tyrosine can also function as a CD45 substrate. Supporting this hypothesis, CD45-deficient macrophages and T cells are abnormally adherent due to enhanced SFK-dependent integrin signaling (72, 73). Despite hyperphosphorylation of the negative regulatory tyrosine, SFK kinase activity is elevated as a result of increased phosphorylation of the autocatalytic site in these cells. Expression of non-oncogenic levels of the LckY505F transgene in CD45−/− thymocytes results in hyperphosphorylation of the Lck autocatalytic tyrosine Y394 and development of thymomas over time, further supporting a role for CD45 as a negative regulator of Lck (74). Taken together, these data suggest that CD45 can modulate signal transduction thresholds by functioning as both a positive and negative regulator of SFKs.

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Which role dominates in TCR signaling? The block in thymic development, hyporesponsiveness to TCR stimulation, and rescue by the Lck Y505F transgene in CD45−/− mice support a predominant positive role for CD45 in antigen receptor signaling. The apparent paradoxical roles of CD45 can be reconciled in a model in which CD45’s net function is determined by its localization relative to its substrate (Figure 3). Thus, in resting T cells CD45 dephosphorylates the negative regulatory and, to a lesser extent, the autocatalytic site, generating a signal-competent pool of Lck. During antigen recognition, TCR clustering in the central region of the immunologic synapse, a process that evolves over time, would functionally segregate CD45 from its substrate and thus facilitate sustained Lck activity during initiation of signal transduction cascades. Consistent with this model, most biochemical and microscopic studies have demonstrated that CD45, but not Lck, is excluded from lipid rafts and the immunologic synapse [reviewed in (75)].

Figure 3 Reciprocal regulation of SFKs by CD45 and Csk. Lck is in a dynamic equilibrium between its inactive and primed conformations due to the reciprocal activity of CD45 and Csk. While CD45 can dephosphorylate both autocatalytic and inhibitory tyrosines of SFKs, the latter is enzymatically favored. This generates a pool of signalcompetent SFK. We hypothesize that Lck may traffic between rafts and other subcellular pools based upon its tyrosine phosphorylation status. Primed Lck, unopposed in the rafts, is able to undergo transphosphorylation of Y394 generating an active kinase that subsequently phosphorylates the ITAMs of the CD3 and ζ chains. The phosphorylated ITAMs provide docking sites for the SH2 domains of ZAP-70, which is then phosphorylated by Lck, allowing propagation of downstream signaling events. SFKs are inactivated by Csk-mediated phosphorylation of the negative regulatory tyrosine. It should be noted that Csk localization is dynamic and that a pool of Csk can be targeted to the raft via the actions of cbp/PAG and PEP family phosphatases.

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In addition to SFKs, CD45 may also negatively regulate cytokine and interferon (IFN) receptor activation by dephosphorylating Janus kinases (JAKs) (76–78). JAKs function as positive regulators of cytokine signaling by phosphorylating the signal transducers and activators of transcription (STATs) family of transcription factors. Phosphorylated STATs translocate to the nucleus and regulate the expression of genes involved in cytokine and chemokine responses. Increased cytokine-dependent myelopoiesis and erythropoiesis and corresponding hyperphosphorylation of JAK2 and STATs 3 and 5 have been reported for CD45−/− mice (76). CD45 deficiency also protects mice from the lethal cardiomyopathy caused by Coxsackievirus B3 (CVB3) infection (76). However, protection from CVB3 infection has also been observed in Lck-deficient mice (79), suggesting that some effects ascribed to CD45’s function as a JAK phosphatase could, in fact, be mediated by CD45’s effects on SFKs. Other possible, but controversial, CD45 substrates include ZAP-70 (80) and CD3ζ (81). Growing evidence suggests that CD45 may function as a survival factor in T cells. CD45−/− thymocytes have enhanced basal apoptosis in situ (54), but not in vitro (82). This phenotype is rescued in CD45−/− LckY505F mice, clearly implicating SFKs as regulators of this process (69). However, it is unclear if the reduction in apoptosis is mediated by restoration of antigen, growth factor, and/or integrin receptor signaling. Interestingly, the regulation of apoptosis appears to require the extracellular domain but not phosphatase activity (83). Several studies have also provided evidence that activated T cells upregulate B220, the CD45 isoform normally found on B cells and a subset of NK cells, immediately prior to apoptosis (63, 84). Whether and how this contributes to death is unknown. It is intriguing that this isoform is the predominant T cell isoform in patients and mice with deficiency in Fas or FasL. While cell line studies have suggested that CD45 and Lck are not involved in Fas-mediated death (85, 86), inactivation of one CD45 allele in Fas-deficient mice results in marked improvement in the lymphoproliferative disorder seen in these mice (87). ISOFORM-SPECIFIC REGULATION OF T CELL SIGNAL TRANSDUCTION Different isoforms of CD45 appear to have identical PTPase activity in vitro (88). Moreover, several cell culture studies have shown that the cytoplasmic tail is sufficient to restore TCR signaling (10, 16, 17). These results would suggest that the PTPase activity is independent of the extracellular CD45 splice variants. However, these findings do not exclude a role for the extracellular domain in vivo. Indeed, CD45deficient BW5147 thymoma cells reconstituted with RO produce more IL-2 after antigenic stimulation than those reconstituted with RABC (89). However, subsequent studies of isoform-specific signaling in other CD45-deficient cell lines and in CD45 exon 6–deficient mice have yielded controversial results. In some cases, ROreconstituted cells signal more efficiently; in other systems, RABC is more effective in restoring TCR signaling (90–96). These inconsistent observations could be due to differences in the CD45-negative cell lines used, the disparate stimuli added, and/or the readouts examined. Moreover, interpretation of the data is complicated

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by the fact that CD45 is one of the most abundant cell surface molecules. Most studies to date have not attained equivalent and physiologic levels of isoform expression, making data interpretation difficult. Titration of CD45 levels can have important physiologic effects as demonstrated by the altered thymic selection in CD45+/− mice (21).


CD45 Function in B Cells B cell fate is determined by the signals emanating from the B cell receptor (BCR), which in turn are influenced by a variety of factors including cell maturation stage, strength of antigen binding, and presence of coreceptors. Lyn, Fyn, and Blk are the primary SFKs in B cells [reviewed in (97)]. Of these, Lyn has a unique role as both a positive and negative regulator of BCR signal transduction. Its positive effects are mediated by phosphorylation of BCR ITAMs. This results in recruitment of the tyrosine kinase Syk and propagation of BCR signals. While Lyn’s positive role is redundant among BCR SFKs, its negative regulatory role is indispensable. After BCR stimulation, Lyn rapidly initiates a negative regulatory feedback loop by phosphorylating ITIMs in the cytoplasmic domains of inhibitory receptors such as CD22 and Fcγ RIIB. This allows recruitment of SHP-1 and SHIP-1, facilitating downregulation of the B cell response [reviewed in (98)]. MODULATION OF SFKS BY CD45 IN B CELLS Similar to T cells, CD45 is an essential and critical modulator of signaling thresholds in B cells. Despite significant expression of CD45 throughout B cell development, B cell maturation appears to proceed normally until the final stage in CD45-deficient mice (27, 54). There is a two-fold increase in the total number of B cells, despite a significant decrease in the number of mature IgMloIgDhi cells in the spleen and circulating in the peripheral blood (27, 43, 54, 99). This discrepancy is accounted for by the accumulation of immature IgMhiIgDlo cells in the spleen (54, 99). CD45 deficiency results in multiple signaling abnormalities in B cells. Whereas CD45−/− B cells have a normal response to LPS and CD40, they are severely hyporesponsive to stimulation with IgM or IgD in vitro (27, 54, 99). Interestingly, they also have decreased responsiveness to stimulation with PMA and ionomycin (54, 99). The latter finding suggests that CD45 may modulate other signal transduction pathways in addition to those governed by the BCR. Despite these signaling defects, CD45-deficient mice have normal immunoglobulin levels and normal responses to T cell–dependent and –independent stimuli in vivo if CD45+/+ CD4 helper cells are provided (100). These observations can be reconciled by a model in which CD45 functions as a rheostat to set the threshold for BCR signaling. In the presence of an increased antigen receptor threshold, ligation of sIgM would fail to induce proliferation of CD45−/− B cells. However, in the context of a strong antigenic stimulus and coreceptor help (e.g., provided during immunization with KLH and CFA) the activation threshold may be reached, allowing productive signaling to occur. Direct evidence for CD45 modulation of antigen receptor threshold stems from an elegant study

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employing the hen egg lysozyme (HEL) transgenic system (101). Normally, circulating HEL autoantigen mediates negative selection of CD45+/+ B cells. However, in the absence of CD45, this strong antigenic stimulus results in positive selection of HEL-binding B cells and promotes their accumulation as mature, long-lived IgDhi cells. Additional evidence that CD45 modulates BCR signaling thresholds is provided by analyses of mice homozygous for CD45 deficiency and the motheaten viable mutation (mev), a catalytically defective form of SHP-1. B cell function in these mice is largely normal, suggesting that these phosphatases normally counterbalance each other (102). The biochemical basis for these observations is beginning to be elucidated. Analyses of CD45−/− B cells demonstrate a profound defect in calcium flux from extracellular, but not intracellular, stores and in stimulation of the Erk-2 pathway (99, 101, 103). Despite these defects, tyrosine phosphorylation events appear largely intact in resting and activated CD45−/− B cells. This may reflect the compensatory activity of Syk, which appears to be appropriately activated and recruited to the BCR in CD45-deficient B cells (104, 105). Alternatively, or in addition, it could reflect balanced attenuation of positive signals mediated by SFKs coupled to the BCR and negative signals mediated by the SFK Lyn (98). Supporting this notion, CD22, a target of the SFK Lyn, has been shown to be hyperphosphorylated and to more efficiently recruit SHP-1 in CD45-deficient cells (106). Studies exploring the regulation of SFK phosphorylation in a variety of CD45deficient B cell lines have focused on the SFK Lyn and have produced mixed results to date. In CD45-deficient DT40 B cells (107), Bal17 B cells (108), and the mature plasmacytoma cell line J5589micron3 (105), Lyn is hypoactive despite hyperphosphorylation at both tyrosines, suggesting that dephosphorylation of the negative regulatory tyrosine is a necessary prerequisite for BCR signaling in these systems. In contrast, while Lyn is also hyperphosphorylated at both sites in the immature B cell line WEHI-231, Lyn activity is enhanced in in vitro kinase assays, suggesting that the autocatalytic site may be dominant in this system (109–111). The authors conclude that CD45 is a negative regulator of BCR signaling in immature B cells and that the stage of B cell development may contribute to CD45’s negative or positive effects, perhaps by altering substrate access. However, it should be noted that CD45-deficient WEHI-231 cells are minimally responsive to BCR stimulation, raising the possibility that the increased Lyn kinase activity is in a BCR-independent pool. It is also unclear in any of these studies if both tyrosines are phosphorylated on the same molecule or if the results reflect differential phosphorylation of independent intracellular pools.

CD45 Function in Other Cell Types Despite abundant expression, the role of CD45 in cells of the myeloid lineage is poorly characterized. While myelopoiesis is increased in CD45-deficient mice, it is unclear if this is a cell-autonomous effect or secondary to alterations in the cytokine/chemokine milieu of the animals (27, 76). Rescue of the mev phenotype

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in CD45−/− mev/mev mice suggests CD45 may also function to oppose SHP-1’s functions in the myeloid lineage (102). As discussed above, CD45 appears important for macrophage-mediated adhesion (73). CD45 is essential for histamine degranulation after immunoglobulin E (IgE) receptor cross-linking in mast cells (112). The majority of studies exploring neutrophil function have relied upon the use of CD45 antibodies to modulate function. Unfortunately, it is often unclear whether these antibodies are activating or inhibitory (reviewed in 2). While effects on chemotaxis have been reported as both positive and negative depending upon the system utilized (113–115), most studies suggest that CD45 negatively regulates the oxidative burst (117). CD45 may also modulate chemokine-induced signaling in neutrophils. Treatment of isolated human neutrophils with antibody to CD45 results in downmodulation of the chemokine receptors CXCR1 and CXCR2 and a corresponding decrease in tyrosine phosphorylation of a 54- to 60-kDa protein (118). Further studies exploring the biochemical pathways regulated by CD45 in myeloid cells are clearly warranted.

REGULATION OF CD45 PHOSPHATASE ACTIVITY Because CD45 plays a pivotal role in signal transduction in leukocytes, elucidating how it is regulated is critical to our understanding of the development of the immune system and the regulation of the immune response. Possible means to regulate CD45 include ligand(s) binding, dimerization, localization and access to substrate, interactions with other proteins, covalent modifications, and the action of specific intracellular inhibitors.

CD45 Ligands? The search for CD45 ligand(s) has been carried out for more than a decade. The existence of a ligand is presumed based upon the observation that the overall features of CD45’s extracellular domain are conserved across evolution and are similar to those of receptor tyrosine kinases (RTKs) such as EGFR and PDGFR. In particular, the cysteine-rich motif in CD45 extracellular portion is analogous to that found in the EGFR, where it is important for ligand binding to the EGFR. The cysteines in this motif are conserved from human to rodent, despite the 35% homology of the entire extracellular domain (2). However, many attempts have failed to identify a ligand for CD45. Both CD22 and galectin-1 have been proposed as possible CD45 ligands (119, 120). However, these lectins bind nonspecifically to T cell glycoproteins (2, 121, 122). More importantly, there is no direct evidence that these lectins modulate CD45 phosphatase activity.

Isoform-Differential Dimerization Dimerization has been proposed as a mechanism to regulate CD45 function based on studies using a chimeric molecule consisting of the extracellular and transmembrane domains of the epidermal growth factor receptor (EGFR) fused

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to the cytoplasmic domain of CD45 (EGFR-CD45) (123). EGFR-CD45 restores TCR signaling in a CD45-deficient T cell line. Strikingly, this activity is abrogated by EGF-induced dimerization of the EGFR-CD45 chimera (123). Similarly, some antibodies that can dimerize CD45 inhibit its function (2). One mechanism for such dimerization-induced inhibition is suggested by the crystal structure of the membrane-proximal phosphatase domain of RPTPα. This protein fragment forms a dimer in which the catalytic site of one molecule is blocked by specific contacts with a structural wedge from the membrane-proximal region of its partner (124). Sequence comparison indicates that amino acid residues within the wedge are conserved among receptor-like PTPs and suggests that CD45 may fold into a similar structure (124). Indeed, mutation of a key residue at the tip of the putative wedge in the context of EGFR-CD45 abolishes the inhibitory effect of EGF (125). The physiological significance of this dimerization model was confirmed by introducing this single mutation into the mouse germ line by homologous recombination (44). The resultant CD45E613R mice developed a lymphoproliferative syndrome and severe autoimmune nephritis with autoantibody production, resulting in early death (44). The dramatic phenotype of the mice demonstrates the importance of negative regulation of CD45 by dimerization in vivo. Dimers of CD45 have been detected by several different methods. A small population of CD45 migrates as dimers after chemical cross-linking in a murine T cell line YAC1 (126). A recombinant protein consisting of the membrane proximal region and D1 of murine CD45 exists primarily as dimers (11). Recombinant proteins of rat CD45 extracellular domain fragments exist as both monomers and dimers (127). Homodimers of RO have been detected by fluorescence resonance energy transfer (FRET) analysis in a CD45-deficient T cell line reconstituted with various isoforms (92). In addition, by engineering a cysteine in the juxtamembrane region of CD45 extracellular domain, it has been shown that full-length CD45 can spontaneously form homodimers in the absence of exogenously added proteins (44). Although the possibility of ligand-mediated regulation cannot be ruled out at this stage, the abundance of CD45 on the cell surface, the detection of CD45 homodimers, and the conserved pattern of highly regulated alternative splicing (see above) suggest an alternative mechanism to modulate CD45: spontaneous and isoform-differential homodimerization. This mechanism has been recently demonstrated using chemical cross-linking and a cysteine dimer-trapping method (128). The alternatively spliced isoforms of CD45 differentially homodimerize in primary T cells. The smallest isoform RO dimerizes more efficiently and rapidly than the larger isoforms. Mechanistic studies have determined that dimerization is impeded by sialylation and O-glycosylation of the alternatively spliced exons. Furthermore, when expressed at the same amount as the endogenous protein, RABC reconstitutes the signaling defect in CD45-deficient T cells more effectively than RO. This finding is consistent with studies using primary human and murine T cells, in which RA+ na¨ıve cells exhibit greater tyrosine phosphorylation, calcium flux, and inositol phosphate generation upon TCR stimulation compared with ROexpressing memory cells (129, 130).

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Figure 4 Negative regulation of CD45 by isoform-differential homodimerization. Larger RA+ isoforms exist predominantly as monomeric active phosphatase, while the smaller size of the RO extracellular domain facilitates dimerization. The efficient homodimerization of RO renders it less active, increasing the signal transduction threshold. In T or B cells this would contribute to cessation of the primary immune response, while in resting monocytes and macrophages this would help maintain the quiescent state (see text for further discussion).

Based on these results, a model has been proposed for the regulation of CD45 (Figure 4). An equilibrium between CD45 monomers and dimers exists on the cell surface for all isoforms. The total CD45 phosphatase activity in a cell is determined by this equilibrium, which is in turn controlled by the isoforms expressed. For example, in na¨ıve T cells, the expression of mixed larger RA+ isoforms would shift the equilibrium toward monomers because the negative charges and bulky O-linked glycoconjugates in their extracellular domain act as a repulsive barrier to homodimerization. In support of this, early FRET analysis suggests that CD45 mainly exists as monomers in resting cells (131). Consequently, total CD45 phosphatase activity would be higher, maintaining SFKs in a “primed” state capable of full activation upon TCR stimulation. During the initial phase of T cell activation, CD45 activity remains high, facilitating sustained TCR signaling during a primary immune response (123). TCR signaling induces the expression of splicing factors that mediate the exclusion of exons A, B, and C in CD45 (30). Due to the slow turnover of surface CD45, it takes several days for RA+ isoforms on the cell surface to be replaced by RO (31, 32). The expression of RO in activated T cells shifts the equilibrium toward dimers due to its more efficient homodimerization, which renders it subject to negative regulation by the inhibitory wedge. Thus, this isoform switch may act as part of a negative feedback loop, contributing to the cessation of the immune response to avoid undesirable tissue injury.

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In addition to isoform switching, cells may also titrate total CD45 phosphatase activity by varying its surface expression level. CD45 expression is upregulated during differentiation of multiple lineages and upon activation of T, B, and myeloid cells (Figure 2). The increased density of CD45 molecules may alter the equilibrium between monomers and dimers by facilitating dimerization. Supporting this notion, positive selection is actually enhanced in mice heterozygous for a CD45 null mutation (21). This hypothesis is also attractive for myeloid cells. The predominance of the RO isoform in resting cells may help maintain the cells in a quiescent state, preventing tissue damage. Upon activation, the expression of RA+ isoforms may tip the balance from dimer to monomer, increasing the amount of primed SFKs and enhancing signals through multiple pathways. This would limit macrophage-mediated cytotoxicity to sites of pathogen entry.

Localization As discussed above, cellular localization and access to substrate may contribute to CD45’s effect on signaling. Intriguingly, redistribution of an intracellular pool of CD45 has been observed upon cellular activation of both T cells and neutrophils, although its functional consequence is unknown (132–136). In the past few years, the localization CD45 with respect to lipid rafts and the immunological synapse has been an area of intensive research. It has been shown that CD45 is absent from membrane lipid rafts in unstimulated Jurkat T cells. TCR engagement promotes the aggregation of lipid rafts, which facilitates aggregation and colocalization of raftassociated proteins, such as Lck, LAT, and TCR, but excludes CD45 (137, 138). A similar phenomenon has been observed in B cells (139). However, other studies have found CD45 weakly partitioning into rafts in mouse thymocytes (140, 141), human leukemic cell lines, and human granulocytes (142). The above discrepancies could be due to the different techniques used to identify raft components, to the heterogeneity in raft components within a cell, and/or to variations in raft composition in different cell lines or cells at different developmental stages. More rigorous criteria for raft localization is required to address this question. CD45’s relationship to the immunological synapse (IS)/supramolecular activation cluster (SMAC) is complex. Because of its large size and the relatively small size of molecules involved in antigen-specific recognition, it is predicted that CD45 is excluded from the IS/SMAC (143). Confocal microscopy examining the IS/SMAC formed between a T cell clone and peptide-pulsed APC supports this prediction (144). However, an earlier study using deconvolution microscopy reports that some CD45 could be found at the T cell–APC contact site (145). In another study, functional TCR signaling in live T lymphocytes was imaged using laser scanning confocal microscopy and planar lipid bilayers containing MHC-peptide and ICAM-1. Upon antigen engagement, CD45 was first excluded from IS/SMAC and later migrated back to the center of IS/SMAC to a distinct region separate from the TCR (146). The discrepancies in these data could be attributed to the limitations of the different methods and techniques employed. For example, it is

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possible that some CD45 attributed to the IS/SMAC could actually be localized to submembraneous vesicular compartments (M. Dustin, personal communication).


Interacting Proteins The function of CD45 may also be modulated through its interactions with other proteins, which may change its substrate access and/or phosphatase activity. Using a bifunctional cross-linker, CD45 was found to associate with multiple cell surface molecules in different cell types. These include Thy-1, the TCR, and CD2 (2). However, these findings must be viewed cautiously. Because CD45 is one of the most abundant surface proteins, it is possible that the above molecules were randomly cross-linked to CD45. An interaction between the transmembrane domains of CD45 and lymphocyte phosphatase-associated phosphoprotein (LPAP) is more clearly established (147, 147a). LPAP was originally identified as a binding partner of CD45 by chemical cross-linking and by co-immunoprecipitation in lymphocytes (126, 148). However, the function of this molecule is still unknown. LPAP-deficient mice generated by three groups show controversial and modest, if any, phenotype (149–151). Interestingly, LPAP seems to preferentially associate with monomers of CD45 (128). Whether LPAP plays a role in regulating CD45 dimerization remains to be determined. Immunoprecipitation experiments have also shown that CD45 may interact with CD100 (152) and the IFN receptor α chain (153). The association between CD45 and CD100 has been implicated in T cell adhesion (152). Interestingly, only the isoforms containing one or two variable exons appear to associate with CD100 in this system. The association of CD45 with the IFN receptor α chain is necessary for transducing IFNα’s growthinhibitory signals in T cells (153). The cytoplasmic domain of CD45 has been reported to bind fodrin and SKAP55. The association of CD45 with the cytoskeletal protein fodrin, as detected by sucrose gradient centrifugation and immunofluorescent microscopy, provides a link between this surface molecule and intracellular microfilament network (154). Binding to fodrin could significantly stimulate the PTPase activity of CD45 in vitro (155). The D1 domain mediates the binding of CD45 to the adaptor protein SKAP55, which may be responsible for coupling CD45 to SFKs for dephosphorylation (156). Initial studies on isoform-specific association of CD45 with other molecules are inconclusive because the cells used express multiple isoforms [reviewed in (2)]. Subsequently, these experiments were repeated in cells transfected with individual isoform of CD45. Compared to larger isoforms, RO was found to preferentially associate with CD4/CD8 and TCR as suggested by co-capping, coimmunoprecipitation, and FRET analysis (92, 94, 157, 158). This may explain why in some situations RO-expressing cells respond more effectively to TCR signaling than RABC-expressing (89, 92). However, the major concern with these, as well as many other studies regarding CD45-associating proteins, is the lack of evidence for direct and specific interactions.

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Other Possible Means of Regulation Serine phosphorylation of CD45 is well documented (reviewed in 2). Mapping studies in the pre-B cell line 70z/3.12 have identified four serine phosphorylation sites, although the kinase(s) responsible for phosphorylating these sites is unknown (159). Two subsequent studies have found four serine residues in the acidic insert in the D2 domain that could be phosphorylated by CK2 (14, 15). Mutating these serine residues results in perturbation of TCR signaling, although there is controversy regarding which downstream events are influenced by the phosphorylation of CD45 (14, 15). Although several tyrosine kinases phosphorylate CD45 on tyrosines and consequently alter its PTPase activity in vitro or in a heterologous system (160, 161), the physiologic relevance of this modification is unclear. Transient tyrosine phosphorylation of CD45 has been detected in vivo only when cells are pretreated with a PTPase inhibitor (160, 161). Lastly, reactive oxygen intermediates generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase may contribute to the inhibition of PTPs such as CD45 during neutrophil activation (116).

CD45 AND DISEASE Due to the correlation between isoform expression and functional status of T cells, many studies have focused on alterations in isoform expression in disease. Significant associations have been reported for infantile cholestasis, malnutrition, systemic lupus erythematosus (SLE), rheumatoid arthritis, and HIV (77 and references within). Perturbations in CD45 function have also been implicated in hematologic malignancies and Alzheimer’s disease (77, 162). Whether these changes reflect alterations in CD45 function that drive disease development or are merely coincidental is largely unknown. Several lines of evidence suggest that perturbation of CD45 activity can contribute to autoimmune disease. Studies of SLE patients have revealed decreased CD45 expression and/or PTPase activity (163–165). While it is unclear if identical cellular subpopulations from patients and controls were compared in these studies, it is intriguing that phosphotyrosine patterns and SHP-1 and Lyn activity were altered (164). These findings support the notion that perturbation of the balance between positive and negative regulation of tyrosine phosphorylation could contribute to the development of autoimmune disease. Alternatively, downregulation of CD45 in patients with autoimmune disease could represent a failed attempt to minimize disease. Interestingly, MRL mice heterozygous for CD45 express less CD45 on the cell surface and have delayed kinetics of disease development and a reduction in autoantibody production (87). Finally, constitutive activation of CD45 in CD45E613R mice results in a lupus-like phenotype (44). Some insight into the role of CD45 in multiple myeloma has recently emerged. CD45 expression is lost in 50% of patients with multiple myeloma. Interestingly, a direct correlation between CD45 expression and IL-6 dependency has been found

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(166, 167). In these patients, IL-6 stimulation leads to induction of CD45 expression and proliferation, while removal of IL-6 results in downregulation of CD45. Moreover, addition of the potent PTP inhibitor vanadate blocks IL-6-induced proliferation in CD45-positive, but not -negative, cells. The molecular mechanism underlying this observation has recently been elucidated. While activation of Stat3 and ERK1 are dispensable, regulation of Lyn by CD45 is essential for IL-6 responsiveness (168). CD45 isoforms may also modulate HIV infection. While the early events in HIV-1 life cycle (budding, fusion, and entry) (169) are independent of CD45 isoform expression, it has long been appreciated that HIV preferentially replicates in CD4+ CD45RO+ memory cells rather than in CD4+ CD45RB+ na¨ıve cells (169– 173). The molecular basis for HIV’s preferential replication in CD4+RO+ cells has recently been explored. Using a CD45-deficient Jurkat T cell line reconstituted with equivalent levels of a single isoform, the authors found HIV-1 preferentially replicated in RO- versus RABC-expressing cells (174). Further analyses have demonstrated that this is due to a stronger NFAT1 response during CD3/CD28 stimulation in RO-expressing cells (175). Consequently, activity of the HIV-1 long terminal repeat (LTR), which contains multiple NFAT binding sites, was enhanced (174).

CD45 AS A THERAPEUTIC TARGET The pivotal role CD45 plays in the hematopoietic system and its close association with various diseases make CD45 an attractive target for drug-design. Modulation of CD45 function could provide a means to control the immune responses in autoimmunity, immunodeficiency, and cancer. There are two major approaches to modulate CD45 function: selective inhibitors and specific anti-CD45 antibodies. CD45 inhibitors fall into two categories. Some are nonselective among PTPs, such as the peptide-based sulfotyrosyl peptides (176) and the small-molecules Peroxynitrite (177) and Nitroarylhydroxymethylphosphonic acids (178). This presumably is because all PTPs utilize a common mechanism for dephosphorylation. Others are specific for CD45. Among these, TU752 inhibits IgE-mediated anaphylaxis and murine contact hypersensitivity reactions in vivo. Thus, it is potentially useful in the treatment of allergic diseases (179). A series of 9,10phenanthrenediones, the most potent small-molecule inhibitors of CD45 known to date, also inhibit T cell proliferation in functional assays (180). An alternative approach is the use of monoclonal antibodies (mAbs) as vehicles to deliver targeted therapy to malignant cells in the form of antibody-mediated cellular toxicity, radiation, or other cytotoxic agents. Studies have demonstrated the antileukemic effect of anti-CD45 antibodies when used either unconjugated or attached to radioactive iodine. For example, 131I-labeled anti-CD45 antibody therapy has been used in bone marrow transplantation to target radiation to hematopoietic cells with the goal of decreasing relapse rates while minimizing toxicity [reviewed in (181)]. Allograft rejection is a significant problem in solid organ transplantation. The anti-CD45RB mAb MG23G2, as well as radioactive anti-CD45 antibodies, have

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been successfully used to prevent allograft rejection in murine models and in humans. In addition, the immunosuppressive ability of MB23G2 has shown efficacy in the treatment of preclinical models of autoimmunity (182). Nevertheless, the precise mechanism of MB23G2 action remains unknown. MB23G2 may induce tolerance by rendering alloreactive T cells anergic; it may also act through the immunoregulatory activity of CD45RBlo, IL-4 and IL-10-producing T cells (182). An unexpected link between MG23G2 treatment and upregulation of CTLA-4 expression may also contribute to the immunosuppressive effect of this mAb (183). Future studies need to address the molecular basis of these antibody-mediated effects.

SYNOPSIS AND FUTURE DIRECTIONS We have learned a great deal about CD45’s role in modulating immune cell function during the past nine years. While it is clear that CD45 can act as both a positive and negative regulator to modulate signaling thresholds, several issues require further clarification. By segregating the functional activities of kinases from phosphatases, regulated subcellular localization is likely to be critical for cellular activation. The biochemical approaches used to date have been limited by the fact that they measure total cellular pools of protein. Novel approaches that allow real time assessment of phosphorylation status and activity of subcellular pools, and ultimately, of individual molecules are needed. Additional clarification of the functional role different CD45 isoforms play should be another key area of focus. Further progress in identifying the elements that regulate CD45 expression and alternative splicing should aid in the development of expression systems that recapitulate endogenous isoform levels and allow manipulation of isoform levels through targeted genetics in mice. Progress in these areas should provide the foundation for examining the mechanisms underlying the role of CD45 in disease and facilitate development of therapeutic agents targeting CD45 and the signal transduction pathways it modulates. The Annual Review of Immunology is online at http://immunol.annualreviews.org

LITERATURE CITED 1. Thomas ML. 1989. The leukocyte common antigen family. Annu. Rev. Immunol. 7:339–69 2. Trowbridge IS, Thomas ML. 1994. CD45: an emerging role as a protein tyrosine phosphatase required for lymphocyte activation and development. Annu. Rev. Immunol. 12:85–116 3. Okumura M, Matthews RJ, Robb B, Litman GW, Bork P, Thomas ML. 1996. Comparison of CD45 extracel-

lular domain sequences from divergent vertebrate species suggests the conservation of three fibronectin type III domains. J. Immunol. 157:1569–75 4. Sato T, Furukawa K, Autero M, Gahmberg CG, Kobata A. 1993. Structural study of the sugar chains of human leukocyte common antigen CD45. Biochemistry 32:12694–704 5. Furukawa K, Funakoshi Y, Autero M, Horejsi V, Kobata A, Gahmberg CG.

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6.

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160. Autero M, Saharinen J, Pessa-Morikawa T, Soula-Rothhut M, Oetken C, et al. 1994. Tyrosine phosphorylation of CD45 phosphotyrosine phosphatase by p50csk kinase creates a binding site for p56lck tyrosine kinase and activates the phosphatase. Mol. Cell. Biol. 14:1308–21 161. Stover DR, Walsh KA. 1994. Proteintyrosine phosphatase activity of CD45 is activated by sequential phosphorylation by two kinases. Mol. Cell. Biol. 14:5523– 32 162. Tan J, Town T, Mori T, Wu Y, Saxe M, et al. 2000. CD45 opposes betaamyloid peptide-induced microglial activation via inhibition of p44/42 mitogenactivated protein kinase. J. Neurosci. 20: 7587–94 163. Takeuchi T, Pang M, Amano K, Koide J, Abe T. 1997. Reduced protein tyrosine phosphatase (PTPase) activity of CD45 on peripheral blood lymphocytes in patients with systemic lupus erythematosus (SLE). Clin. Exp. Immunol. 109:20–26 164. Huck S, Le Corre R, Youinou P, Zouali M. 2001. Expression of B cell receptorassociated signaling molecules in human lupus. Autoimmunity 33:213–24 165. Blasini AM, Alonzo E, Chacon R, Riera R, Stekman IL, Rodriguez MA. 1998. Abnormal pattern of tyrosine phosphorylation in unstimulated peripheral blood T lymphocytes from patients with systemic lupus erythematosus. Lupus 7:515–23 166. Mahmoud MS, Ishikawa H, Fujii R, Kawano MM. 1998. Induction of CD45 expression and proliferation in U-266 myeloma cell line by interleukin-6. Blood 92:3887–97 167. Ishikawa H, Mahmoud MS, Fujii R, Abroun S, Kawano MM. 2000. Proliferation of immature myeloma cells by interleukin-6 is associated with CD45 expression in human multiple myeloma. Leukoc. Lymphoma 39:51–55 168. Ishikawa H, Tsuyama N, Abroun S, Liu S, Li FJ, et al. 2002. Requirements of src family kinase activity associated

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ACKNOWLEDGMENTS We thank our colleagues and members of the Weiss lab for valuable discussions. This work was supported by NIH grant A135297 (to A.W.) and the Rosalind Russell Medical Research Center for Arthritis.

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ON NATURAL AND ARTIFICIAL VACCINATIONS, Rolf M. Zinkernagel COLLECTINS AND FICOLINS: HUMORAL LECTINS OF THE INNATE IMMUNE DEFENSE, Uffe Holmskov, Steffen Thiel, and Jens C. Jensenius THE BIOLOGY OF IGE AND THE BASIS OF ALLERGIC DISEASE, Hannah J. Gould, Brian J. Sutton, Andrew J. Beavil, Rebecca L. Beavil, Natalie McCloskey, Heather A. Coker, David Fear, and Lyn Smurthwaite

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POSITIVE AND NEGATIVE SELECTION OF T CELLS


Timothy K. Starr, Stephen C. Jameson, and Kristin A. Hogquist Center for Immunology and the Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota 55455; email: [email protected], [email protected], [email protected]

Key Words T cell development, thymocyte, thymus, allelic exclusion, differential signaling ■ Abstract A functional immune system requires the selection of T lymphocytes expressing receptors that are major histocompatibility complex restricted but tolerant to self-antigens. This selection occurs predominantly in the thymus, where lymphocyte precursors first assemble a surface receptor. In this review we summarize the current state of the field regarding the natural ligands and molecular factors required for positive and negative selection and discuss a model for how these disparate outcomes can be signaled via the same receptor. We also discuss emerging data on the selection of regulatory T cells. Such cells require a high-affinity interaction with self-antigens, yet differentiate into regulatory cells instead of being eliminated.

INTRODUCTION Five hundred million years ago, as the jawed vertebrates diverged from the jawless fish, an ancestor of the jawed vertebrates acquired the ability to present antigen via major histocompatibility complex (MHC) molecules and to somatically rearrange antigen receptors using the recombinase activating genes (RAGs), thus giving birth to the adaptive immune response (1). Over time the vertebrate adaptive immune system has evolved a highly coordinated interplay of cells specialized for antigen presentation, antibody production, and cytotoxicity and has developed the means to direct this powerful response against pathogens. In this review we look at what is known and what remains unknown about the ontogeny of one of the major players in the adaptive immune response, the thymus-dependent α/β T cell.

Overview of Thymic Development and Anatomy T cells arise from hematopoietic stem cells that migrate to the thymus. Their development does not occur cell autonomously but requires signals from nonhematopoietic stromal cells including various types of thymic epithelial cells (TECs) and mesenchymal fibroblasts. These cells reside in distinct anatomic locations in the 0732-0582/03/0407-0139$14.00

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Figure 1 Bone marrow progenitors (DN1: CD44+, CD25−) enter the adult thymus near the cortico-medullary junction. As these cells migrate toward the subcapsular epithelium they upregulate CD25 (DN2) then downregulate CD44 (DN3). At this point TCRβ rearrangement occurs, and successful pairing with pre-Tα results in downregulation of CD25 (DN4) and extensive proliferation and expression of CD4 and CD8 (DP). Next, rearrangement of TCRα occurs and cortical thymocytes are subject to positive and negative selection. Cells surviving these checkpoints migrate through the medulla before exiting the thymus.

thymus, and movement of precursor cells between these microenvironments is critical for the perception of differentiative signals (Figure 1) [reviewed in (2)]. Differentiation is characterized by the temporally coordinated expression of cell surface proteins on the thymocyte, including CD4, CD8, CD44, and CD25 (Figure 1). Upon entry into the thymus, precursors lack expression of CD4 and CD8 and are called double negative (DN). Lind et al. (3) have shown that progenitor cells enter the thymus at the cortico-medullary junction (CMJ) and move through the cortex as the cells progress through DN1 (CD44+/CD25−) and DN2 (CD44+/CD25+), arriving at the subcapsular zone as they enter DN3 (CD44−/CD25+) (Figure 1). Rearrangement of the T cell receptor (TCR) γ , δ, and β loci is evident at this stage. The two lineages of T cells—α/β and γ /δ—diverge at or prior to this stage (4). Although the precise details of the γ δ/αβ commitment process are not well understood, it is generally accepted that signals that emanate from a correctly assembled receptor are required for further survival and differentiation along the appropriate lineage. For γ /δ cells, this is the γ /δ/CD3 complex, for α/β T cells it is a complex composed of CD3, TCRβ, and the invariant pre-TCRα chain (pTα). The assembly of a functional β/pTα complex leads to α/β lineage commitment and involves expansion, expression of CD4 and CD8 coreceptors, and rearrangement of the TCR α locus. As thymocytes enter this stage they travel back through the cortex (Figure 1). Thus, double-positive (DP) thymocytes that reside in the cortex

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are the first population capable of expressing α/β heterodimeric T cell receptors at the cell surface. The ligand specificity of the receptor dictates the cell’s fate and is the subject of this review. Positive and negative selection allow only cells with functional TCRs that will not be self-reactive in the periphery to pass these checkpoints. This small fraction of cells (less than 5%) will downregulate one of the coreceptors, becoming either CD4 single positive (SP) or CD8 SP and, within a few days, will leave the thymus to circulate as mature T cells.


POSITIVE SELECTION The TCRα Locus Undergoes Processive Rearrangement Until Positive Selection DN precursors expressing only a TCR β chain initiate α locus rearrangement immediately after pre-TCR signaling, and Vα/Jα joins can be detected at low levels in DN cells in the final stage of differentiation (DN4). Nonetheless, full-scale α locus rearrangement is not in place until the cell becomes a quiescent DP cell (5). The productive rearrangement of an α chain, however, is not sufficient to trigger the termination of recombination. Instead, only α chains that form an MHC-restricted receptor when paired with the β chain will trigger termination (6, 7). The generation of an MHC-restricted receptor from random α/β pairs is thought to occur relatively infrequently, despite an inherent propensity of α/β T cell receptors to bind MHC (8). Thus, the majority of DP thymocytes express a surface TCR but remain in an undifferentiated state and express a high level of the RAG recombinase. The α locus is structured such that multiple V/J recombination events can occur on the same allele, each time resulting in excision of the prior recombined DNA. Additionally, it was shown that precursors begin α locus recombination at the 50 end of the J locus and proceed to the 30 end (9, 10). This processivity means that multiple different productive TCR α gene rearrangements can be tested per cell and presumably provides an efficient means to screen for rare progenitors while expending a minimal amount of metabolic energy. This process is finite, however, as evidence suggests that the average DP cell has a life span of 3–4 days. Several factors contribute to the regulation of this life span, including the steroid transcription factor RORγ (13) and transcription factors in the wnt signaling pathway, TCF-1 and LEF-1 (11, 12). Both are linked to controlling expression of the anti-apoptotic factor, Bcl-XL (12, 13). Without RORγ or TCF-1 activity, Bcl-XL is not expressed and DP thymocytes die by neglect before having the opportunity to be positively selected. Interestingly, the lifespan of the cell determines the processivity of α locus recombination. Short-lived precursors only get a short way into α locus recombination; long-lived precursors exhaust the α locus (14). Thus, the lifespan of the DP seems to be, on average, sufficient to allow recombination down through the TCR α locus. The fact that TCR assembly itself is not sufficient to trigger differentiation and RAG downregulation is one way in which developing T cells differ from developing B cells. In developing B cells it is presumed that most properly assembled

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H/L chain pairs will trigger allelic exclusion and differentiation, although there are some potential exceptions (15, 16). This difference between T and B cells presumably reflects the fact that T cells recognize antigen in an MHC-restricted fashion, whereas B cells do not. Therefore having a checkpoint for MHC restriction, not merely assembly, during selection maximizes the utility of the T cell repertoire. DP precursors expressing an MHC Class I– or Class II–restricted receptor produce a signal that results in RAG gene repression, long-term survival, migration into the medulla, and differentiation into mature T cells. This process, termed positive selection, is initiated in cortical DP thymocytes and takes several days to finalize. Interestingly, evidence suggests that the TCR must remain engaged and sustain signaling for the duration of this process (17).

The Specific Ligands for Positive Selection An important issue in the field regards the precise TCR ligand that stimulates positive selection. The receptor must be MHC restricted, but most MHC molecules display self-peptides, and numerous studies have shown that positive selection involves combinatorial recognition of peptide and MHC, as does T cell activation [reviewed in (18)]. However, the number and nature of the self-peptides involved, and their relationship to antigenic peptides has been controversial. Early experiments used TCR transgenics and studied synthetic peptides that were or were not related to the antigenic peptide. Several reports suggested that a subthreshold density of the antigenic peptide itself was capable of promoting positive selection (19–22). This finding was subsequently reinterpreted when it became clear that selected cells, although phenotypically mature, failed to respond normally to the same antigen at high doses (20, 23, 24). Peptides that antagonize mature T cell responses were also shown to induce positive selection in fetal thymus organ cultures (FTOC) (25–27) and in vivo (28). However, others have shown that the presence of an antagonist peptide failed to promote (29, 30) or impaired (20, 31, 32) positive selection. Finally, three studies showed that apparently unrelated peptide/MHC complexes induced positive selection (33–35). Whether the selecting peptides were related to the antigenic peptide or not, affinity measurements support the idea that TCR affinity for positive selection ligands is lower than for negative selection ligands (36–38). However, all of these studies suffered from the same drawback: Whereas they described ligands that can promote positive selection in experimental situations, this may not accurately reflect the properties of the natural self-peptide/MHC ligands that drive positive selection in vivo. Three general approaches have identified naturally occurring self-peptides that support positive selection: random sequencing and testing, purification and sequencing of bioactive peptides, and identification of candidates by database search strategies. In one study three random, abundant, naturally occurring H-2Db bound self-peptides were sequenced. Two of these induced positive selection in Class I– restricted TCRs: F5 (39) and H-Y (40), but only at very high peptide concentrations. Given the existence of several putative self-peptides with higher homology

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to the male antigen (41), one might expect that the former self-peptides, while abundant, may not be the most relevant self-peptide ligands in vivo. Two groups studying H2-M3-restricted TCR transgenics also tested random H2-M3-binding peptides. In this case the potential repertoire of self-peptides capable of binding H2-M3 is much smaller since this MHC molecule requires a formylated methionine at position 1, and only 13 mitochondrial genes are thought to give rise to formylated peptides in the mouse. In one system a peptide derived from the mitochondrial protein ND1 was highly effective at inducing positive selection, and three others could do so at high concentrations (requiring at least 100 times more peptide) (42). However, for another H2-M3-restricted TCR, the ND1 peptide was not particularly effective at inducing positive selection (43). Other groups have identified selecting self-peptides based entirely on bioactivity. A thymocyte “dulling” assay was used to screen high-performance liquid chromatography fractions of peptides eluted from purified MHC molecules, and the sequences of individual peptides were obtained using tandem mass spectrometry (44). For the TCR transgenic OT-I this led to the identification of two peptides with high homology to the antigenic peptide (45). In the case of the N15 TCR transgenic, only one high-performance liquid chromatography fraction showed bioactivity, and a peptide derived from this fraction showed no homology to the viral antigenic peptide for the N15 receptor, except at the MHC anchor position (46). Finally, database search strategies have been successful in identifying putative self-peptides capable of inducing positive selection (41, 47; P. Ohashi, personal communication). This approach will not detect peptides that are unrelated to the antigenic peptide, and the processing and presentation of such candidates in vivo must be confirmed. One question addressed by these approaches relates to the abundance of the selecting peptides for a given TCR. In the case of the OT-I receptor, only two peptides were identified, although three peaks of bioactivity were reproducibly observed (45). This stands in contrast to another report, which found that 2 out of 3 randomly sequenced H-2Db-binding peptides were able to select specific CD8 T cells in another TCR transgenic system (39). However, peptide doses were quite high in that report. Indeed, a separate study found that even among putative selfpeptides chosen for their homology to the H-Y male peptide, only 1 out of 10 was capable of efficiently promoting thymocyte selection (41). Likewise, among H-2Db-binding peptides chosen for their homology to the viral antigenic peptide recognized by the P14 TCR, only 2 out of 16 were capable of promoting thymocyte selection (47). Finally, in a screen of all peptides extracted from thymic Kb molecules, Sasada and colleagues were only able to detect one self-peptide capable of initiating DP dulling and positive selection in N15 TCR transgenic thymocytes (46). These results suggest that the number of functionally relevant self-peptide ligands for a typical Class I–restricted TCR is low. If, indeed, selecting self-peptides are rare, one would predict that the absence of an appropriate positive-selecting self-peptide could result in deficient selection of individual antigen specificities. Consistent with this notion, the lack of responsiveness to an ovalbumin antigen

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in certain mice can be explained by the absence of appropriate selecting selfpeptide(s) (48, 49). Lack of available self-peptides may underlie other cases of positive-selection deficiency as well (50). Another issue addressed by these studies is whether similarity to antigen is a universal feature of self-peptides that are involved in positive selection. For OT-I a homology-based search strategy identified the same two peptides as a bioactivity-based approach (45). Other studies have also found such homology, although as discussed above, there was one exception. Another approach, using “single-complex” transgenic mice, has suggested that positive selection ligands do not need to be structurally related to the antigenic peptide ligand. C57BL/6 DM-deficient mice, which predominantly present the CLIP peptide via I-Ab, produce a diverse repertoire of CD4 T cells capable of responding to Class II peptides unrelated to CLIP (51, 52). However, it was subsequently shown that many CD4 T cells in these mice are selected by the diverse self-peptides presented by I-Ab at very low levels (53, 54). Therefore the studies on selection specificity in DM-deficient mice cannot be interpreted in a straightforward fashion. In mice with peptides covalently linked to Class II, this “leaky” self-peptide presentation occurs much less frequently. Indeed, evidence suggests that the majority of CD4 T cells present in IAb-Eαp transgenic mice are selected on IAb-Eαp complexes, as their development in organ cultures can be blocked by the YAE antibody specific for IAb-Eαp (55). In these mice, although the CD4 T cell repertoire is significantly less diverse, immune responses to antigens unrelated to Eαp can be observed (33). This suggests that structural homology is not a mandatory feature of positive selection ligands. However, these results cannot be interpreted to mean structural homology does not play an important role in normal mice, in which individual self-peptide/MHC complexes are present among a diverse mix of peptides at levels much lower than in single-complex mice. Consistent with this, it was found that TCRs cloned from IAb-Eαp mice were not selected normally when expressed as TCR transgenes in vivo (56). Finally, regarding the question of whether positive selection can occur in response to low concentrations of a high-affinity ligand, none of the naturally identified self-peptide ligands were stimulatory for mature T cells, with the exception of the ND1 peptide ligand for an H2-M3-restricted TCR (57). Even this ligand, however, was only weakly stimulatory. No studies on naturally occurring Class II self-peptides in positive selection have been reported. This may reflect the fact that organ culture of β 2-microglobulindeficient or TAP-deficient mice, a tool widely used by investigators studying Class I–restricted receptors, has no equivalent in the Class II system. Nonetheless, two groups have reported that antibodies specific to a particular antigenic peptide/MHC complex were able to block positive selection of T cells specific for that antigen (58, 59) but not other T cells. This block was observed on a selecting background, i.e., in the absence of the antigenic peptide. A blocking effect was observed despite the fact that these antibodies did not show dramatic staining of self-MHC in the absence of antigen. This result was interpreted to mean that the relevant

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self-peptides for positive selection of those two Class II–restricted receptors must be structurally related to the antigenic peptide and occur at low abundance in normal cells. Altogether, the data accumulated to date generally support the notion that relatively rare, low-affinity self-peptides promote positive selection. This gives rise to mature T cells having high affinity for foreign peptides that are generally structurally related to the self-peptides involved in selection.

Signals and Transcription Factors Required for Positive Selection Gene-deficient mice have been highly instructive in understanding the signals involved in positive selection (Figure 2). Positive selection is initiated by TCR ligation of low-affinity self-peptide/MHC complexes; thus, structural components of the TCR are required. Indeed, TCR α chain–deficient mice do not develop past the DP stage. The role of TCRβ, CD3γ , CD3ε, and TCRζ in positive selection was more difficult to study because deficiency in these genes resulted in an earlier block in the generation of DP cells (60–64). Interestingly, CD3δ deficiency did not impair the generation of DP cells but profoundly blocked positive selection (65). The differing requirement for CD3δ in β selection and positive selection is intriguing because β selection is thought to result from ligand-independent signaling (66) and positive selection involves ligand-dependent signaling. The precise role that CD3δ plays in TCR triggering, for example by transducing a conformational change or mediating oligomerization, remains unknown. Interestingly, it was recently reported that γ /δ TCR complexes do not contain CD3δ (67), and the development of γ /δ T cells is not dependent on CD3δ. A specific motif is found in the TCRα chain connecting peptide and not in the corresponding region of TCRδ (68). This motif is required for positive selection of α/β T cells and is important for retaining CD3δ in the TCR complex (69). Deficiency of specific immunoreceptor tyrosinebased activation motifs (ITAMs) in the TCRζ chain did not impair β selection and allowed their role in positive selection to be studied. Surprisingly, the overall process of positive selection could occur in the absence of TCRζ chain ITAMs. However, the TCR repertoire was skewed by ITAM deficiency, as judged by the effect on selection of individual TCR transgenic strains (70). Several Src and Syk family kinase proteins proximal to TCR signaling are critical in positive selection. Although the kinase Lck is required for β selection, its importance in positive selection was addressed by temporally controlled expression of a dominant interfering and constitutively active form of Lck (71, 72). These studies demonstrated that Lck is required for positive selection. Several proteins negatively regulate TCR signaling at a proximal point in the signaling pathway. Some of these, including c-Cbl, SLAP, and Csk, oppose the process of positive selection, and their deficiency results in enhanced selection. c-Cbl (73) and SLAP (74) appear to regulate surface TCR levels and thereby influence TCR signaling. Csk is a kinase that negatively regulates Lck activity. Deficiency in Csk resulted in

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Figure 2 Selected molecules identified as critical regulators of selection checkpoints in T cell development. The function of most of these molecules was established by analysis of gene-deficient mice unless indicated by (∗ ). We list certain molecules required for the DN3 → DN4 transition, as they may also be involved in positive and negative selection, but their function at the DP stage has not been evaluated. Molecules listed before the fork are required for both positive and negative selection. Molecules listed on far right adjust the threshold of thymic selection.

positive selection of mature CD4 T cells in the absence of MHC or even a surface TCR, highlighting the importance of Lck in initiating TCR signals in the thymus (75). The phenotype of the ZAP-70-deficient mouse showed strongly impaired positive selection (76). One of the main targets for ZAP-70 kinase activity is the adaptor protein LAT. LAT is assumed to be critical for positive selection, but again deficiency resulted in profoundly impaired β selection (77), precluding direct assessment of positive and negative selection with standard gene knockout approaches. LAT nucleates the recruitment of several components of the TCR

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signal transduction pathway including GADs, SLP-76, Grb2, and PLCγ 1 [reviewed in (78)]. Of these, PLCγ 1 is central to the generation of a Ca++ flux. The importance of signals generated by a Ca++ flux have been appreciated for some time, because positive selection can be abrogated by cyclosporin A or FK506, inhibitors of the Ca++-dependent calcineurin pathway (79, 80). A direct role for PLCγ 1 has not been tested because mice lacking this protein die in utero (81). Two groups reported the phenotype of mice expressing a mutant form of LAT that cannot recruit PLCγ 1. This mouse had a severe defect in pre-TCR signaling, albeit not as complete as the LAT-deficient mouse (82, 83). The Tec family of kinases, recruited to TCR signaling complexes by SLP-76/GADs/LAT, are also critical in generating a Ca++ flux and, not surprisingly, are required for optimal positive selection (84, 85). Likewise, mice lacking the GADs adaptor have impaired positive selection (86). Grb2 is the only LAT-recruited protein that does not seem to be critical for positive selection (87). Activation of PLCγ results in the generation of two important mediators: diacylglycerol (DAG) and IP3. IP3 generates an elevated intracellular Ca++ level resulting in activation of the calcineurin pathway. Whereas calcineurin Aα o mice did not show a defect in positive selection (88, 89), calcineurin Aβ o mice did (90). The NFAT family of transcription factors represents a primary target of the Ca++-activated calcinuerin protein (91). DP thymocytes express a high level of NFAT4, and genetic deficiency resulted in impaired positive selection (92). Thus, all of these results are consistent in highlighting the significance of TCR-mediated NFAT4 activation for positive selection (Figure 3, below). DAG, however, is an important factor for activating PKC family members. Given the importance of PKC activation pathways in mature T cells, it is surprising that data do not suggest a significant role in positive selection (93, 94). However, it was recently discovered that DAG is also a mediator of ras activation in some circumstances. The RasGRP class of guanine nucleotide exchange factors contains a DAG binding domain and can be directly activated by phorbol esters. Mice lacking RasGRP1 display profoundly impaired positive selection (95). Thymocytes from such mice did not activate extracellular signal-related kinase (ERK) after TCR stimulation, suggesting that in thymocytes TCR signals lead to Ras activation via a RasGRP-dependent manner (Figure 3, below). The significance of ERK activation was appreciated early on, when dominant interfering forms of ras and MEK were found to block positive selection (96, 97), and this was confirmed upon analysis of ERK1-deficient mice (98). In thymocytes a likely nuclear target of activated ERK is the Egr-1 transcription factor (99). Enforced expression of Egr1 resulted in positive selection of HY T cells, even on a “nonselecting” background (100), and Egr1-deficient mice have impaired positive selection (101). The helix-loop-helix (HLH) family of transcription factors is also important in positive selection [reviewed in (102)]. This family consists of “E” proteins that bind DNA and “Id” proteins that heterodimerize with E proteins and oppose their function. E2A-deficient mice displayed enhanced positive selection, suggesting that E2A normally functions to attenuate positive selection (103). TCR stimulation

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Figure 3 Model of differential signaling in the thymus. (A) Positive selection: A low-affinity ligand stimulates the TCR, which transmits a signal via the TCR αCPM, CD3γ -ITAM, and CD3δ. Partial LAT phosphorylation recruits PLCγ 1 and Gads/Slp76/Itk, resulting in activation of PLCγ 1 and production of diacylglycerol (DAG) and calcium signals. RasGRP is turned on by DAG and stimulates sustained, low-level ERK activation while the calcium flux activates NFAT. (B) Negative selection: A high-affinity TCR ligand along with costimulation results in fully phosphorylated LAT, recruitment of Grb2/SOS1, and strong, transient activation of ERK along with p38 and JNK activation.

of DP thymocytes induced ERK activation and egr-1 gene expression, resulting in an increase in Id3 expression (104). Id3 inhibits E protein function, and Id3deficient mice showed reduced positive selection (105). Because E proteins are known to regulate RAG gene expression and TCR locus accessibility (106), one likely consequence of Id3 expression and E protein inhibition in positive selection is to discontinue V/J recombination and close the TCRα locus. Another transcription factor was recently identified as playing a critical role in positive selection. Schnurri-2 (Shn-2) is a zinc finger transcription factor assumed to function in TGFβ superfamily signaling. Studies in Drosophila have linked Shn to decapentaplegic signaling, which is homologous to bone morphogenetic protein (BMP) signaling in vertebrates. Takagi et al. generated a Shn-2-deficient mouse strain and found a significant block in positive selection, whereas negative selection, tested by superantigen and anti-CD3 stimulation, was normal (107). Bone marrow chimeras demonstrated that this defect was intrinsic to thymocytes, and that the phenotype was gene-dosage dependent, as heterozygotes had a partial block in positive selection. More recently, Graf and colleagues identified the secreted protein, Twisted gastrulation (a modulator of BMP signaling), as a protein

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upregulated during positive selection (108). Although functional studies focused on its role in the DN → DP transition, its expression pattern indicates a possible role in positive selection.


The Role of Epithelial Cells in Positive Selection It has long been appreciated that efficient positive selection requires an intact three-dimensional microenvironment and that cortical epithelial cells are a key cellular component of this (2). Indeed, thymic epithelial cells provide specialized accessory interactions that MHC+ epithelial cells from other tissues do not (109). Several genes unique to cortical epithelial cells have been identified by subtractive hybridization techniques. A 12-transmembrane-spanning protein, TSOIC12, is specifically expressed in cortical epithelial cells but has no identified function (110). Thymus LIM protein (Tlp) was expressed in an interesting pattern in cortical epithelial cells (111). A knockout of Tlp showed reduced thymic cellularity, though the basis of this effect remains unclear. The MT-SP1 serine protease is also expressed in MHC Class II+ thymic stroma (112), and a knock-out showed increased thymocyte apoptosis, although it was unclear if the phenotype was secondary to a severely compromised epidermal barrier function (113). Finally, as discussed above, BMP signaling appears to be important for positive selection based on the Shn-2−-deficient mice (107). Because thymic stromal cells express BMP 2, 4, and 7 (108), these could represent essential factors contributed by epithelial cells during selection. Further experiments will be required to test this. Additional functional studies, however, have shown that the essential stromal factors for positive selection are cell-surface or cell-associated molecules that are downregulated upon monolayer culture (114, 115), properties that should aid in their identification. Whereas proper T cell development requires epithelial cells, proper epithelial differentiation also requires T cells—a phenomenon termed “crosstalk” [reviewed in (2)]. For example, in mice with a block at the DN1 stage owing to ectopic expression of a CD3ε transgene, cortical epithelial cells lack the normal three-dimensional network. Likewise, in recombination-deficient mice, thymic medullary epithelial cells are disorganized. Recent research on thymic epithelial cells has focused on the differentiation of cortical and medullary epithelial cells from a common precursor. Two studies showed that the surface glycoprotein MTS24 identifies a common epithelial progenitor cell present in abundance in the embryonic thymus but only rarely in the adult (116, 117). MTS24+ cells were able to fully reconstitute the thymic microenvironment and support T cell development when grafted ectopically. This finding should provide a useful model system to study epithelial cell function, particularly if MTS24+ cell lines can be established. Using advanced microscopy techniques, two groups observed thymocytes interacting with thymic epithelial cells in real time (118, 119). Both groups used reaggregate cultures of thymic epithelial cells, which mimic the in vivo process of positive and negative selection (120). Using two-photon laser scanning microscopy

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Bousso et al. observed thymocytes vigorously moving around their environment, apparently sampling signals from neighboring cells (118). When the reaggregate cultures were made with thymic epithelial cells expressing MHC peptide ligands for positive selection, some thymocytes stuck to epithelial cells for an estimated 6–12 h. Using a different imaging technique, Richie et al. watched in real time thymocytes transfected with green fluorescent protein–labeled signaling molecules interact with thymic epithelial cells expressing MHC peptide ligands for negative selection (119). Subtle differences were noted in the arrangement of molecules at the interface between a thymocyte and a thymic epithelial cell from that observed in mature T cell–antigen-presenting cell (APC) interactions. Future development of these techniques will provide valuable insight into the behavior of thymocytes and their partners during positive and negative selection.

NEGATIVE SELECTION Models Although survival and differentiation are clear outcomes of TCR ligation in DP thymocytes, not all TCR ligation events lead to this end. As discussed above, the ligands for positive selection are generally not stimulatory for mature T cells. Stimulatory ligands for a given T cell (generally referred to as foreign, agonist, or antigenic peptides) cause clonal deletion if present in the thymus during development. This is a rapid and sensitive process thought to play a major role in eliminating self-reactivity in the T cell repertoire. Despite intensive investigations over the past decade, the molecular mechanisms of clonal deletion remain ill defined (121). In part this may be attributed to the fact that a plethora of models exist for testing negative selection and not all of these involve the same or similar mechanisms. These issues are summarized in Table 1 and the discussion below. The first experiments describing clonal deletion showed the elimination of Vβ17a T cells in animals that expressed an endogenous superantigen and the I-E MHC molecule (122). Endogenous superantigen–mediated deletion generally occurred late in development, at the single-positive (SP) stage in the medulla (Figure 1), even in TCR transgenic animals (123). This is presumed to be due to the medullary location of superantigen expression. Endogenous superantigens are naturally occurring ligands, and the normal T cell repertoire can be studied without the manipulation of TCR transgenesis. Nonetheless, utilization of this model remains problematic because robust biological effects are not seen in H-2b mice, making the analysis of various knockouts cumbersome because of the backcrossing involved. TCR transgenic mice expressing a receptor specific for a self-antigen along with that self-antigen represent the most widely used model for negative selection. Many systems were designed such that both the TCR and the antigen are expressed as transgenes, although in some cases such as the HY or C5 systems the antigen is a

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TABLE 1 The plethora of clonal deletion models Model

Developmental stage

Advantages

Drawbacks

Anti CD3 in vivo

DP

Ease, cost

Cross-linking antibodies may not mimic peptide/MHC in TCR signaling Mature T cells stimulated at high frequency produce cytokines and stress hormones that induce nonspecific death of DP thymocytes

Endogenous superantigen (e.g., Vβ8.1/Mlsa)

DP → SP

Natural ligand Can detect responding T cells with Vβ specific antibody

Superantigen stimulation may not be equivalent to MHC/peptide antigen? Strain combinations required make it less easy to evaluate the effects of gene knock-outs

TCR Tg treated with peptide in vivo or in vitro

DP

Many models available

Mature T cells stimulated at high frequency produce cytokines and stress hormones that induce nonspecific death of DP thymocytes High rate of nonspecific death of thymocytes in vitro Thymocytes may respond differently in absence of stromal cells

TCR Tg with endogenous ligand (e.g., HY)

DN → DP

Natural ligand, widely used Many models available

TCR is expressed and ligated earlier in development than in normal mice High frequency of responders may yield nonphysiologic results

TCR Tg with endogenous ligand (e.g., 6.5)

DP → SP

Natural ligand Many models available

High frequency of responders may yield nonphysiologic results (TCR is still expressed early, but ligation does not occur until late)

TCRβ Tg + endogenous ligand (e.g., 5C.C7β)

DP

Frequency of responders is closer to normal TCR is expressed at appropriate stage

Requires analysis of specific T cells with tetramers Not all TCRβ Tgs display a high enough frequency of tetramer binding to study DP thymocytes

Abbreviations: DP, double positive; MHC, major histocompatibility complex; TCR, T cell receptor; SP, single positive; DN, double negative.

naturally expressed protein. Model systems of this nature can be generally divided into those in which clonal deletion occurs early [at the double-negative (DN) stage as cells progress to the double-positive (DP) stage] or those in which it occurs late (at the SP stage, or as cells progress to the SP stage). There are several possible explanations for why some transgenics delete early and others delete late (124). First, the anatomy of self-antigen expression might determine the developmental stage of deletion, because DN cells reside in the subcapsular area or outer cortex and SP cells reside in the medulla (Figure 1). Second, the type of APC that presents the particular self-antigen in each microenvironment could determine whether the cell dies or not. Finally, the affinity or avidity of the TCR for self-antigen might

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determine the stage of deletion; DP thymocytes, which express a lower level of TCR than SP thymocytes express, would require a stronger TCR stimulus to die (125, 126). However, the experimental systems that addressed this did not rule out that the specific peptide antigen could be expressed by different APCs in these systems. Additionally, DP thymocytes are as sensitive to peptide/MHC stimuli as SP cells are, despite their lower receptor level (127, 128). Finally, peptide titration experiments did not demonstrate preferential SP deletion at low doses (129, 130). Thus, it is unclear what determines the stage of clonal deletion in the thymus. It should be noted that there is some controversy as to whether TCR ligation at an early stage impairs the generation of DP cells, as opposed to deleting cells that developed to the DP stage (131). This is an important distinction because the two processes may have different molecular mechanisms. Also of note, clonal deletion in normal (non-TCR transgenic) animals is more likely to occur at later stages because most DN and early DP cells do not yet express an intact α/β TCR at the cell surface. Injection of anti-CD3 into animals causes a profound death of DP thymocytes. This provides an easy way to induce synchronous death and was extensively utilized early on as a model of clonal deletion. However, it is not generally favored now because of the finding that activation of a high frequency of T cells results in the production of inflammatory cytokines and steroid hormones that cause nonspecific death of thymocytes (132–134). Treatment of DP thymocytes in vitro circumvents this problem to some extent but is not a favored model because apoptosis in thymocytes may be critically influenced by the thymic microenvironment. This is apparent from the high level of nonspecific apoptosis that occurs in DP thymocytes upon removal from the organ. Additionally, it was found using this assay that clonal deletion requires costimulatory factors other than TCR ligation that are presumably provided by stromal cells in vivo (135, 136). A final drawback to this model is the concern that TCR stimulation via cross-linking antibodies induces a qualitatively distinct TCR signal from peptide/MHC ligation. The injection of specific peptide into TCR transgenic animals also induces massive apoptosis of DP thymocytes (137). This model eliminates the concern about the artificial nature of cross-linking antibodies as TCR ligands. However, other concerns about the overwhelming activation that results when a majority of the cells respond to the stimulus hold here as well. For example, adoptive transfer of mature TCR transgenic T cells into a B6 mouse followed by peptide injection resulted in death of the polyclonal (non-antigen-specific) DP thymocytes (134). Thus, activation of a high enough frequency of mature T cells can cause nonspecific death of thymocytes. In support of this notion, injection of exogenous superantigen (SEB) or a specific peptide into TCRβ or TCRα/β transgenic adult animals (with mature T cells) causes death of DP thymocytes, whereas injection into neonates (without mature T cells) leaves DP cells largely intact and induces selective death of immature SP thymocytes (138). Also, TCR stimulation of a high frequency of DP thymocytes can cause a cytokine-mediated stromal cell–activation process (139), recruitment of eosinophils (140), and collapse of

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the thymic architecture (141), all of which may contribute to nonspecific effects. One study attempted to get around these issues by eliminating mature T cells or reducing the frequency of responders using mixed bone marrow chimeras, but antigen stimulation still caused death of thymocytes (142). This study shows that DP thymocytes undergo clonal deletion when triggered through their TCR, but the unfortunate fact remains that in many models this clonal deletion is overlaid by one or more nonspecific death pathways, making mechanistic studies difficult to interpret. One of the more physiologic models for antigen-induced clonal deletion described to date involves the use of a TCR β transgenic strain (143). In such mice the surface α/β receptor is not expressed earlier than normal, nor is the frequency of responders overwhelmingly high. In the case of the 5C.C7β transgenic, 0.25% of DP cells could bind the MHC/peptide complex, I-Ek/PCC. This allowed the authors to study the fate of tetramer-binding cells in the presence or absence of a transgene expressing PCC as a self-antigen. In the presence of antigen, the percentage of DP cells that bound tetramer was reduced by half and the percentage of SP cells was reduced by tenfold, suggesting that deletion occurs more efficiently at later stages of development. Whereas this model does not have the problem of nonspecific activation that many other models have, the reduced frequency makes the responders difficult to detect and study. Additionally, not all TCRβ transgenics show such a high frequency of cells capable of binding antigen in the preselection repertoire. For example, in Vβ8 transgenics, H-2Db-restricted male reactivity was only observed when rare Vα9 chains were paired (144). Overall, we caution that candidate proteins or processes involved in clonal deletion should be evaluated in multiple models before making an interpretation. In the next section we review studies of the signals and transcription factors involved in clonal deletion and note which models were used to make each assessment.

The Signals and Transcription Factors Involved in Clonal Deletion Many of the proteins involved in TCR proximal signal transduction are required for both positive and negative selection (Figure 2). We focus on genes and pathways that appear to be uniquely involved in negative selection resulting in death of the thymocyte by apoptosis. There are generally considered to be two initiator pathways of apoptosis in cells, one via surface receptors such as tumor necrosis factor receptor (TNFR) and Fas, which contain the Fas-associated death domain (FADD), and a second via activation of Bcl-2 family members and subsequent activation of caspase 9, although there is cross-talk between these pathways [reviewed in (145)]. Most studies showed that Fas-Fas ligand interactions are not necessary for negative selection [reviewed in (146)]. Nonetheless, it may play a role in some cases, particularly at high antigen doses (138). Analysis of mice deficient for both TNFR1 and TNFR2 revealed that TNF signaling is also dispensable for negative selection induced by endogenous peptides, although TCR cross-linking-induced

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deletion was reduced in these mice (132). Strasser and colleagues used a dominant negative construct of FADD to simultaneously inhibit downstream signaling from multiple death receptors including Fas, TNFR1, DR3, and TRAILR2. Surprisingly, negative selection in response to superantigen was normal, whereas negative selection in response to TCR cross-linking or in the HY male model was actually increased, suggesting a role for FADD signaling in survival (147). In the Bcl-2 apoptosis pathway, cytochrome C is released from the mitochondria, binds to apoptosis protease activation factor-1 (APAF-1), and activates caspase 9 [reviewed in (148)]. Caspase 9 initiates a cascade of cleavage and activation of other caspases, resulting in eventual death of the cell. The Bcl-2 family consists of several interacting proteins that are either pro- or antiapoptotic. Overexpression of one of the main antiapoptotic members, Bcl-2, in thymocytes did not hamper negative selection by superantigen (149) but did modestly reduce the level of negative selection in the HY model (150). Mice deficient in BIM, a proapoptotic BH-3-only member of the Bcl-2 family, revealed a striking inhibition in negative selection in response to anti-CD3, SEB, or peptide administration. HY male mice on a BIM-deficient background also showed a modest block in negative selection (151). Given this evidence for the involvement of BIM in negative selection, it was surprising that negative selection was normal in APAF-1-deficient thymocytes using the HY and superantigen models (152). Thus, it is unclear if BIM’s role in negative selection involves the classic caspase 9/APAF-1–dependent pathway of death. That caspase activation occurred during anti-CD3 or peptide-induced thymocyte death implied that caspase activation might be required for negative selection (153). Transgenic expression of the bacculovirus p35 caspase inhibitor (known to block BIM-mediated death in other systems) partially blocked death in response to anti-CD3 or peptide injection. However, it did not block endogenous superantigen-mediated negative selection or death of HY transgenic male thymocytes (154, 155). Thus, the basic death pathway involved in negative selection in vivo remains ill defined. Despite the lack of understanding of the ultimate death effector molecules involved in negative selection, many studies have shown the involvement of specific intracellular signals and transcription factors. An adaptor protein associated with TCR signaling, Grb2, was recently shown to be important in negative but not positive selection (87). Ironically, Grb2 was initially thought to be important for positive selection because of its assumed role in TCR-induced ras activation. Grb2 recruits the ras-activator SOS1 to the membrane, which is dependent upon TCRinduced phosphorylation of LAT [reviewed in (78)]. Thus, it was logical to assume that ERK activation, which is required for positive selection, was dependent upon Grb2/SOS1. Surprisingly, analysis of Grb2 heterozygous mutant animals showed that positive selection and ERK activation were unaffected, whereas negative selection was impaired in response to superantigen, TCR cross-linking, and in HY male mice (87). As discussed above, the ERK MAP kinase pathway is important for positive selection. Two other MAP kinase pathways, JNK and p38, have been implicated in negative selection [reviewed in (157)]. In the Grb2 heterozygous mice mentioned

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above, antibody cross-linking of the TCR resulted in normal phosphorylation of ERK2, whereas levels of phosphorylated JNK and p38 were decreased (87). TCR transgenic mice with a dominant negative JNK1 transgene showed decreased levels of apoptosis after in vivo injection of an agonist peptide (158), and JNK2-deficient thymocytes were resistant to apoptosis induced by antibody cross-linking of CD3. However, the concerns noted above about these models leave open the possibility that JNK is required for peripheral T cell activation and not for thymocyte negative selection. Nonetheless, overexpression of the main upstream activator of JNK, MKK7 (158), increased negative selection in HY male mice (159). Using a pharmacological inhibitor of p38 MAP kinase, Sugawara et al. saw diminished negative selection but no effect on positive selection in HY TCR transgenic mice (159). However, JNK1 knockout mice had normal thymopoiesis (160), and JNK2 knockout mice with a dominant negative JNK1 transgene also had apparently normal thymopoiesis (161). MKK7-deficient mice are embryonic lethal. By making chimeric embryos with MKK7−/− cells one can generate viable mice whose lymphoid cells all derive from the MKK7-deficient cells. These mice also had normal thymopoiesis (161). Another upstream activator of JNK, MKK4, produced a similar phenotype in MKK4-deficient chimeras (162). Likewise, the knockout of MKK3, an upstream activator of p38, had normal thymopoiesis (163). Apparently normal thymopoiesis in polyclonal mice does not imply the lack of an influence on negative selection, however, and analysis in TCR transgenic models is needed to further discern the roles of MKKs, JNK, and p38 MAP kinases in negative selection. It has been shown that negative selection in thymocytes requires a second signal apart from the TCR-MHC (135, 136, 164, 165). For example, CD28 can provide a costimulatory signal required for negative selection (135). CD28-deficient mice were shown to be resistant to anti-CD3 or peptide-induced apoptosis (166). However, this may reflect an impaired peripheral T cell activation process and corresponding decrease in nonspecific death of thymocytes, because CD28 deficiency did not impair endogenous antigen-specific negative selection in 2C mice or endogenous superantigen-mediated deletion (167). Nonetheless, a recent study employing perinatal antibody blockade of CD28’s binding partners, B71 and B72, demonstrated that negative selection was impaired in two models: endogenous superantigen and TCR transgenic with endogenous antigen (168). In addition, adoptive transfer of polyclonal T cells from B7-blockaded mice into syngeneic mice resulted in severe and rapid autoimmunity, suggesting that auto-reactive cells are not deleted if CD28-B7 signaling is blocked. CD40-deficient mice showed a striking block in negative selection in models in which endogenous antigen caused deletion (169). Interestingly, in models in which exogenous stimuli caused death, CD40 deficiency was not protective, suggesting that exogenous and endogenous models of negative selection involve different molecular mechanisms. Another TNF family member, LIGHT, was recently suggested to be important for T cell costimulation (170). LIGHT is expressed on thymocytes and binds to LTβR and HVEM (171). Wang et al. used various methods of blocking LIGHT in TCR Tg organ cultures and showed a pronounced block in death mediated by exogenous

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peptide and a lesser effect in HY males in which endogenous antigen mediates death (172). Normal thymopoeisis was observed in LIGHT-deficient mice, although negative selection was not directly tested (173). It seems likely that some of these molecules could serve in a redundant manner as costimulators for negative selection. Analysis of endogenous superantigen-mediated negative selection in mice with combined genetic deficiencies supports this idea (174, 175). In addition, the mechanisms that operate at the early double-positive (DP) stage versus a later single-positive (SP) stage may operate through different pathways (176). Evidence suggests that clonal deletion requires RNA and protein synthesis (177). Searching for the downstream regulators of TCR-induced apoptosis, two groups identified the orphan steroid receptor transcription factor, Nur77 (178, 179). Dominant negative and constitutively active mutants of Nur77 resulted in decreased and increased negative selection, respectively, using the HY model, in vivo TCR cross-linking, and in vivo peptide injection into a TCR transgenic mouse (180, 181), implicating Nur77 as an important transcription factor in negative selection of thymocytes. Nur77 deficiency, however, had no effect on negative selection (182), but this may be due to functional redundancy with two other Nur77 family transcription factors (183). Little is known about the upstream activation or downstream targets of Nur77. Another gene highly upregulated during negative selection is a recently identified inhibitor of NFκB, IκBNS. Clayton and colleagues showed that IκBNS when overexpressed caused increased apoptosis in thymocytes stimulated with antiCD3ε (184). Several studies indicated that NFκB activation protects thymocytes from apoptosis during the pre-TCR DN3 stage (185, 186) and lack of activation was correlated with negative selection (187). However, single deficiencies of NFκB family members had little impact on thymocyte selection (188). NFκB is mainly regulated by sequestration in the cytosol by binding to IκB [reviewed in (189)], which is ubiquitinated and destroyed after TCR stimulation, allowing NFκB family members to move into the nucleus and form hetero- and homodimers that can act as transcriptional activators. Using a super-inhibitory IκB transgene that tightly sequesters NFκB in the cytosol, Hettmann et al. showed reduced positive selection but no effect on negative selection using the HY model (190), whereas a different transgenic line using the same inhibitor showed a block in negative selection using the HY model (191). Interpreting the role of NFκB has been difficult. One reason, undoubtedly, is that the five NFκB members can form hetero- and homodimers that have different effects on transcription. The IκB super-inhibitor and single knockouts may disrupt the balance between various hetero- and homodimers, thus giving phenotypes that are complex. Another explanation could be due to NFκB modification as it moves to the nucleus. Clayton and colleagues (184) found that IκBNS binds to nuclear RelA/p65 but not cytosolic RelA/p65, indicating there may be modification of NFκB members that could alter their transcrptional activity. The study of more sophisticated NFκB mutants using multiple models of negative selection will be needed to clarify the roles of NFκB family members. Autoimmunity is often attributed to defects in peripheral tolerance [reviewed in (192)]. Using the nonobese diabetic (NOD) mouse model of organ-specific

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autoimmunity, which was thought to be caused by defects in peripheral tolerance, two groups have shown a direct link between impaired thymic negative selection and autoimmunity (193, 194). Kishimoto et al. demonstrated that thymocyte apoptosis was reduced in NOD mice after in vivo injection of TCR cross-linking antibodies or superantigen (193). Using a TCR transgenic model of induced autoimmunity on the NOD background, Lesage et al. found that autoreactive CD4+ T cells escaped clonal deletion owing to a difference in non-MHC chromosomal regions among the susceptible and resistant strains (194). Locating and identifying the specific non-MHC, non-TCR genes responsible for inheritance of NOD susceptibility will hopefully shed light on the factors required for negative selection in the thymus. One can assume that evolution would provide a robust system for eliminating autoreactive thymocytes. Synthesis of the findings from the numerous studies of negative selection supports this assumption as evidenced by the overlapping and redundant molecular mechanisms involved in negative selection.

Getting Past Clonal Deletion: Receptor Editing In some models clonal deletion was not readily apparent when TCR transgenics were crossed to animals expressing the high-affinity ligand (195, 196). In both cases thymocytes expressing endogenously rearranged T cell receptors preferentially developed. Such a finding could be explained by preferential survival of cells that had undergone secondary rearrangement prior to antigen encounter (197) or by a specific upregulation or maintenance of gene rearrangement signaled via the TCR (196). The latter is similar to the receptor editing process that occurs in developing B cells at an analogous developmental stage (15). We have proposed that the two explanations are related, that antigen-specific receptor editing could result from TCR internalization, resulting in the continuation of rearrangement at the TCRα locus (described above) (198). It is unclear why deletion predominates in some models but not others and could involve factors such as the timing and level of TCR expressed in different transgenics. Thus, a key question is whether antigen-specific receptor editing occurs in normal animals and whether some of the 30% of peripheral T cells that express two functional TCRα rearrangements could be autoreactive.

DIFFERENTIAL TCR SIGNALING IN THYMIC SELECTION A long-standing issue in thymocyte development is how signals through the same receptor can generate such opposing outcomes as death and differentiation. As discussed in Positive Selection, above, the data accumulated to date generally support the notion that self-peptides that promote positive selection are low-affinity TCR ligands, whereas those that promote negative selection are high-affinity ligands. How then, do short-lived TCR/ligand interactions generate a signal of differentiation and survival, whereas more stable interactions trigger death by apoptosis? One model gathering increased support is a differential MAP kinase activation model. As depicted in Figure 3, this model predicts that positive selection results

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from a low, sustained level of ERK activation, whereas negative selection occurs in response to a large burst of ERK activation concomitant with JNK and p38 activation. Evidence for this model comes from several studies. First, analysis of animals deficient for ERK activation yet having normal JNK and p38 activation had impaired positive but not negative selection (96, 97). Conversely, when analyzing the Grb2 heterozygous animals that have normal positive but impaired negative selection (see above), Gong et al. (87) found that activation of ERK was normal but JNK and p38 activation was inhibited. Further correlative support comes from analysis of the kinetics of ERK activation, in which negative selection peptides generate a short burst of ERK activity while positive selection peptides generate a lower but more long-lived pattern of ERK phosphorylation (199, 200). One study suggested that blocking ERK phosphorylation using a pharmacological inhibitor can switch a negative selection signal into a positive selection signal (201). A clue as to how differential activation of the MAP kinase pathways might occur comes from the discovery of a second Ras activator used by T cells. For many years it was known that the Grb2/SOS1 complex was capable of activating Ras (202), yet the finding by Gong et al. discussed above suggests that reduced levels of Grb2 do not translate into reduced levels of ERK phosphorylation after TCR stimulation. A second Ras activator, RasGRP, was recently identified in T cells and shown to be important for TCR-induced ERK activation and positive selection (95, 203). An undoubtedly oversimplified model can now be postulated (Figure 3), whereby a low-affinity TCR interaction results in Ras activation via RasGRP and generates a low, sustained level of ERK activation, resulting in differentiation. High-affinity interactions coupled with costimulation would result in Grb2/SOS1mediated ERK activation of higher magnitude and shorter duration and in JNK and p38 activation, leading to negative selection. That Grb2 is an intermediate in JNK and p38 activation in thymocytes is clear, but whether this involves a Ras-mediated mechanism is not known. Additionally, as discussed above, it is not clear if and how negative selection results from JNK or p38 activation. Such a “GRP versus GRB” model of selection might have its basis in differential phosphorylation of the adaptor protein LAT, because the tyrosine residues required for recruitment of Grb2 and PLCγ (needed for RasGRP activation) are different (78). Alternatively, costimulatory signals could result in amplification of the Grb2-dependent pathway of p38 and JNK activation (Figure 3). The task for thymocyte biologists is to understand how TCR binding kinetics can alter the molecular pathway used. Much is known about what occurs immediately upon TCR engagement with peptide/MHC in mature T cells [reviewed in (204)]. One of the primary events is the recruitment of Lck via the coreceptors CD4 or CD8 and activation of Lck via removal of an inhibitory phosphate group by CD45. Activated Lck phosphorylates the ITAMs on CD3 and TCRζ , causing recruitment of Zap70 and its subsequent activation. Initial studies with TCR antagonists in peripheral T cells showed that these peptides, which are typically low-affinity ligands, resulted in accumulation of the p21 form of phospho-ζ chain. High-affinity stimulatory ligands, on the other hand, resulted in full

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phosphorylation, generating the p23 form of ζ (205, 206). This intriguing result provided a potential model for differential recruitment of downstream activators. However, careful studies of thymocytes showed that low-affinity ligands did not result in stable qualitative differences in phosphorylation of ζ , but rather just lower levels of p21 and p23 (27, 128). This, together with studies of selective ITAM deficiencies (70), did not support the idea that differential phosphorylation of TCRζ underlies thymic selection. Alternatively, there is good genetic evidence that differential activation of MAP kinases may be controlled by CD3δ linked to the TCRα chain via its α-connecting peptide motif (α-CPM). When the TCR α-CPM motif was mutated there was a block in positive but not negative selection (69, 199), ERK activity was generated in response to negative but not positive selection ligands, and CD3δ no longer coimmunoprecipitated with the TCR. Furthermore, TCR cross-linking in CD3 δ-deficient animals resulted in decreased ERK phosphorylation but normal JNK and p38 phosphorylation (207). Thus, it is possible that positive selection ligands cause ERK activation through the TCRα-CPM and CD3δ. Positive selection is also impaired in mice lacking the ITAM motif in CD3γ (208). Peptide titration experiments revealed a bona fide positive selection defect, as opposed to a selection threshold shift, like that observed in TCRζ ITAM–deficient mice. Gil and colleagues recently reported that the kinase Nck binds to a motif in the CD3ε chain that is revealed upon TCR ligation (209). Whether this conformation change would occur upon TCR ligation of low-affinity ligands remains to be determined. However, it raises the interesting possibility that TCR binding kinetics might dictate which CD3 signaling motifs are revealed and utilized in the response, and this feature could underlie the different pathways activated during positive and negative selection.

Developmental Attenuation of the Response to Low-Affinity Self Peptides Whereas it is clear that low-affinity ligands are required to initiate positive selection at the DP stage, it has been shown that after selection developing T cells preferentially lose their sensitivity to low-affinity ligands (127, 128, 210). At the same time, the ability to respond to high-affinity agonist peptides remains the same or even increases. It is unclear what adjustments are made within the cell that selectively affect this regulation. Several possible explanations have been proposed including increased TCR levels (211), a reduction in the constitutive association of Lck and/or TCRζ with the TCR complex (128), and upregulation of the inhibitory receptors CD2 and CD5 (210). Developmentally regulated levels of surface glycosylation may also explain part of the phenomenon of developmental attenuation. It has been shown that surface levels of sialylation increase as T cells mature owing to upregulation of the sialyltransferase, St3Gal-1 (212). One effect of the increased sialylation is a decrease in the ability of CD8 to bind directly to MHC class I (213, 214), which could also affect TCR responsiveness. Indeed, desialylation of

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mature T cells restores their ability to respond to low-affinity ligand (T.K. Starr, S.C. Jameson & K.A. Hogquist, unpublished observations). An intriguing hypothesis proposed by Grossman & Paul posits that a T cell behaves similarly to a neuron undergoing adaptation to repeated signaling (215). Their tunable activation threshold hypothesis predicts that repeated subactivationthreshold TCR stimulation results in raising the actual activation threshold. This hypothesis is difficult to test in thymocytes undergoing selection, though some supporting evidence exists. Using a triple transgenic mouse engineered to express three main peptides with low levels of endogenous peptides, Rudensky and colleagues (210) showed that thymocytes in a nonselecting environment would still respond to subactivation-threshold peptide/MHC ligands by upregulating CD2 and CD5, effectively raising the activation threshold required for TCR stimulation. However, it is difficult to explain why the TCR changes its sensitivity to low-affinity but not high-affinity stimulation using the tunable activation threshold hypothesis without also postulating that costimulatory “booster” factors are upregulated during development (215). As in most biological phenomena, developmental attenuation is probably a result of several interrelated molecular mechanisms, and the proposed mechanisms above are not mutually exclusive. Teleologically, it would seem that developmental attenuation could be important in broadening the “safety net” that prevents mature T cell autoreactivity.

AGONIST SELECTION OF REGULATORY CELLS Whereas a differential signaling model of thymic selection is thought to apply to the majority of α/β T cells, there are populations whose development does not fit these rules. These include NKT cells, CD8αα+ intraepithelial T cells of the gut, and CD25+ CD4+ T cells. For these cells, not only does a high affinity or agonistic interaction with self-antigen not result in clonal deletion but it is required for differentiation. NKT cells are α/β T cells that express the NK1.1 antigen and are thought to regulate conventional T cell responses through the secretion of cytokines (216). A predominant population expresses a unique Vα14 TCR and is selected on the nonclassical class I molecule CD1 (217). Using specific CD1d/glycolipid tetramers, Benlagha and colleagues recently identified a thymic precursor in normal mice and showed that it is expanded and acquires activation markers and regulatory properties over time, suggesting an antigen-driven fate-specification process that occurs in the thymus (218). Although the natural ligand for these NKT cells has not been identified, it is assumed to be an agonistic or stimulatory lipid ligand because of the activated phenotype of NKT cells and because a Vα14 TCRα chain transgenic showed a thymic deletion phenotype (reduced cellularity) (219). Not all NKT cells express a Vα14 TCR. Interestingly, conventional Class I– and Class II–restricted TCR transgenics give rise to CD4 or double-negative (DN) NKT cells

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when agonist ligand is present (220, 221). These results suggest that NKT cells are a subset of α/β T cells selected from the normal repertoire via an agonistic interaction with self-antigen. CD8αα+ T cells are another specialized subset of α/β T cells thought to have regulatory function (222). They are numerous in the gut, where they represent over 50% of intraepithelial T cells. They can express either classical or nonclassical Class I–restricted T cell receptors. Like NKT cells, this population has an activated phenotype (223). Indeed the finding that CD8αα homodimers can be upregulated on activated T cells suggests that they, like NK1.1 and CD25, are activation markers and not lineage markers. CD8αα+ IEL are expanded in Class I– or Class II– restricted TCR transgenics when agonist ligand is present in the thymus (224–226), again suggesting an antigen-driven fate specification. CD25+CD4+ cells have been extensively studied in the past few years (227). They exhibit an activated phenotype and inhibit T cell proliferation in vitro and prevent the development of gastritis, colitis, and diabetes in vivo. It was predicted some time ago that agonist interactions with self-antigens in the thymus would promote the development of regulatory cells, which could then contribute in a dominant fashion to self-tolerance in the animal (228). This was shown directly in the Class II–restricted 6.5 TCR transgenic model, where the presence of viral HA antigen caused variable levels of deletion but a striking increase in CD25+CD4 T cells (229–231). Development occurred in the thymus, was directed by radioresistant antigen-presenting cells (APCs), and required high-affinity interaction with self-antigen (230). Interestingly, Apostolou and colleagues showed, in a similar system, that hematopoietic cells could direct the development of regulatory CD4 T cells but that they did not express CD25 (231). These three populations—NKT cells, CD8αα+ T cells, and CD25+CD4+ T cells—have several features in common: (a) They all display an activated or partially activated phenotype, (b) agonistic interactions with self-antigens are required for development, and (c) they display regulatory function. Although these commonalties are intriguing, it is not clear to what extent they represent similar differentiation processes. For example, the surface phenotype of CD8αα+ (223) and other nonclassical Class I–restricted cells (232) is distinct from that of NKT cells (218) and regulatory CD4 T cells. However, it is not clear if such populations are truly unique lineages or if they represent particular activation states of cells responding to particular MHC/self-antigens. For example, in mice lacking conventional Class I molecules (232), the majority of peripheral CD8 T cells, which presumably were selected by interaction with nonclassical Class I molecules, express an activated phenotype. However, several transgenic strains expressing foreign antigen-specific, nonclassical Class I–restricted TCRs did not show an activated phenotype (42, 43, 233). Likewise, in neonates CD1 tetramer-binding NKT cells did not show an activated phenotype, but this changed during the first few weeks of life. Finally, naive antigen-specific CD4 T cells acquired a stable CD25+ phenotype and regulatory function after adoptive transfer into antigen-bearing hosts (234). Therefore, it is possible that the surface phenotype identifies a subpopulation

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of α/β T cells that have previously encountered antigen and does not represent a lineage marker, per se. Differences in surface phenotype may simply reflect where and when the cells encountered agonist ligand and could predominate among small populations by clonal expansion (218). From the above-cited studies it is quite clear that encounter with self-antigen and acquisition of an activated phenotype and regulatory function can occur during development in the thymus. Therefore an important question is why such cells are not deleted from the repertoire. Two explanations have been put forward. First, differences in avidity could underlie whether agonist selection or deletion will prevail. In 6.5 TCR transgenic mice expressing a higher amount of antigen, deletion was more pronounced than the generation of CD25+CD4+ cells (229). Also, Gavin and colleagues found a correlation between peptide/MHC complex density and development of CD25+CD4+ T cells in mice expressing single peptide/MHC Class II complexes (235). It should be stressed that both studies were correlative and did not directly demonstrate that avidity was the determining factor. A second possibility is that APC type may determine which mechanism prevails. Laufer and colleagues showed that cortical epithelial expression of MHC Class II could support the differentiation of CD25+ regulatory T cells (236). Interestingly, in an in vitro culture system, antigen-specific DP thymocytes exposed to antigenbearing epithelial cells did not die but differentiated into DN cells with regulatory function (237). The elegant work of Kyewski and colleagues has identified a population of medullary epithelial cells that express otherwise tissue-specific genes in a promiscuous fashion, resulting in T cell tolerance to those proteins (238, 239). It is intriguing to think that this population could be specialized for the generation of antigen-specific regulatory T cells. However, other regulatory populations such as NKT cells required antigen expression on hematopoietic cells for optimal differentiation (217). Apostolou and colleagues further showed that hematopoietic expression resulted in generation of CD25−CD4+ regulatory cell differentiation, whereas epithelial expression promoted CD25+CD4+ regulatory cells (231). Thus, it is not clear if APC type dictates the death versus differentiation decision. One possibility is that both deletion and differentiation occur in response to self-antigens, but with stochastic determination. It is interesting to note that in several TCR transgenics in which efficient development of regulatory cells occurred, the thymus also showed vastly reduced numbers of mature cells—historically considered the hallmark of “deletion” (219, 221, 231, 240). Alternatively, stimulation of immature thymocytes with high-affinity ligands could trigger the upregulation of molecules that alter the threshold of T cell signaling (215). Only the fraction of cells that upregulated such molecules in time to prevent clonal elimination would continue development. If this were true, one might predict both the avidity and costimulatory contexts to alter the outcome. Surprisingly, the antigendependent development of regulatory cells required the α−CPM motif of the TCR (221, 240) like conventional α/β T cells did (69), even though the deletion observed in the same model occurred independently of this motif. The α−CPM motif has been described as important in distinguishing high- and low-affinity ligands (199).

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However, these results point more toward a role for α−CPM in distinguishing differentiative from deletional signals, independent of affinity. This is an interesting result, but until we know more about what unique signals are contributed by the α−CPM motif, it is difficult to interpret this finding.


CONCLUDING REMARKS The recent past has seen steady progress on the topic of thymic selection. Studies of peptide specificity generally concur that the ligands for positive selection are low-affinity self-peptide ligands, often structurally related to foreign antigenicpeptide ligands. Such ligands have been shown to induce TCR signaling and ERK activation, consistent with the genetic studies of the positive selection process. TCR (αCPM) and CD3 (γ ITAM) mutants will be useful tools in dissecting how TCR ligation of low-affinity ligands triggers a unique differentiative signal in immature thymocytes. Despite outstanding questions about how TCR ligation triggers a response, genetic studies have developed to a point at which we can link the TCR directly to transcription factors in the nucleus (TCR → Lck → ZAP-70 → LAT → PLCγ → DAG → RasGRP → Ras → MEK → ERK → Egr-1 → Id3). The negative selection response has been more difficult to understand and may involve different molecular processes at different stages in development. Future refinement of clonal deletion models, or at least consensus within the field as to the validity of the current models, is needed to clarify and further define the molecular mechanisms of negative selection. Finally, recent data provide a new perspective on central tolerance, where it appears that both dominant (T regulatory cell generation) and recessive (clonal deletion) mechanisms operate in the thymus. Understanding the molecular mediators that distinguish agonist selection from clonal deletion and finding ways to generate T regulatory cells for therapeutic purposes will be important areas of investigation in the future. ACKNOWLEDGMENTS We thank Charlly Kao for insightful discussion and critical reading of the manuscript. The Annual Review of Immunology is online at http://immunol.annualreviews.org

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210. Wong P, Barton GM, Forbush KA, Rudensky AY. 2001. Dynamic tuning of T cell reactivity by self-peptide-major histocompatibility complex ligands. J. Exp. Med. 193:1179–87 211. Dautigny N, Lucas B. 2000. Developmental regulation of TCR efficiency. Eur. J. Immunol. 30:2472–78 212. Priatel JJ, Chui D, Hiraoka N, Simmons CJ, Richardson KB, et al. 2000. The ST3Gal-I sialyltransferase controls CD8+ T lymphocyte homeostasis by modulating O-glycan biosynthesis. Immunity 12:273–83 213. Moody AM, Chui D, Reche PA, Priatel JJ, Marth JD, Reinherz EL. 2001. Developmentally regulated glycosylation of the CD8alphabeta coreceptor stalk modulates ligand binding. Cell 107:501–12 214. Daniels MA, Devine L, Miller JD, Moser JM, Lukacher AE, et al. 2001. CD8 binding to MHC class I molecules is influenced by T cell maturation and glycosylation. Immunity 15:1051–61 215. Grossman Z, Paul WE. 2001. Autoreactivity, dynamic tuning and selectivity. Curr. Opin. Immunol. 13:687–98 216. Bendelac A, Bonneville M, Kearney JF. 2001. Autoreactivity by design: innate B and T lymphocytes. Nat. Rev. Immunol. 1:177–86 217. Bendelac A, Rivera MN, Park SH, Roark JH. 1997. Mouse CD1-specific NK1 T cells: development, specificity, and function. Annu. Rev. Immunol. 15:535–62 218. Benlagha K, Kyin T, Beavis A, Teyton L, Bendelac A. 2002. A thymic precursor to the NK T cell lineage. Science 296:553– 55 219. Bendelac A, Hunziker RD, Lantz O. 1996. Increased interleukin 4 and immunoglobulin E production in transgenic mice overexpressing NK1 T cells. J. Exp. Med. 184:1285–93 220. Curnow SJ, Boyer C, Buferne M, SchmittVerhulst AM. 1995. TCR-associated zetaFc epsilon RI gamma heterodimers on CD4-CD8-NK1.1+ T cells selected by

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240. Leishman AJ, Gapin L, Capone M, Palmer E, MacDonald HR, et al. 2002. Precursors of functional MHC class I- or class II-restricted CD8alphaalpha(+) T cells are positively selected in the thymus by agonist self-peptides. Immunity 16:355– 64

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Annu. Rev. Immunol. 2003. 21:177–204 doi: 10.1146/annurev.immunol.21.120601.141011 c 2003 by Annual Reviews. All rights reserved Copyright ° First published online as a Review in Advance on January 28, 2003

IGA FC RECEPTORS Renato C. Monteiro1 and Jan G. J. van de Winkel2,3 Annu. Rev. Immunol. 2003.21:177-204. Downloaded from arjournals.annualreviews.org by HINARI on 09/01/07. For personal use only.

1

INSERM E0225, Bichat Medical School, 16 rue Henri Huchard, Paris 75870, France, and 2Immunotherapy Laboratory, Department of Immunology, University Medical Center Utrecht, and 3Genmab, Jenalaan 18a, 3584 CK Utrecht, The Netherlands; email: [email protected]; [email protected]

Key Words IgA, FcR, mucosal defense, inflammation, glomerulonephritis ■ Abstract The IgA receptor family comprises a number of surface receptors including the polymeric Ig receptor involved in epithelial transport of IgA/IgM, the myeloid specific IgA Fc receptor (FcαRI or CD89), the Fcα/µR, and at least two alternative IgA receptors. These are the asialoglycoprotein receptor and the transferrin receptor, which have been implicated in IgA catabolism, and tissue IgA deposition. In this review we focus on the biology of FcαRI (CD89). FcαRI is expressed on neutrophils, eosinophils, monocytes/macrophages, dendritic cells, and Kupffer cells. This receptor represents a heterogeneously glycosylated transmembrane protein that binds both IgA subclasses with low affinity. A single gene encoding FcαRI has been isolated, which is located within the leukocyte receptor cluster on chromosome 19. The FcαRI α chain lacks canonical signal transduction domains but can associate with the FcR γ -chain that bears an activation motif (ITAM) in the cytoplasmic domain, allowing activatory functions. FcαRI expressed alone mediates endocytosis and recyling of IgA. No FcαRI homologue has been defined in the mouse, and progress in defining the in vivo role of FcαRI has been made using human FcαRI transgenic (Tg) mice. FcαRI-Tg mice demonstrated FcαRI expression on Kupffer cells and so defined a key role for the receptor in mucosal defense. The receptor functions as a second line of antibacterial defense involving serum IgA rather than secretory IgA. Studies in FcαRI-Tg mice, furthermore, defined an essential role for soluble FcαRI in the development of IgA nephropathy by formation of circulating IgA-FcαRI complexes. Finally, recent work points out a role for human IgA in treatment of infectious and neoplastic diseases.

INTRODUCTION Fc receptors (FcR) belong to the immunoreceptor family, including T cell receptors, B cell receptors, and NK receptors, and their function is to recognize antigens. FcR are present on many cells and provide an essential link between humoral and cellular branches of the immune system. The interaction between antibodies and FcR provides antigen-(Ag) specific recognition to cells that express a given FcR. This interaction can initiate a variety of responses, varying from endocytosis, phagocytosis, transcytosis, exocytosis, superoxide generation, antibody-dependent cell 0732-0582/03/0407-0177$14.00

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cytotoxicity (ADCC), and release of cytokine inflammatory mediators to modulation of cell survival. FcR have been recognized for all five human antibody classes. Receptors for immunoglobulin A (IgA) were described more than 25 years ago by Lawrence et al. who showed binding of an IgA1 myeloma protein and secretory IgA to blood neutrophils (1). Others, using red cell rosettes, identified IgA receptors on a subpopulation of lymphocytes (30%) (2–4), on blood monocytes, and on neutrophils (5). In this article, we address five types of receptors for IgA and focus on the biology of the myeloid cell FcαRI (or CD89).

Biosynthesis and Structure of IgA IgA-bearing B cells appear first during the eleventh week after birth, contrasting with those bearing IgG and IgM that are earlier in development (6). While both IgM and IgG plasma cells can usually be found by the fifteenth week of gestation, IgA-producing cells have not been observed before the thirty-second week (7). Serum IgA is usually undetectable at birth, and adult serum levels are not attained until around the time of puberty. In adults, the majority of human plasma cells are committed to produce IgA, and IgA is thus by far the most abundant immunoglobulin (Ig) (9). More IgA is produced per day (66 mg/kg/d) than all other classes combined (10). IgA is also the most heterogeneous among the Ig, and IgA displays a T-shaped structure, which differs from the common Y-shape of other Ig (11). IgA is divided into closely related subclasses, IgA1 and IgA2, that basically differ by the absence of a 13-amino acid sequence in the hinge region of the IgA2 molecule (12). This difference explains resistance of IgA2 against the action of bacterial proteases (i.e., from Streptococcus mutans, Neisseria meningitidis, and Haemophilus influenzae) (13) and may underly the predominance of IgA2 in mucosal secretions. In serum, IgA constitutes one fifth of the total Ig pool due to a fast catabolism (half-life: 3– 6 days), where it exists mainly in monomeric form and of the IgA1 subclass, with a minor percentage of polymeric IgA (pIgA). Serum IgA is generated by B lymphocytes in the bone marrow and in some peripheral lymphoid organs (14, 15). In mucosal secretions (saliva, tears, colostrum, gastrointestinal fluids, nasal bronchial secretion, and urine), however, local plasma cells produce IgA as pIgA. This pIgA exists almost exclusively as dimers, joined by a polypeptide termed J-chain, and is linked to the secretory component (secretory IgA, SIgA). Recently, it has been proposed that secretory IgA comes from two sources, the B1 and B2 lymphocytes (16). The first one contributes about 25% of secretory IgA and is produced by B1 lymphocytes that develop in the peritoneal cavity. IgA derived from B1 lymphocytes has been proposed to represent a primitive system, a T lymphocyte–independent source of IgA, recognizing commensal bacteria. The second source, the B2 lymphocytes, represents the majority (75%) of lymphocytes in organized germinal centers of mucosal-associated lymphoid tissues (MALT) such as Peyer’s patches. This IgA against exotoxins is T lymphocyte dependent. It should be noted, however, that animals deficient in

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lymphotoxin α and lymphotoxin β receptors are devoid of both mesenteric lymph nodes and Peyer’s patches, but can still produce IgA (17). Mucosal IgA is produced by plasma cells and transported from the baso-lateral epithelial compartment to the apical/luminal side. Dimeric IgA, containing the J-chain, is secreted in the lamina propria. It binds to and forms covalent complexes with the membrane-associated polymeric Ig receptor (pIgR) on the baso-lateral side of mucosal epithelial cells (18). The complex is actively transported through the epithelial cell to the apical/luminal side, where bound IgA is released by proteolytic cleavage from the pIgR, generating the so-called secretory component, which remains associated with dimeric IgA, forming altogether SIgA (19). Interestingly, the IgA system differs substantially between three species studied in detail, human, mouse, and rabbit. Two IgA subclasses are recognized in humans, one class in mice, and 13 subclasses in rabbits (9, 20). Serum IgA is mostly monomeric in humans and polymeric in mice. Clearance via the hepatobiliary route plays an important role in mice but not in humans (9).

Functions of IgA The mucosal surface encompasses more than 400 m2 that is permanently in contact with multiple bacterial strains and other microorganisms. More than 70% of immune cells are mobilized daily to resist systemic infections, including antibodysecreting cells. SIgA plays a major role in the innate immune system preventing microorganisms and foreign proteins from penetrating the mucosal surfaces (21). It also neutralizes toxins and infectious organisms. SIgA antibodies have been proposed to act at three levels in the mucosal compartment. The first level is at the luminal side via a mechanism called immune exclusion (22, 23). SIgA can inhibit adherence of microorganisms by surrounding pathogens with a hydrophilic shell that is repelled by the mucin glycocalix at mucosal surfaces (9, 24). In addition to this exclusion mechanism, two additional activities have been defined; one is the transport of IgA complexed with antigens that cross the epithelial cell barrier to the luminal side; the second is intracellular interception of viral antigens during transepithelial IgA transport (25–29). The third mechanism of protection by SIgA has been documented to be active at the stromal side. IgA/Ag complexes can be eliminated via the pIgR at the baso-lateral side of epithelial cells by transcytosis (30) or by FcαR-bearing phagocytes (31, 32). The inability of SIgA to fix complement efficiently or to act as an opsonin is an advantage in secretions, where initiation of an inflammatory reaction would likely affect the most important component of local defense, the integrity of the mucosal surface (9). Whereas the role of secretory IgA is established in mucosal immunology, the function of serum IgA antibodies is mostly unknown. Studies on the ability of IgA antibodies to regulate humoral response are scarce. IgA was shown only in one report to enhance the induction of immunological memory to soluble Ag (34). IgG antibodies represent the most prominent component of secondary systemic immune responses to Ag, whereas IgA is rarely observed. The specificity of serum IgA in the human

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antibody repertoire and IgA antigen selection remain poorly defined. Serum IgA is considered a “discrete housekeeper” because IgA immune complexes can be removed by the phagocytic system with little or no resulting inflammation. Moreover, monomeric serum IgA displays anti-inflammatory activity and is capable of inhibiting functions such as IgG-induced phagocytosis, bactericidal activity, oxidative burst, and cytokine release (9). Another argument in favor of anti-inflammatory properties of serum IgA is provided by selective IgA-deficient patients, the most common Ig deficiency (35). IgA deficiency has frequently been associated with allergy and autoimmunity. The molecular basis for this is not understood. Polymeric IgA and IgA-containing immune complexes (IC), in contrast, can efficiently trigger immune effector functions on blood leukocytes through IgA Fc receptors. In this context, interaction of serum IgA with FcαRI on tissue phagocytic cells can act as a second line of defense in the case of bacterial infections following penetration through the mucosal barrier (12).

TYPES OF IgA RECEPTORS Fc receptors are defined by their specificity for the Fc fragment of immunoglobulin isotypes, and receptors for IgA are referred to as FcαR (36). Although they are not structurally related, five types of IgA receptors are now recognized (Figure 1). Three of them are considered bona fide FcαR. The first one, the polymeric Ig receptor, is involved in transport of IgM and polymeric IgA across epithelial barriers [reviewed in (18, 37)]. The second type is designated FcαRI (or CD89) and is a receptor specific for IgA, capable of binding both human IgA1 and IgA2 subclasses (38, 39). The third receptor type is the recently described Fcα/µR (40). The two alternative IgA receptors are the asialoglycoprotein receptor and the transferrin receptor (41, 42).

FcαRI Expression, Modulation, and Tissue Distribution Expression of FcαRI/CD89 begins at least as early as the promyelocyte stage in differentiation (43, 44). FcαRI expression is restricted to cells of the myeloid lineage including neutrophils, eosinophils, most of monocytes/macrophages, interstitial dendritic cells, Kuppfer cells, and cell lines corresponding to these cell types (31, 33, 43, 45, 46). Tonsilar, splenic, and alveolar macrophages do all express FcαRI (43, 47, 48), in contrast to intestinal and genitourinary mucosal macrophages (44, 49). FcαRI is neither expressed on cord blood–derived mast cells, erythrocytes, platelets, nor lymphoid cells, even after polyclonal or mitogenic stimulation (B. Pasquier, M. Arock & R. Monteiro, unpublished data). FcαRI expression is constitutive and independent of the presence of IgA ligand because the receptor is expressed at similar levels on cells from patients deficient in IgA (50). Several anti-FcαRI (CD89) mouse and human monoclonal antibodies (mAb) have been generated (43, 51). Most of them recognize nonpolymorphic

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TABLE 1 Location of IgA- and CD89 mAb binding sites within the FcαRI extracellular domains EC1

EC2

IgA-binding site

+

−

anti-FcαRI mAb

My43, 2E6, 2D11, 7G4, 2H8

A59, A62, A77, 7D7

determinants on FcαRI (43). The CD89 mAb epitopes on FcαRI have been characterized (52). Monoclonal Ab that bind in the EC1 domain of FcαRI (e.g., My43) can block IgA binding, whereas those that bind in EC2 do not (Table 1). A3 mAb may recognize a binding site between both FcαRI extracellular domains. The level of FcαRI expression on cells is estimated to be 57,000 per monocyte and 66,000 per neutrophil (44). A number of cytokines and other agents modulate FcαRI expression, as summarized in Table 2. FcαRI expression levels are upregulated on neutrophils in response to formyl-methionyl-leucyl-phenylalanine (FMLP), interleukin 8, and tumor necrosis factor α (TNF-α) (53–55). Receptor upregulation on neutrophils is rapid and results mainly from recruitment from intracellular pools (53). FcαRI upregulation has been defined to occur via a Ca2+dependent signaling pathway on neutrophils and eosinophils (45, 53); ionomycin upregulates FcαRI expression on eosinophils but not on U937 cells. Expression of FcαRI on monocytes and monocyte-like cell lines can be upregulated by phorbol esters, calcitriol, lipopolysaccharide (LPS), TNF-α, granulocyte-macrophage colony stimulating factor (GM-CSF), and IL-1β (38, 54–57). FcαRI is downregulated by transforming growth factor (TGF-β), interferon γ , suramin, and by its ligand (57–61). Indeed, in contrast to other FcR (such as FcεRI), FcαRI expression is downregulated by polymeric IgA (61).

Genetics Expression cloning of a cDNA encoding FcαRI (CD89) was performed using a library made from U937 cells and the anti-FcαRI mAb My43 (39). This clone was 1.6 kb long including an 861-bp open reading frame and a 711-bp 30 -UTR ending in a poly-A stretch. The latter includes an Alu sequence but lacks a classical

TABLE 2 Modulation of FcαRI expression Cell type

Increased expression

Decreased expression

Monocytes/macrophages

Calcitriol, PMA, TNF-α, IL-1β, GM-CSF, LPS

TGF-β, IFN-γ , suramin, pIgA

Neutrophils

IL-8, TNF-α, GM-CSF, FMLP, ZAS, ionomycin

Eosinophils

Ionomycin

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polyadenylation signal (39). The FcαRI gene consists of 5 exons spanning approximately 12 kb (63). The first exon (S1) includes the 50 -UTR, an ATG translation initiation codon, and part of the leader peptide. The exon 2 (S2) is a mini-exon only 36 bp long, which codes for the leader peptide, including the predicted signal peptidase cleavage site. Exons EC1 and EC2 each encode a single extracellular Ig-like domain of 206 amino acids. The last exon, called TM/C, encodes the transmembrane domain and the cytoplasmic tail of 19 and 41 amino acids, respectively. This FcαRI single gene is located in the distal part of the q-arm on chromosome 19, at 19q13.4 (64) (Figure 2A). The molecular structure of FcαRI classified this FcR as a member of the Ig gene superfamily (39). It is distantly related to other FcR genes (∼20% homology), such as the Fcγ R and FcεRI genes, that are all located on chromosome 1 (65). Interestingly, FcαRI is more homologous (∼35%) to another family of receptors, the so-called leukocyte receptor cluster, that includes the killerinhibitory/activatory (KIR/KAR)-related immunoreceptors, the Ig-like transcripts (ILTs), the leukocyte and monocyte/macrophage Ig-like receptors (LIRs, MIRs) (66–75). FcαRI is also closely related to the bovine Fcγ 2R and human and mouse platelet-specific collagen receptor (GPVI) (77). FcαRI and Fcγ 2R in fact constitute a separate group of FcR evolving from a common ancestral gene. These genes seem to have diverged from each other before the divergence of humans and cattle (76). It is noteworthy that no murine homologue for FcαRI has been identified, in spite of intensive efforts to find one. Hybridization of murine cDNA libraries with a human FcαRI cDNA did result in description of two new receptors called paired Ig-like receptors, PIR-A and PIR-B (78, 79). PIR ligands are so far unknown. Moreover, other uncharacterized IgA-binding molecules have been described on rat macrophages and rabbit lymphocytes (80, 81). A 929 bp fragment of the FcαRI promoter region has been characterized (82). Sequences between 59 and 197 bp downstream of the major transcription start site were shown to be essential for promoter activity. This sequence contains multiple consensus binding sites for transcription factors that function in myelo¨ıd gene expression, including three CCAAT enhancer-binding protein binding sites, an NFκB binding site, an Spl site, an Ets family protein binding site, and a Myb-binding site. Two polymorphisms have been identified (C-T transitions) at positions 114 bp upstream and 56 bp downstream of the transcription start site. FcαRI promoter region carrying both –114T and +56T alleles exhibits a lower promoter activity than promoters harboring the C alleles at both sites (82).

Transcripts, Protein Structure, and Ligand Binding Several alternatively spliced FcαRI transcripts have been identified by using RTPCR (48, 83–85). Figure 2B summarizes the different FcαRI transcripts. Fulllength transcripts are denominated FcαRI a.1, whereas spliced variants are defined as a.2, a.3, . . .. Two of these transcripts, a.2 and a.3, specified proteins in in vitro translation experiments (48). In vivo, FcαRI exists as at least two isoforms (a.1 and a.2) differing by a deletion in the extracellular domain (48). Whereas the a.1

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Figure 2 A. Location of the FcαRI (CD89) gene within the leukocyte receptor complex (LRC) on chromosome 19. B. Phylogenic analysis of LCR. C. Schematic representation of FcαRI (CD89) gene intron-exon organization and FcαRI transcripts. ILT, Ig-like transcripts; LIR, MIR, the leukocyte and monocyte/macrophage Ig-like receptors; PIR, paired Ig-like receptors; KIR, killer inhibitory receptors; NCR1, natural cytotoxicity receptors; CHIR, chicken Ig-like receptors.


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isoform is expressed on blood monocytes, neutrophils, eosinophils, cultured blood macrophages, and peritoneal macrophages, the a.2 isoform is found exclusively on alveolar macrophages (48). Another FcαRI isoform, named FcαRIb, is the product of an alternate splicing that skips the 30 splice site at the end of the EC2 exon, resulting in an extension of 23 new amino acids before reaching the stop codon, and thus potentially generating a “TM/C-less protein” (86). An FcαRIb protein product was shown to represent a soluble protein in transfectants and was unable to associate with the FcR γ -chain. Whether this isoform is related to previously described FcαRI soluble proteins is unclear. Native proteins corresponding to any of the other splice variants have not been identified. Whether this is due to “sterile transcripts” or to proteins not detected by CD89 antibodies is unknown. FcαRI a.1 represents a type I, 287-amino acid protein containing a 21 amino acid hydrophobic leader that is removed during processing to form the mature 266 amino acid FcαRI a.1 full-length glycoprotein (39). FcαRI is composed of two extracellular Ig-like domains, a predicted transmembrane region and a cytoplasmic tail devoid of recognized signaling motifs (Figure 3A). The protein core has a predicted Mr of 30 kDa and bears five potential N-linked glycosylation sites and several putative O-glycosylation sites. Mature cell surface FcαRI display heterogeneous glycosylation with Mr ranging from 50 to 100 kDa, depending on the cell type (38, 43). Deglycosylation experiments using endoglycosydase F or O, indeed, confirmed a heterogeneous FcαRI glycosylation with two molecular species, one of 32 and a second of 34 kDa, possibly attributable to inaccessibility of some carbohydrates (38, 43, 45). FcαRI a.2 has a deletion in the extracellular domain of 22 amino acids and a backbone of 28 kDa (48). Another indication that FcαRI a.1 exists in different glycosylated forms was obtained using the CD89 mAb A62 (43). This mAb recognizes a subpopulation of FcαRI proteins with lower Mr (55–65 kDa) than the whole FcαRI population (55–75 kDa) recognized by other CD89 mAb (A3, A59, A77, My43) on monocytes and neutrophils. The FcαRI binding site has been located in the membrane-distal EC1 domain (52, 65). This was demonstrated in experiments where point-mutations within EC1 greatly reduce IgA binding. A number of residues are potentially involved in IgA binding, located in the C strand (Y35), the C0 -E region (R52), and the F-G loop (Y81, R82, I83, G84, H85, and Y86) (Figure 3B). This model predicts the F-G loop to be located at the bottom of EC1, apparently in a position close to the cell membrane. This represents a unique feature among the two-domain type FcR because Fcγ R and FcεRI both bind their respective Ig ligands via the membraneproximal EC2 domains (88–90). It is noteworthy that the closely related bovine Fcγ 2R and p58 KIR molecules also bind ligand (bovine IgG2, and HLA molecules, respectively) via their EC1 domain (52, 91). The high degree of similarity between FcαRI and p58 KIR proteins allowed a three-dimensional model of CD89 to be proposed based on the solved structure of KIR. FcαRI is a low-affinity receptor for IgA (Ka approximately 106 M−1). Rapid dissociation of the FcαRI:IgA complex (t1/2 ∼25 s) using recombinant soluble FcαRI

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suggests that monomeric IgA bind only transiently to cellular FcαRI, whereas IgA immune complexes bind avidly (65). This confirms previous monocyte data showing polymeric IgA and IgA-IC to bind more efficiently to FcαRI than monomeric IgA (50). FcαRI binds IgA1 and IgA2 molecules at the boundary between the Cα2 and Cα3 domains (Figure 3B) (92, 93). FcαRI glycosylation may also play a role because desialylated receptors bind five times more IgA (38). Another study documented full IgA glycosylation not to influence FcαRI binding. Indeed, absence of N-glycans in Cα2 constructs does not impede binding to human neutrophil FcαRI (94). The observation that SIgA binds transiently but specifically to FcαRI (95) suggests that the binding site on IgA is not obstructed by a bound secretory component. Recent work defined a crucial role for complement receptor 3 (Mac-1, CD11b/CD18) in FcαRI’s capacity to bind SIgA, but not serum IgA (95a). Cytokines can influence IgA binding to FcαRI. Both IL-4 and IL-5 increase IgA ligand binding to FcαRI, without effects on receptor expression, suggesting that cytokine stimulation regulates FcαRI avidity (96). The increase in FcαRI’s avidity for IgA induced by cytokines seems mediated by a cytokine-induced inside-out signaling mechanism. This involves PI 3 kinase and phosphorylation of a serine residue (S263) in FcαRI’s cytoplasmic tail (97, 98). Whether or not mouse IgA binds to human FcαRI is controversial. Initial studies used erythrocytes coated with mouse IgA myeloma MOPC-315 to detect human IgA receptors (4). Later studies by others failed to observe binding of mouse IgA to human FcαRI (93). However, recent evidence for binding of dimeric, but not monomeric, mouse IgA to human FcαRI comes from experiments using macrophages from human CD89 transgenic (Tg) mice (99). Macrophages from human CD89-Tg SCID mice allow detection of mouse IgA binding, which is inhibited by the CD89 mAb My43 (99). These Tg mice, furthermore, form IgA complexes with soluble human FcαRI, culminating in the development of IgA nephropathy in six-month-old mice, supporting interaction between mouse IgA and human FcαRI. Two types of soluble FcαRI have been described. The first type is generated by proteolysis via an FcR γ -chain–dependent pathway (100). This soluble form, a slightly glycosylated 30-kDa protein with a 25-kDa backbone, was shown to be covalently associated with polymeric IgA, which circulates in serum of normal individuals (101). The molecular nature and function of this 30-kDa FcαRI remains unclear. A second soluble FcαRI type was described in serum from patients with IgA nephropathy (IgAN) (99). Studies with metabolically labeled cells from IgAN patients revealed a glycosylated soluble FcαRI form of 50–70 kDa with a 24-kDa protein core (99). Production of this latter soluble FcαRI is induced by polymeric IgA from FcαRI transfected cells. IgA-induced shedding was indicated by the loss of reactivity with an antibody raised against FcαRI cytoplasmic tail. These data indicate that cleavage of the FcαRI extracellular domain may occur, resulting in release of IgA/FcαRI complexes into circulation.

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Signal Transduction FcαRI is a member of the multichain immune recognition receptor (MIRR) family. Signaling is dependent on association of FcαRI with the FcR γ−chain subunit, forming the trimer FcαRIα/γ γ (102). The FcR γ -chain was initially described as a component of FcεRI and Fcγ RI, expressed on mast cells and monocytes, respectively (36). The FcR γ−chain contains a so-called ITAM (immunoreceptor tyrosine-based activation motif ) signaling motif (103). The FcαRIα-FcRγ interaction is strong and depends on oppositely charged residues in their transmembrane regions (104, 105). However, FcαRI can be expressed either associated, or non-associated with FcRγ (so called γ -less receptor) on monocytes or neutrophils (Figure 4). Although the basis for this partial association of FcαRI to FcRγ remains unknown, it is possible that due to the positively charged Arginine at position 209 FcαRI may associate with another—as yet uncharacterized— molecule. Notably, colostral neutrophils express only γ -less FcαRI, despite large amounts of intracellular FcR γ -chain (95). While γ -less FcαRI represent the majority of cell surface receptors, the level of FcαRI-γ 2 is upregulated by phorbol esters and interferon-γ on monocytes (105). Importantly, human FcαRI cannot be expressed in vivo in mice deficient in the FcR γ−chain, contrasting with in vitro data using transfectants (106) (M. Arcos-Fajardo and R. Monteiro, unpublished). This discrepancy may be attributable to species-specific differences in FcαRI assembly. Cross-linking of IgA bound to FcαRI triggers the receptor’s redistribution into glycosphingolipid- and cholesterol-rich domains or “rafts” in the cell membrane that serve as signaling platforms important for recruitment of signaling effectors (107, 108). FcRγ -ITAM’s are initially phosphorylated by the Src kinase lyn, which leads to recruitment of a number of tyrosine kinases including Syk, Blk, Btk, PI-3 kinase, and PLC-γ 2 (Figure 4). Recruitment and phosphorylation of Syk and Btk are modulated by cell stimulation with interferon-γ and/or phorbol ester, indicating that activation of these tyrosine kinases through FcαRI may depend on the level of cell priming at inflammatory sites (110). FcαRI cross-linking triggers calcium release from intracellular stores in neutrophils (112) and induction of NADPH oxidase activity that is sensitive to inhibition by PI 3-kinase inhibitors (113). FcαRI can also associate with Grb2, Shc, SHIP (SH2-containing inositol phosphatase-1), and SLP-76 (SH2-containing leukocyte protein of 76 kDa), suggesting the formation of adaptor complexes to regulate signalling (111). Recently, it has been shown that IgA can also activate the ERK1/2 MAP kinase pathway on PMA-treated alveolar macrophages (114) and serine/threonine kinases such as protein kinase C (PKC)α, PKCε, and protein kinase B (PKB) α (115).

Biological Function FcR participate in many aspects of host defense through engagement with antibodies complexed to antigens. FcR ligation by immunoglobulins can initiate a plethora of biological processes, including phagocytosis, antigen presentation,

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ADCC, superoxide generation, and the release of cytokines and inflammatory mediators (116). FcR ligation can also modulate the activation status of cells, and consequently immune responses (117). The cellular functions promoted by FcαRI depend mostly on FcR γ -chainmediated tyrosine kinase activation. In this context, it has been shown using transfectants that only FcαRI molecules associated with FcRγ can induce Ca2+ release, IL-2 release, degranulation, or IgA degradation (104, 105). However, both types of FcαRI, FcαRI-γ 2, and γ -less FcαRI perform endocytosis at similar rates (105). Although γ -less FcαRI are unable to mediate downstream functions, they recycle internalized IgA complexes and protect them from degradation. Cells expressing the two forms of FcαRI (FcαRI-γ 2 and γ -less FcαRI) may play a regulatory role, either by degrading IgA antibody complexes or by recycling serum IgA to achieve serum homeostasis, possibly depending on receptor clustering size (105). Recently, it has been shown that FcαRI-γ 2 complexes mediate antigen presentation in IIA1.6-cell transfectants expressing FcαRI plus either wild-type FcR γ−chain, a γ−chain in which the ITAM was altered by a Y to F mutation, or a γ -chain in which the ITAM was substituted with the ITAM of Fcγ RIIA (118). The results indicated that signaling-competent ITAM was not required for endocytosis of IgA-ovalbumin. Antigen presentation, however, was impaired by ITAM changes. Signaling-competent FcR γ -chain ITAM appeared necessary for transport of ligated FcαRI to lamp-1(+) late endocytic compartments, for remodeling and/or activation of those compartments, and also for efficient degradation of IgA complexes. Moreover, FcαRI ligation activated efficient processing of nonreceptortargeted antigen. The results suggest FcR γ -chain signaling to activate the antigen processing (118). FcαRI mediates phagocytosis of IgA-opsonized bacteria and yeast particles, and priming of neutrophils and monocytes represents an essential step in phagocytosis of IgA-coated particles. This has been demonstrated using GM-CSF; and IL-8 on neutrophils (55, 119–122); IL-1, TNF-α, GM-CSF, or LPS on monocytes (57); and GM-CSF, IL-4, or IL-5 on eosinophils (96). Priming-induced increases in IgAmediated phagocytosis have been attributed to either modulation of the number of FcαRI molecules on the cell surface or an increase of FcαRI avidity for ligand. Another function initiated by FcαRI is antibody-dependent cell-mediated cytotoxicity (or ADCC). Cells primed by IgA antibodies mediate lysis of target cells such as bacteria, Schistosoma mansoni schistosomula, erythrocytes, as well tumor cells (123–126). This has been the basis for the development of therapeutic approaches targeted to FcαRI. CD89-targeted bispecific antibodies direct highly effective reverse ADCC and phagocytosis of tumor cells by FcαRI expressing cells (127–129). In vitro data indicate that under certain conditions FcαRI cooperates with complement receptors CR1 and CR3 to improve the efficiency of different cellular effector functions. Immune complexes containing IgA and C3b/iC3b induce faster release of lactoferrin from neutrophils than do IgA immune complexes alone (130). In another study, leukocytes required IgA and complement to kill Streptococcus pneumoniae (131). More recently it was demonstrated that human FcαRI

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transgenic mice back-crossed to CR3−/− animals were unable to initiate extracellular lysis of target cells (106, 132). Activation through FcαRI depends on receptor clustering on the cell surface. Studies with unprimed monocytes showed FcαRI cross-linking to result in induction of IL-6 and TNF-α release (133). Also, FcαRI triggering on monocytes by CD89 mAb or by IgA immune complexes induces secretion of IL-1β, IL-8, leukotrienes C4 and B4, and prostaglandin E2, as well as superoxide release (134– 138). Similarly, aggregated serum IgA and cross-linked monomeric, polymeric IgA, SIgA, or CD89 mAb My43 trigger a respiratory burst inducing superoxide release in neutrophils (120, 139–142). Both subclasses of IgA, IgA1 and IgA2, can initiate these functions. It is noteworthy that IgA2 immune complexes trigger neutrophil activation more efficiently than do IgG complexes (143). A similar trend was observed in studies with FcR-directed bispecific antibodies, documenting FcαRI-directed antibodies to be superior to Fcγ R-directed BsAb in facilitating lysis of CD20-positive tumor cells (129). IgA antibodies potently induce lysis of lymphoma and solid tumor targets and are far more effective than IgG anti-tumor antibodies in recruiting neutrophils, the most populous type of tumor-cytolytic cells in blood (144, 145). IgA therapeutic molecules also do not interact with down-modulatory types of FcR, such as Fcγ RIIb (117), in contrast to IgG antibodies. In addition, work with recombinant SIgA molecules directed to S. mutans documented these molecules to be longer lived at mucosal sites than IgG, and to effectively prevent oral bacterial colonization in humans (146). These data suggest IgA antibodies to represent attractive candidates for immunotherapy of neoplastic and infectious disorders (147). On eosinophils, FcαRI aggregation seems required for degranulation and release of eosinophil-derived neurotoxin following triggering by SIgA-coated beads (148). However, since other less-well characterized receptors for secretory component have been described on eosinophils (149), it is difficult at this time to attribute these effects conclusively to FcαRI.

Role of FcαRI in Mucosal Defense The observation that intestinal macrophages fail to express FcαRI (49) may point to a programmed anti-inflammatory system for protection of mucosal integrity. In this context, CD15+ colostrum neutrophils express FcαRI at levels similar to those on blood neutrophils. Most colostral neutrophils (70%), however, bear SIgA on their surface, whereas blood cells do not. The former cells do express FcαRI alone and fail to release superoxide products or kill bacteria in an FcαRI-dependent way, indicating that γ -less FcαRI exhibit anti-inflammatory properties (95). By contrast, colostral mononuclear cells are able to kill enteropathogenic Escherichia coli opsonized with colostral IgA via FcαRI (150). Recently a differential regulation has been documented for the FcαRI a.2 isoform on human alveolar macrophages at the level of the ERK1/2 pathway (114). It was observed that S-IgA and p-IgA downregulate the LPS-increased

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respiratory burst in alveolar macrophages through an inhibition of ERK1/2 activity. Both these IgA ligands, however, induce an increase in respiratory bursts associated with upregulated ERK1/2 phosphorylation on PMA-treated cells. Analysis of the in vivo role of FcαRI was made possible by generation of two types of transgenic mice. One model, in which the FcαRI transgene was placed under control of a CD11b promoter, resulted in high human FcαRI expression levels on monocytes and macrophages (99). In a second model, created with the use of a cosmid clone bearing the human FcαRI gene under its own regulation, a preferential expression on neutrophils was observed (106). In both models, FcαRI transgenic mice express CD89 only on myeloid cells, similarly as in humans. However, because the cosmid-generated FcαRI transgenic (Tg) mice contain the endogenous regulatory sequences, it was possible to modulate FcαRI expression on macrophages and Kupffer cells (33, 151). These mice helped to determine the role of FcαRI in mucosal infections including studies with S. pneumoniae and Bordetella pertussis. In both cases, FcαRI transgenic mice could be protected against pneumonia and sepsis (152) (G. Vidarsson & J.G.J. van de Winkel, unpublished). Kupffer cells express FcαRI in humans and have been proposed as essential for the clearance of bacteria passing the mucosal barrier (33). The FcαRI-Tg mice demonstrated that FcαRI plays a key role in mucosal defense. Indeed, in vivo studies show that FcαRI-expressing Kupffer cells vigorously ingest E. coli, opsonized with human serum IgA. Notably, these studies revealed human SIgA to be incapable of initiating phagocytosis. This was also observed in vitro for other pathogens, including Staphylococcus aureus, Candida albicans, B. pertussis, and S. pneumoniae (33, 153–155, 155a). Based on these results one can propose that SIgA may function as an “antiseptic coating” at the mucosa by avoiding bacterial adherence and invasion of microorganisms. Binding of antigens to SIgA, thus, does not initiate inflammatory processes, qualifying this class of antibody as noninflammatory (Figure 5A). Under pathological conditions, characterized by disruption of the mucosal barrier with production of inflammatory mediators, primed Kupffer cells or dendritic cells expressing FcαRI may play a important role either as a second line of defense against bacterial infection or by arming the immune system to respond to external antigens (Figure 5B) (33). In this context, upregulation of FcαRI associated with FcR γ -chain on monocytes from patients with septic shock by gram-negative bacteremia has been reported. This confirms a possible role of this receptor in immunity against bacterial infections (156). FcαRI is expressed on immature dendritic cells but is undetectable by immuno histochemical methods on human epithelial LC (31), suggesting that LC may neglect IgA immune complexes within the epithelium in the absence of a breakdown of the epithelial barrier. FcαRI downregulation may be mediated by TGFß1, which surrounds mucosal areas. This is demonstrated when in vitro (31, 60) FcαRI is active in Ag-binding and Ag-uptake, permitting internalization of the ligand by immature DC, which triggers overexpression of the costimulatory molecule CD86,

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and of MHC class II molecules at the plasma membrane, which increases their allostimulatory activity and triggers IL-10 production (31) (Figure 5C). IL-10 has been found to mediate IgA1 and 2 isotype switching (157). FcαRI+ interstitial-type dendritic cells may well play a role in mounting specific immune responses against mucosa-derived antigens bound to SIgA; activation of dendritic cells via FcαRI may positively feed-back on IgA production. It should be mentioned, however, that a recent report confirms FcαRI expression by immature dendritic cells but claims SIgA binding to these cells is mediated not by FcαRI, but rather via carbohydraterecognizing receptors such as mannose receptor (46).

Fcα/µR A mouse Fc receptor for IgA and IgM, designated Fcα/µR, recently described (40, 158), represents a type I transmembrane protein of 503 amino acids containing four potential sites for N-linked glycosylation. The receptor has only one extracellular loop, which contains a conserved motif present in the first EC loop of human, bovine, and murine pIgR, suggesting an ancestral link. Fcα/µR has a human homologue, and both bind IgM as well as IgA with intermediate affinity. The human Fcα/µR gene is located at chromosome 1q 32.3 near several other FcR genes. Fcα/µR is constitutively expressed on the majority of murine B lymphocytes and macrophages. Cross-linking Fcα/µR by soluble IgM or IgM-coated microparticles triggers receptor internalization. Fcα/µR, furthermore, mediates B lymphocyte endocytosis of IgM-coated S. aureus. Fcα/µR has been proposed to play a role in the primary stages of antimicrobial immune responses (40). Moreover, Fcα/µR is expressed on mature, but not immature, B lymphocytes and acquires the ability to bind IgA and IgM antibodies after B lymphocyte stimulation (158). This receptor is abundantly expressed in secondary lymphoid organs such as lymph nodes, appendix, and intestine, suggesting a role in systemic and mucosal immunity (158). More recently, Fcα/µR transcripts have been shown in human mesangial cells, which were in addition markedly upregulated by proinflammatory cytokines such as IL-1 (159), suggesting a regulatory role for this receptor during inflammation.

ALTERNATIVE IgA RECEPTORS Asialoglycoprotein Receptor (ASGP-R) The liver plays an important role in maintaining homeostasis through regulation of IgA catabolism. The ASGP-R expressed on hepatocytes (41, 160) recognizes terminal Gal residues on serum glycoproteins, including IgA, and conveys bound ligand for intracellular degradation. Studies performed in primates revealed that a minority of proteins bound and internalized by ASGP-R escape degradation and are transported into bile in intact form (161). The ASGP-R is proposed to be involved in IgA clearance from the blood (162). The major pathway for IgA2 clearance was recently shown to be mediated by the liver ASGP-R. Liver-mediated

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uptake through the ASGP-R was suppressed in ASGP-R-deficient mice. Notably, only a small percentage of IgA1 is cleared through this pathway. Clearance of IgA1 lacking the hinge region, with its associated O-linked carbohydrate, was more rapid than that of wild-type IgA1. The rapid clearance of IgA2, and not IgA1, through the liver may contribute to the higher serum levels of IgA1, relative to IgA2 (158).


Transferrin Receptor (TfR) Transferrin receptor, TfR (or CD71), selectively binds IgA1 (42). In contrast to FcαRI, this IgA receptor is not fully expressed on mature blood leukocytes, but it is well-expressed on cultured renal mesangial cells. Highly purified polymeric IgA1 (free of transferrin) binds better to TfR than does monomeric IgA1 (I.C. Moura & R. Monteiro, unpublished). Human T and B lymphocytes have been observed to bind human IgA (2–4, 164–166). IgA binding proved dependent on T lymphocyte proliferation, and the TfR mediated the interaction with IgA (42). TfR is an IgA receptor expressed on B lymphocyte cell lines, such as Daudi cells (42). It is possible that the TfR mediates the earlier described IgA1 binding to T cells, mediated by O-linked carbohydrate moities within the IgA1 (and IgD) hinge region (167, 168). It remains unclear whether TfR binds IgD (169).

Secretory Component Receptor (SCR) A receptor specific for secretory component (SC) with a Mr of 15 kDa has been isolated from eosinophils (149). This molecule binds SC and SIgA, but not serum IgA, and triggers degranulation and release of eosinophil cationic protein and peroxidase. Thus, the existence of two types of IgA receptors capable of binding SIgA, FcαRI and the SC receptor, may underlie SIgA’s potency to trigger eosinophil degranulation (148). SIgA also induces basophil degranulation in an FcαRI-independent manner and may express SCR as well (170).

Other IgA Receptors Recently, a mouse IgA receptor was described on Peyer’s patch M cells, an epithelial cell located exclusively within the follicle-associated epithelium overlying mucosa-associated lymphoid tissues (170a). Although the molecular nature of this receptor remains unknown, it is interesting that this murine molecule can bind human IgA2 but not human IgA1. Furthermore, in contrast to FcαRI, the IgA binding to the M-cell receptor was dependent on Cα1 and Cα2 domains.

INVOLVEMENT OF IgA RECEPTORS IN PATHOLOGY IgA-associated diseases are characterized by increased serum IgA levels, often paralleled by IgA tissue deposition. These disorders include IgA nephropathy (IgAN), ankylosing spondylitis, Sjögren’s syndrome, alcoholic liver cirrhosis (ALC), HIV infection, and dermatitis herpetiformis.

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Abnormal FcαRI endocytosis is potentially harmful due to impaired removal of IgA-containing immune complexes from blood. Defective FcαRI endocytosis rates, and increased IgA recycling toward the cell surface, have been demonstrated on blood phagocytes from patients with IgAN and ALC (171, 172). As a consequence, cells from these patients express high IgA levels. In IgAN, an altered O-linked glycosylation, a decreased galactosylation and altered sialylation, has been observed in a subpopulation of serum IgA1 (173). Macromolecular IgA complexes that escape clearance by IgA receptors may be trapped in the mesangium in IgAN (173). It remains unclear, however, whether abnormally glycosylated IgA1 in IgAN patients influences its capacity to bind to FcαRI, ASGPR, or TfR. Some studies suggest IgA binding to FcαRI to be favored in IgAN patients because increased amounts of IgA bound to blood monocytes and neutrophils were observed. In addition, purified IgA from these patients binds better to normal monocytes than IgA from healthy volunteers (61). Notably, another study documented a reduced affinity of patient IgA for FcαRI-transfected B cells (174). The fact that mouse B cells may express up to three IgA receptor types, pIgR (175), Fcα/µR (40), and TfR (42), complicates interpretation of these latter data. Studies in IgAN patients, and patients with other IgA-associated disease including HIV infection, ALC, and spondyloarthropathies, indicate reduced FcαRI expression levels on circulating monocytes and (to a lesser degree) on neutrophils, (61, 172, 176, 177). Other investigators did not observe a decreased monocyte FcαRI expression using indirect immuno fluorescence (178). Addition of IgA has a negative effect on FcαRI expression, possibly due to shedding of FcαRI’s extracellular domain (99). The demonstration of soluble FcαRI in serum of IgAN patients and not in serum from healthy controls supports this hypothesis. In addition, metabolically labeled cells from IgAN patients released a glycosylated FcαRI form of 50–70 kDa with a 24-kDa protein core. Production of soluble FcαRI was also induced by polymeric IgA from FcαRI-transfected cells. IgA-induced FcαRI-shedding was indicated by the loss of reactivity with an antibody specific for FcαRI’s cytoplasmic tail. These results indicate that cleavage of the FcαRI extracellular domain can occur, resulting in release of IgA/FcαRI complexes into the circulation. Cleavage may be promoted by FcαRI aggregation with cell surface protease(s). IgA-induced receptor shedding in IgAN patients may amplify the molecular size of immune complexes and could include IgA–IgG rheumatoid factors, or IgA-fibronectin complexes (173). Recent work in human FcαRI Tg mice modeled the development of IgAN (99). Human FcαRI interacts with mouse polymeric IgA to form complexes that are deposited in the renal mesangium of FcαRI Tg mice. Whereas other animal models, such as the ddY, HIGA mice, and uterogloblin knockout mice, only show some signs of IgAN (173), human FcαRI transgenic mice developed mesangial IgA deposition, hematuria, mild proteinuria, and macrophage infiltration around the renal glomeruli (99). The disease can be transferred to wild-type recipients by infusion of serum IgA/soluble FcαRI complexes from Tg mice. To examine the

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contribution of IgA, a model of SCID-FcαRI Tg mice was created. These mice do not develop IgAN spontaneously, but develop manifestations of IgAN upon injection of IgA from IgAN patients. Interestingly, IgA from healthy subjects did not result in IgAN in SCID-FcαRI Tg mice, suggesting that abnormally glycosylated IgA coupled with FcαRI participates in IgAN pathogenesis. Circulating complexes containing IgA and soluble FcαRI may well be involved in the development of IgAN (99, 179). Overexpression of a mesangial IgA1 receptor, the TfR (CD71), has also been found in patients with IgAN (42), and may well mediate the (selective) deposition of IgA1 complexes in kidneys. Deposited mesangial IgA1 complexes may trigger inflammation via the release of pro-inflammatory cytokines such as IL-1, IL6, and TNF-α, with consequent fibrosis and renal impairment (179, 180). This hypothetical cycle of events could thus account for progression and chronicity of disease. This picture may yet be more complicated, as Fcα/µR transcripts have also been found in human mesangial cells, which are upregulated by pro-inflammatory cytokines such as IL-1 (150). Enhanced FcαRI surface expression has been observed on eosinophils of allergic patients (45) and on monocytes from patients with gram-negative bacteremia (156). Increased levels of IgA antibodies against allergen and bacterial antigens have been documented in sputum of atopic asthmatic individuals (181). Whether increased FcαRI expression exerts a protective or harmful role in these diseases remains to be established.

CONCLUSIONS Receptors for IgA play a significant role in vivo in maintaining the integrity of immune responses in systemic and mucosal compartments. In this review we summarized the current knowledge of five types of IgA receptors, focusing on FcαRI (CD89). This receptor appears to play an important role in immunity by linking the IgA response to powerful cellular effector mechanisms. A role for select IgA receptors has been implicated in a variety of pathological conditions. Recent studies support a role for IgA antibodies and FcαRI-directed molecules as therapeutics for human disease. ACKNOWLEDGMENTS The authors thank B. Pasquier and M. Benhamou for critical reading of the manuscript, E. Broug for stimulating discussions, and M. van Egmond for help with Figure 5. RCM’s work was supported by grants from INSERM, Fondation pour la Recherche Medicale, Association pour la Recherche contre le Cancer, and Ligue contre le Cancer. JVDW’s work was supported by grants from the Netherlands Organization for Scientific Research (NWO) and the Dutch Cancer Society (KWF).

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L, Trabulsi LR, Carneiro-Sampaio MM. 1997. Colostral mononuclear phagocytes are able to kill enteropathogenic Escherichia coli opsonized with colostral IgA. Scand. J. Immunol. 46:59–66 van Egmond M, Hanneke van Vuuren AJ, van de Winkel JG. 1999. The human Fc receptor for IgA (FcαRI, CD89) on transgenic peritoneal macrophages triggers phagocytosis and tumor cell lysis. Immunol. Lett. 68:83–87 Hellwig SM, van Spriel AB, Schellekens JF, Mooi FR, van de Winkel JG. 2001. Immunoglobulin A-mediated protection against Bordetella pertussis infection. Infect. Immun. 69:4846–50 van Spriel AB, van den Herik-Oudijk IE, van Sorge NM, Vile HA, van Strijp JA, van de JA, Winkel JG. 1999. Effective phagocytosis and killing of Candida albicans via targeting FcαRI (CD64) or FcαRI (CD89) on neutrophils. J. Infect. Dis. 179:661–69 van der Pol W, Vidarsson G, Vile HA, van de Winkel JG, Rodriguez ME. 2000. Pneumococcal capsular polysaccharidespecific IgA triggers efficient neutrophil effector functions via FcαRI (CD89). J Infect. Dis. 182:1139–45 Rodriguez ME, Hellwig SM, Hozbor DF, Leusen J, van der Pol WL, van de Winkel JG. 2001. Fc receptor-mediated immunity against Bordetella pertussis. J. Immunol. 167:6545–51 Vidarsson G, van der Pol WL, van den Elsen JMH, Vilé, Jansen M, et al. 2001. Activity of human IgG and IgA subclasses in immune defense against meningitidis serogroup B. J. Immmunol. 166:6250–56 Chiamolera M, Launay P, Montenegro V, Rivero MC, Velasco IT, Monteiro RC. 2001. Enhanced expression of Fcα receptor I on blood phagocytes of patients with gram-negative bacteremia is associated with tyrosine phosphorylation of the FcR-γ subunit. Shock 16:344–48 Fayette J, Dubois B, Vandenabeele S,

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167. Rudd PM, Fortune F, Patel T, Parekh RB, Dwek RA, Lehner T. 1994. A human Tcell receptor recognizes ‘O’-linked sugars from the hinge region of human IgA1 and IgD. Immunology 83:99–106 168. Swenson CD, Patel T, Parekh RB, Tamma SM, Coico RF, et al. 1998. Human T cell IgD receptors react with Oglycans on both human IgD and IgA1. Eur. J. Immunol. 28:2366–72 169. Coico RF, Xue B, Wallace D, Pernis B, Siskind GW, Thorbecke GJ. 1985. T cells with receptors for IgD. Nature 316:744– 46 170. Iikura M, Yamaguchi M, Fujisawa T, Miyamasu M, Takaishi T, et al. 1998. Secretory IgA induces degranulation of IL-3-primed basophils. J. Immunol. 161: 1510–15 170a. Mantis NJ, Cheung MC, Chintalacharuvu KR, Rey J, Corthésy B, Neutra MR. 2002. Selective adherence of IgA to murine Peyer’s patch M cells: evidence for a novel IgA receptor. J. Immunol. 169:1844–51 171. Monteiro RC, Grossetete B, Nguyen AT, Jungers P, Lehuen A. 1995. Dysfunctions of Fcα receptors by blood phagocytic cells in IgA nephropathy. Contrib. Nephrol. 111:116–22 172. Silvain C, Patry C, Launay P, Lehuen A, Monteiro RC. 1995. Altered expression of monocyte IgA Fc receptors is associated with defective endocytosis in patients with alcoholic cirrhosis. Potential role for IFN-γ . J. Immunol. 155:1606– 18 173. Novak J, Julian BA, Tomana M, Mestecky J. 2001. Progress in molecular and genetic studies of IgA nephropathy. J. Clin. Immunol. 21:310–27 174. van Zandbergen G, van Kooten C, Mohamad NK, Reterink TJ, de Fijter JW, et al. 1998. Reduced binding of immunoglobulin A (IgA) from patients with primary IgA nephropathy to the myeloid IgA Fc-receptor, CD89. Nephrol. Dial. Transplant. 13:3058–64

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178. Kashem A, Endoh M, Nomoto Y, Sakai H, Nakazawa H. 1996. Monocyte superoxide generation and its IgA-receptor in IgA nephropathy. Clin. Nephrol. 45: 1–9 179. Monteiro RC, Moura IC, Launay P, Tsuge T, Haddad E, et al. 2002. Pathogenic significance of IgA receptor interactions in IgA nephropathy. Trends Mol. Med. 8:464–68 180. Couser WG. 1999. Glomerulonephritis. Lancet 353:1509–15 181. Nahm DH, Kim HY, Park HS. 1998. Elevation of specific immunoglobulin A antibodies to both allergen and bacterial antigen in induced sputum from asthmatics. Eur. Respir. J. 12:540–45

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Figure 1 The human IgA receptor family. Orange circles are schematic representations of extracellular Ig-like domains (for an example of a three-dimensional structure see CD89 in Figure 3B). pIgR, polymeric Ig receptor; FcαRI, the myeloid IgA Fc receptor; Fcα/µR, the IgA/IgM Fc receptor; ASGP-R, the asialoglycoprotein receptor; TfR, the transferrin receptor.


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Figure 3 (A) Schematic model for FcαRI (CD89) structure. FcαRI a.1 can be expressed associated or not associated with the FcR γ -chain. FcαRI a.2 has a deletion of 22 amino acids in the EC domain near the membrane. (B ) Automated protein-modeling for FcαRI (CD89) indicating residues important for ligand binding [modified from Wines et al. (87)]. Copyright 2001. The American Association of Immunologists, Inc.


Figure 4 Signaling pathways triggered by FcαRI (CD89). Schematic model for IgA-mediated cellular activation depending on clustering of FcαRI-γ 2 complexes. Signaling through FcRγ -unassociated FcαRI molecules is unclear. IgA IC, IgA immune complexes.


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Figure 5 Model for the role of FcαRI in mucosal defence. (A) Protection of mucosal surface by SIgA. (B ) FcαRI as a gatekeeper against bacterial infections. Upon disruption of the epithelial barrier, pathogens are exposed to serum IgA. Inflammatory cytokines induce Kupffer cell FcαRI, which filter the portal blood via FcαRI-mediated phagocytosis. P, portal vein, H, hepatic vein. Small red circles represent bacteria. (C ) FcαRI-mounted immune response following disruption of epithelial barrier. Small blue circles represent environmental antigens and yellow triangles, the FcαRI. MHC, major complex of histocompatibility; B7, costimulatory molecule; LC, Langerhans cells; DC, dendritic cells.


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REGULATORY MECHANISMS THAT DETERMINE THE DEVELOPMENT AND FUNCTION OF PLASMA CELLS Annu. Rev. Immunol. 2003.21:205-230. Downloaded from arjournals.annualreviews.org by HINARI on 09/01/07. For personal use only.

Kathryn L. Calame, Kuo-I Lin, and Chainarong Tunyaplin Departments of Microbiology and Biochemistry and Molecular Biophysics, Columbia University College of Physicians and Surgeons, New York, New York 10032; email: [email protected], [email protected], [email protected]

Key Words XBP-1, Blimp-1, IRF4, Bcl-6, Pax5 ■ Abstract Plasma cells are terminally differentiated final effectors of the humoral immune response. Plasma cells that result from antigen activation of B-1 and marginal zone B cells provide the first, rapid response to antigen. Plasma cells that develop after a germinal center reaction provide higher-affinity antibody and often survive many months in the bone marrow. Transcription factors Bcl-6 and Pax5, which are required for germinal center B cells, block plasmacytic differentiation and repress Blimp-1 and XBP-1, respectively. When Bcl-6-dependent repression of Blimp-1 is relieved, Blimp-1 ensures that plasmacytic development is irreversible by repressing BCL-6 and PAX5. In plasma cells, Blimp-1, XBP-1, IRF4, and other regulators cause cessation of cell cycle, decrease signaling from the B cell receptor and communication with T cells, inhibit isotype switching and somatic hypermutation, downregulate CXCR5, and induce copious immunoglobulin synthesis and secretion. Thus, commitment to plasmacytic differentiation involves inhibition of activities associated with earlier B cell developmental stages as well as expression of the plasma cell phenotype.

INTRODUCTION A bad beginning makes a bad ending. Euripides A hard beginning maketh a good ending. John Heywood Terminally differentiated, antibody-secreting plasma cells are the end-stage effectors of the humoral immune response. From 1950 to1970 they received attention primarily because of their roles in multiple myeloma and autoimmunity. Development of techniques to produce plasma cell tumors in mice immunized with specific haptens made plasmacytomas a critical tool for systematic studies on antibody proteins (1) and, in the late 1970s, provided a basis for classic studies elucidating the rearrangement of immunoglobulin light chain (2) and heavy chain (3) genes. However, to understand humoral immunity in both normal responses 0732-0582/03/0407-0205$14.00

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and in disease states, one must understand the biology of normal plasma cells. Obviously, developmental decisions at earlier B-cell stages are critical for the ultimate generation of plasma cells. Regulation of B-cell development has received much attention (4–9), but plasmacytic differentiation has remained poorly understood. Recently this situation has improved, primarily because of studies with genetically altered mice and data from DNA microarray analyses. In this review we summarize the characteristics of plasma cells and discuss anatomical sites and times during a humoral response when plasma cells form. We then review recent work that provides new insights into the regulatory mechanisms that are involved in commitment to terminal B-cell differentiation and expression of the plasma cell phenotype. The focus is primarily but not exclusively on transcriptional regulatory mechanisms.

Overview of Plasma Cells PROLIFERATION Although terminal differentiation is usually preceded by robust proliferation of plasmablasts (10), cessation of cell division is a prerequisite for plasmacytic differentiation. In the mouse lymphoma cell line BCL1, where treatment with cytokines induces plasmacytic differentiation, ectopic expression of either c-Myc or cyclin E to enforce proliferation blocks cytokine-driven differentiation to immunoglobulin (Ig)-secreting plasma cells (11). Although required, cessation of cell cycle is not sufficient to drive activated B cells to become plasma cells in the BCL1 model (11). The requirement for cessation of cycle prior to terminal differentiation is conserved in human B cells because IL-6-dependent plasmacytic differentiation of lymphoblastoid cells is accompanied by enhanced expression of cdk inhibitor p18INK4c, leading to cell cycle arrest (12). Mice deficient in p18 are defective in their ability to form Ig-secreting plasma cells (198). The lifespan of nonproliferating plasma cells varies from a few days to many months. IgM-secreting plasma cells formed early in a primary response by antigen activation of marginal zone B cells often survive only a few days and undergo apoptosis in situ (13). Long-lived plasma cells have been found primarily in the bone marrow (BM) (14), although there is evidence that some plasma cells in the spleen may also be long-lived (15). Several proteins appear to be involved in plasma cell apoptosis. Expression of an anti-apoptotic Bcl2 family member, A1, is important for determining the survival capacity of activated B cells (16). Expression of A1, via a CD40/NF-κBdependent mechanism, protects germinal center (GC) B cells from receptor ligationinduced apoptosis (17–19). A1 also decreases during plasmacytic differentiation induced by ectopic expression of the transcriptional repressor B lymphocyte induced maturation protein-1 (Blimp-1) (20, 21). Ectopic expression of A1 reverses Blimp-1-dependent apoptosis in a lymphoma line (20), providing evidence that A1 levels are important for plasma cell survival. On the other hand, Blimp-1 induces pro-apoptotic proteins GADD45 and GADD153 (21), which may decrease plasma cell survival.

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Bone marrow stromal cells provide signals that promote plasma cell survival and possibly final differentiation of plasmablasts (22, 23). One critical differentiation and survival factor for plasma cells provided by bone marrow stromal cells is IL-6, although the nature of the survival signal is not understood. Blimp-1 is present in plasma cells in the bone marrow (24), but the expression of A1, GADD153, or GADD45 has not been studied in these cells. ANTIBODY SECRETION The sole function of plasma cells is to secrete soluble immunoglobulin molecules; in fact, membrane Ig and the B cell receptor (BCR) are low or absent on plasma cells. During plasmacytic differentiation, steady-state immunoglobulin heavy and light chain mRNAs increase to superabundant levels, apparently due to both increased transcription and increased mRNA stability (25, 26). The ratio of secreted to membrane heavy chain mRNA, determined by use of different polyadenylation sites (27), increases, consistent with Ig secretion by the plasma cell. Differential polyadenylation appears to involve the cleavage stimulatory factor Cst-64 (28), a member of a heterotrimeric complex responsible for endonucleolytic RNA cleavage (29). In chicken B cells, induction of Cst-64 causes an increase in secreted µ mRNA (30). However, in other studies Cst-64 did not increase in B cells expressing secreted µ mRNA, suggesting a role for additional factors (31). A recent report suggests that U1A binding to motifs upstream of the secreted polyA site is important in regulating secreted µ mRNA (32). To accommodate translation and secretion of the abundant Ig mRNAs, plasma cells have an increased cytoplasmic-to-nuclear ratio and prominent amounts of rough endoplasmic reticulum and secretory vacuoles (33–36). The presence of unfolded proteins in the endoplasmic reticulum (ER) is associated with an ER stress or unfolded protein response (UPR), which includes induction of the heat shock protein homologue BiP/GPR78, a member of the heat shock protein HSP 70 family (37, 38). Folding of the nascent immunoglobulin heavy and light chains requires association with BiP prior to disulfide bond formation (39), subsequent association with GPR94, a homologue of HSP90, thiol-dependent interactions with other ER proteins, and glycosylation prior to secretion (40, 41). Activation of the UPR in murine plasma cells also causes IRE1-dependent splicing of a small intron from XBP-1 mRNA, thus encoding a more stable form of this transcriptional activator (42). CHANGES IN CELL SURFACE PROTEINS Numerous surface proteins change upon plasma cell differentiation: Class II major histocompatibility complex (MHCII), B220/CD45, CD19, CD21, CD22, and CXCR5 decrease, whereas Syndecan-1 increases. Loss of CD19, CD21, CD22, and CD45, all modulators of BCR signaling, is consistent with lack of BCR in plasma cells. Syndecan-1, a proteoglycan that recognizes extracellular matrix and growth factors, is present on antibodysecreting B cells and is often used as a marker of plasma cells (43). However, Syndecan-1 is also found on a subset of germinal center B cells that are probably plasmablasts or cells committed to become plasmablasts (24, 44), and thus it is expressed prior to the fully differentiated plasma cell state. VLA-4 is the most

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predominant integrin family protein on plasma cells (45) and is important for cell-cell contact between plasma cells and bone marrow stromal cells. CD38, an adhesion and signaling molecule that recognizes the ligand CD31, is expressed on human plasma cells (46, 47) but not on murine plasma cells. Class II MHC, which is required for antigen presentation to T cells, is absent from the surface of plasma cells because plasma cells do not require signals from T cells. Class II MHC, DM, and invariant chain genes fail to be transcribed in plasma cells due to the absence of class II transactivator (CIITA), a coactivator required for their transcription (48). Chemokine receptors CXCR5 and CCR7 are decreased on plasma cells, reducing responsiveness to chemokines CXCL13, CCL19, and CCL21 in the B and T cell zones of spleen. Expression of CXCR4, which recognizes CXCL12 present in splenic red pulp, lymph node medullary cords, and in bone marrow, remains high (49). These changes mediate movement of plasma cells from the follicles to red pulp and the bone marrow.

PLASMACYTIC DIFFERENTIATION DURING THE HUMORAL IMMUNE RESPONSE Soluble antigen or particulate antigen associated with dendritic cells activates longlived na¨ıve B cells in the spleen or lymph nodes to proliferate as plasmablasts and to differentiate into plasma cells. Specialized myeloid dendritic cells appear to be required for survival and differentiation of plasmablasts (50). Plasma cells develop in different locations and from different B cell precursors depending on the antigen and stage of the humoral response.

Plasma Cells from B-1 Cells and Marginal Zone B Cells B-1 cells and marginal zone (MZ) B cells provide the first response to antigen, and in mice they persist throughout life in the absence of cell division (51). B-1 cells, found primarily in peritoneal and pleural cavities, are a B-cell subset identifiable by their unique surface proteins and self-renewal capacity (52–55). They secrete natural antibodies important for innate immunity and develop into plasma cells that provide mucosal immunity and T-independent production of mucosal IgA in response to commensal bacteria in the gut (56). B-1 cells develop primarily from fetal progenitors (57), and the specificity and surface density of BCR has been shown to be critical for determining B-1 versus B-2 (conventional) development (58). However, the lineage relationship between B-1 and B-2 B cells remains controversial (52, 59, 60). Marginal zone (MZ) B cells are the first B cells encountered by blood-borne antigens because of their location adjacent to the marginal sinuses in spleen and their proximity to antigen-trapping macrophages and dendritic cells. Thus, MZ B cells provide an early humoral response to antigen (61, 62). Na¨ıve MZ B cells are distinguished from na¨ıve follicular B cells by lower surface expression of IgD and CD23, higher surface expression of CD21, and inability to circulate to lymph

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nodes. Repertoire selection appears to favor MZ location for B cell clones with specificities for T cell–independent (TI) bacterial antigens (63). MZB cells are also known to provide a rapid response to particulate, T cell–independent antigens (63, 64) owing to their location in the MZ, where they are the first to encounter blood-borne antigen, as well as their inherent ability to generate effector cells more rapidly than follicular B cells (65). Following antigen activation, MZ B cells become plasmablasts and rapidly exit the MZ. They undergo a burst of proliferation and form plasma cell foci in extrafollicular regions along the periphery of the periarterial lymphatic sheath [reviewed in (63)]. These early plasma cells usually secrete IgM, and while many undergo apoptosis in situ within a few days (13), others can survive longer, yet are limited by the intrinsic capacity of the spleen to support plasma cells (15). Following activation by T-dependent antigens, MZ B cells may travel to follicles to participate in germinal center reactions prior to final differentiation to plasma cells.

Postgerminal Center Plasma Cells A later plasma cell response to primary immunization with a T cell–dependent (TD) antigen occurs after the germinal center reactions are complete. The GC reaction has been intensively studied (66) and is only briefly summarized here. Centroblasts in the dark zone are highly proliferative and undergo somatic hypermutation of their rearranged Ig V genes. Centrocytes in the light zone are programmed to undergo apoptotic death, unless they are rescued by signals from T cells and antigen. Isotype switch recombination also occurs in centrocytes. Iterative cycles of proliferation, somatic hypermutation, and apoptosis in the GC result in selection of B cells making antibody with increased affinity for antigen and switched isotypes. The GC reaction usually peaks approximately 10–12 days following immunization, giving rise to two types of B cells: Ig surface positive, hypermutated, nonsecreting memory B cells and antibody-secreting plasmablasts. Plasmablasts exit the GC and develop into terminally differentiated plasma cells secreting high-affinity antibodies. Recently the Noelle group identified a heterogenous population of post-GC, short-lived precursors in BM that give rise to short- or long-lived plasma cells (67), establishing that plasma cell precursors can migrate from GCs to the BM prior to terminal differentiation. Intriguingly, these precursors divide, and cell division, but not antigen, is required for their differentiation into plasma cells. BM stromal cells provide survival signals to plasma cells (23); adoptive transfers have revealed that long-lived plasma cells in BM secrete antibody for many months in the absence of antigen or cell proliferation (68–70). They account for more than 80% of antibody in immune serum. The biology of memory B cells remains somewhat mysterious although it is known that they are long-lived, even in the absence of antigen (71), and do not secrete antibody [reviewed in (72)]. Upon exiting the GC, memory B cells recirculate or home to draining areas of lymph node and spleen, including the MZ of spleen. Recent studies have identified a distinct population of B220 memory cells that undergoes affinity maturation in the splenic red pulp, is maintained long-term in spleen and bone marrow, and is thought to be a major component of the splenic

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memory cell compartment (73). Upon secondary exposure to antigen, rapid and massive clonal expansion of memory B cells occurs, generating 8- to 10-fold more plasma cells than in a primary response (72). A secondary GC response can also occur. In addition, some memory cells persist to replenish the memory compartment.


Developmental Decisions that Determine Plasma Cell Fate In the generation of plasma cells from B-2 (conventional) B cells, there are at least three critical developmental decisions: (a) the decision of na¨ıve follicular precursors to enter the MZ or mature in the follicle, (b) the decision of GC B cells to become memory cells or plasmablasts, and (c) the decision of memory cells, upon secondary encounter with antigen, to become plasma cells or retain the memory phenotype. MARGINAL ZONE VERSUS FOLLICULAR B CELL DECISION BCR signals, probably from self-antigens or antigens from endogenous bacterial flora, are critical for B cell development, establishment of tolerance, and survival of all na¨ıve B cells (74). This has been called tickling of the BCR by self-antigen and is different from subsequent activation by foreign antigen (62, 75). Mice deficient for genes affecting BCR signal strength indicate that weaker tickles from self-antigen favor migration of precursors to the MZ rather than maturation in the follicles (62, 75). Tumor necrosis factor (TNF) family ligands and TNF receptors (TNFRs) also play important roles in determining splenic architecture and B cell development (76–79). Lymphotoxins are necessary for establishment of splenic T and B cell zones, and TNF is important for formation of marginal sinuses and GCs. Two ligands, B lymphocyte stimulator (BLyS or BAFF) and APRIL, and three receptors, TACI and BCMA (recognizing both ligands) and BAFF-R (recognizing only BLyS) (80), have roles in development of mature follicular B cells, GC B cells, and Ig production (81). BLyS is required for early maturation of B cells in spleen, and BLyS−/− mice lack follicular and MZ B cells (82, 83). TACI−/− mice specifically lack TI-2 B cells responses, whereas BAFF-R−/− mice have a milder phenotype [reviewed in (79)]. Further studies on TNF/TNFR proteins are likely to reveal additional roles for them in B lymphopoiesis and to refine our understanding of how they determine MZ and follicular B cell biology. EXITING THE GERMINAL CENTER AND THE MEMORY VERSUS PLASMA CELL DECISION

What signals tell a GC B cell to exit the GC and what determines memory versus plasma cell fate? There is evidence that plasma cell commitment occurs in the GC. A subset of germinal center B cells with a partial plasma cell phenotype has been described (24, 84). These cells express Syndecan-1, Blimp-1, and IRF4, but not Bcl-6 or Pax5, and they exit the germinal center more rapidly than the bulk of germinal center B cells, suggesting that plasma cell commitment is associated with termination of the GC reaction (24). At present, the relationship of this GC subset to the newly identified plasma cell precursors in BM (67) is not known, but it seems likely that the BM precursors may have arisen from plasmablasts that exit the GC and migrate to the BM.

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Because somatic mutation, which drives affinity maturation, is a random process, it seems unlikely that a given number of cell divisions determines when a B cell exits the GC. One model is that signaling via high-affinity antibody causes an end to the iterative cycles of mutation and selection. However, a recent study has provided evidence that GC B cells undergo a fixed mutation program, regardless of their initial BCR affinity (85), so this question remains unresolved. There is evidence that high-affinity BCR favors plasma cell differentiation over memory cell fate. Analysis of single, antigen-specific B cells from both memory and plasma cell compartments indicated that in a primary response there is selective differentiation of high-affinity variants into plasma cells (86). In mice constitutively expressing Bcl-2 to enhance B cell survival, excessive numbers of GC and memory B cells were observed, but the number of high-affinity plasma cells in bone marrow was not altered (87), providing further indirect evidence that plasma cell development depends on high antigen affinity of the BCR. Additional data suggest that the memory versus plasma cell decision is instructed rather than stochastic. CD40 signals favor a memory phenotype, while another TNF family receptor, OX40L, favors a plasma cell fate (88). Signals via CD27, triggered by CD70 on T cells, appear to drive human memory cells to a plasma cell phenotype (89, 90), although there is a contrasting report showing that CD27 signals block plasmacytic development of murine B cells (91). Cytokines are important: Plasma cell differentiation in culture occurs in IL-2 and IL-10 and in the absence of CD40L (92). IL-10 interrupts memory B cell expansion (93) and induces CD27 (94) and drives plasma cell development (95) while IL-4 directs B cells to a memory fate (95). IL-6 is required for differentiation of plasmablasts and antibody secretion (22), in part due to induction of the cyclin-dependent kinase inhibitor p18 (12). ACTIVATION OF MEMORY B CELLS Upon secondary antigen encounter, memory B cells are biased to become plasma cells (96). Although CD40 is important for development of memory B cells, it does not appear to be required for the development of plasma cells from memory cells in a secondary response (97). Recent studies show that the heavy chain isotype of the BCR can influence the fate of the B cell (98), with IgG triggering a significantly larger proliferative burst than IgM. This proliferative advantage of IgG B cells helps explain the dominance of IgG isotypes in memory responses and may also play a role in determining plasma cell fate in a primary response.

REGULATION OF GERMINAL CENTER AND POSTGERMINAL CENTER DEVELOPMENTAL DECISIONS In the germinal center, B cells have at least three developmental options: to continue further rounds of mutation and selection as GC B cells, to become memory B cells, or to become plasma cells. Mechanisms regulating these decisions are intertwined, and thus understanding plasma cell development requires understanding regulation

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of GC and memory development as well. Our understanding of transcriptional regulators involved in GC, plasma cell, and memory fate decisions is discussed below and summarized in Figure 1.

Bcl-6


Mice lacking the transcriptional repressor Bcl-6 have B cells that develop normally but fail to undergo a GC reaction when activated (99, 100). Reconstitution studies showed that Bcl-6 is required in a B-cell autonomous manner for the GC reaction (99–101). Although Bcl-6 protein is present ubiquitously at low levels, it is elevated in GC B cells (24, 102). Regulation of Bcl-6 expression is complex, involving both transcriptional and posttranscriptional mechanisms and including negative autoregulation of transcription (103–105). Bcl-6 mRNA is expressed ubiquitously (103), and transcriptional regulation of the gene is only partially understood. Bcl-6 represses its own transcription via two regulatory elements within intron 1 (105). Also, Blimp-1 downregulates Bcl-6 mRNA, but it is not known if BCL-6 is a direct target of Blimp-1 (21). Although resting B cells and germinal center B cells express comparable levels of Bcl-6 mRNA, germinal center B cells express 3- to 34-fold more Bcl-6 protein compared to resting B cells (103). In quasimonoclonal BCR mice, delivering a strong signal via BCR induces Bcl-6 and a GC reaction in vivo (106). It would be interesting to determine if Bcl-6 is also induced via BCR signals from these B cells in vitro, possibly providing a system for further study on posttranscriptional regulation of Bcl-6 during the initiation phase of the GC reaction. When microarrays were used to identify genes repressed by Bcl-6 in GC B cells, PRDM1 (the gene encoding Blimp-1) was identified as a target (107). Two Bcl-6 response elements have been located in the murine prdm1 gene within introns 3 and 5 (C. Tunyaplin & K. Calame, unpublished), confirming that prdm1 is a direct target of Bcl-6-dependent transcriptional repression. Because Blimp-1 induces plasmacytic differentiation (108), Bcl-6-dependent repression of Blimp-1 in GC B cells appears to prevent premature plasma cell differentiation. Indeed, enforced expression of Bcl-6 inhibits plasma cell differentiation, possibly by inhibiting the activity of STAT3 (109).

Pax5 Pax5 (also called B-cell lineage-specific activator protein or BSAP) is required not only for early commitment to the B lineage (110), but also for maintenance of B cell identity later in development (111). However, enforced expression of Pax5, achieved either experimentally (112, 113) or as a result of chromosomal translocation (114), blocks plasma cell differentiation and Ig secretion. Thus, like Bcl-6, Pax5 appears to be required for GC B cells but is inhibitory for plasma cell differentiation. Pax5 is a dual-function transcriptional activator and repressor (115, 116). Many genes activated by Pax5, including CD19, CD79A, syk, and BLNK (111,

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116–118), are highly expressed in germinal center B cells (119). Because these genes participate in BCR signaling, this is consistent with the idea that BCR signaling is critical for GC B cells. Pax5 represses genes involved in Ig secretion. Including XBP-1 (118), J chain (117, 120, 121), IgH (122), and possibly Igκ (123–125), consistent with the requirement for its repression in plasmacytic differentiation.


Termination of the Germinal Center Reaction Somatic hypermutation and selection during the germinal center reaction result in B cells expressing Ig with high affinity for antigen. As discussed above, strong BCR signals may be important for termination of the GC reaction and commitment to a plasma cell fate. This involves decreased Bcl-6 expression, relieving Bcl-6dependent repression of prdm1. In Ramos cells, BCR stimulation causes Bcl-6 to be phosphorylated by MAP kinase, ubiquitinated, and degraded via the proteasome pathway (104). Because GC cells constantly receive BCR signals, it seems likely that in vivo only strong BCR signals cause enough Bcl-6 degradation to relieve repression of prdm1. Consistent with this idea, B cells incapable of BCR affinity maturation (due to absence of activation induced cytidine deaminase, or AID) are trapped at the germinal center stage (126, 127). Also, de-regulated Bcl-6 expression occurs in many diffuse large B-cell lymphomas that have a GC phenotype (128), consistent with the idea that absence of Bcl-6 is required for B cells to develop beyond the GC stage. Once Bcl-6-dependent repression is relieved, expression of Blimp-1 ensures the irreversibility of plasma cell differentiation by repressing BCL-6 (21). Double negative feedback between these two important transcriptional regulators is probably critical for both GC and plasma cell fate decisions (Figure 1). Cytokines secreted by follicular dendritic cells and/or T cells may also induce Blimp-1. Blimp-1 is induced in many mature B cell lines by IL-2, IL-5, or IL-6, all of which activate STAT3 (108; K.L. Lin, unpublished data). In addition, IL-10, which also activates STAT3 (129), promotes in vitro differentiation of GC B cells into plasma cells (93). An alternate fate at the end of the GC reaction is to become a memory B cell. Memory B cells do not express Blimp-1 (24), but Bcl-6 is present in human memory B cells (L.M. Staudt, personal communication), although levels are lower than in GC B cells and similar to those in na¨ıve B cells (G. Cattoretti, personal communication). Expression of Bcl-6 in memory B cells may be sufficient to repress prdm1 and prohibit plasma cell differentiation, just as it does in germinal center B cells. Expression of XBP-1 and Pax5 has not been studied in memory B cells, and it is currently unclear what determines the memory B cell fate. BCR affinity for antigen has been proposed to be involved in the decision (87). One model posits that germinal center B cells with high-affinity BCR are selected to differentiate into plasma cells; cells with intermediate affinity differentiate into memory B cells; and cells with low-affinity BCR, which cannot compete for the survival signal, die from apoptosis. However, two recent studies show that, in the absence of competition, B cells expressing receptors with low affinity can mature into Ig secreting cells (85, 130), showing that a high-affinity BCR is not an absolute

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requirement to exit the GC and become a plasma cell, even though it does provide a competitive advantage for GC selection and survival (85). In addition to signals from the BCR, cytokines influence the development of memory B cells. IL-4 represses Blimp-1 expression (131), induces Bcl-6 mRNA and protein in a STAT-6-dependent manner (132), and promotes memory B cell development in vitro (95, 133). Engagement of accessory molecules such as CD40 (92) or Fcγ RIIb (134) on GC B cells also appears to favor memory B cell fate. Interestingly, engagement of Fcγ RIIb on B cells results in decreased Ras and MAPK activity (135). This may block Bcl-6 phosphorylation and degradation and stabilize Bcl-6 enough to keep prdm-1 repressed, thus pushing the cells to a memory fate.

ROLES FOR SPECIFIC TRANSCRIPTION FACTORS IN PLASMA CELL DIFFERENTIATION AND FUNCTION Commitment to a plasma cell fate causes a sea change in the gene expression program and phenotypic characteristics of B cells. Below we discuss our current understanding of transcriptional regulators required for the plasma cell phenotype. Some of this information is summarized in Figure 2.

XBP-1 An absolute requirement for XBP-1 (X-box-binding protein-1) in plasmacytic differentiation was demonstrated by analysis of XBP-1-deficient lymphocytes in the RAG-2 complementation system (136). XBP-1 is a basic–region leucine zipper protein in the ATF/CREB family. XBP-1−/− embryos die due to liver hypoplasia and anemia (137). Although XBP-1 is expressed ubiquitously, XBP-1 transcripts increase during plasmacytic differentiation (136, 138). Strikingly, chimeric RAG−/− mice lacking XBP-1 in their lymphocytes have severely impaired Ig secretion, even though their T and B cells develop normally and GC formation and cytokine secretion are normal (136). XBP-1 is intimately involved in the unfolded protein response (UPR), which is activated during endoplasmic reticulum (ER) stress. XBP-1 is induced by ATF6, which is activated in response to ER stress (139). In addition, a mammalian form of IRE1 is activated during the UPR and splices XBP-1 mRNA to encode a more stable protein (42, 140, 141). This stable form of XBP-1 protein is highly induced in response to LPS stimulation of splenic B cells (42), suggesting it is the primary form of XBP-1 in plasma cells. Although no target genes regulated by XBP-1 in plasma cells have been identified yet, its association with the UPR suggests XBP-1 may activate genes required for Ig secretion in plasma cells. XBP-1 is repressed by Pax5 (118). Blimp-1-dependent repression of Pax5 is required for induction of XBP-1 during plasmacytic differentiation but is not sufficient for full induction (21, 142). Signals from IL-6 (138) and cues from the UPR that activate ATF6 (139) also induce XBP-1 transcription.

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Blimp-1 Blimp-1 was identified based on its induction during cytokine-dependent plasmacytic differentiation of the BCL-1 B cell lymphoma line (108). Blimp-1 is a 98-kDa transcriptional repressor containing five zinc finger motifs that confer DNA binding ability (143). A 12 bp consensus binding site, GTAGTGAAAGTG, has been determined for Blimp-1 (T. Kuo & K. Calame, unpublished). A proline-rich region N-terminal to the zinc finger motifs is required for transcriptional repression via association with hGroucho and class I histone deacetylases (144, 145). In vivo, Blimp-1 is found in all plasma cells, including those formed in a primary response to either a TI or TD antigen and those formed from memory cells in a secondary response and in long-lived plasma cells in human bone marrow (24). In addition, it is expressed in a subset (5%–15%) of germinal center B cells but not found in memory B cells (24). Consistent with its expression pattern in plasma cells, Blimp-1 has the unique ability to drive plasmacytic differentiation upon enforced expression in BCL-1 cells (108) or primary splenic B cells (146, 147). An inhibitory form of Blimp-1 (TBlimp) blocks cytokine-dependent plasmacytic differentiation in cell culture or LPS-dependent plasmacytic differentiation of primary splenic B cells (21, 142, 148), suggesting an essential role for Blimp-1 in plasma cell differentiation. However, since Blimp-1-deficient mice die as embryos (M. Davis, personal communication; M. Shapiro & K. Calame, unpublished), this has not yet been confirmed using gene targeting. A recent microarray study showed that Blimp-1 represses more than 225 genes and induces more than 30 (21). More than 10% of named genes regulated by Blimp-1 in this study were transcription factors (Table 1), suggesting a cascade of gene regulation initiated by Blimp-1 (21). The five known direct targets of Blimp-1 repression, c-myc, CIITA, Pax5, SpiB, and Id3, are all transcription factors. Blimp1 is the previously described plasmacytoma repressor factor binding in the c-myc promoter (149). Ectopic expression of either c-Myc or cyclin E blocked BCL-1 differentiation, establishing a requirement for c-myc repression and cessation of cell cycle in plasmacytic differentiation (11). However, repression of c-myc is not sufficient to differentiate BCL-1 cells because a dominant negative form of c-Myc cannot initiate the differentiation program (11). Blimp-1 represses promoter III of CIITA, a transcription coactivator that is critical in regulating the expression of class II MHC and two genes, invariant chain (Ii) and DM, whose products facilitate the presentation of exogenous peptides in the context of class II MHC (147, 150, 151). Identification of CIITA as Blimp-1 target extended earlier studies implicating a plasma cell–specific soluble repressor for CIITA (48, 152). Pax5 (also called B-lineage specific activator protein or BSAP) is repressed by Blimp-1. Pax5 is a transcription factor that is critical for commitment to and maintenance of the B-lineage (110, 153) and for B cell function through the GC stage (154). Pax5 activates genes involved in B cell identity (111), such as CD19, CD79A,

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TABLE 1 Transcription factors shown to be downregulated by Blimp-1 by microarray analysis Target Activated A/BMyb BLC-6 Annu. Rev. Immunol. 2003.21:205-230. Downloaded from arjournals.annualreviews.org by HINARI on 09/01/07. For personal use only.

Repressed

Sox4 CDC2 Id2

CIITA

Class II MHC Invariant chain

c-Myc

RCL1 DHFR ODC LDH-A CDC2

EBF

CD79a Pax5

a

E2A

AID

E2F-1

c-Myc DHFR B-Myb PCNA cdc47 CDC2

Oct2/OBF

CD79a CXCR5 Class II MHC

Pax5

CD19 CD79a CD20 BLNK CIITA

Spi-B

Btk

Gadd 153 Gadd45 p21

J chain IgH XBP-1

Several targets, such as CD79a and Class II MHC, are regulated by more than one transcription factor. Other targets, such as CIITA and c-Myc, are regulated by both direct and indirect mechanisms. a

A recent study has shown that E2A activates AID (C. Murre, personal communication).

CIITA and BLNK (111, 115), and Pax5 represses genes involved in antibody secretion such as J chain (117, 120, 121), IgH (122), possibly Igκ (124, 125), and XBP-1 (118). Pax5 is required for GC B cells and for proliferation of splenic B cells treated with LPS ex vivo (155). Although Pax5 inhibits plasmacytic differentiation and Ig secretion when ectopically expressed in plasma cell lines (112) and in

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splenic B cells (142), deletion of Pax5 in late-stage B cells is not sufficient to induce Blimp-1 and J chain or to trigger plasmacytic differentiation (111). XBP-1 may be a particularly important target of Pax5 in the context of plasma cell biology since XBP-1 is critical for plasma cell formation and Ig secretion (136). Blimp-1 binds in the Pax5 promoter and represses Pax5 transcription (142). Repression of Pax5 by Blimp-1 is required for IgM secretion by LPS-treated splenocytes because it can be inhibited by ectopic expression of TBlimp, a blocking form of Blimp-1 (142). Blimp-1 binds to evolutionarily conserved sites in regulatory regions of both Spi-B and Id3 in vitro and in vivo (21), providing evidence that the downregulation of SpiB and Id3 observed in microarrays is due to direct repression by Blimp-1. These transcription factors are required for effective BCR signaling (156–158). Overall, the microarray study showed that genes regulated by Blimp-1 comprise three programs: proliferation, Ig secretion and GC function and B cell identity (21). The proliferation program repressed by Blimp-1 includes repression of c-myc (and its targets such as RCL1, ODC, LDH-A, and DHFRI) as well as E2F-1 (and its targets such as c-myc, DHFR, PCNA, and CDC2), and the anti-apoptotic gene A1. The program also includes induction of cdk inhibitor p18, required for plasmacytic differentiation (12), and pro-apoptotic genes GADD45 and GADD153. Secondly, Blimp-1 induces expression of genes required for Ig secrection including XBP-1, J chain. and HSP70. Finally, Blimp-1 represses multiple genes required for B cell identity and GC function. Genes encoding BCR signaling components, including CD79A, BLNK, btk, PKCβ, lyn, syk, BRDG-1, CD45, and CD19 (159–163) are downregulated by Blimp-1 (21). BCR signaling inhibits differentiation of mature B cells into plasma cells, and this response involves repression of Blimp-1 (131, 146). Among the genes required for GC function, Blimp-1 downregulates BCL-6, AID, M17, AMYB, and SIAH-2 and CXCR5 (responsible for B cell homing to follicles). Blimp1 also represses genes required for isotype switch recombination and somatic hypermutation including AID, STAT6, Ku70, Ku86, and DNAPKcs. Apparently, inhibition of BCL-6, PAX5, AID, and other genes critical for GC function ensures that plasmacytic differentiation initiated by Blimp-1 is irreversible.

IRF4 Expression of IRF4, a member of the interferon regulatory factor family, is primarily restricted to lymphocytes (164), although expression is also seen in macrophages (165). IRF4−/− mice exhibit severe defects in mature T and B lymphocyte function and have no detectable antibody response or serum Ig. Peripheral B-cell development is blocked at the GC stage, and no plasma cells are present (166). This phenotype correlates well with the fact that in B cells IRF4 is highly expressed on a subset of cells in the light zone of the GC (84, 167) that also expresses Syndecan1 and Blimp-1 (24) and appears to be committed to a plasma cell fate. IRF4 is induced in vitro following stimulation of splenic B cells with anti-CD40 and IL-4

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(168). Thus, IRF4 appears to play a role at the developmental stage when plasma cell commitment occurs. It will be interesting to learn whether IRF4 is part of the Blimp-1/XBP-1 pathway or whether it works by an independent pathway. The ability of IRF4 to associate with other proteins is important for determining its binding and transcriptional activity. IRF4 binds to ISREs (interferon-stimulated response elements) or interferon-γ -activated sequences (GAS). IRF4 alone binds the ISRE motif and represses transcription of class I MHC (H2-Ld) (169). With other partners, such as a phosphorylated PU.1, IRF4 binds distinct sites (170). For example, in association with PU.1, IRF4 activates both the κ and λ light chain enhancers, which partially explains the defect in the production of Igs in IRF4-deficient mice (170–172). Association with PU.1 is also important for IRF4 activation of CD20 (173), toll-like receptor 4 (TLR4) (174), and IL-1β (175). IRF4 associates with STAT-6 to activate the human CD23 promoter via a GAS element, but activation in a cotransfection study was inhibited by Bcl-6 and Blimp-1 space (168, 176). These data emphasize the importance of determining how IRF4, Bcl-6, and Blimp-1 may interact functionally in vivo.

NF-ATc The nuclear factor of activated T cells (NF-AT) family is composed of at least four calcium-regulated members (NF-ATc1, NF-ATc2, NF-ATc3, and NF-ATc4), among which NF-ATc1 and NF-ATc2 are highly expressed in lymphocytes (177). NF-ATs are activated via signaling through TCR, BCR, and CD40 (178). Calcium signaling activates the phosphatase calcineurin and induces movement of NFATc proteins into the nucleus, where they interact with other proteins such as AP-1 to regulate genes [for recent review, see (179, 180)]. Although NFAT proteins were originally studied in the context of T cell activation, where they induce a variety of cytokine genes (177) and are required for effector differentiation (181), homeostasis, and Th2 differentiation (182), some recent studies suggest they also play a role in plasma cells. Mice lacking NF-ATc1 and/or NF-ATc2 in their lymphocytes have been studied in the RAG complementation system (181). IgG1 and IgE decreased in NF-ATc1/Rag2−/− chimeras but were elevated in the NF-ATc1 and NF-ATc2 double knockout chimeras, demonstrating complexity in the roles of individual family members. B cell hyperactivation and plasma cell expansion in the double knockout chimeras suggest a role for NFATc1 and 2 in plasma cell homeostasis and differentiation. However, it remains to be determined whether this is secondary to the T cell defects in these mice. In T cells NF-ATc2 enhances transcriptional activation by IRF4 (183), and similar mechanisms may also occur in B cells or plasma cells expressing both proteins.

Octamer proteins Octamer sites are highly conserved (184) and functionally important (185, 186) regulatory elements of Ig heavy and light chain promoters and the IgH 30 Cα enhancer. Octamer factors also activate CD79A and CD79B promoters (187–189).

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Oct-1, expressed ubiquitously, and Oct-2, which has more lymphoid-restricted expression, bind these sites in association with a B-cell specific cofactor OCA-B [reviewed in (190)]. Early on these observations suggested a critical role for octamer proteins in Ig transcription. Alternatively or in addition, the role of octamer proteins in Ig promoters and the 30 Cα enhancer might mean that Oct-1 and/or Oct-2, along with OCA-B, would be important in plasma cells, which require robust Ig transcription. Recent data do not fully support either model, although several possibilities remain untested. Oct-2−/− mice show normal Ig transcription and early B-cell development; however, they have decreased numbers of IgM+ B cells, and their splenic B cells are defective for Ig secretion following LPS treatment ex vivo (191). Studies with altered specificity mutants suggest, however, that Oct-1, rather than Oct-2, may be the critical regulator of Ig gene transcription during B cell development (192), and the phenotype of B cells lacking Oct-1 or both Oct-1 and Oct-2 has not been reported. Mice lacking OCA-B fail to make GCs and have reduced serum levels of switched isotypes. The development of B1 cells and plasma cells is normal (193, 194), a finding not consistent with a requirement for octamer proteins in plasma cells. However, there have been suggestions of additional, currently unidentified, octamer cofactors (195), which could be important in plasma cells. Octamer proteins also activate CXCR5 (196), which is expressed in GC B cells but is downregulated in plasmablasts and plasma cells (197). Blimp-1 downregulates the expression of Oct-2 and CXCR5 (21). Loss of CXCR5 is important for allowing plasma cells to leave the follicles and home to sites in the red pulp, medullary cords, and bone marrow (49). Thus, it may be important to turn off Oct-2 in plasma cells. If so, it will be particularly interesting to determine the role of Oct-1 in plasma cells.

CONCLUDING REMARKS Understanding the critical roles of Bcl-6 and Pax5 in inhibiting plasma cell differentiation and the importance of Blimp-1 and XBP-1 in the differentiation and function of plasma cells provides a rational basis for designing detailed studies to elucidate more fully the regulatory mechanisms that determine GC B cell, memory B cell, and plasma cell fate decisions. Given the development of powerful genomic and proteomic techniques, along with more sophisticated culture systems for primary B cells, we can expect rapid progress in the next few years. ACKNOWLEDGMENTS We are grateful to our colleagues and members of the Calame laboratory for helpful discussions and for communicating recent results. We thank Dr. Karen Hinrichs Sterling for critically reading the manuscript. This work was supported by RO1AI43576 and RO1AI50659 to K.C. K.L. is a Fellow of the Leukemia and Lymphoma Society (5332-00).

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191. Corcoran LM, Karvelas M, Nossal GJ, Ye ZS, Jacks T, Baltimore D. 1993. Oct-2, although not required for early B-cell development, is critical for later B-cell maturation and for postnatal survival. Genes Dev. 7:570–82 192. Shah PC, Bertolino E, Singh H. 1997. Using altered specificity Oct-1 and Oct-2 mutants to analyze the regulation of immunoglobulin gene transcription. EMBO J. 16:7105–1 193. Nielsen PJ, Georgiev O, Lorenz B, Schaffner W. 1996. B lymphocytes are impaired in mice lacking the transcriptional co-activator Bob1/OCA-B/OBF1. Eur. J. Immunol. 26:3214–18 194. Qin XF, Reichlin A, Luo Y, Roeder RG, Nussenzweig MC. 1998. OCA-B integrates B cell antigen receptor-, CD40Land IL 4-mediated signals for the germinal center pathway of B cell development. EMBO J. 17:5066–75 195. Schubart K, Massa S, Schubart D, Corcoran LM, Rolink AG, Matthias P. 2001. B cell development and immunoglobulin gene transcription in the absence of Oct-2 and OBF-1. Nat. Immunol. 2:69– 74 196. Wolf I, Pevzner V, Kaiser E, Bernhardt G, Claudio E, Siebenlist U, Forster R, Lipp M. 1998. Downstream activation of a TATA-less promoter by Oct-2, Bob1, and NF- kappaB directs expression of the homing receptor BLR1 to mature B cells. J. Biol. Chem. 273:28831–36 197. Wehrli N, Legler DF, Finke D, Toellner KM, Loetscher P, Baggiolini M, MacLennan IC, Acha-Orbea H. 2001. Changing responsiveness to chemokines allows medullary plasmablasts to leave lymph nodes. Eur. J. Immunol. 31:609–16 198. Tourigny MR, Ursini-Siegel J, Lee H, Toellner KM, Cunningham AF, Franklin DS, Ely S, Chen M, Qin XF, Xiong Y, MacLennan IC, Chen-Kiang S. 2002. CDK inhibitor p18(INK4c) is required for the generation of functional plasma cells. Immunity 17:179–89


Figure 1 Transcriptional mechanisms determining germinal center B cell, memory B cell and plasma cell fates. Solid black indicates proteins that are expressed and the regulation they effect. Light gray indicates proteins that are not present and regulation that does not occur. Blue indicates Bcl-6 present at an intermediate level with unknown function in memory cells. Expression and activity of Pax5 and XBP-1 in memory cells, indicated by parentheses, is inferred but not known.


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Figure 2 Transcriptional cascades and their outcomes in plasma cells. Direct targets of Blimp-1 are shown in red. Although XBP-1 is required, no target genes in plasma cells are currently known.


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Annu. Rev. Immunol. 2003. 21:231–64 doi: 10.1146/annurev.immunol.21.120601.141152 c 2003 by Annual Reviews. All rights reserved Copyright ° First published online as a Review in Advance on November 7, 2002

BAFF AND APRIL: A Tutorial on B Cell Survival Fabienne Mackay,1 Pascal Schneider,2 Paul Rennert,3 and Jeffrey Browning3 Annu. Rev. Immunol. 2003.21:231-264. Downloaded from arjournals.annualreviews.org by HINARI on 09/01/07. For personal use only.

1

Garvan Institute of Medical Research, Department of Arthritis and Inflammation, Darlinghurst, Australia; email: [email protected] 2 Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland; email: [email protected] 3 Biogen, Inc., Departments of Immunology and Exploratory Sciences, Cambridge, Massachusetts; email: paul [email protected], jeff [email protected]

Key Words B lymphocyte, TNF family, autoimmunity, maturation, spleen ■ Abstract BAFF, a member of the TNF family, is a fundamental survival factor for transitional and mature B cells. BAFF overexpression leads to an expanded B cell compartment and autoimmunity in mice, and elevated amounts of BAFF can be found in the serum of autoimmune patients. APRIL is a related factor that shares receptors with BAFF yet appears to play a different biological role. The BAFF system provides not only potential insight into the development of autoreactive B cells but a relatively simple paradigm to begin considering the balancing act between survival, growth, and death that affects all cells.

INTRODUCTION When lacking appropriate growth and survival signals, most cells die. Death as the default pathway provides insurance against inappropriate events such as cell transformation. In the immune system, the balance between survival and death underlies the controlled cell expansion in response to pathogens, subsequent diminution of the response, tolerance to self, and homeostasis of the compartment size (1). The control of cell survival is believed to involve regulation of the anti-apoptotic machinery. This control is exquisite, as evidenced by the profound dysregulation of the immune system that occurs upon alteration of this mechanism. The elements that affect lymphocyte survival have been the focus of much attention in recent years. The problem has been difficult to unravel because the immune system can be either quiescent or vigorously proliferating; in addition, survival elements are often embedded within the context of cell activation and proliferation. A factor called BAFF was recently discovered that is clearly a survival factor for most B cells. APRIL is a second molecule related to BAFF and shares some of the BAFF receptors. APRIL may play a different biological role but because it shares some features with BAFF, it is discussed in parallel. BAFF and its receptor interactions 0732-0582/03/0407-0231$14.00

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have been reviewed extensively (2–12). Here we have focused on the survival aspect of this system as it applies to B cell biology and to other TNF family members.

BIOCHEMISTRY OF THE BAFF AND APRIL SYSTEM


BAFF/APRIL Ligands BAFF (B cell activation factor of the TNF family, TNFSF 13b) and APRIL (a proliferation-inducing ligand, TNFSF 13a) are TNF ligand family members displaying typical features of type II transmembrane proteins that were discovered by homology searches of the nucleic acid databases (13, 14). BAFF is also known as TALL-1, THANK, BLyS, and zTNF4 (15–18). The gene for BAFF is located on human chromosome 13q34 and on mouse chromosome 8 (13, 17, 19). The 13q34 locus is frequently involved in chromosomal translocations in patients with Burkitt lymphoma-leukemia (20). In the mouse genome, the Baff gene is identical in six common strains, but several polymorphisms have been detected in the NZB autoimmune prone strain (19). APRIL, also known as TRDL-1, is found close to the telomeric end of human chromosome 17p13.1 and in a similar syntenic position on mouse chromosome 11 (21). In both the human and mouse genomes, the APRIL gene is flanked within 1–2 kb by another TNF family member called TWEAK (TNFSF 12) and on the other side by Smt3ip1/Senp-3, a member of the sentrin/SUMO specific protease family (22). The exon organization and position of these three genes is very closely retained in both species. In humans, APRIL lies about 200 kb telomeric from the p53 tumor suppressor gene and Fg f-11 is about 14 kb distal to APRIL. This orientation is roughly preserved in mice. TNF family ligands are characterized by a C-terminal domain coined THD (for TNF homology domain). Consensus sequences critical for beta sheet formation and trimerization of this domain earmark the TNF family. Within the THD, BAFF shares roughly 20 to 30% similarity with 16 other family members, namely TNF, LTα, LTβ, LIGHT, FasL, TRAIL, RANKL/ODF/TRANCE, TL1A/VEGI, GITRL/AITRL, EDA, TWEAK, CD40L, CD27L/CD70, CD30L, 4-1BBL, and OX40L, and up to 50% similarity with APRIL, a ligand that may share several biological activities with BAFF (23). BAFF and APRIL are also related at the level of their genomic organization. Alternative splicing of exon 3 has been found in both the BAFF and APRIL genes, although these splice variant forms of APRIL and BAFF are not found on the cell surface nor are they secreted in transient expression studies (21; Ambrose & Rennert, unpublished data). Other striking features of the chromosomal organization of the BAFF and APRIL genes are the occurrence of an intron in the middle of the THD (within sheet C) and the presence of a short exon encompassing a four-amino-acid residue consensus sequence that corresponds to the specificity of furin-type proteases (Arg-Xxx-Arg/Lys-Arg). In contrast, other members of the family have most of their C-terminal domain encoded within a single exon and lack the exon containing the furin site (24).

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The furin cleavage sites are located on the N-terminal side of the TNF homology domain and permit the release of soluble forms of BAFF, APRIL, EDA, and TWEAK (13, 17, 25–29). Cleavage at the furin consensus site is essential for the function of EDA, and mutations that abolish the furin site are functionally as severe as those leading to a complete absence of the ligand (27, 28). The cleavage and secretion of BAFF was unequivocally demonstrated in a BAFF-transgenic (Tg) animal in which the full-length form was overexpressed in the liver. Soluble BAFF was detected in the blood and B cell hyperplasia was observed globally, indicating transfer of the soluble BAFF from the liver into organs in which the transgene was not expressed (30). Soluble BAFF can be detected in the blood of patients with various autoimmune diseases (18, 31–33). Although BAFF, APRIL, EDA, and TWEAK are biologically active in a soluble form, it is not known whether the processing of BAFF is absolutely required for its activity or whether the membrane-bound form alone could suffice. In this respect, it is noteworthy that membrane-bound BAFF has been detected on the surface of human monocytes and murine dendritic cells, as well as on cells infiltrating the salivary glands of patients suffering from Sjögren’s syndrome (17, 31, 34). In general, BAFF appears to be expressed primarily by monocytes and dendritic cells, although the precise picture is still ill defined. Although the BAFF convertase has not been described, a more fundamental question remains: What are the physiological and pathological consequences of global soluble versus local membrane-bound BAFF? If the TNF example is any indication, this problem will baffle investigators for some time. A further structural link between BAFF, APRIL, EDA, and TWEAK is the presence of a characteristic intramolecular disulfide bridge linking β-strands E and F within the TNF homology domain, which is apparent in three independent X-ray structures of BAFF (35–37). Interestingly, this disulfide bridge is also present in Eiger, the only TNF homologue in Drosophila species (38). Furthermore, the Eiger receptor, called Wengen, is a small, single-domain TNF family receptor that resembles Fn14 and BAFF-R, the receptors for TWEAK and BAFF respectively (39). These considerations suggest that the BAFF, APRIL, EDA, and TWEAK clade is derived from a primordial or Ur-TNF and raises the intriguing possibility that they control related biological functions predating the emergence of the adaptive immune system.

BAFF Structure Crystal structures of BAFF reveal a trimeric organization similar to that of TNF, LTα, CD40L, RANKL, and TRAIL, except that the shape of the molecule is slightly flatter and wider (35–37). A unique feature of BAFF is the presence of two magnesium ions that bind within the trimer along the threefold axis of symmetry and interact with the side chains of Gln234, Asn235, and Asn243 (36). An analogous situation occurs in TRAIL, where the Cys230 residues of each monomer coordinate a zinc atom at the center of the trimer (40). Because only one of the

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three chelating side chains is conserved in APRIL, it is unclear whether this ligand will chelate a metal ion. Another structural hallmark of BAFF is the insertion of five amino acid residues between β-strands D and E. This creates an especially deep cleft at the subunit junction in the lower half of the molecule. Several interpretations regarding the impact of this loop with respect to receptor binding have been made. In one model, this flexible loop is part of a tight binding site compensating for the smaller contact region provided by a single domain receptor (35). In addition, the BAFF cleft region is very acidic while the surface of the BAFF-R appears to be basic. These surface charge interactions may underlie the inability of APRIL to bind to BAFF-R since the corresponding cleft region was found to be basic in a computational model of APRIL (35). In a second model, the DE loop obstructs half of the receptor-binding groove, favoring the binding of short, BCMA-type receptors to the remaining portion of the cleft (36). (BCMA receptors are discussed below.) Crystal structures and deletion experiments suggest that the DE loop of BAFF is not involved in receptor binding but rather promotes intertrimer interactions that can ultimately lead to the pH-dependent formation of an ordered and symmetric structure comprising no less than 20 trimers (37). Because of geometrical constraints, such a structure can be observed only after the processing of membrane-bound BAFF to its soluble form. High valency engagement of receptors by oligomeric BAFF could enhance its signaling ability, as shown for some other TNF receptor (TNF-R) family members (41). However, soluble, nonaggregated, recombinant, eukaryotically derived, trimeric BAFF appears to be fully capable of inducing B cell proliferation in the anti-immunoglobulin M (anti-IgM) costimulation assay and, moreover, in our hands the DE loop deletion still retains activity (J. Thompson & F. Qian, unpublished observations). Further experiments will be required to clarify the biological significance of highly oligomerized BAFF.

BAFF and APRIL Receptors Both BAFF and APRIL bind to two receptors called BCMA (B cell maturation antigen, TNFRSF 17) and TACI (transmembrane activator and CAML interactor, TNFRSF 13b) (18, 42–47). BCMA was identified as a translocation event in a human T cell lymphoma, yet its expression is mostly limited to mature B cells (48). TACI was initially cloned using a two-hybrid screen for proteins that could interact with calcium-modulator and cyclophilin ligand (CAML) and is found on some T and B cells (49). Interactions of both BCMA and TACI with BAFF and APRIL were uncovered in deorphanization screens using panels of recombinant ligands and receptors, as well as by expression cloning. The lack of appreciable BCMA or TACI expression on the BAFF-binding BJAB cell line, and the inconsistent phenotypes of the receptor knockout mice, led to the search for and expression cloning of the third BAFF receptor, called BAFF-R (TNFRSF 13C) (50, 51). This receptor selectively binds BAFF and not APRIL. BCMA, TACI, and BAFF-R are located on human chromosomes 16p13.1, 17p11.2, and 22q13.2, and mouse

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Figure 1 Illustration of the receptors that bind to BAFF and APRIL. BAFF and APRIL have consensus furin protease cleavage sites in the stalk region that allow for their secretion. Each box in the extracellular domain of the receptors represents a distinct module.

chromosomes 16, 11, and 15, respectively. Four haplotypes of BCMA have been noted, but none were linked to autoimmune disease (52). The binding interactions of APRIL and BAFF are outlined in Figure 1. Multiple receptor and ligand interactions are frequent in the TNF field, where 30% of the receptors and 40% of the ligands share more than one interaction partner. One needs to approach this biochemical complexity with caution. It appears that as receptors and ligands duplicate and evolve to encompass new functions, biochemical cross reactivity can be retained. Even though promiscuity is observed in biochemical analyses, the temporal expression patterns and the cellular geography in vivo can severely limit the relevant signaling events. Therefore, the biology may be simpler than the biochemistry suggests. For instance, the related ligands

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TNF and LTα share two TNF receptors, yet the biology of LTα is dominated by the heteromeric LTα/β ligand acting through the LTβ receptor. The decoy receptor, OPG, inhibits RANKL activity and binds the ligand TRAIL, yet obvious interactions between the RANK and TRAIL systems have not emerged to date. Genetic analyses outlined below clearly indicate that BAFF:BAFF-R interactions control B cell survival and that APRIL, BCMA, and TACI do not deliver such a signal. Despite the similarities between BAFF and APRIL, their biological roles must be distinct.

Receptor Structure The signature of the TNF-R family resides in the cysteine-rich extracellular moiety, which typically contains two to eight elementary structural modules, each stabilized by one or two disulfide bridges. Two modules form the conventional cysteine-rich domain (coined CRD) that forms the basic ligand binding unit of this structural class (23). BCMA is a very small receptor containing a single TNFreceptor CRD domain. Only the TWEAK-binding Fn14 and the Eiger-binding Wengen receptors are similar to BCMA in this respect (39, 53). TACI contains two such domains encoded by distinct exons that may have appeared by duplication. A distinct module not commonly found in other receptors characterizes the CRDs of BCMA and TACI. The third receptor for BAFF, BAFF-R, is even more unusual because it contains only a partial, atypical TNF-type module. This sequence was not recognized by search algorithms and thus its identification required expression cloning (50, 51). Examination of the two available crystallized receptor-ligand complexes reveals that three out of five (TRAIL-R2) or three out of eight (TNF-R1) modules are directly involved in ligand binding (40, 54). In the case of BCMA and BAFF-R (and probably TACI), only one or two modules must be sufficient to form a high affinity contact with the ligand. Other structural aspects of this group of receptors extend the limits of receptor diversity in the family. First, BAFF-R, TACI, and BCMA along with X-linked EDA receptor (XEDAR) are likely to be members of the type III class of membrane proteins. First noted in viral genomes, these unique membrane proteins are oriented like type I membrane proteins, yet lack leader sequences (55). BAFF-R and TACI appear to be conventional plasma membrane receptors; however, BCMA is clearly unusual in that it is localized in the Golgi compartment (56). In general, the localization of TNF receptors has not been well examined, and in several cases, the bulk of the protein is retained in intracellular organelles. For example, TNF-RI is predominantly localized in the endoplasmic reticulum, which is interesting since excessive surface expression is probably lethal (57). Likewise, some TRAIL receptors are found in an intracellular location and relocalized to different compartments upon TRAIL-initiated signaling (58). Transient transfection with BCMA constructs can force the appearance of BCMA on the surface, yet examples of genuine surface expression are rare. As detailed below, BCMA is not relevant to BAFF signaling, so why does this apparently intracellular receptor

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form exist? Some intracellular receptor signaling events have been described; fibroblast growth factor is one of the most developed examples. Perhaps surface expression is tightly regulated and a specific conventional ligand-receptor interaction will emerge. Alternatively, one may need to entertain a more unusual role, e.g., a chaperone or transport function. Human BCMA, TACI, and BAFF-R bind both mouse and human ligands relatively well, i.e., with the nanomolar affinities expected for biologically relevant interactions in this family (44; Thompson & Ambrose, unpublished observations). Murine BCMA binds murine APRIL effectively but not murine BAFF, while murine TACI binds both ligands. Mice expressing a murine BCMA-Ig as a transgene lacked an obvious phenotype, while the TACI-Ig transgenic mouse had a reduced B cell compartment similar to that observed in BAFF−/− mice (59). The ligand-binding domain of TACI is linked to the transmembrane domain by a relatively long stalk that can be proteolytically processed at various basic sites, potentially releasing a decoy form of the receptor (59). Although processing of endogenous TACI has not been demonstrated, such a mechanism would be in line with the unexpected observation that TACI−/− mice have an expanded B cell compartment (60). A cleaved form of TNF-R1 is known to buffer the activity of TNF, and impaired processing leads to a periodic fever syndrome in humans (61). Alternatively, signaling through the membrane-bound TACI exerts a negative regulatory effect.

BAFF Receptor Signaling The signal transduction pathways utilized by BCMA, TACI, and BAFF-R are only partially characterized. TNF-R family members rely on interactions with death domain–containing proteins, TNF receptor–associated factors (TRAFs), and several receptor-specific associated proteins in order to trigger apoptotic or survival signals. Death domain motifs are absent in the three BAFF binding receptors. TRAF proteins can activate NF-κB and MAPK pathways, and the potent antiapoptotic activity associated with NF-κB activation would be consistent with the survival function of BAFF (62, 63). The intracellular domain of BCMA contains two consensus TRAF binding sites (TVEE and AMEE), and deletion studies indicate that the first site is largely responsible for the binding of TRAFs (1–3) and for TRAF-dependent activation of NF-κB, p38 MAPK, and JNK, but not ERK (Figure 1) (64). TACI activates the transcription factors NF-κB, AP-1, and NF-AT; the latter is dependent on AP-1 (49). The intracellular domain of TACI has a classical consensus TRAF-binding sequence (PTQE) that is conserved across species, and an upstream TRAF-6 binding site with a minimal consensus sequence (PXE). TRAF-2, -5, and -6 were found to interact with these sites in a yeast two-hybrid screen (65). In addition, the intracellular membrane-proximal portion of TACI interacts with the N-terminal part of CAML, a positive regulator of the calcium-dependent phosphatase calcineurin (49, 65). Activated calcineurin dephosphorylates the NF-AT transcription factors,

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allowing their dissociation from the cytoplasmic sequestering protein 14-3-3 and translocation to the nucleus. The cytoplasmic tail of BAFF-R is devoid of obvious TRAF-binding consensus sites but shares a short stretch of homology with BCMA. An insertion of 21 amino acids in place of the last 8 amino acids occurs in the A/WySnJ strain, leading to a loss of BAFF-R function (50, 51). Therefore the final C terminus of BAFF-R is crucial for survival signaling. Stimulation of tonsillar B cells with BAFF in the presence of a polyclonal B cell activator results in the induction of the mRNA for Polo-like kinase, a Ser/Thr kinase that controls separation of sister chromatids during the prophase of mitosis (25, 66). BAFF also activated the transcription factor ELF-1, which belongs to the ETS family of transcription factors that modulates the activity of other transcription factors (25, 67). ELF-1 specifically binds Jun and NFκB transcription factors and regulates several genes related to immune response, including that for CD25/interleukin-2 receptor alpha subunit (IL-2Rα) (25). It is not known whether these responses are mediated through TACI or BAFF-R. The phenotypes of BAFF-, BCMA-, TACI-, and BAFF-R-deficient mice clearly indicate that the BAFF survival signal in transitional and mature B cells is mediated by BAFF-R in mice, and not through BCMA and TACI (46, 50, 68–70). One can speculate that the survival function of BAFF-R signaling results from enhanced anti-apoptotic or diminished pro-apoptotic activity of the Bcl family members. Indeed, Tg expression of Bcl-2 in B cells, or the removal of the Bcl-2 antagonist Bim, led to phenotypes remarkably similar to that of BAFF-Tg mice, specifically B cell hyperplasia and autoimmunity (30, 71–73). In addition, increased Bcl-2 levels were noted in a fluorescence activated cell sorter (FACS) analysis of B cells from BAFF-Tg mice (30). Slightly different results were obtained in studies using cultured cells, yet in these cases BAFF regulated anti-apoptotic and proapoptotic Bcl family elements in a manner consistent with a prosurvival effect (74, 75). Abrogation of NF-κB signaling in lymphocytes (c-Rel−/− and RelA−/− double knockout) led to the failure to upregulate Bcl-2 and A1, and A1 is expressed during B cell maturation at the point where BAFF is required (76, 77). The doubly deficient c-Rel/RelA mice had a block in B cell maturation very similar to that observed in mice lacking BAFF or BAFF-R, and Bcl-2 expression rescued the B cell deficiency (69, 70, 76, 78). In the A/WySnJ strain, which has a dysfunctional BAFF-R gene, transitional B cells have elevated expression of a pro-apoptotic Bik-like killer gene (Blk) (79). BAFF-R signaling may negatively regulate Blk. It is possible that BAFF-R is coupled to the Bcl family via NF-κB activation, yet how this NF-κB activation occurs is unclear. One example lies in the LTβR system, in which LTβR signaling induces NF-κB inducing kinase (NIK) to activate IKKα (79a). Mature B cell numbers are reduced in mice lacking NIK or p52 and in irradiated mice reconstituted with IKKα −/− lymphocytes (80–82). BAFF did activate NF-κB in U937 or TACI transfected 293 cells, although this activation does not relate to the obligate BAFF-R activation events (16, 83). However, BAFF induced p50/RelB in resting primary B cells, and this event is more likely to be mediated by BAFF-R (74). Table 1 lists the effects on the size of the mature B cell compartment of manipulation of genes possibly relevant to the BAFF system. In

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TABLE 1 Effects of genetic manipulation on size of mature B cell compartment


References Manipulations resulting in expansion BAFF-Tga Bcl-2 Tg Bim−/−

(18, 30, 71) (72) (73)

Manipulations resulting in major loss TACI-Tg BAFF−/− BAFF-R−/− A/WySnJ strain p50 (NF-κB1)/p52(NF-κB2) c Rel/RelA−/− p50(NF-κB1)/RelB−/− Bcl-xl−/−

(70) (69, 70) Unpublisheda (50, 79) (154) (76) (145)b (146)

Manipulations resulting in partial loss NIK−/− IKKα −/− P52 (NF-κB2) Bcl-3−/− RANK or RANKL−/− CD19−/−

(82) (80) (147, 148) (149) (139) (150)

Manipulations resulting in no loss BCMA−/− TACI−/− TACI/BCMA−/− RelB−/− p50 (NF-κB1)−/− CD21−/−

(46, 69) (60, 68) Unpublisheda (151) (152) (150)

a

M. Dobles & M. Scott.

b

Bone marrow defect.

IKKα −/− and NIK-deficient B cells, p52, which is one of the five proteins involved in the formation of homo- and heterodimers of NF-κB, fails to be processed from its p100 precursor and impairs transcription of several NF-κB responsive genes (80, 84). It is therefore tempting to speculate that BAFF-R controls the activity of NIK and IKKα in B cells and promotes survival by upregulation of Bcl-2 family members. On the other hand, the disruption of the mature B cell compartment by NIK and IKKα deficiency is not as severe as that resulting from the loss of BAFF; moreover, NIK−/− mice have reduced populations of immature B cells in the bone marrow, pointing to other possible BAFF-independent defects (Table 1). Perhaps an IKKα axis is only partially involved in BAFF-R signaling. It will be important to define the BAFF-R signal transduction pathway that is so critical for B cell survival.

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BAFF IN THE IMMUNE SYSTEM


BAFF is a B Cell Survival Factor During maturation, immature B cells generated in the bone marrow (BM) enter the spleen, where they go through two or three intermediate stages, namely transitional type 1 (T1), type 2 (T2), and type 3 (T3), prior to maturity (85, 86). Figure 2 outlines the development and maturation of B cells in the periphery. The first indication that BAFF may be a survival factor came from the vastly expanded B cell compartments found in mice overproducing BAFF (18, 30, 71). Furthermore, in vitro survival assays using recombinant soluble BAFF confirmed the direct survival effect of BAFF on B cells (87). This in vitro experiment has been done in several formats with slightly differing results (44, 74, 87, 88). The experiments have used various media with or without 2-mercaptoethanol or the anti-apoptotic additive primatone, which may affect survival or receptor expression (89). One probably needs to rely primarily on in vivo observations to define BAFF requirements.

Figure 2 Stages in B cell maturation. B cell lymphopoiesis occurs exclusively in the bone marrow, and immature cells transit to the spleen for final maturation. Follicular (mature), germinal center, and plasma B cells compose the B-2 lineage. A second lineage found in the peritoneum is called B-1. The presence of this cell requires the spleen, which suggests possible derivation from the transitional or marginal zone B cell stage, or perhaps the splenic microenvironment is required for B-1 lineage maturation (153). Germinal center formation leading to memory and plasma cells can occur in the spleen, lymph nodes, or mucosa associated lymphoid tissue (MALT). Plasma cells disperse and are found in the bone marrow, peripheral tissues, and spleen.

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Genetic analysis of the gain and loss of BAFF function in mice does not support a role for BAFF in B cell development in the BM, nor in the survival of immature or splenic T1 B cells (59, 69, 70). However, the subsequent stages of B cell differentiation, namely T2, mature, and marginal zone (MZ), were BAFF dependent. These observations indicate a fundamental role for BAFF in the survival of splenic T2 B cells, and without this activity there can be little further differentiation into mature and MZ-B cells. This notion is supported by analysis of BAFF-treated and BAFFTg mice, in which the BM and T1 compartments remained normal, but the subsequent differentiation stages were expanded, especially the T2 and MZ populations (75, 87). Clearly BAFF is required to maintain survival, but it is difficult to determine whether BAFF acts directly on transitional B cells to induce differentiation/ maturation. Transitional B cells cultured in the presence of BAFF acquire mature B cell markers, which suggests that BAFF may also be a maturation factor (87, 88). Does BAFF maintain the survival of mature naive B cells? Treatment of mice with BCMA-Ig rapidly induced the loss of mature B cells as well as T2 and MZ-B cells (44; S. Kalled, in preparation). Under these conditions, the rate of elimination of mature B cells exceeds the normal life span of these cells, suggesting that the loss of mature B cells reflects their dependence on BAFF-mediated survival signals rather than just an impaired supply of new mature B cells. Further support for this concept was provided by the results with mixed BALB/c + A/WySNJ BM (∼BAFF-R−/−) chimeras (90). Lastly, direct examination of cell cycling using BrdU labeling showed that BAFF did not affect cell proliferation but was required for survival (75, 88). Therefore, the loss of mature B cells following BAFF inhibition reflects a continual dependence on BAFF for survival as well as the lack of further B cell replenishment. MZ-B cells are a subset of mature B cells that display unique markers and respond quickly to antigenic stimulation (91). They arise from the transitional or mature B cell pool, yet reside in a unique anatomical compartment that provides specialized antigen presenting cell (APC) input, access to bloodborne antigens, and survival signals (92, 93). The MZ-B cells disappear in the absence of BAFF, which suggests that they also need BAFF. It is also possible that the loss of BAFF could perturb the MZ microenvironment, leading to the secondary loss of the MZB population, or that when deprived of support signals the MZ-B cells then lack the surface markers that render them experimentally visible. The stage at which MZ-B cells differentiate from the T1-T2-mature lineage is not clear. Given that BAFF−/− mice have T1 cells, but no MZ-B cells, T1 cells are unlikely precursors of MZ-B cells provided that the MZ-B cell maturation step is BAFF independent. Btk-deficient CBA/N mice have arrested B cell maturation at the T2 B cell stage and have a normal MZ-B cell population (85). Using a conditional RAG-2 knockout, B-2 B cell numbers were RAG-2 dependent, but B-1 and MZ-B cells were not (94). These observations are consistent with the hypothesis that T2 B cells are precursors for MZ-B cells and may at least partially explain the BAFF requirement.

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B-1 B cells are a unique population of cells found primarily in the peritoneum. Whether they are derived from a fetal lineage and self-renew or they emerge from conventional B-2 cells remains a lively debate (95). If B-2 B cells are the precursors for B-1 B cells, then mutations such as the deletion of the Baff gene, which reduces the size of the B-2 compartment, should also affect the B-1 B cell compartment. However, mice deficient in IL-7-, BAFF-, and BAFF-R (i.e., the A/WySnJ strain), which lack a normal B-2 B cell compartment, all have normal B-1 populations (59, 69, 70, 78, 96). These data tend to support an alternative developmental pathway for B-1 cells, yet they do not entirely exclude activation-dependent shaping of the B-1 B cell pool out of the B-2 B cell population. In general, the genetic analyses do not support an essential role for BAFF or APRIL in B-1 development and survival (59, 69, 70, 97). Another critical question concerns the role of BAFF in the survival of proliferating mature B cells, which includes cells participating in extrafollicular responses and the centroblasts in the germinal center. In vitro experiments indicated that BAFF increased proliferation in CD40L-stimulated B cell cultures and that decreased apoptosis accounted for the apparent increase in proliferation rates (74). Whether proliferating B cells need BAFF in vivo to survive is unresolved. It is possible that proliferating B cells utilize CD40 instead of BAFF-R for survival input, but such a simple distinction between quiescent and cycling cells awaits more experimentation. The role of BAFF in the generation and survival of memory cells is also currently unexplored. Likewise, plasma B cells are another terminally differentiated form of B cells whose BAFF dependence needs to be established. Preliminary data indicate that expansion of an antigen-specific plasma cell population requires BAFF (L. Gorelik, unpublished observations). Increased numbers of plasma cells are also detected in BAFF-treated and BAFF-Tg mice, yet it is unknown whether this reflects increased production or cell survival (30, 74). Table 2 lists the various categories of B cells and their BAFF dependence. Genetic dissection has pinpointed BAFF-R as the sole mediator of the BAFF survival signal (Table 1). B cell maturation in TACI-, BCMA-, and double BCMA/ TACI-deficient mice is normal, excluding a dominant role for these two receptors in this process (46, 51, 68, 69; M. Scott & M. Dobles, unpublished data). In contrast, the mutation of the BAFF-R gene that occurs in the A/WySnJ strain of mice closely resembles the BAFF−/− mouse (50, 51). Complete knockout of the BAFF-R gene resembles the BAFF deficiency even more closely, indicating that the A/WySnJ strain may retain residual BAFF-R function (M. Scott & M. Dobles, unpublished observations). Although lack of TACI expression did not prevent B cell maturation, it increased mature B cell numbers, which suggests negative regulation of B cell homeostasis via signaling through TACI (51, 68). Alternatively, shed TACI may be a negative regulator of BAFF activity. Analysis of receptor expression patterns is also consistent with complete control of B cell maturation by the BAFF-BAFF-R axis. BAFF binding to B cells increases as B cells mature, and onset of expression of BAFF-R appears consistent with acquisition of BAFF sensitivity (75; S. Kalled, unpublished observations).

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TABLE 2 B cell types dependent upon BAFF for survival B cell type

Location

BAFF dependence

Pre/Pro

Bone marrow

No

Immature

Bone marrow

No

Transitional T1

Spleen

No

Transitional T2

Spleen

Yes b

Yes

Mature follicular

Spleen, LN, MALT

Marginal zone

Spleen

Likely

Extrafollicular ASC

Spleen, LN, MALT

?

Germinal center T dependent T independent

Spleen, LN, MALT

Memory

Spleen, periphery?

?

Plasma

Spleen, periphery, bone marrow

Likely

Long-lived plasma

Bone marrow

?

a

No? ?

Mucosal IgA secreting

MALT

?

B-1

Peritoneum, lung, spleen

No

a

Antibody secreting cells.

b

Mucosa associated lymphoid tissue.

BAFF and Immune Responses Early in vitro experiments demonstrated that BAFF had a costimulatory effect in standard B cell activation assays using anti-IgM antibodies (13). In these assays BAFF behaves in a qualitatively similar fashion to IL-4 or CD40L; however, BAFF alone does not induce proliferation of B cells in most experiments (13, 17, 74, 75, 83, 88). Recombinant BAFF treatment boosted the primary in vivo immunoglobulin response to various antigens, e.g., Pneumovax, DNP-BSA, and NP-CGG (74, 98). In the first 4–5 days, only IgM and IgA titers were increased, but with longer term treatment, IgG levels also increased (98). After 15 days of exposure, even salivary IgA output was increased (98). In general, the increases in IgG and IgM titers were quite modest, on the order of two- to fourfold, but these results did indicate that BAFF could have an adjuvant-like effect. Only in the case of the IgA response to Pneumovax immunization did it appear that more dramatic events were at play (74). Likewise, murine BCMA-Ig and TACI-Ig could inhibit the responses to NP-CGG, KLH, and Pneumovax; however, TACI-Ig appeared more effective, perhaps because it binds better to murine BAFF (47, 59, 65, 83). Barring the IgA response, one simple interpretation of these observations is that BAFF-induced survival of B cells allows proportionally more cells to be activated and to remain alive after activation (74).

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A comparison of T-dependent (TD) vs T-independent (TI) immunoglobulin responses reveals some complexity. In the long term, BAFF is required for both types of responses, probably because of effects on the B cell pool size (69, 74). It should be noted that B cell numbers begin to drop quickly within 2–4 days after inhibition of BAFF (L. Gorelik, unpublished observations). Therefore, effects on immunoglobulin responses using inhibitors such as BCMA-Ig or BAFF-R-Ig may be further obfuscated by partial pool size reduction even during the course of a short-term experiment. Curiously, TACI-deficient mice have diminished responses to TI antigens, yet retain normal TD responses (60, 68). This observation is inconsistent with a model whereby shed TACI buffers cells from BAFF; instead it suggests that BAFF, APRIL, or a potential further TACI ligand binds to TACI and regulates TI responses. The local microenvironments are crucial especially for TI responses to microbial polysaccharides (91). As the exact status of various myeloid elements following genetic disruption or injection of BAFF or various inhibitors is undefined, it is still difficult to analyze the exact nature of TACI-mediated regulation of TI responses. The role of BAFF signaling in the germinal center (GC) reaction has been partially assessed. One report noted that TACI-Ig was able to block GC reactions, presumably via BAFF inhibition, although effects on T cell help could be involved (83). Likewise, spleens from nonimmunized BAFF-Tg mice contain many GCs (30). However, several observations call into question the involvement of BAFF in the GC reaction. First, CD40-induced B cell proliferation in cultures in vitro does not require BAFF (65). Second, spleens from immunized BAFF-deficient mice, or wild-type mice treated extensively with human BCMAIg to block BAFF, retained a high proportion of peanut agglutinin–positive cells resembling GC-B cells, and more direct assessments failed to reveal a critical role for BAFF (M. Scott, S. Kalled, F. Mackay, unpublished data). Whether GCs are fully functional in the absence of BAFF is unclear since impaired GCs can form even in the absence of follicular dendritic cell networks or as short-lived structures in the TI-GC reaction (99–101). The proliferating centroblasts in the GC require CD40 signaling to survive. In this complex case, CD40 is likely to provide both an activational costimulus, i.e., T cell help and a survival signal. Thus BAFF may not be required for cycling GC-B cells and CD40 signaling is sufficient. The role of BAFF in T cell responses remains unresolved at this point. Since TACI can be found on some T cells, a role for BAFF or APRIL in T cell responses has been speculated. TACI-Ig can inhibit in vivo and in vitro T cell responses quite effectively (102). BAFF and APRIL can costimulate T cells in vitro, consistent with a role for TACI on these cells (47, 103). However, T-dependent immunoglobulin responses in the TACI-deficient mouse appeared to be normal, suggesting that T cell help is not dependent upon TACI signaling (60, 68). APRIL-Tg mice appear also to have relatively normal responses to superantigen and T-dependent antigens (97). More work will be required to investigate these aspects of T cell biology.

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BAFF IN DISEASE


BAFF Transgenic Mice Immune tolerance ensures an inability to react to self-antigens while preserving defenses against pathogens. Many mechanisms have been identified that can neutralize autoreactive lymphocytes and protect individuals against the emergence of autoimmunity (104, 105). The control of cell survival is one critical factor determining immune tolerance, and its dysregulation can promote autoimmune diseases. It is therefore not surprising that BAFF-Tg mice also developed an immunoglobulin-based autoimmune disorder (18, 30, 71). Three different lines of BAFF-transgenic strains were generated, utilizing liver-based α1-antitrypsin, immunoglobulin heavy chain, and β-actin promoters, and all lines show clear signs of B cell hyperplasia and hyperglobulinemia. In these mice, serum IgG, IgM, IgE, and IgA levels were elevated to varying degrees; IgA levels in one case were increased 130-fold (71). These mice had enlarged spleens, Peyer’s patches, and lymph nodes; circulating immune complexes, rheumatoid factors, and anti-doublestranded and single-stranded DNA, anti-nuclear and anti-histone autoantibodies. The kidneys of BAFF-Tg mice were diseased; high protein levels were detected in the urine and extensive leukocytic infiltrates, signs of vasculitis, abnormal and enlarged glomeruli, and protein casts were present (18, 30, 71). These features are observed in patients with systemic lupus erythematosus (SLE). As BAFF-Tg mice get older, they develop a secondary condition similar to Sjögren’s syndrome in humans. This condition is characterized by enlarged salivary glands due to inflammation and leukocytic infiltrates and reduced saliva production as a consequence of acinar cell destruction (31). The BAFF-Tg mice did not exhibit intestinal inflammation, indicating a lack of one of the more typical manifestations of altered T cell homeostasis.

BAFF and Peripheral Tolerance Immune tolerance is controlled at critical stages of B cell development, either centrally in the BM or in the periphery (105). Based on various observations, it is unlikely that central B cell immune tolerance in the BM is affected by excess BAFF. For example, overexpression of BAFF does not alter developing B cell numbers in the BM (18, 30, 71). The BM of BAFF-deficient mice is also normal, and negative selection in an in vitro model system was BAFF independent (69, 70, 88). Therefore, it is probable that excess BAFF alters immune tolerance in the periphery. Since BAFF plays an essential role in B cell maturation, one can envision that elevated BAFF alters immune tolerance while B cells transit from the immature to the mature stage. Signaling mechanisms associated with the B cell receptor (BCR) are different in immature and mature B cells and as a consequence, immature B cells are extremely vulnerable to apoptosis induced by strong antigenic stimulation

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while mature B cells proliferate (106). This mechanism has an obvious advantage, as it will eliminate strongly self-reactive B cells before they enter the mature B cell pool. Therefore a logical hypothesis to explain the autoreactivity in BAFF-Tg mice is the aberrant survival of maturing autoreactive T2 B cells and their emergence into the antigen-responding B cell pool. Viewed differently, survival during maturation in the spleen may rely on the balance between survival input and the nature of the BCR signal. Excess BAFF may lower the threshold for BCR signaling and maintain survival when a normal autoreactive B cell would undergo death (2). The expression of several surface molecules that modulate BCR signaling (e.g., CD19/21) changes during the transition from an immature to a mature B cell. BAFF may directly affect their expression, thereby modulating the BCR threshold rather than providing a direct survival signal (S. Kalled, unpublished observations). Such a model would not apply to the survival effects of BAFF in assays in vitro. Another possible explanation for the disease symptoms observed in the BAFFTg mice is polyclonal B cell activation. Since some autoreactive B cells are present in normal mice, polyclonal activation could increase their numbers to a level where disease manifestations are noted. This scenario is probably unlikely since BAFF does not directly promote B cell activation, i.e., its role does not appear to be costimulatory. Animal models that induce excessive costimulatory activity provide a comparison point. For example, mice with an inactivating point mutation in the CD45 gene lack the negative regulatory influence of the CD45 tyrosine phosphatase on cell activation (107). Like the BAFF-Tg mice, these mice have lymphoid hyperplasia and autoimmune nephritis. However, a high percentage of the B cells display the CD69 activation markers, which has not been observed in BAFF-Tg mice. In contrast to the BAFF-Tg mice, these mice do not have elevated serum IgM or IgG concentrations, but curiously only increased IgA levels. Small changes in CD19 expression can shift the signal transduction threshold (108). CD19 overexpression increased the peritoneal B-1 and decreased the peripheral B cell populations. These changes do not resemble those observed in the BAFF-Tg mice. Based on these comparisons, the BAFF-Tg mouse better resembles a basic deficiency in peripheral tolerance than a shift toward general polyclonal B cell activation.

BAFF and the Marginal Zone B Cell Compartment Since BAFF levels appear to alter peripheral tolerance, a fundamental question is which cellular compartments contribute to the emergence of autoreactive B cells. This question is important not only in the BAFF-Tg mouse but in other conventional mouse models of SLE and in human disease. Although all peripheral B cell subsets were larger in BAFF-Tg mice, the splenic T2 and MZ-B cell subsets were preferentially enlarged (87). These cells were also abnormally present in the blood and lymph nodes of BAFF-Tg mice (87). BAFF-induced expansion of the MZ-B compartment could have complex underpinnings, as the size of the MZ is under genetic control, varying substantially between mouse strains and species. Additionally, MZ-B expansion appears to be linked to bottlenecks in

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B cell development (91). One can imagine that excess survival signals triggered by BAFF in T2 B cells may lead to the emergence of more B cells with an increased resistance to censoring BCR signals, and these cells could be MZ-B candidates. Whether BAFF itself plays a role in skewing the differentiation of B cells into the MZ compartment and away from the follicle remains an interesting question. There is some evidence linking the MZ-B compartment and autoimmunity (91). In the mouse (NZB × NZW)F1 model of lupus, the CD1high B cell population contains autoreactive B cells and MZ-B cells are CD1 bright (109). Also, increased estrogen levels exacerbated disease in a model of SLE utilizing an immunoglobulin transgene encoding a pathogenic anti-double-stranded-DNA heavy chain, and under these conditions, transgenic B cells were found to display a MZ-B phenotype (110). Lastly, some autoreactive B cells are more likely to differentiate into MZ-B cells and to locate in the MZ (111, 112). MZ-B cells are designed to mediate a fast response to microbial antigens, and these responses can cross-react with self-antigens (91, 113). Consistent with this hypothesis, the response to infection is sometimes associated with increased and transient levels of so-called natural anti-DNA autoantibodies and rheumatoid factors (114). Thus, the expanded MZB cell compartment could be linked to autoreactivity in the BAFF-Tg mice. More direct evidence arose from analysis of B cells infiltrating the inflamed salivary gland of BAFF-Tg mice and the thyroids of people with Graves’ disease, where many infiltrating B cells had a MZ-B-like phenotype (31, 115). Cells with a B-1 phenotype were also found in the salivary gland of BAFF-Tg mice and may be involved in autoimmune disorders (116). Whether infiltrating B cells have an active pathogenic role in tissues via secretion of autoantibodies or trigger damage simply by inflammatory mediator release is still unclear (117). An inhibitor of the lymphotoxin pathway, LTβR-Ig, selectively eliminates the MZ-B cell compartment, and using it to treat BAFF-Tg mice with established kidney disease decreased proteinuria scores (J. Gommerman & F. Mackay, unpublished results). As this agent also ablates follicular dendritic cell networks, there was the potential for impoverished GC reactions. However, treatment of BAFF-Tg mice with an anti-CD40L antibody had no effect on proteinuria, excluding GC involvement at this stage. CD40L blockade does, however, prevent or ameliorate SLE-like symptoms in two conventional lupus models, (SWR × NZB)F1 and (NZB × NZW)F1 (118, 119). This difference in the pathological process in these two types of rodent SLE models may reflect separate components of human SLE and may be a source of heterogeneity in this disease. Whether the beneficial effects of LTβR-Ig treatment stem directly from elimination of the MZ-B compartment will require further exploration.

BAFF and IgA-Mediated Disease There are reasons to suspect IgA involvement in the BAFF-Tg mice. BAFF-Tg mice have elevated populations of IgA-secreting cells in the bone marrow, lymph nodes, and kidney (J. Gommerman, unpublished observations). Furthermore, there was a preferential impact of BAFF on the IgA response to Pneumovax vaccine as well

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as the elevated serum IgA levels in the BAFF-Tg mice (71, 74). The improvement in kidney function in BAFF-Tg mice upon inhibition of the lymphotoxin pathway probably requires the removal of the preestablished immune complexes. Since the treatment spanned only 5 weeks, it is perhaps unlikely that the circulating IgG levels would decrease substantially in a short time frame, but IgA and IgM titers would be lowered. IgA antibodies arise from conventional adaptive B-2 responses and these switching events are probably mostly limited to mucosal microenvironments and their draining lymph nodes (LN), although IgA-secreting B cells can be found in the spleen, peripheral LN, and bone marrow. Another source of IgA is from B-1 cells that migrate to the gut and undergo class switching (120). B-1-derived IgA responses arise in the gut in response to the microbial flora. These responses are driven in a T-independent manner and these responses are probably associated with substantial innate signaling components (113). One hypothesis is that autoreactive IgA-secreting cells arise in the mucosal compartment where excess BAFF signaling has lowered the BCR threshold even further. In this case, the BAFF-Tg mouse may actually be an IgA-centric autoimmune disease model. In view of the highly similar functions of MZ and B-1 B cells, it is also tempting to speculate that both MZ-B and mucosal IgA+ plasma cells contribute to disease in BAFF-Tg mice. Therefore, IgA nephropathy or Berger’s disease may be a good candidate for BAFF blockade. Considerable effort will be required to tease apart the relative roles of these components, e.g., plasma cells, GC reactions, MZ-B cells, and the mucosal IgA compartment; yet a precise picture will have a major impact on our views of systemic immunoglobulin-based autoimmunity in humans.

Disease Models A critical role for BAFF in the progression of autoimmune diseases was confirmed in mouse disease models. Treatment with TACI-Ig, used as a decoy receptor, reduced symptoms in the (NZB × NZW)F1 mouse model of SLE (18, 70). The development of collagen-induced arthritis was also inhibited by the BAFF blockers TACI-Ig or BCMA-Ig (70, 102; F. Mackay, unpublished data). This model relies on both T cell and B cell responses; however, parallels to the K/B × N model would indicate that this is primarily an immunoglobulin/B cell–based disease at the effector phase (121, 122). TACI-Ig administered after the collagen boost suppressed both immunoglobulin and T cell responses (102). Both studies showed a surprising rapid decrease in anticollagen IgG levels given the 2- to 3-week half-life of IgG antibodies in the blood. It will be interesting to determine which aspects of the anticollagen responses are blunted in this system or whether this effect is due to impaired T cell help at a critical juncture. Established disease was not blocked, indicating that once anticollagen antibodies are formed, effector site manifestations are BAFF independent (F. Mackay, unpublished data). Does BAFF play a major role in the progression of human autoimmune disorders? Analysis of BAFF levels in sera from patients with severe B cell disorders, i.e., SLE, rheumatoid arthritis, and Sjögren’s syndrome, revealed higher levels of

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BAFF in the blood (31–33). This was particularly true for Sjögren’s syndrome, a condition characterized by a severe alteration of B cell numbers and very high hyperglobulinemia (123). The elevated levels in some human patients could reflect a direct role as a disease driver, or elevated BAFF may simply be symptomatic of chronic inflammation. Expression of BAFF by macrophages and dendritic cells is stimulated by gamma interferon (IFN-γ ) and IL-10, factors often produced during inflammation and infection (13, 17, 34). Chronic infection has been associated with the development of rheumatoid arthritis (124). Higher levels of BAFF are found in the sera of human immunodeficiency virus (HIV) patients and there is also a pathogenic relationship between HIV and SLE (125, 125a). Chronic infection may lead to the sustained release of BAFF and thus the emergence of autoreactivity, especially in people with autoimmune susceptibility genes. A juvenile polyarthritis and IgA deficiency syndrome appear to accompany 22q11 deletions, which may affect BAFF-R expression at 22q13.2 (126). The current picture of BAFF indicates that it can be critically involved in immunoglobulin-based autoimmune diseases. Blockade of BAFF is likely to have a different activity spectrum compared to the elimination of CD40 signaling and hence may help to control distinct aspects of autoimmune disease.

BAFF and B Cell Tumors Whether BAFF contributes to the survival of B cell tumors will have implications for oncology. Expression of a BAFF receptor will be a requirement for BAFF sensitivity. In fluorescence activated cell sorter (FACS) analyses, BAFF was shown to bind to various diffuse large cell, mantle, and marginal zone B lymphomas at levels similar to those observed in normal mature B cells, while lower levels were found in follicular non-Hodgkin’s lymphoma (NHL) and B-chronic lymphocytic leukemia (B-CLL) (127). Most B-CLL cells express BAFF-R RNA and a subset actually displayed BAFF on their surfaces, raising the exciting possibility that autocrine BAFF may be a component of B-CLL survival (128). Elevated levels of BAFF were detected in serum from NHL patients (127). Occasionally, Sjögren’s syndrome is complicated by the emergence of B cell lymphomas, which, incidentally, are often described as of MZ origin (123, 129). Interestingly, a small number of older BAFF-Tg mice develop a submaxillary gland tumor that is composed essentially of hyperplastic B cells (31). Neoplastic B cells may emerge constantly and are probably eliminated by immunological mechanisms. Excess BAFF-mediated survival may impair this process and facilitate the progression of these cells to full lymphomas. It is possible that a subset of B cell tumors will rely at least partially on BAFF for their survival in vivo.

APRIL IN THE IMMUNE SYSTEM The ability of APRIL to bind to both BCMA and TACI led many to believe that APRIL and BAFF shared biological roles. Since BAFF knockout mice displayed the full reduction in the peripheral follicular B cell compartment, APRIL is not

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involved in this function (69, 70). A lack of involvement of APRIL in B cell survival was also indicated by the normal B cell compartment in a mouse expressing murine BCMA-Ig as a transgene (59). Murine BCMA, in contrast to human BCMA, binds very poorly to murine BAFF but well to murine APRIL, so murine BCMA-Ig-Tg mice will be effectively APRIL-deficient mice. Moreover, mice expressing human APRIL under the lck promoter resulted in T cells that contained APRIL, and soluble APRIL was present in the blood at reasonable levels (97). These mice are a counterpart to the BAFF-Tg mice, yet they do not have B cell hyperplasia or autoimmune nephropathy. Thus there is no obvious role for APRIL in B cell survival. T cells from APRIL-Tg mice survived better in vitro and there was a small impact on TD IgM and TI antibody responses. Overall, these mice lacked profound changes in the immunological functions that were examined. Since APRIL appears to bind to a third receptor besides TACI and BCMA, this receptor could also be related to the effects on T cell survival. However, in our hands, a recombinant preparation of APRIL that could drive NIH 3T3 proliferation at 1–5 ng/ml required 500 ng/ml to efficiently affect T cell proliferation in experiments in vitro (K.Vora & P. Rennert, unpublished experiments). For this reason, we suspect that APRIL’s physiological activity may rest in other, nonimmunological arenas.

APRIL AND NONHEMATOPOIETIC LINEAGE CELLS APRIL and Growth Regulation Overexpression of APRIL in NIH 3T3 cells accelerated tumor growth in vivo and in vitro (14, 43). These observations were verified using a tetracycline-inducible expression system to exclude clonal variation as a possible complication in the original study. APRIL does not induce NIH 3T3 cells to form foci in a conventional transformation format (P. Rennert, unpublished data), but other genes involved in oncogenic processes such as the Wnt family are also unable to induce foci (130). Since APRIL expression can enhance tumor growth rates, its action appears to lie intermediate between that of an oncogene and the effects of Wnt overexpression. The growth of three APRIL-transfected human glioma cell lines was not altered by APRIL expression; however, when cells were treated with FasL, survival was modestly improved by APRIL (131). Importantly, NIH 3T3 cells completely lack TACI or BCMA, indicating the existence of a third APRIL receptor that could be APRIL specific (43). While it is likely that all canonical TNF ligands and receptors have been discovered, noncanonical or poorly homologous receptors may exist. This structural untidiness is perhaps not surprising, as the first defined TNF receptor-ligand interaction was p75 NGF receptor binding to NGF, a dimeric protein with a TGF-β folding pattern. Most likely, some further expansion of the family can be anticipated. APRIL-mediated regulation of cell growth is in part mediated by its ability to induce the expression of anti-apoptotic proteins. In glioblastoma cell lines, APRIL

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induced expression of XIAP and caspase activity was reduced, but Bcl-2 and Bcl-xl expression was not affected (131). APRIL induced expression of Bcl-2 and Bcl-xl in NIH 3T3 fibroblast and HT29 adenocarcinoma cells (P. Rennert, unpublished data). In the fibroblast system, APRIL stimulation also led to an increased response to growth factor–induced cell proliferation (P. Rennert, unpublished data). This result suggests that the role of APRIL in this setting is to provide a survival signal creating a permissive environment for cell growth.


APRIL-Deficient Mice Deletion of the APRIL gene led to embryonic lethality; notably, this is the first such observation in the TNF field (L. Runkel, unpublished observations). While not lethal, knockout of the LTβR, EDA, and RANK systems in mice impaired lymph node, hair, tooth, and mammary gland development. Given the close proximity of the TWEAK and Senp-3 genes, the Neo gene was flanked by Lox/Cre sites and subsequently deleted to avoid interference. Since TWEAK-deficient animals are viable, interference with TWEAK regulation cannot account for the lethality in APRIL-deficient mice (L. Runkel, unpublished observations). Deficiencies in the development of the fetal heart myocardium and neural crest cell migration into the emerging heart valve (cushion) structures were noted. Given that the embryos died at days E9–12, a heart defect is the likely culprit. Mice lacking FADD, FLIP, and caspase 8 have similar developmentally lethal defects in heart structure (132–134). Curiously, no knockout of a death domain containing TNF family receptor has been reported to be lethal. Therefore, if an as yet unidentified receptor is required to couple to FADD, FLIP, and caspase 8 during heart development, APRIL may be its ligand. Another APRIL−/− mouse has been made that is viable (A. Ashkenazi, personal communication). Potential explanations could lie in alternative splicing of TWEAK elements into residual APRIL exons, varying perturbations on chromatin structure, complex compensation between TWEAK and APRIL expression, and altered expression patterns of the Senp-3 gene.

APRIL IN DISEASE The role of APRIL in disease has focused on T cell activity and solid tumor growth. RNA-based studies have identified monocytes and tumor cells as the primary sources of APRIL expression. Examination of expression databases showed that approximately 50% of all APRIL “hits” were in solid tumor samples, of which the majority were adenocarcinomas (P. Rennert & M. Lukashev, unpublished). APRIL RNA was expressed in 5 of 12 glioblastoma cell lines and elevated levels were detected by in situ hybridization analysis expression in some adenocarcinomas (14, 131). Notably, lymphoma samples only rarely scored positive for APRIL message RNA. Other significant sources of APRIL in these studies were circulating monocyte samples (10%) and tissues harvested from inflammatory settings (10%).

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Blockade of APRIL activity using a BCMA-Ig fusion protein was shown to slow the growth of several tumor lines in human xenograft models. These effects were more prominent when tumors were treated from the point of implantation rather than with established tumors. During the early events in tumor implantation, APRIL’s prosurvival activities may play a critical role. We hypothesize that as the tumor receives effective support from the stroma and neovasculature, APRIL plays a less critical role in regulating survival.


TNF FAMILY MEMBERS AND SURVIVAL ACTIVITIES The concept of a survival factor is theoretically quite simple. Cells enter into apoptosis by default and are prevented from death by survival and growth signals that act to dampen the pro-apoptotic signaling pathways. A pure survival factor should simply maintain sufficient anti-apoptotic machinery. In reality, such activity is often interwoven with entry into cell cycle or a differentiation program; hence these events are difficult to deconvolute. Moreover, it is probable that survival signals differ depending on whether the cells are quiescent or in cell cycle, e.g., resting vs activated lymphocytes or the lobular epithelial cells of the breast in the involuted vs lactating states. BAFF-R signaling is instructive since there does not appear to be a strong proliferation-inducing component. Since B cells have evolved such a dominant system, it may be useful to reexamine other cell types for comparable factors. Many cytokines have been shown to control lymphocyte survival throughout their life, such as CD40 ligand. Factors that maintain survival are perhaps best described in the bone marrow, where IL-7 is required. Here, however, the situation is complex, as cells are rapidly dividing and differentiating into specialized leukocytes. Other than the stem and progenitor cells and the long-lived plasma cells, most of these cells complete their maturation within days and egress into the periphery. Therefore, molecules like IL-7 may also be competency factors for continued progression. Elements that control compartment size homeostasis may be survival factors. For example, in the presence of excess BAFF, the overall size of the B cell compartment expands. It is not clear whether a linkage between compartment size and survival factors can be generalized. The search for T cell survival factors has focused on interleukins 2, 4, 7, 9, and 15 and their links to the bcl family (1). In particular, IL-15 appears to be a survival factor for CD8 cells, although the interplay with cell cycling is complex in this case (135). Several other enigmatic TNF family members may also deserve attention as possible T cell counterparts to BAFF. For example, 4-1BB signaling has been described as a survival signal for activated CD8 and CD4 T cells (136, 137). Immunologists tend to focus on the costimulatory properties of new factors, and since the readout can be dependent on the size and robustness of the output population, survival factors may look like costimulatory factors. Moreover, effects such as that of BAFF in the costimulation assay in vitro may appear to be minor and may be misleading. Other complex systems such as DR6, CD27, CD30, and OX-40 may benefit from reanalysis in the context of lymphocyte survival signals.

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In contrast to the traditional expectation that TNF family members have immunomodulatory roles, a general theme has emerged in which some TNF family members are required for the survival of certain epithelial and stromal tissues. For example, LTβR most likely affects survival/differentiation of stromal elements during lymph node anlage formation (138). RANK appears to promote the survival of ductal epithelial cells in the lactating mammary gland, although direct effects on cell cycling may complicate the picture (139, 140). Among myeloid lineage cells, RANK signaling triggers osteoclast differentiation, but these data could also be interpreted as promoting the survival of M-CSF differentiated osteoclasts (139). EDAR and XEDAR signaling are likely to be critical for the survival of ectodermal lineage cells during hair follicle and tooth development (141, 142). As hair follicle, tooth, and mammary gland development are well defined, one can look to these systems for more precise details on the working of this survival theme. Viewed within the context of the RANK, EDA, and BAFF systems, APRIL may be a potential survival factor. APRIL augments thymidine incorporation and can potentiate the activity of some growth factors (43; P. Rennert, unpublished observations). This activity resembles the ability of TWEAK to potentiate FGFor VEGF-induced endothelial cell proliferation, although APRIL has no effect on endothelial cells and TWEAK cannot substitute for APRIL in the NIH 3T3 proliferation assay (143, 144). The ability to enhance fibroblast growth is probably not a very direct monitor of APRIL’s true function and therefore the proliferation data appear unremarkable when compared to an actual growth factor. Nonetheless, in settings where growth factors are limited, such activity may be pivotal. Slow growth is more typical of human tumors and their survival could be very sensitive to factors such as APRIL; however, slow-growing tumors are not favored in experimental settings. In general, the analysis of these various TNF systems from the viewpoint of survival may be instructive. If one could peel away the proliferation components of many of these systems, tissue-specific activation of cell survival may emerge as a general theme for at least a subset of death-domain-less TNF family members.

CONCLUSIONS The biochemical and genetic dissection of the BAFF system has yielded a clear and relatively unambiguous picture of an obligate survival signal for both maturing and fully differentiated B cells. Such clarity is relatively unusual in immunology. As such it has provided immunologists with a foundation upon which one can explore how B cells integrate the signaling provided by the BCR, costimulatory elements, and BAFF receptors during selection in the periphery. Comprehension of how BAFF levels are regulated should lead to some insight as to how B cells can survive in various locales in both physiological and pathological conditions. Perhaps most importantly, BAFF is a relatively simple system, tutoring us in how to view survival signaling in more complex scenarios where the unraveling of proliferation from survival signals is much murkier. This paradigm may also be

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pivotal to understanding other potential survival systems, e.g., APRIL, TWEAK, and EDA. Lastly, the BAFF system provides a powerful tool to manipulate the immune system in humans (11, 12). Historically, down-modulation of the B cell arm with biological agents has been limited to the exogenous addition of excessive amounts of IgG, tolerization with excess antigen, and wholesale depletion of all B cells. BAFF inhibitors would be a more selective means of damping the immune system, especially considering that some elements of B cell function such as bone marrow output and GC formation may be preserved. Alongside CD40, complement intervention, and Fc receptor regulation, BAFF inhibitors may give clinical immunologists an additional tool to deal with complex autoimmune diseases such as lupus and rheumatoid arthritis. ACKNOWLEDGMENTS We wish to thank members of the BAFF and APRIL teams at Biogen for their data in advance of publication and critical advice, especially S. Kalled, L. Gorelik, M. Scott, M. Dobles, L. Runkel, K. Vora, C. Ambrose, J. Thompson, Y-M. Hsu, T. Cachero, K. Farrington, M. Lukashev, E. Notidis, and J. Gommerman. F. Mackay is a Wellcome Trust Senior Fellow. The Annual Review of Immunology is online at http://immunol.annualreviews.org

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Annu. Rev. Immunol. 2003. 21:265–304 doi: 10.1146/annurev.immunol.21.120601.141053 First published online as a Review in Advance on January 8, 2003

T CELL DYNAMICS IN HIV-1 INFECTION∗ Daniel C. Douek1, Louis J. Picker2, and Richard A. Koup3 Annu. Rev. Immunol. 2003.21:265-304. Downloaded from arjournals.annualreviews.org by HINARI on 09/01/07. For personal use only.

1

Human Immunology Section and 3Immunology Laboratory, Vaccine Research Center, NIAID, NIH, Bethesda, Maryland 20892; email: [email protected], [email protected] 2 Vaccine and Gene Therapy Institute, Oregon Health Sciences University, Beaverton, Oregon 97006; email: [email protected]

Key Words T cells, activation, lymphopenia ■ Abstract In the absence of antiretroviral treatment, HIV-1 establishes a chronic, progressive infection of the human immune system that invariably, over the course of years, leads to its destruction and fatal immunodeficiency. Paradoxically, while viral replication is extensive throughout the course of infection, deterioration of conventional measures of immunity is slow, including the characteristic loss of CD4+ T cells that is thought to play a key role in the development of immunodeficiency. This conundrum suggests that CD4+ T cell–directed viral cytopathicity alone cannot explain the course of disease. Indeed, recent advances now indicate that HIV-1 pathogenesis is likely to result from a complex interplay between the virus and the immune system, particularly the mechanisms responsible for T cell homeostasis and regeneration. We review these data and present a model of HIV-1 pathogenesis in which the protracted loss of CD4+ T cells results from early viral destruction of selected memory T cell populations, followed by a combination of profound increases in overall memory T cell turnover, damage to the thymus and other lymphoid tissues, and physiological limitations in peripheral CD4+ T cell renewal.

INTRODUCTION The hallmark of HIV-1 infection is the progressive depletion of CD4+ T cells. Yet the extent and nature of this depletion, and the mechanisms by which it arises, remain highly controversial (1–3). HIV-1 infection also induces profound qualitative changes in CD4+ T cells, and in most other elements of the immune system too, yet the mechanisms responsible for immunodeficiency are still not well characterized. No viral infection in humans, either acute resolving or chronic persistent, whatever the viral load, is known to cause such profound and inevitable CD4+ T cell loss, except HIV-1. Even HTLV-I, which exhibits the same fastidious tropism ∗ The U.S. Government has the right to retain a nonexclusive, royalty-free license in and to any copyright covering this paper.

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for CD4+ T cells and renders them targets for HTLV-specific CD8+ T cells, results in CD4+ T cell lymphocytosis rather than lymphopenia (4). HTLV, however, is not cytopathic, whereas HIV-1 is well known to infect and kill primary CD4+ T cells (5). Yet, as we discuss below, cytopathicity alone is unlikely to provide a satisfying explanation for the course of the disease. A more complete explanation would undoubtedly highlight T cell dynamics as a central factor in HIV-1 pathogenesis, affecting virtually all aspects of the infection, including (a) viral dynamics, via the regulation of viral target densities; (b) the development, maintenance, and effectiveness of HIV-1-specific cellular immunity; and (c) the mechanisms that maintain the integrity of the naive and memory T cell pools. What singular feature that sets this infection apart from other viral infections underlies the dynamics of T cells and virus at the microscopic level? Is it the unique combination of the destructive potential of the virus with the intimate, multifaceted relationship between T cell activation and virus replication? Introduction of virus into the host results at first in explosive activation and replication. The dynamics of both T cells and virus then appear to stabilize, but the initial events that establish the chronic infection leave a profound and long-lasting impact on the immune system that may set the stage for the subsequent slow progression of the disease. This review examines HIV-1 infection from the perspective of lymphocyte dynamics, linking recent empirical and conceptual advances in HIV-1 biology with new insights into T cell dynamics to provide an immunological view of the pathogenesis of this deadly infection. Notably, most studies of such dynamics have been performed only in peripheral blood during the chronic stage of HIV-1 infection. Therefore, we attempt to deconstruct HIV-1 disease into its acute and chronic phases, which manifest markedly distinctive viral and lymphocyte dynamics. We address the contributions of the virus itself, T cell activation, T cell reconstitution, and target cell availability in the shaping of these dynamics during each phase. We highlight the major role of persistent immune activation during the chronic phase, but also suggest that profound memory CD4+ T cell destruction occurring during the acute phase may have a crucial impact on the subsequent course of the infection.

THE PHASES OF HIV INFECTION The course of untreated HIV-1 infection in the majority of individuals is illustrated in Figure 1. It begins with an acute symptomatic illness, lasting only a few weeks, which is associated with a high viremia, a sharp drop in peripheral blood CD4+ T cell counts (6–14), establishment of a reservoir of latently infected CD4+ T cells (15–18), and development of an HIV-1-specific immune response (19–22). This is followed by a 100- to 1000-fold fall in viral load, a partial rise in peripheral blood CD4+ T cell counts, and then a generally asymptomatic phase of chronic infection, lasting on average 10 years, which is marked by slowly falling peripheral blood CD4+ T cell counts and slowly rising viremia. As peripheral

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blood CD4+ T cells decline to less than 200 cells/µl, when the total number of CD4+ T cells in the body has been reduced by at least half (23), the opportunistic tumors and infections that characterize AIDS beset the individual. This often occurs concomitantly with a precipitous rise in viremia and a crash in peripheral blood CD4+ T cell counts (24). Although these phases are very different in terms of their viral and T cell dynamics, they are obviously interdependent in many of their pathogenic mechanisms, and one cannot be considered in the absence of the other.

THE ACUTE PHASE OF INFECTION Establishment of the Infection Exposure to HIV-1 is primarily through the mucosal route, either gastrointestinal or reproductive, which is thought to result in initial local replication of the virus within target cells of the mucosal tissue. The establishment of infection is dependent on the target cells’ expression of CD4 and a chemokine receptor (CCR) (25). Although a variety of CCRs can serve as coreceptors in vitro, CCR5 and CXCR4 are likely to be the major receptors used by HIV-1 in vivo, with CCR5 almost always being the initial target coreceptor for naturally transmitted virus (25–30). Although HIV-1 usage of CXCR4 develops over time in many individuals, significantly expanding the target cell repertoire of the virus and accelerating disease progression (31–33), R5-tropic strains predominate also in chronically HIV-1-infected patients (34) and cause CD4+ T cell depletion. Little is known about the primary pathophysiological events of lymphocyte and viral dynamics in acute, as opposed to chronic, HIV-1 infection in humans. Thus, we may turn to recent studies that have shed light on the earliest events in SIV infection of rhesus macaque monkeys. Whereas the kinetics of viral replication and disease progression differ somewhat in these two systems, and indeed in different viral strains within each system, the fundamental pattern remains the same (12, 35). CXCR4 conversion is rare in most commonly studied SIV strains but is also not a prerequisite for peripheral CD4+ T cell depletion and progression to AIDS (28, 36, 37). After intravenous, intrarectal, or oral inoculation of SIV, it appears that the major, and possibly the earliest, cellular substrates for the initial burst in viral replication are CD4+ T cells in the lamina propria of the mucosal tissues, rather than nonlymphocyte targets such as dendritic cells (38–44). Similarly, 2–3 days after intravaginal inoculation of SIV, the majority of productively infected cells are CD4+ T cells in the lamina propria of the endocervix (45). This is not altogether surprising, considering that viral replication in dendritic cells is considerably less efficient than in CD4+ T cells and is actually blocked in mature dendritic cells (46). However, whereas dendritic cells may not be the initial target for viral replication, they may act as local facilitators for productive infection. The binding and internalization of intact virions via DC-SIGN enhances infection in trans of CD4+ T cells (47–49). In transmission of SIV across the vaginal epithelium, which is considerably thicker than the friable rectal and cervical epithelia, the picture may be

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different (50). Intraepithelial Langerhans cells can become productively infected with SIV at a low level by 18 h postinoculation, after which they may mature and migrate directly to local lymph nodes where the virus can be propagated to resident CD4+ T cells (51).


The Tissue Distribution of Target Cells Directs Viral Replication After establishment of infection in the mucosa, SIV is rapidly disseminated over the next 2 weeks, infecting increasing numbers of CD4+ T cells in both local and distant lymphoid tissue including lymph node, thymus, spleen, and mucosal tracts (41, 42, 45, 52). There is also evidence from lymph node biopsies from small cohorts of acutely infected humans that T cells are the major target for and source of HIV-1 (45, 53), resulting in the early establishment of a pool of latently infected CD4+ T cells (15). Indeed, in situ hybridization for SIV and HIV-1 RNA in combination with quantitative image analysis shows that the frequencies of infected T cells in lymph node and mucosal tissue are considerably higher than during the asymptomatic phase (23, 44, 45, 53). Furthermore, HIV-1 can accumulate to very high levels in the pool of lymph node follicular dendritic cells during acute infection (54) and may become a major reservoir of infectious HIV-1 in the late stages of infection (23, 55–59). Direct tracking of HIV-1 viral genotypes in local microenvironments indicates that cell to cell transmission of virus is a local phenomenon, wherein an infected cell releases virus that efficiently infects only nearby targets (60). This implies that propagation of infection depends upon local cell interaction and local target cell densities, rather than on the overall number of targets in the body (61). During acute infection the mucosal tissues presumably offer, in effect, a continuum of target cells through which the virus rapidly propagates and multiplies. The target cells for this explosive dissemination are, as discussed above, predominantly CCR5+ CD4+ T cells, a memory T cell subset that is infrequent in peripheral blood, lymph node, and spleen but that accounts for almost all CD4+ T cells in other tissues including the mucosal surfaces of the intestinal, respiratory, and reproductive tracts (40, 62–67). Figure 2 shows the distribution of CCR5 expression on peripheral blood T cells and lung lavage T cells, as an example of a mucosal site, in a healthy rhesus macaque (L.J. Picker, unpublished observation). CXCR4 levels tend to be low on mucosal tissue memory CD4+ T cells and much higher on memory CD4+ T cells in peripheral blood, lymph node, spleen, naive CD4+ T cells, and thymocytes (40, 63, 65, 66, 68). Mucosal CCR5+ T cells qualify as targets for virus replication also by virtue of their activation phenotype. Many of these cells express surface antigens such as CD69 and HLA-DR (40, 43, 65) that have also been defined on peripheral blood T cells as markers of T cell receptor (TCR)–mediated activation, a cellular state generally thought to be a prerequisite for high-level lentiviral replication (69–71). However, the ubiquity of CCR5 expression on extra-lymphoid T cells suggests that

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Figure 2 CCR5 is preferentially expressed by mucosal memory T cells. The phenotypic profiles of CD4+ T cells from peripheral blood and bronchoalveolar lavage specimens from a healthy rhesus macaque are shown (5000 events gated on total CD4+ T cells or CD4+, CD95hi memory T cells). Note that peripheral blood contains both naive (CD95lo) and memory (CD95hi) CD4+ T cell subsets (106), the latter including a majority of the CD28+/CCR7+ “central” (lymph node homing) memory subset and a minority of CD28−/CCR7− “effector” (extra-lymphoid tissue homing) memory subset. Whereas both central and effector memory T cells in blood can express CCR5, the frequency of this expression is about fivefold higher in the latter population (56% versus 11%). In keeping with this, lung T cells are essentially 100% memory, the vast majority (∼80%) lacking CCR7 and expressing CCR5.


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it may be a general phenotype of tissue-infiltrating T cells (65, 72, 73), which often express so-called activation markers induced by environmental signals unrelated to suprathreshold TCR signaling. Indeed, the mucosal environment is rich in inflammatory cytokines, some of which activate T cells (74) and promote the infection and propagation of HIV-1 in resting CD4+ T cells (75, 76). Furthermore, HIV-1 gene products themselves such as Nef, Tat, Vpr, and even Env induce TCR-independent T cell activation programs and/or viral transcription (77–85). Thus, it appears that even in the absence of full antigen-induced TCR-mediated activation, the phenotype and environment of mucosal CD4+ T cells makes them excellent substrates for HIV-1 infection. Together, the effects of antigen- and cytokine-mediated stimulation, as well as of HIV-1 proteins with the innate ability to activate T cells, will result in efficient production and propagation of the virus from infected cells to adjacent CCR5+ CD4+ T cells. One final point to bear in mind in considering the impact of acute HIV-1 infection is that the gastrointestinal tract may be the largest lymphoid organ in the body. Indeed, it has been estimated that it contains, at steady state, at least 60% of the total body T cell load (86, 87). Furthermore, it has been shown that, after infectious antigenic challenge in mice, the epithelia of the gastrointestinal and respiratory tracts are the major site for the sequestration of memory CD4+ and CD8+ T cells (88–90). Thus, a large fraction of CD4+ T cells in the body reside in the mucosal tissues, express CCR5, and are prime targets for R5-tropic HIV-1 infection and replication.

The Dynamic Consequences of Being Targeted The abundance of mucosal substrates for viral replication accounts for a profound impact of the virus on the immune system early after infection. In macaques infected with SIV, intravenously or via a mucosal route, there is a rapid and profound loss of intestinal CD4+ T cells, such that their numbers are almost entirely depleted by 3 weeks after infection (38, 39, 41, 42). Furthermore, because this loss is specific to CCR5+ CD4+ T cells, which are underrepresented in peripheral lymphoid organs compared with extra-lymphoid tissues, mucosal CD4+ T cell depletion is far greater in proportion than that seen in peripheral blood, lymph node, or spleen, and occurs sooner (39–41). However, those CD4+ T cells in peripheral lymphoid organs that do express CCR5 are indeed selectively depleted during acute infection (40). Interestingly, studies of lymph nodes in acutely SIV-infected macaques and early HIV-1 infection in humans have shown that many of the infected CD4+ T cells are not in an “activated state” by their lack of expression of Ki67 and HLA-DR (45). However, as discussed above, such markers may not fully reflect the actual activation state of T cells in their local environment of cytokines and viral gene products. Figure 3 shows the depletion of CD4+ T cells in peripheral blood and lung lavage following intravenous infection of a rhesus macaque with the pathogenic SIV isolate SIVmac239 (L.J. Picker, unpublished observation). The analysis of pulmonary lavages following infection reveals that lung CD4+ T cells (which are

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Figure 3 Loss of T cells in pathogenic CCR5-tropic SIV infection is rapid and reflects the distribution of the CD4+, CCR5+ memory T cell subset. CD4+ T cell population dynamics are shown following infection of the same animal shown in Figure 2 with pathogenic CCR5tropic SIV. Note that the frequencies of CD4+ T cell in lung plummet in the first 3 weeks of infection, accompanied by only a very modest decline in the representation of the overall CD4+ T cell subset in peripheral blood (left panel). However, closer examination of the subset composition of the circulating CD4+ T cells (right panel) reveals a substantial decline of total memory CD4+ T cells and profound depletion of the CD4+, CCR5+ memory subset.

essentially all CCR5+) (Figure 2) are so rapidly depleted that they are almost entirely lost by day 21 of infection. By contrast, there is only a very modest decline in the representation of the overall CD4+ T cell subset in peripheral blood (Figure 3, left panel). However, closer examination of the subset composition of the circulating CD4+ T cells (right panel) reveals a profound depletion of the CD4+, CCR5+ memory subset. Thus, the primary SIV target population is systemically depleted in the first weeks of infection, and despite increased T cell proliferation (see below), these populations are not reconstituted. This confirms previous studies showing that regardless of the systemic or mucosal route of inoculation with SIV there is rapid and massive mucosal CD4+ T cell depletion in both gastrointestinal and respiratory tracts well before significant depletion occurs in lymph node and peripheral blood (41). That the dynamics of CD4+ T cell depletion are determined by coreceptor usage of the virus has been strikingly illustrated by the use of chimeric viruses (SHIVs) consisting of an SIV backbone with an HIV-1 envelope. SHIVs have been constructed with envelopes that are exclusive in their usage of either CCR5 or CXCR4. As expected from infection with natural SIV, intravaginal R5-tropic SHIV infection results in rapid and profound depletion of intestinal CD4+ T cells, with little effect on those in peripheral blood and lymph node (91–93). X4-tropic SHIV infection, however, results in a similarly rapid and profound depletion of

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CD4+ T cells but affects mainly those in peripheral blood and lymph node, while leaving intestinal CD4+ T cell populations intact (91). Furthermore, the target cell population of X4-tropic SHIV is significantly expanded to include naive CD4+ T cells and thymocytes (94). The effect of HIV-1 coreceptor usage on target cell depletion has also been examined in vitro with lymph node (95, 96) and thymus (97) tissue-culture explants and in SCID-hu chimeric mice (mice implanted with human fetal thymus and liver) (98–102). Lymph node CD4+ T cells and thymocytes in these models are profoundly depleted by X4-tropic HIV-1 strains but only mildly depleted by R5-tropic HIV-1 strains.

The Dynamics of Depletion and Activation Taken together, these data indicate that R5-tropic virus infection results in a systemic depletion of CD4+ memory T cells that is out of proportion to the decline in peripheral blood CD4+ T cell counts and is characterized by a particularly striking decline in the CCR5+ CD4+ memory T cell subset. This suggests that CD4+ T cell loss is essentially biphasic, with a rapid, massive but somewhat concealed depletion of preexistent T cell targets during acute infection, followed by the well-known slow decline of the remaining CD4+ T cells that characterizes chronic infection. Of the several hypotheses raised to explain CD4+ T cell loss in HIV-1 infection, those that invoke impaired production or chronic activation-induced cell death may have validity for the chronic phase of the infection but would clearly operate too slowly to account for the rapid kinetics of this depletion in acute infection. The almost complete elimination of mucosal CCR5+ CD4+ T cells within 2–3 weeks of infection suggests that SIV removes cells that would not have ordinarily died and may also remove cells that are mobilized to replace them. It is possible to examine the proliferation and turnover of the relevant T cell subsets in acute SIV infection by flow cytometric analysis of Ki67 expression and BrdU uptake and decay (103–105). Cellular BrdU incorporation after in vivo pulsing allows determination of the fraction of cells in S-phase during the pulse and provides a measure of their survival and proliferation. Expression of the cell cycle–associated nuclear antigen Ki67 is associated with cells that have predominantly undergone DNA synthesis in the past 3–4 days (106). Figure 4 shows that after an initial slight decrease, by day 14 after infection with SIV there is a marked and sustained increase in the fraction of CD4+ and CD8+ memory T cells expressing Ki67 (L.J. Picker, unpublished observation). Subsequently, the frequency of proliferating CD4+ and CD8+ memory T cells are closely matched and slowly increase with time. Rhesus CMV infection elicits an analogous proliferative burst, but in this situation the virus is controlled and proliferation returns to baseline by day 56 postinfection. In both acute SIV and RhCMV infection, BrdU incorporation is correspondingly high in CD4+ and CD8+ memory subsets. However, although the decay kinetics of BrdU label (owing to death and proliferative dilution) are similar within the CD8+ and CD4+ subsets in RhCMV infection and the CD8+ subset in SIV infection, essentially

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all circulating CD4+ BrdU-labeled memory T cells have disappeared from the SIV-infected macaque within one week. This disproportionate loss of proliferating CD4+ memory T cells in acute SIV infection—over and above that expected for a given level of proliferation—is consistent with excess cell death, which is likely to be due to direct or indirect viral cytopathicity (5, 107–111) and CD8+ T cell–mediated destruction of infected CD4+ T cells (112). Additionally, in both CMV and SIV-infected macaques there was a marked increase in the number of peripheral blood and lymph node CCR5+ proliferating CD4+ T cells, but significantly, in SIV infection such CCR5 expression is observed in the setting of progressive depletion of preexistent CCR5+ memory T cells (L.J. Picker, unpublished observation). Thus, the proliferative generation of new CCR5+ CD4+ memory T cells may constantly provide new targets for viral replication in the postacute phase. Interestingly, it has been shown that in acute or very early HIV-1 infection, although peripheral blood CCR5+ CD4+ T cell proliferation increases dramatically, their total number remains in the normal range. In striking contrast, in acute EBV infection, proliferating CCR5+ CD4+ T cells accumulate to very high levels (113), which suggests that CCR5+ CD4+ T cells are rapidly removed in primary HIV-1 infection. Thus, although much of the evidence for profound and rapid depletion of CCR5+ CD4+ T cells during acute infection presented above is derived from studies in SIV infection of macaques, it is likely that a similar series of events occurs in HIV-1 infection of humans. Indeed, although studies have not been performed in the most acute stages of HIV-1 infection in humans, it is clear that a profound and rapid loss of intestinal CD4+ T cells occurs in early infection and that this loss is maintained at later stages (114–116).

T Cell Activation Alters T Cell Trafficking The movement of T cells between blood, lymphoid, and extralymphoid tissues is directed by the interplay of homing receptors and their endothelial ligands, adhesion molecule pairs, and cytokines, chemokines, and their receptors, all of which are profoundly altered in the setting of immune activation, with marked differences between naive and memory T cell subsets (64, 117–119). Thus, in HIV-1/SIV infection both the specific activation of T cells by the virus and the generalized immune activation, which are evident very early in infection (Figure 4), necessarily affect the lymphocyte homing and recirculation patterns. In conjunction with the greater expansion of CD8+ T cells in lymphoid tissue, such changes in T cell distribution could account in part for the observation of decreased and falling peripheral blood CD4+ T cell counts, CD8+ lymphocytosis, and inversion of the CD4/CD8 ratio in peripheral blood both in the acute and chronic phases of infection (23, 120–123). In fact, the early rise in peripheral blood CD4+ T cell counts with highly active antiretroviral therapy (HAART) is generally thought to be due to a reversal of this activation state, leading to normalization of lymphocyte distribution (123–129). Indeed, dramatic antigen- and cytokine-mediated changes

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in T cell trafficking and sequestration no doubt occur (23, 130–134), and it has been reported that the decline in peripheral blood CD4+ T cell numbers overestimates the actual CD4+ T cell loss in some lymphoid tissue (135). While these considerations and evidence indicate the inappropriateness of the customary extrapolation from peripheral blood cell counts to total-body T cell numbers, the evidence discussed above from early SIV and HIV-1 infection suggests that peripheral blood CD4+ T cell counts actually underestimate the total-body depletion of CD4+ T cells during acute infection.

T Cell Renewal After Profound Depletion To understand the consequences of the rapid and profound depletion of CD4+ T cells described above, we digress briefly from acute HIV-1 infection to establish a framework for understanding the situation in which the immune system finds itself as viral loads diminish and the infection enters its chronic phase. The human immune system seems to have an impressive ability to reconstitute itself after profound depletion, through both thymic-dependent and thymicindependent (antigen-driven peripheral expansion) pathways (136–140). After all, one of the most successful treatments for leukemia/lymphoma involves myeloablative chemotherapy, often with total body irradiation, followed by the intravenous infusion of hematopoietic stem cells and mature T cells. Reconstitution is often robust, such that many of the complications arise from graft-versus-host disease or recurrence of the malignancy, rather than immunodeficiency. However, if we take a closer look at the reconstitution of T cell numbers in the first 2 years after chemotherapy-induced depletion, a consistent theme becomes apparent: Whereas reconstitution of CD8+ T cell numbers is rapid and occurs by peripheral expansion, recovery of CD4+ T cell numbers is limited and delayed (141, 142) and is constrained by the age-dependent decline in thymopoiesis. There is compelling evidence that successful CD4+ T cell reconstitution after chemotherapy, or under HAART in HIV-1 infection, is determined to a large degree by thymic output (136, 140, 143–151). In contrast, CD8+ T cell reconstitution is dependent neither on age (152), nor on thymic output and can reach prechemotherapy baseline levels by 3 months posttherapy, a time when CD4+ T cell numbers average only 35% of pretherapy levels (153, 154). Furthermore, expanded populations of memory CD4+ T cells may subsequently decrease in number owing to an increased susceptibility to apoptosis (154). Even after autologous peripheral blood stem cell transplantation in children and adults, mature CD4+ T cells in the graft do not contribute substantially to CD4+ T cell reconstitution, and CD4+ T cell lymphopenia is prolonged (155, 156) and correlates with increasing age (156). When CD4+ T cells are specifically depleted with monoclonal antibodies in the treatment of multiple sclerosis and rheumatoid arthritis, their depletion is both profound and sustained (157, 158), with no effect on the numbers of circulating CD8+ T cells (157). Thus, the CD4+ and CD8+ T cell regenerative pathways appear to be distinct and, unlike CD8+ T cells, reconstitution of memory CD4+ T cell numbers

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after profound depletion is dependent on the reconstitution of naive CD4+ T cell numbers, which in turn depends on thymic output. Indeed, such data from human studies confirm what we are beginning to understand of the homeostatic mechanisms that maintain naive and memory T cell numbers in mice after lymphopenia and during antigenic stimulation. First, in lymphopenic states it appears that reconstitution and maintenance of the naive T cell pool depends on continued thymic output (159, 160) and that naive and memory T cells can supply the resting memory pool by peptide/MHC-dependent expansion (159–166). Second, antigen-driven memory CD4+ T cell expansion is limited, in contrast to the extensive expansion seen with CD8+ T cells (167–172). Third, whereas a significant proportion of activated expanded CD8+ T cells may “rest down” to resupply the resting memory CD8+ T cell pool, the fates of CD4+ T cells are very different after activation, and fewer seem to survive (170, 172–177). Indeed CD4+ T cells appear to have an intrinsically lower capacity for survival (165). Finally, maintenance and homeostatic expansion of the naive CD4+, but not CD8+, T cell pool is critically dependent on the presence of normal secondary lymphoid tissue (178). Taken together, these data suggest there is a substantial difference in the homeostasis and regenerative capacity of the CD4+ and CD8+ memory T cell pools, and that maintenance of the memory CD4+ T cell pool may be critically dependent on input from the naive CD4+ T cell pool. Figure 5 illustrates the dynamics of normal T cell homeostasis. The main principles, all based on experimental evidence, are (a) More CD4+ than CD8+ naive T cells exit the thymus; (b) turnover within the naive T cell pools is minimal; (c) naive T cells may be activated to enter the memory T cell pools, where they may remain activated or return to a resting memory state; (d) the expansion of the activated CD8+ T cell pool is much greater than that of the activated CD4+ T cell pool; (e) many more activated CD8+ T cells reenter the resting memory T cell pool than activated CD4+ T cells; and ( f ) the vast majority of activated T cells die, far more so for activated CD4+ T cells than activated CD8+ T cells. In a healthy individual over the course of a lifetime sufficient CD4+ and CD8+ T cell numbers are maintained to ensure immune competence. However, the proportions of the various pools change dramatically and differentially with age: Naive T cells decrease with respect to memory T cells, and CD4+ T cells decrease with respect to CD8+ T cells (179–186). Thus, as the brief period of acute HIV-1 infection wanes, the immune system is faced with two problems, both caused by the virus and both particular to CD4+ T cells. The first is to reconstitute a profoundly depleted memory CD4+ T cell pool in the face of age-attenuated thymic output and ineffective peripheral expansion— renewal mechanisms that are wanting, irrespective of ongoing viral replication. The second problem, discussed in the next section, is that ongoing viral replication aggravates those renewal limitations by impairing thymic output, disrupting lymph node architecture, and inducing CD4+ T cell activation that serves only to propagate the virus while placing additional homeostatic strain on maintenance of the “resting” naive and memory CD4+ T cell pools.

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THE CHRONIC PHASE OF INFECTION


Chronic Activation and T Cell Death Whereas acute HIV-1 infection is marked by rapidly increasing (and then decreasing) viral load and rapid CD4+ T cell depletion, the chronic phase of the infection manifests markedly different viral and T cell dynamics. Plasma viral loads are orders of magnitude lower but then rise slowly; peripheral blood CD4+ T cell counts often recover partially from their nadir during acute infection but then fall slowly over a period of, on average, 10 years before the onset of AIDS (14). However, even though things look quieter in this phase, it is actually characterized by a high level of activation of both CD4+ and CD8+ T cells. Thus, chronic infection is a state of chronic immune activation (187). Indeed, the chronic activation that results from persistent viral replication may be a better predictor of disease progression than the plasma viral load itself (188–191). Multiple studies of T cell turnover have resulted in various, often contradictory, interpretations of the T cell dynamics in chronic HIV-1 infection. Generally, these studies have shown a state of “high turnover” affecting all T cells, both CD4+ and CD8+ (129, 192–197). High turnover may imply simply moderately increased rates of division and death for most slowly dividing cells or activation of a small fraction of cells at any given time and their subsequent rapid expansion followed by death. The latter concept of high turnover involves varying degrees of increased T cell activation, expansion, movement through the naive to memory to effector axis, and ultimately, activation-induced cell death. Usually, however, only average turnover rates are observed. Studies of SIV-infected rhesus macaques have shown increased rates of in vivo incorporation and loss of BrdU in all T cell populations, with memory T cells being affected far more than naive (193, 195). Studies using Ki67 expression as a marker of cell proliferation have indicated that CD4+ and CD8+ T cell turnover increases in both naive and memory subsets during HIV-1 infection (129, 198, 199). Yet another study has shown that proliferation within the naive CD4+ T cell compartment is not increased in chronic HIV-1 infection (200). Quantitative image analysis of lymph nodes from HIV-1-infected individuals has revealed CD4+ T cell depletion in an environment of increased T cell proliferation and apoptosis (201). Finally, the measurement of T cell half-lives using in vivo labeling with stable deuterium isotope has shown that HIV-1 infection causes a decrease in memory (but not naive) CD4+ and CD8+ T cell half-life (192, 194). Notably, these studies have also shown that the number of proliferating and dying CD4+ and CD8+ peripheral blood T cells decreases rapidly with HAART (129, 192, 196–198), and other studies have measured a similar decrease in the number of T cells expressing markers of activation in both peripheral blood and lymph node (123–125, 128, 201–205). The interpretation of these data in terms of cause and effect has been the subject of considerable controversy: Does the virus cause massive CD4+ T cell death for which the immune system attempts to compensate with an impressive homeostatic proliferative response, or does the

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virus cause massive T cell activation and proliferation, with death being the natural immunological consequence? First, it is unlikely that the virus directly causes massive death of CD4+ T cells in chronic infection because the degree of their productive infection appears to be very low in both peripheral blood and lymph node, with estimates ranging from ∼0.01–1% (18, 23, 57, 206–210). Second, HIV-1 preferentially infects expanding populations of activated CD4+ T cells (211), the majority of which are already destined to die rapidly after proliferation and elaboration of effector function (212). Third, CD8+ T cell death occurs at the same rate as that of CD4+ T cells in kinetic models, yet their numbers are not significantly depleted until late in the course of infection (192, 194–196). In vivo BrdU pulse-labeling in HIV-1-infected individuals has shown no significant differences between CD4+ and CD8+ T cells in their high proliferation and death rates, and when viral replication is suppressed with HAART, T cell death rates do not change, implying that the death of recently divided cells is independent of HIV-1 (196). This effect can also be observed in SIV-infected macaques. Figure 2 shows that whereas decay of BrdU-labeled memory CD4+ T cells is more rapid than their CD8+ counterparts in acute infection, the decay rates of these two populations overlap during the plateau phase of chronic infection. The high rates of T cell death in chronic infection therefore seem to be the consequence, rather than the cause, of T cell activation and expansion. Thus, it is more likely that high lymphocyte turnover rates in HIV-1-infected individuals are caused by T cell activation, either virus-specific or owing to nonspecific “bystander” activation (3, 196, 211, 213). Notably, the predominant mechanism for CD4+ cell death, during the quasi–steady state dynamics of chronic infection, differs completely from that during acute SIV infection in macaques discussed above where the dynamics are entirely different.

Chronic Activation and T Cell Depletion If chronic immune activation accounts for the high death rates of both CD4+ and CD8+ T cells, these high rates per se do not explain what causes CD4+ T cell depletion. However, evidence has been accumulating in support of the concept that chronic activation can effect T cell depletion (3, 187, 206, 214). In HIV-1 infection the degree of chronic activation may be the best predictor of disease progression (188–191). SIV-infected sooty mangabeys and African green monkeys—natural hosts of this virus—develop high viral loads but neither profound CD4+ T cell depletion nor progressive disease (215–218), even though the virus is cytopathic (216, 218–220). The striking finding in SIV-infected sooty mangabeys that might explain this is that there is no generalized increase in T cell activation (218). Furthermore, HIV-2 infection is associated with lower viral loads and a slower decline in CD4+ T cell counts than in HIV-1 infection, but when CD4+ depletion does reach similar levels, equivalent levels of immune activation are found in individuals infected with either virus (221). Consistent with this concept is the finding of substantially decreased levels of immune activation in individuals whose

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viremia is not controlled by HAART but who nevertheless maintain increasing CD4+ T cell counts (205). It has been proposed that the decisive events that result in CD4+ T cell depletion are exerted through various mechanisms on the populations of resting T cells—both memory and naive (3, 23). These mechanisms include (a) attrition of the memory T cell pools by repeated immune activation events, (b) broad activation of naive T cells to enter the memory T cell pools, (c) reduced steady-state numbers of resting T cells owing to recurrent subthreshold stimulation, and (d) impaired supply of naive T cells from lymphopoietic sources and destruction of stromal elements involved in peripheral homeostatic functions owing to chronic infection and activation. These mechanisms involve direct, destructive effects of the virus as well as nondestructive effects. Obviously, CD4+ T cells are far more vulnerable than CD8+ cells to the directly destructive effects. However, as discussed above, the proliferative responses to antigen and the regenerative capacities of the CD4+ and CD8+ memory T cell pools differ markedly, by physiological design, which should result in differential susceptibility of the two subsets to the other detrimental effects of chronic immune activation. The greater clonal expansion of CD8+ cells than CD4+ cells is associated with larger steady-state numbers of activated cells, which may explain in part the increases in total CD8+ T cell numbers but not CD4+ numbers in the chronic phase (3). In addition, the unremitting rounds of memory T cell expansion and death would place a greater strain on maintenance of the resting CD4+ memory T cell pool because of its inherently greater dependency on the differentiation of activated naive cells. Moreover, although persistent homeostatic and antigen/inflammation-driven flow of naive T cells into the memory T cell pools, in the context of age-attenuated thymic output (185), would “drain” both the CD4+ and CD8+ naive T cell pools (222), the pressure may be greater on the naive CD4+ T cell pool because of the higher dependency of the CD4+ memory pool on input from the naive compartment. It is not surprising, therefore, that naive T cells play a major role in the long-term immune reconstitution of individuals on HAART (124–126), and that overall CD4+ T cell reconstitution may depend to a large degree on naive CD4+ T cell reconstitution via the thymus (143–149, 152, 223).

Infection and Inflammation: The Effects of HIV-1 on the Thymus, Bone Marrow and Lymph Nodes We have so far discussed the effects of HIV-1 infection on peripheral T cell pools; however, the primary supply route for naive T cells, before they settle in the lymph node, is the bone marrow–thymus axis, which is also a target for HIV-1-mediated suppression. Whereas mature T cells in the periphery seem able to adjust their proliferation and death rates to maintain homeostasis of numbers during normal aging, it is unclear whether this is the case under the strain of HIV infection. It is also unclear how the thymus responds to depletion of the peripheral T cell compartment.

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Naive CD4+ T cell increases following chemotherapy in children are associated with enlargement of the thymus above baseline—a phenomenon termed “thymic rebound” (224). Increased plasma levels of interleukin-7 (IL-7) in the context of lymphopenia owing to genetic abnormalities, chemotherapy (225, 226), or HIV-1infection (226, 227) have been suggested to indicate an IL-7-mediated homeostatic response to augment both thymic and peripheral pathways of T cell renewal (227). However, it is more likely that plasma IL-7 levels simply reflect the dynamics of binding of secreted IL-7 to T cells expressing IL-7 receptors: The fewer circulating T cells, the more free IL-7 (225). Indeed, the mouse thymus varies neither the export rate nor the composition of emigrant thymocytes in response to changes in either the size or composition of the peripheral T cell compartment (228–230). Evidence from mice suggests that thymocytes are exported at a fixed rate of ∼1–2% of total thymocytes per day, implying that the absolute amount of emigrants is determined by thymic mass (228). This is supported by the positive correlation between thymic size and the rate of naive CD4+ T cell reconstitution after HAART for HIV-1 infection in humans (143–149). Clearly, the thymus functions in adults and can contribute substantially to immune reconstitution (150, 231), a contribution that is crucial for producing a broad T cell receptor repertoire (136, 140, 151). However, age takes a considerable toll on thymic output (185, 186). Thus, it makes sense that the age-associated decrease in thymic size and output (185, 186) would, even in the absence of any inhibitory effects of the virus, render it increasingly difficult for the thymus to keep up with the constant drain on the peripheral naive T cell pool in chronic HIV-1 infection engendered by the processes described above (23). It also makes sense that the significance of inhibition of thymic output by HIV-1 depends on age. The reliance upon thymic output for the generation of an immune-competent memory T cell pool, both in terms of size and T cell receptor (TCR) diversity, is greater at younger ages when the memory pool is smaller. Thus, the consequences of thymic inhibition would be large in children but almost negligible in old age. It is generally accepted, through a considerable body of evidence, that HIV-1 infection adversely affects the thymus in both children and adults (186, 232, 233). Clinical studies have shown that the thymuses of HIV-1infected children and adults undergo abnormal morphological changes including thymocyte depletion and advanced involution (186, 234–237). Thymic dysfunction has been associated with early progression of disease in perinatally infected infants (238–240). SIV infection in rhesus macaques causes similar changes (241, 242). Studies using reaggregate thymic cultures, thymic organ cultures, and SCID-hu mice have shown that thymocytes at almost all stages of maturation are targets for HIV-1 infection (97–102, 243–248). Finally, the observation that peripheral blood TREC levels are decreased in untreated chronically HIV-1-infected children and adults is thought to reflect, at least in part, diminished thymic output (1, 147, 185, 200, 249–254). The effects of in vitro infection depend to a large degree on coreceptor tropism, as CD4 and CXCR4 are expressed on nearly all thymocytes, whereas CCR5 is expressed only at low levels on mature thymocytes (68, 255, 256). X4-tropic strains

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are highly cytopathic in vitro and rapidly deplete thymocytes, whereas R5 strains cause less thymocyte depletion but result in stromal cell abnormalities (100, 255). A recent study suggested that children infected with X4-tropic HIV-1 had lower thymic output than those infected with R5-tropic virus (257). However, analysis of coreceptor usage of thymic primary HIV-1 isolates from neonates has shown that X4- and R5-tropic isolates were both present and equally cytopathic for thymocytes (258). Either way, the thymus may act as a fertile ground for the replication of HIV-1, particularly X4-tropic viruses, owing to the high number of proliferating thymocytes (259) and the local production of cytokines such as IL-7 (248, 258, 260). Indeed this may explain the higher set-point viral loads in children and the observations that HIV-1-infected children often progress to AIDS more rapidly than adults (238, 261–263). In addition, it has been shown in the SCID-hu mouse model that HIV-1-infected thymocytes may survive to be exported into the periphery, where the virus remains latent until T cell receptor stimulation, indicating that the thymus might also be a source of latent HIV-1 in humans (264). It has long been known that HIV-1 infection may also inhibit the production of hematopoietic lineages other than CD4+ T cells, by directly or indirectly suppressing the maturation of hematopoietic progenitor cells (265, 266). Indeed, HIV1-infected individuals are often pancytopenic. Bone marrow architecture and cellularity is usually abnormal (267). However, the inhibitory effect of HIV-1 does not seem to involve direct infection of progenitor cells. The evidence clearly suggests that the progenitor populations are intact and largely uninfected and that the growth and differentiation of the few infected cells is unaffected. It is rather the stromal auxiliary cells of the bone marrow that are persistently infected and dysfunctional (268–270). Thus, as the memory, naive, and thymic compartments are progressively exhausted in chronic HIV-1 infection, the supportive functions of the hematopoietic stromal tissues fail as well and the primary source of all lymphocyte founders. The ongoing cycles of viral replication and chronic immune activation are also likely to be the cause of observed pathological changes in lymph node architecture in HIV-1 infected individuals (23, 271–273). As HIV-1 infection progresses, the lymph node CD4+ T cell population becomes depleted with, eventually, loss of all recognizable anatomic structures. This niche must preserve its structural integrity to ensure (a) proper function of the homeostatic mechanisms that maintain the naive CD4+ T cell pool (119, 178, 274–276), (b) productive interactions between T cells and antigen-presenting cells that are involved in generating and maintaining the memory CD4+ T cell pool, and (c) that cytokine signals reach target cells not only within their own microenvironment, but in other microenvironments within lymphoid tissue as well (64, 277–280). Importantly, studies of mice have shown that CD4+ T cell homeostasis is far more dependent on the presence of lymph node than CD8+ T cell homeostasis (178). Thus, over time, damage by the virus to the supply routes and anatomical niches that maintain the resting CD4+ T cell compartments act together with the homeostatic strains imposed by chronic

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immune activation to exacerbate further the progressive net loss in CD4+ T cell numbers.


What Makes HIV Infection Unique? Why does HIV-1 infection differ from all other infections? Other chronic viral infections in humans do not result in a substantial depletion of CD4+ T cell numbers and certainly do not cause AIDS (281–284). Whereas chronic parasitic infections may cause chronic high-level T cell activation, inversion of CD4+/CD8+ T cell ratios, and reduction of naive T cell numbers in peripheral blood, they do not result in profound CD4+ T cell loss unless there is co-infection with HIV-1 (285, 286). When addressing the roles of chronic activation and the limited capacity for renewal of CD4+ T cells in disease progression, one should not overlook the fact that HIV-1 has additionally an exquisite predilection for infecting CD4+ T cells, is cytopathic, and targets infected cells for killing by HIV-1-specific CD8+ T cells. The consequences of this were starkly illustrated in the preceding section on acute infection, which suggested that an HIV-1-infected individual enters the chronic phase of the disease with an already profoundly depleted total CD4+ T cell compartment, with the memory T cells of the mucosa mainly affected. Indeed, in SIV and HIV-1 infection the depletion of mucosal CD4+ T cells persists throughout the course of the disease (39, 114–116). Such profound depletion would place an added strain on CD4+ T cell renewal mechanisms, as the lymphopenia would impose an even greater homeostatic pressure on naive CD4+ T cells to enter and maintain the memory CD4+ T cell compartment (159–162). Thus, HIV-1 infection differs from other chronic infections in that, in addition to attrition of the resting memory and naive T cell pools caused by persistent immune activation, such activation is coupled to a state of severe memory CD4+ T cell lymphopenia almost from the outset. Given the destructive potential of the virus so clearly evident during the acute phase, it seems ironic that in the chronic phase of HIV-1 infection it is so difficult to pinpoint and evaluate the consequences of infection by the virus on its well-known target cells. Clearly, HIV-1 can also cause the lytic death of CD4+ T cells during the chronic phase. Yet even though a small number of the rapidly proliferating, activated CD4+ T cells identified in turnover studies is likely to be the major source of virus production, the consequence of this on the size of that T cell pool is moot, because the vast majority is destined to die anyway as a direct result of such activation (3). However, those cells that contribute to the maintenance of the resting memory CD4+ T cell pool—the small proportion of activated naive and memory CD4+ T cells that survive to enter or reenter the resting memory CD4+ T cell compartment—are also subject to HIV-1-mediated killing. In particular, studies show that naive T cells that have been activated are exquisitely sensitive to HIV1 infection (210) and that cytokine signals in the absence of full TCR-mediated activation are sufficient for HIV-1 infection of resting naive and memory CD4+ T cells in vitro and in vivo (76, 287, 288). Thus, in the midst of the homeostatic

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upheaval and CD4+ T cell depletion caused by chronic activation, the virus itself slowly chips away at those cells attempting to sustain the resting CD4+ T cell pool.


T Cell Dynamics During the Chronic Phase: The Overall Picture Whereas the dissemination of HIV-1 in acute infection is explosive and suggests rapid propagation of virus between many available target cells (23, 45), observations of HIV-1 viral genotypes in local microenvironments (60, 289), and other studies (210), support a concept of “proximal immune activation and virus transmission” (61). The implications of this proximal activation are that during much of the chronic phase, when viral loads are low, efficient transmission of the virus from cell to cell is largely limited to local immune activation bursts in lymphoid tissue that may be provoked by antigenic and inflammatory stimuli. Given that HIV-1 infection itself induces immune activation, it has been proposed that immune activation is the engine driving viral replication (3). Thus, the virus constantly generates its own targets. As most of these target cells are, by nature, short-lived and “expendable,” their massive subsequent death does not immediately affect, in the chronic phase, the crucial cellular resources of the immune system, namely, the naive and resting memory T cell pools. However, we have described how chronic immune activation may slowly but progressively drain these pools. In addition, the virus directly interferes destructively with the supply routes of naive and memory T cells and destroys the organs that maintain them. All of these events affect CD4+ T cells more than CD8+ T cells, owing both to physiological differences between the two subsets and the early established lymphopenia affecting the first, hence their preferential loss. Under such conditions the immune system eventually collapses. The scheme shown in Figure 6 summarizes T cell dynamics during the chronic phase of HIV-1 infection. The main principles depicted are that (a) there are pathological changes in bone marrow, thymus, and lymph node architecture, and there is a decrease in thymic output; (b) the memory CD4+ T cell pool is already decreased in size after the acute phase; (c) the CD4+ and CD8+ naive and resting memory T cell pools become chronically activated; (d) increased T cell activation results in increased T cell death; (e) the expansion of the activated CD8+ T cell pool is much greater than that of the activated CD4+ T cell pool; ( f ) more activated CD8+ T cells than activated CD4+ T cells re-enter the resting memory T cell pool; (g) the vast majority of activated T cells die, more so for activated CD4+ T cells than activated CD8+ T cells; (h) the main source of virus is the activated CD4+ T cell pool; (i) the majority of infected activated CD4+ T cells are physiologically destined to die simply owing to their activation; and ( j) a fraction of the already small proportion of infected CD4+ T cells destined to enter or reenter the resting memory CD4+ T cell pool will fail to contribute to that pool.

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In essence, the chronic phase of HIV-1 infection is a quasi-stable steady state, a slowly decaying unstable equilibrium between T cell activation, death, and renewal and production and removal of virus. Exponential spread of the virus by self-perpetuating rounds of activation and infection is kept in check by the transient and local nature of viral bursts, by the short life span of most virus-producing cells, and by physiological constraints on the accumulation of the longer-lived infected memory T cells (61). Eventually the equilibrium breaks down completely as progressive immune degradation ultimately results in clinical immunodeficiency manifested by the development of the opportunistic infections and associated malignancies that characterize overt AIDS (14, 24). Loss of HIV-1-specific immunity might play a role in the acceleration of the viral replication that can be observed in late disease (290, 291). Furthermore, the mutability and genetic flexibility of HIV-1 and SIV allow these viruses to respond to selection pressure, adapt to changing host conditions, and evolve into viral variants with an expanded repertoire of target cells and enhanced pathogenicity (26–28, 100, 292, 293) or diminished susceptibility to HIV-specific T cell immune responses (112).

HIV-1-Specific T Cell Immune Responses: Part of the Solution but Also Part of the Problem Despite a rapid fall in HIV-1 load after acute infection, this quasi-stable steady state often takes months to become established, usually preceded by a period of strong viral load fluctuation. The factors involved in this initial partial control of viral replication remain controversial. Indeed, it has been suggested that the main determinant of the decrease in viral load is the rapidly diminishing availability of activated CD4+ T cell substrates for infection (294). The profound depletion that has now been observed in the mucosal tissues, as we have described, is largely consistent with this interpretation. However, a significant body of evidence suggests that the immune response is actively involved in viral control. There is early circumstantial evidence in humans showing a temporal correlation between the initial drop in viremia and the appearance of HIV-1-specific CD8+ T cells but not neutralizing antibodies (19, 20). Several studies have also shown a direct association between class I HLA types and rates of HIV-1 disease progression (295–299). In acute SIV infection of rhesus macaques, the emergence of SIV-specific CD8+ T cells coincides with clearance of virus (52, 300), and transient depletion of CD8+ T cells in chronically SIV-infected rhesus macaques leads to a rapid increase in viral replication (301– 303). Finally, viral sequence changes allowing escape from virus-specific CD8+ T cells develop very rapidly in acute infection in humans (304) and monkeys (305– 307), indicating that these responses exert sufficient antiviral pressure to drive the outgrowth of minor viral species. The interplay between newly generated T cell responses and rapid viral escape that occurs during and after acute infection may be partially responsible for the period of viral load fluctuation that precedes the establishment of a viral set point.

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Much of the T cell activation in HIV-1 infection is likely to be due to virusspecific responses, as evidenced by the high frequency of circulating HIV-1-specific T cells (particularly CD8+ T cells) throughout the course of infection before the onset of AIDS (21, 22, 308–313). However, although strong and broad HIV-1-specific responses that are clearly able to reduce viral loads are elicited early in infection, they fail to prevent disease progression. Studies that show absence of correlation or positive correlation between viral load and the overall frequency or breadth of circulating HIV-1-specific CD8+ T cells (308– 310, 314, 315) suggest that HIV-1-specific CD8+ T cells respond to the virus, but these studies fail to indicate the effectiveness of this response. Recruitment of naive HIV-1-specific CD4+ T-cells, along with HIV-1-specific CD8+ T cells, into infected lymphoid sites may affect the rate of viral clearance (316) but may also provide cellular substrates for viral replication (289). Indeed, rapidly expanding CD4+ T cells, in transition from naive to fully activated phenotype, are exquisitely susceptible to productive HIV-1 infection (210). Thus, the initial antigen-specific activation of HIV-1-specific T cells may augment viral replication, which will in turn stimulate more HIV-1-specific T cells, leading to further activation and expansion in a positive feedback loop (61). With this in mind, we have confirmed that HIV-1-specific CD4+ T cells are preferentially infected by HIV-1 at all stages of disease (210). Indeed, we might speculate that it is the HIV-1-specific response itself that provides both the initial thrust and continued momentum for the maintenance of chronic activation. The other side of the coin is that recurring cycles of activation and infection may result in the progressive loss of HIV-specific CD4+ T cell responses that begins during acute infection and continues throughout its course (21, 22, 311, 317), although circulating HIV-1-specific CD4+ T cells are clearly present in the chronic phase (21, 308, 312). In addition to the actual reduction in the specific CD4+ T cell population, the postactivation state of many of the remaining HIV-1-specific CD4 cells and their continued stimulation during a state of high viremia may be associated with a transient inability to proliferate in response to activation signals (312), as occurs in other acute or chronic viral infections (318–324). A similar form of unresponsiveness is apparently also exhibited by many of the HIV-1-specific CD8+ T cells. Peripheral blood HIV-1-specific CD8+ T cells are highly sensitive to Fas-induced apoptosis (291), have low expression of perforin (325), may not produce cytokines in response to antigen (326, 327), often have TCRs of low functional avidity (328) and inappropriate signaling (327), exhibit skewed maturation profiles (329–331), and are often unable to proliferate in response to antigen (339). However, CD8+ T cells with these phenotypes are often the dominant subset at particular stages of protective immune responses against other viruses such as CMV and EBV (332–334), and may also arise in other situations of chronic antigenic stimulation (120, 335–337). As such, these “defective” HIV-1-specific CD8+ T cells, trafficking between blood and lymphoid tissue, are likely to reflect the consequences of chronic antigen stimulation rather than being the cause of inadequate control of viral replication (334). The viral

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epitope escape mutants that develop rapidly during acute infection (304–306), particularly from epitopes that elicited high-avidity CD8+ T cells (307), could still maintain high frequencies of those HIV-1-specific CD8+ T cells, the majority of which may be impaired in their ability to clear the predominant circulating viruses (338). Thus CD4+ and CD8+ HIV-1-specific T cells make a valiant and partially successful attempt to control virus production and halt the disease. However, from the earliest stages of the infection, by driving CD4+ and CD8+ T cell activation, by providing substrates for viral replication, and by unwittingly being targeted for preferential infection, the HIV-1-specific response becomes part of the problem as well as part of the solution. With the emergence of escape mutants, a selfdestructive situation develops in which the virus may persistently activate the immune system at little cost to itself but with the benefit that the activation increases virus production.

CONCLUSION We began this review by asking what sets HIV-1 infection apart from other viral infections and suggesting that the answer rests in understanding the underlying dynamics of T cells and virus. The data we have reviewed do not provide definitive answers but offer promising clues. New observations suggest that considerable damage is caused to the immune system during the acute phase of the infection, resulting in a selective but substantial lymphopenia. Several other observations reveal a unique strategy in which HIV-1 induces immune activation to generate replaceable target cells in order to sustain its replication. Most of the target cells are short-lived and “expendable,” by physiological design. Nevertheless, as we have described, chronic activation can strain homeostasis of the naive and resting memory T cell pools indirectly in a number of ways and, when coupled to the combined impact of ongoing, low-level destructive events mediated by virus and of the “historic” lymphopenia resulting from acute infection, this strain leads to the progressive depletion of the more vulnerable CD4+ T cell pools. We do not think destroying the immune system is part of this strategy, but rather that AIDS is a consequence of incomplete adaptation of the virus to its relatively new host. Better understanding of the causes and effects underlying the unstable dynamics that we call disease progression will undoubtedly contribute to our ability to intervene and treat this unique infection.

ACKNOWLEDGMENTS We thank the following for informal discussions and kind advice: David Price, Tim Schacker, Ashley Haase, Robert Seder, Mike Lederman, Martin Meier-Schellerscheim, and Zvi Grossman.

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LITERATURE CITED 1. McCune JM. 2001. The dynamics of CD4+ T-cell depletion in HIV disease. Nature 410:974–79 2. Rowland-Jones S. 1999. HIV infection: Where have all the T cells gone? Lancet 354:5–7 3. Grossman Z, Meier-Schellersheim M, Sousa AE, Victorino RM, Paul WE. 2002. CD4+ T-cell depletion in HIV infection: Are we closer to understanding the cause? Nat. Med. 8:319–23 4. Asquith B, Hanon E, Taylor GP, Bangham CR. 2000. Is human T-cell lymphotropic virus type I really silent? Philos. Trans. R. Soc. London Ser. B 355:1013–19 5. Lenardo MJ, Angleman SB, Bounkeua V, Dimas J, Duvall MG, et al. 2002. Cytopathic killing of peripheral blood CD4(+) T lymphocytes by human immunodeficiency virus type 1 appears necrotic rather than apoptotic and does not require env. J. Virol. 76:5082–93 6. Cooper DA, Gold J, MacLean P, Donovan B, Finlayson R, et al. 1985. Acute AIDS retrovirus infection: definition of a clinical illness associated with seroconversion. Lancet 1:537–40 7. Ho DD, Sargadharan MG, Resnick L, DiMarzo-Veronese F, Rota TR, et al. 1985. Primary human T-lymphotropic virus type III infection. Ann. Int. Med. 103:880–83 8. Henrard DR, Daar E, Farzadegan H, Clark SJ, Phillips J, et al. 1995. Virologic and immunologic characterization of symptomatic and asymptomatic primary HIV-1 infection. J. Acquired Immune Defic. Syndr. Hum. Retrovirol. 9:305–10 9. Daar ES, Moudgil T, Meyer RD, Ho DD. 1991. Transient high levels of viremia in patients with primary human immunodeficiency virus type 1 infection. N. Engl. J. Med. 324:961–64 10. Clark SJ, Saag MS, Decker WD, Cam-

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Figure 1 The natural history of typical HIV-1 infection depicting changes in plasma HIV-1 viral load, peripheral blood CD4+ T cell count, and HIV-1-specific CD8+ T cell response.


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Figure 4 Memory T cell dynamics in progressive SIV and nonprogressive CMV infection. CD4+ and CD8+ memory T cells were followed for their expression of the proliferation marker Ki-67 or their incorporation of the thymidine analogue BrdU by multiparameter flow cytometry after primary infection with pathogenic SIV (A) or rhesus CMV (B ). BrdU was given in single daily IV injections on postinfection days 10–13 (A and B) and days 115–118 (A only). CD4+ memory T cells are shown in blue, CD8+ memory T cells in red.


Figure 5 Diagrammatic representation of T cell dynamics in a healthy individual. Resting cells along the bone marrow to naive T cell axis are shown in yellow. Activated T cells (due to any stimulus) are shown in green. Resting memory T cells are shown in blue. Dying cells are shown in black. The arrows depict movement of cells between the pools. The sizes of the boxes depicting the pools and the number of cells therein represent the relative magnitude of those cellular pools, but they are not to scale. The “activated” box would include so-called effector memory cells—those elaborating effector functions. The peripheral T cells shown comprise those in lymphoid and extra-lymphoid tissues.


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Figure 6 Diagrammatic representation of T cell dynamics in an individual in the chronic phase of HIV-1 infection. Resting cells along the bone marrow to naive T cell axis are shown in yellow. Activated T cells (due to any stimulus) are shown in green. Resting memory T cells are shown in blue. Dying cells are shown in black. HIV-1-infected T cells are shown in red and are considered to have a reduced life-span. The peripheral T cells shown comprise those in lymphoid and extra-lymphoid tissues. The red arrows depict direct and indirect negative effects of HIV-1 on T cell production and/or survival, including destruction of lymphoid organ architecture. The green arrows depict HIV-specific and “bystander” HIV-1-induced T cell activation.


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Annu. Rev. Immunol. 2003. 21:305–34 doi: 10.1146/annurev.immunol.21.120601.141110 First published online as a Review in Advance on December 5, 2002

T CELL ANERGY∗ Ronald H. Schwartz Annu. Rev. Immunol. 2003.21:305-334. Downloaded from arjournals.annualreviews.org by HINARI on 09/01/07. For personal use only.

Laboratory of Cellular and Molecular Immunology, National Institutes of Health, Bethesda, Maryland 20892-0420; email: [email protected]

Key Words clonal anergy, adaptive tolerance, regulatory T cells, CD28/CTLA-4, IL-2/IL-2R ■ Abstract T cell anergy is a tolerance mechanism in which the lymphocyte is intrinsically functionally inactivated following an antigen encounter, but remains alive for an extended period of time in a hyporesponsive state. Models of T cell anergy affecting both CD4+ and CD8+ cells fall into two broad categories. One, clonal anergy, is principally a growth arrest state, whereas the other, adaptive tolerance or in vivo anergy, represents a more generalized inhibition of proliferation and effector functions. The former arises from incomplete T cell activation, is mostly observed in previously activated T cells, is maintained by a block in the Ras/MAP kinase pathway, can be reversed by IL-2 or anti-OX40 signaling, and usually does not result in the inhibition of effector functions. The latter is most often initiated in na¨ıve T cells in vivo by stimulation in an environment deficient in costimulation or high in coinhibition. Adaptive tolerance can be induced in the thymus or in the periphery. The cells proliferate and differentiate to varying degrees and then downregulate both functions in the face of persistent antigen. The state involves an early block in tyrosine kinase activation, which predominantly inhibits calcium mobilization, and an independent mechanism that blocks signaling through the IL-2 receptor. Adaptive tolerance reverses in the absence of antigen. Aspects of both of the anergic states are found in regulatory T cells, possibly preventing them from dominating initial immune responses to foreign antigens and shutting down such responses prematurely.

NOMENCLATURE The term anergy was first coined by Von Pirquet in 1908 (1) to describe the loss of delayed-type hypersensitivity to tuberculin in individuals infected with measles virus. In 1980, Nossal & Pike (2) borrowed this word to describe the functional inactivation of B cells that they observed in mice administered doses of antigen that induce tolerance. Because their phenomenon was antigen-specific, the phrase clonal anergy was adopted to distinguish it from the nonspecific effect on skin tests observed during acute infectious diseases. This new term was subsequently applied ∗ The U.S. Government has the right to retain a nonexclusive, royalty-free license in and to any copyright covering this paper.

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to describe T cell phenomena in which modified forms of antigen presentation to T cell clones produced a hyporesponsive state affecting subsequent IL-2 production and proliferation on restimulation (3). By the early 1990s this area of immunology research had become so popular and promiscuous that the term anergy was used to describe almost any tolerance phenomenon in which the lymphocytes survived and appeared to be functionally unresponsive! Perhaps this is the inevitable drift for terminology when a word is used to describe complex biological phenomena for which there is no good understanding of the underlying molecular events. This dilemma makes a review on T cell anergy difficult to execute because it is not always straightforward to pigeonhole all of the experimental results into neatly defined functional categories. Nonetheless, I attempt in this review to lay out a few working definitions to try and clarify the large body of sometimes seemingly contradictory experiments that have been published. I start with a global definition for T cell anergy that ignores its historical roots. It is a tolerance mechanism in which the lymphocyte is intrinsically functionally inactivated following an antigen encounter, but remains alive for an extended period of time in the hyporesponsive state. The anergic cell’s survival is not necessarily the normal life span of the cell, but it must be greater than the standard 8–24 h period observed for cells that have initiated an apoptotic death process. Although some of the biochemical steps in antigen-induced apoptosis and anergy overlap, caspase activation represents a distinct physiological response and plays no role in anergy (4, 5). A variety of functional limitations characterize the anergic state, including cell division, cell differentiation, and cytokine production. The only agreed-upon characteristic is that the state itself must be cell-autonomous, distinguishing it from immunoregulation mediated by other cells in the population (regulatory cells), although the induction might involve such cells. Maintenance of the anergic state may or may not require the persistence of antigen, and the state may or may not be reversible upon the addition of IL-2, although these properties are selectively associated with certain types of anergy. It is important to distinguish anergy from differentiation of cells to a Th-2 effector phenotype, which is typically done by showing that the cells in question fail to make IL-4 on restimulation, and in one model by a failure of the cells to proliferate to IL-4 (6). In some cases, however, the production of cytokines such as IL-10 is associated with the anergic state and may in fact synergize to create a negative regulatory phenotype. The intimate relationship between suppression and anergy is discussed in detail at the end of this review. Finally, the antigen encounter required to induce the state can occur either during development or after the cells have matured. The basic types of T cell anergy fall into two broad (but not perfect) categories. One is principally a growth arrest state, which I call T cell clonal anergy, and the other represents a more generalized inhibition of proliferative and effector functions, which my lab has called adaptive tolerance, but is sometimes referred to by others as in vivo anergy (7). The former arises from incomplete T cell activation, is mostly observed in previously activated T cells, and usually does not result in the inhibition of effector functions. The latter is most often initiated in

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na¨ıve T cells in vivo by stimulation in an environment deficient in costimulation or high in coinhibition [e.g., cytotoxic T lymphocyte antigen-4 (CTLA-4)]. The cells proliferate and differentiate to varying degrees and then downregulate both activities in the face of persistent antigen. Although the molecular states underlying these two forms of anergy are not yet fully understood, preliminary biochemical characterization suggests that they are quite distinct. At this time, nothing precludes the possibility that the two states could coexist in the same cell, which leads to some difficulty in sorting out their independent nature and possibly separate biologic functions.

T CELL CLONAL ANERGY Induction of Clonal Anergy The induction and characterization of this tolerant state has been reviewed a number of times (3, 8–10). In brief, the state can be induced in CD4+ T cell clones in about 6–12 h by delivery of a strong T cell receptor (TCR) signal in the absence of costimulation or by stimulation with a low-affinity ligand in the presence of costimulation. It was recently demonstrated that very low doses of a full agonist can also induce anergy in the presence of costimulation, but only with memory T cells (11). All of these conditions result in a weak or incomplete activation of the cell, but one that is strong enough to induce new protein synthesis (12) and the production of proteins required to initiate and maintain the anergic state. The state can also be induced in human T cells under normal activating conditions in the presence of IL-10 (13) and possibly in human T cell clones following T cell antigen presentation (14). The CD28/B7 pathway of costimulation seems to be a critical variable in preventing clonal anergy induction. Two mechanisms have been proposed. One is a direct inhibitory effect of CD28 signaling on the production or function of anergic factors (15). The other is an indirect effect on cell-cycle progression via stimulation of growth factors such as IL-2 (16, 17). Experimental support for the latter hypothesis is very strong. For example, full stimulation of T cell clones in the presence of rapamycin (which blocks cell-cycle progression in G1 by inhibiting activation of the kinase mTor) leads to an anergic state, even though the CD28 signaling induces copious amounts of IL-2 (18). Treatment of T cells with hydroxyurea, which blocks the cell cycle in early S phase does not induce anergy following full T cell activation. This suggests that the transition from G1 to S is the critical stage during which costimulation exerts its effect. Acceptance of the cell-cycle model, however, does not preclude a direct effect of CD28 signaling in preventing anergy; it just makes it harder to prove the existence of such an activity. For example, Wells et al. (19) isolated carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled cells that had divided two times in response to anti-CD3 in the absence of B7 costimulation (CTLA4-Ig blockade) and showed in a restimulation assay that these cells had become clonally anergic. This result suggests that

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cell-cycle progression is not sufficent to avoid anergy induction. The question that arises, however, is whether these cells represent the ones that were optimally stimulated for anergy induction in the first place and thus require more total time spent in G1 to S phase transition (i.e., more cell cycles) in order to circumvent the state? To rule this out, one needs to isolate the cells that divided 3 or 4 times and see whether they are equally anergic. The role of other forms of costimulation in the decision-making process of the T cell, i.e., whether to respond or become anergic, has been studied less. One interesting example is the role of intercellular adhesion molecule-1/leukocyte function antigen-1 (ICAM-1/LFA-1) interactions. Webb and colleagues (20) set up an antigen presentation system in which CD4+ T cells from a TCR transgenic mouse were stimulated with transfected Drosophila cells expressing major histocompatibility complex (MHC) class II molecules and either ICAM-1 or B7-2. Antigenpresenting cells (APCs) expressing the MHC molecules alone did not elicit a T cell response in the presence of the antigenic peptide, other than phosphorylation of the TCRζ chain to the p23 state. These cells did not become anergic or show an effector/memory phenotype upon restimulation. If the APCs expressed both B7-2 and MHC molecules, the T cells were fully activated, i.e., they proliferated, differentiated, and acquired an increased sensitivity to antigen on restimulation. In contrast, APCs expressing ICAM-1 plus MHC class II molecules induced clonal anergy. During the initial stimulation, the cells responded by increasing their activation markers, CD25 and CD69, but they produced no detectable cytokines and proliferated only slightly (2–3 divisions as detected by CFSE). On restimulation with peptide and spleen cells following 3 days of rest, the secondary proliferative response to antigen was greatly diminished with an increase in the dose response curve of 10–100 fold. IL-2 production was also impaired. It is interesting that this anergy induction could be prevented if a combination of IL-1 and IL-6 was added in the primary culture. IL-2 addition did not suffice, which suggests a previously unrecognized role for cytokine costimulation in the prevention of anergy. The best way to understand the role of ICAM-1 in facilitating the induction of anergy is to consider its role in enhancing TCR signaling by increasing the adhesion of the na¨ıve T cell and the APC. In the absence of ICAM-1, TCR occupancy alone under these conditions is adequate only to give activation of src family tyrosine kinases and no other downstream events. Addition of ICAM-1 boosts TCR signaling to a level sufficient for the synthesis of anergic factors and other inductive events such as expression of CD25 and CD69. However, the stimulus is not strong enough to counteract the anergic factors. Hence, this form of costimulation favors anergy induction. In this regard, CD28/B7 interactions can also facilitate TCR signaling by prolonging tyrosine kinase activation following mobilization of CD28 to the synapse (21). Thus, B7 costimulation could in principle work in a similar manner to favor anergy induction. However, there seems to be a qualitative difference between the B7 and ICAM-1 forms of costimulation. No matter how much antigen was used in the in vitro cultures, ICAM-1 expressing APCs never primed, whereas B7-2 expressing APCs always did. This might simply be a

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consequence of the potency of B7 costimulation and the difficulty in achieving a low enough density to obtain only weak activation (especially in the presence of CTLA4, which may preferentially dampen weak signals—see further discussion below). On the other hand, these data might reflect a unique function of CD28 signaling required to induce cell-cycle progression or inactivate anergic factors. In the original experiments on anergy induction in CD4+ T cell clones, peptide presentation by MHC molecules incorporated into planar membranes was sufficient (without ICAM-1) to induce size enlargement and anergy, but never proliferation (12). Na¨ıve T cells, in contrast, are not anergized by the same form of signaling (22). Several groups have reported that na¨ıve T cells also cannot be anergized by other means (23, 24). In addition, anergy induction of na¨ıve cells in vivo is always preceded by cell division (although I argue later that this is probably a different type of anergic state). Finally, in the cases where anergy induction in na¨ıve T cell populations has been reported, either with plate-bound anti-CD3 stimulation (5, 25) or presentation of peptide/MHC complexes with ICAM-1 expressed in Drosophila cells (20), CFSE-labeling studies have shown that the cells also divide several times during the induction (20, 26). Thus, the cells may in fact have been primed first and then anergized. These observations have raised the question of whether T cells must divide before they can be induced into a state of clonal anergy. The best study arguing against this possibility involved CFSElabeled T cells stimulated with anti-CD3 plus APCs in the presence of CTLA4-Ig to block B7 costimulation (19). When isolated four days later, the cells that had not divided failed to proliferate on restimulation with anti-CD3 and anti-CD28. This unresponsiveness could be overcome by the addition of IL-2. Thus, the nondivided cells appeared to be clonally anergic. The one problem with this experiment is that the starting population of T cells came from a normal mouse, which contains a significant population of memory cells. If there were selective survival of the memory cells in the primary culture, then the undivided anergic cells may have all been induced from this pool. Thus, the definitive experiment to prove that clonal anergy can be induced in na¨ıve, undivided cells remains to be done by using CFSE-labeled na¨ıve Rag2−/− TCR transgenic CD4+ T cells stimulated in vitro in the absence of costimulation, followed by sorting and restimulation of the nondivided cells in the presence or absence of IL-2. The role of the coinhibitory receptor CTLA-4 in clonal anergy induction in vitro is complex. CD4+ T cell clones express CTLA-4, although most of the protein is intracellular. Nonetheless, blocking antibodies (which had inhibitory effects in vivo) or genetic CTLA-4 deficiency did not influence the induction of anergy in vitro or allow clonally anergic cells to respond to antigen stimulation (5; N. Nabavi & R.H. Schwartz, unpublished data). In one recently described model system, however, CTLA-4 seems to play a critical role in the induction of unresponsiveness. Wells et al. (19) noticed that a significant percentage (up to 30%) of normal CD4+ T cells fail to divide following stimulation with anti-CD3 and APC, although all the cells become activated as delineated by CD69 expression. When they isolated the nondividing cells using CFSE, they found the cells unable

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to proliferate following restimulation, even though 30% of the cells could make IL-2. Addition of exogenous IL-2 during restimulation confirmed that the failure of the cells to divide was not due to a lack of growth factors. They called this state division arrest anergy. Because this state was not observed when B7 molecules were blocked with CTLA4-Ig (see above), they tested to see if CTLA-4 was playing a role in its induction. Addition of anti-CTLA-4 Fab fragments to the primary stimulation cultures prevented the development of division-arrest anergy. Instead, the cells were clonally anergic, i.e., they divided on restimulation only in the presence of IL-2. These observations demonstrate that CTLA-4 signaling is required for the induction of division arrest anergy but not for clonal anergy, which suggests that the former is a more complicated version of the latter in which CTLA-4 signaling has superimposed an additional block. The fact that clonal anergy develops under the blocking conditions in undivided cells also indicates that B7/CD28 interactions don’t always prevent anergy induction, and argues in favor of a “Goldilocks” (just right) level of TCR signaling model for clonal anergy induction (see discussion above). Finally, these experiments show that not all underlying clonal anergy states need be reversible upon the addition of IL-2, if additional independent blocks are superimposed. In this sense then, division arrest anergy would be simply a deeper substate of clonal anergy. The biochemical signaling events that give rise to the clonal anergic state are poorly understood. Early on, it was demonstrated that cyclosporine A could block anergy induction, suggesting that the calcium/calmodulin/calcineurin pathway was critical (28). In addition, treating T cell clones with a calcium ionophore alone can induce a transient unresponsive state that resembles clonal anergy; however, the state takes longer to induce than with anti-TCR antibody, suppresses all cytokine and chemokine production, and is not stable, i.e., by 4 days the cells regain responsiveness (29, 30). Recently, Rao and colleagues (30) showed that Th1 cells derived from a nuclear factor of activated T cells 1 (NFAT1) gene-targeted mouse were resistant to anti-CD3-mediated induction of clonal anergy, whereas cells transfected with a constitutively active NFAT1 (without overexpression) showed reduced IL-2 production following anti-CD3 and anti-CD28 stimulation, even when the transcription factor was engineered not to interact with AP-1. These results show that NFAT movement to the nucleus and transactivation of genes with NFAT response elements not involving AP-1 as a cofactor are critical parts of anergy induction. One candidate for an NFAT-inducible gene of this nature is early growth response (Egr) 2/3, which is elevated in CD4+ T cell clones following signal 1 alone (M. Safford, J. Powell & R.H. Schwartz, unpublished data). This transcription factor is known to play a critical role in FasL upregulation (31). Egr2/3 may also be an important player in anergy induction. Because calcium ionophores alone cannot completely mimic TCR-induced anergy, there must be other TCR-signaling pathways involved. The drug SB203580, which blocks the p38 mitogen-activated protein (MAP) kinase pathway, and the drug PD90859, which blocks MAP/ERK (MEK) in the extracellular signalregulated kinase (ERK) pathway, do not prevent the induction of anergy, either

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alone or in combination. Furthermore, a T cell line derived from the Jun N-terminal kinase 2 (JNK2−/−), JNK1 dominant negative mouse created by Dong & Flavell could be anergized with anti-TCR antibody (L. Luu, J. Powell & R.H. Schwartz, unpublished data). These preliminary results suggest that no single part of the MAP kinase pathway is necessary for clonal anergy induction, although the possibility of redundancy in this pathway has not yet been fully ruled out. The only other pharmacological reagent we have found to consistently block anergy induction is Go6976, an ATP analog that inhibits classical protein kinase C (PKC) activity (J. Powell & R.H. Schwartz, unpublished data). However, other classical PKC inhibitors have had no effect, leaving in doubt the exact target of this drug. Thus, to date, only the calcium/NFAT pathway has been shown unequivocally to be involved in clonal anergy induction.

Maintenance of Clonal Anergy Clonal anergy in murine T cell clones is characterized by an unusual pattern of cytokine production in response to TCR stimulation, which suggests that the state is predominantly an antiproliferative one (6, 24, 29, 32). On restimulation of the T cell with antigen and APCs, IL-2 production is the most profoundly affected, whereas IL-4 and IFN-γ production are only slightly impaired. Inflammatory chemokine production is also unaffected, while IL-3 production and upregulation of CD40L are intermediate in their responses. The block in IL-3 production may be rationalized in the context of an antiproliferative model of clonal anergy based on the ability of this cytokine to synergize with IL-4 to stimulate cell division among previously activated T cells (33). However, the most persuasive argument that the clonally anergic cell is in a state of growth arrest is the finding that Th-0 cells in this state fail to proliferate to IL-4 following reactivation, even though they can make IL-4 (6). This state is to be distinguished from Th-2 cell differentiation in which the cell utilizes IL-4 as its primary growth factor. Consistent with this argument, clonal anergy has been induced as an additional state in Th-2 cells by stimulating them with altered peptide ligands (34). The biochemical block to reactivation in clonally anergic mouse T cells is still not clearly understood. Somatic cell hybrids of anergic and normal T cells have shown that the anergic state is dominant (35). This suggests the presence of some protein(s) is inhibiting T cell activation. For the IL-2 gene, whose expression is prevented at the level of transcription, defects have been demonstrated in activation of the AP-1 response elements (36). There is less transcription factor binding at the proximal AP-1 site following activation, and the so-called distal AP-1 site (−180 site) shows preferential binding of inhibitory cAMP response elementbinding protein/cAMP response element modulator (CREB/CREM) transcription factors (37). In contrast, mobilization and binding of NFAT transcription factors are normal (38). Consistent with these latter observations, activation of the calcium/ calcineurin pathway in the cell is intact. The block instead appears to be in activation of the Ras/MAP kinase pathway (39, 40). Both the ERK and JNK pathways

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are inhibited, and there is one report of a block in the p38 pathway (41). Direct examination of p21 Ras activation showed a failure to induce the GTP-bound form, yet there was no defect in the activation of son of sevenless (SOS), the guanine exchange factor responsible for facilitating GTP loading (40). These results suggest that the underlying problem might be the constitutive activation of a GTP activating protein (GAP), which would keep p21 Ras in an inactive state by facilitating the conversion of Ras-GTP to Ras-GDP. An alternative model was suggested for the anergic state in human T cell clones in which it was discovered that the pathway leading to Rap-1 activation was enhanced (42). Because Rap-1 in its GTP-bound form can interact with Raf-1 protein kinase and prevent it from being activated by Ras-GTP, a block in the ERK pathway could be affected by this mechanism. However, recent studies (43) involving transfection of constitutively active Rap-1 into T cells showed no inhibition of T cell proliferation, although there was an effect on cell adhesion. Thus, at the present time the activated Ras-GAP hypothesis still seems the most likely explanation for the block in the Ras/MAP kinase pathway. But which are the proteins synthesized during anergy induction that are eventually responsible for maintaining the unresponsive state? Early on, Quill and colleagues (44) noted that the amount and activity of the Src family tyrosine kinase Fyn were elevated in clonally anergic T cells. This was confirmed in anergic human T cell clones (42). However, T cell lines established from Fyn-deficient mice can be anergized (N. Nabavi & R.H. Schwartz, unpublished data). Another proposed anergic factor is the cell-cycle inhibitor p27Kip1 (45). As mentioned earlier, anergy appears to be a growth arrest state, and drugs that inhibit cell-cycle progression, such as rapamycin, favor anergy induction. Boussiotis and colleagues (45) made the surprising observation that overexpression of p27Kip1 in T cell clones inhibited IL-2 production. They also reported that p27Kip1 levels were increased during anergy induction. This correlation, however, could not be confirmed in murine T cell clones, and physiologic levels of p27Kip1 had no effect on IL-2 transcription (46). Furthermore, clonal anergy could be induced in cell lines derived from p27Kip1 knock-out mice. Thus, currently there is no convincing evidence for any particular protein subserving the role of an anergic factor. Efforts are currently underway to use microarray analysis and subtractive hybridization approaches to identify such factors. Because cycloheximide treatment blocks clonal anergy induction (12), it is suspected that at least one of these anergic factors will be newly synthesized and thus identifiable as a difference in mRNA expression. Preliminary reports from the Fathman laboratory have identified a molecule called gene related to anergy in lymphocytes (GRAIL), which has some characteristics consistent with an anergic factor; however, its production is inhibited by CD28 costimulation, which is something that would not have been predicted by the rapamycin experiments described above. Rao and colleagues (30) have identified 18 genes whose expression changes in cells undergoing ionomycin-induced anergy, and 4 of these changed in the presence of constitutively active NFAT-1, but none has yet been tested in expression experiments for an effect on IL-2 production.

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Reversal of Clonal Anergy Exposure of clonally anergic CD4+ T cells to a phorbol ester and a calcium ionophore, e.g., phorbol 12, 13-dibutyrate (PMA) and ionomycin, usually induces IL-2 production and normal proliferation (39, 40, 44). PMA activates protein kinase C, which in turn can downregulate Ras-GAP activity (47), thus facilitating activation of the MAP kinase pathways. However, PMA in conjunction with TCR stimulation only partially reconstitutes the proliferative responsiveness, suggesting that the block in clonal anergy may be more complicated. The complete reversal of the anergic state was first accomplished for human clones by the addition of IL-2 (48). Similar results were obtained in limiting dilution experiments using mouse cells, which showed that the reversal was at the single cell level and did not result from the outgrowth of unanergized cells (17). Mouse CD4+ T cell clones express low levels of the high affinity IL-2 receptor (29) and hence can be reversed simply by the addition of IL-2. In other circumstances, the IL-2 receptor has to be induced by TCR occupancy before reversal can take place (13). Hence, if IL-2R signaling is blocked in addition, as seems to be the case in division arrest anergy, then addition of IL-2 will not reverse the anergic state (19). Activation of Janus kinase (JAK)-3 using an antibody against the common gamma chain of the IL-2 receptor was reported to block clonal anergy induction in human T cell clones (49). However, reversal of the state once it was induced required both CD2 stimulation and IL-2 in order to regain a proliferative response, which suggests that the reversal requires additional biochemical signals (50). For murine clones, IL-2 was sufficient, and the reversal could be blocked with the G1 to S phase cell-cycle inhibitor rapamycin, but not the early S phase inhibitor hydroxyurea (46). This suggests that entry into cell cycle is the critical process for reversing clonal anergy, just as it is for the prevention of its induction. The reversal of murine anergy, however, was only partial. Complete reversal required several days, during which time the cells divided a number of times (17). Why should this be? If the reversal involves the degradation of the proteins maintaining the anergic state, and if this process is initiated by cell cycle–regulated kinases, then the amount of time the cell spends in the G1 to S transition could determine how much degradation takes place. Complete reversal might thus require multiple cell cycles to remove all the inhibitors. Preliminary lab data with drugs that inhibit proteosome function have shown that they can block the IL-2-induced reversal of clonal anergy, making this scenario worthy of further consideration (J. Powell & R.H. Schwartz, unpublished data). Alternatively, low level transcription of a “reversal” gene in late G1 might also bring this about. Finally, a recent report by Croft and colleagues describes the reversal of clonal anergy with anti-OX40 antibody stimulation (51). The A.E7 T cell clone was induced into an anergic state by stimulation with plate-bound anti-CD3. The T cells expressed significant amounts of OX40 on their surface, although somewhat lower than what was found on a nonanergic population of A.E7 T cells. After five days of rest, the cells were restimulated with antigen and APC plus anti-OX40. After

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a further four days, the cells were rechallenged (for the third time) with peptide and APC. On this last stimulation their proliferative response was equivalent to nonanergized cells that had originally been stimulated with anti-CD3 plus antiCD28. Whether this reversal was accompanied by a proliferative response in the presence of anti-OX40 was not determined, and the mechanism at present is not understood.


CLONAL ANERGY OF CD8+ T CELLS The first demonstration of clonal anergy in CD8+ T cells was made in CD8+ clones by Otten & Germain (52). They used APCs lacking costimulatory molecules for induction and read out a phenotype of inhibition of IL-2 production and proliferation with little effect on IFN-γ production or cytotoxic T lymphocyte (CTL) activity. Thus, similar to CD4+ T cell clonal anergy, the state appeared to be predominantly antiproliferative. More recently another form of clonal anergy referred to as activation-induced nonresponsiveness (AINR) has been described by Mescher and colleagues (53). In this case optimal stimulation of na¨ıve TCR transgenic CD8+ T cells with anti-TCR mAb or peptide/MHC complexes in conjunction with B7 costimulation leads to proliferation and IL-2 production, but after 3–4 days the cells lose their capacity to proliferate in response to antigen. They also lose their ability to make IL-2 but retain effector functions such as IFN-γ production and CTL activity (54). AINR resembles the clonal anergy of CD4+ T cells biochemically in that activation of all three MAP kinase pathways (ERK, JNK, and p38) is inhibited, and the block can be bypassed by stimulation with PMA and ionomycin (54). Also similar is the fact that AINR can be overcome if large enough quantities of IL-2 are provided either by CD4+ T cell help or if IL-2 is added exogenously during the course of the immune response. The induction of AINR even in the presence of B7 costimulation and some division is presumably because the CD8+ T cells cannot produce enough IL-2 or other growth factors to drive sufficient rounds of cell-cycle progression to reverse the unresponsive state (see earlier discussion for CD4+ T cells). This phenomenon makes longterm CD8+ T cell expansion (after the initial burst) dependent on CD4+ T cell help.

ADAPTIVE TOLERANCE OR IN VIVO ANERGY Peripheral Models The first observation of anergic T cells in vivo was made by Rammensee and colleagues in 1989 (55) when they injected Mls-1a spleen cells into Mls-1b mice. Some of the Vβ6+ T cells that responded to this Mtv-7 superantigen stimulation persisted in the host, but they did not proliferate when restimulated in vitro with fresh Mtv-7+ APCs. The recovered T cells did increase their expression of CD25 but failed to make IL-2, i.e., they manifested some traits typical of clonal anergy.

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Similar results were subsequently obtained with bacterial superantigens (56, 57) and following certain persistent viral infections (58, 59). In these examples it was observed that the relevant Vβ + and antigen-specific T cells first expanded and that a significant portion of these responding cells eventually died. However, there usually remained a cohort of cells that were unresponsive. They could not produce IL-2 in vitro when restimulated with either the superantigen or antibodies against the particular Vβ. In the Staphylococcal enterotoxin B (SEB) model the unresponsive state was transient, lasting about a month. Adult thymectomy experiments demonstrated that the unresponsive cells could still be found at late time points, ruling out a contribution from newly emerging T cells (60) and suggesting instead that the antigen needed to persist to maintain the state. Nonetheless, many investigators were still reluctant to accept the idea that the cells were anergic. In some cases it was argued that their unresponsiveness was due to a failure to be stimulated by the superantigen in the first place because of the particular TCR α chain that they expressed (60). In other cases the Vβ + cells were seen to slowly disappear on repeated stimulation with superantigen (61), and so it was argued that the process was just a slow deletion. Finally, in the Mls-1 model, Bandeira and colleagues (62) challenged the notion that these cells were even tolerant at all because they found that effector helper activity was generally enhanced, and there was accelerated rejection of Mls-1a B cells following a second injection. More decisive experiments revealing the existence of anergy in vivo were made possible by the introduction of models for peripheral tolerance in which homogeneous populations of T cells from TCR transgenic mice were transferred into hosts that constitutively express the antigen recognized by the transferred T cells (63). The first such model studied by Rocha & Von Boehmer (64) involved CD8+ T cells specific for the H-Y antigen. When these cells were transferred into syngeneic male nude (athymic) recipients, they behaved similarly to cells exposed to superantigens, i.e., the T cells rapidly expanded and then slowly declined in numbers, and the remaining T cells did not proliferate upon restimulation in vitro with either male spleen cells or an anti-TCR idiotypic antibody. If the T cells were removed from the host and parked in a second female nude recipient, then the cells recovered their ability to proliferate to antigen, which suggests that antigen persistence was required to maintain the unresponsive state (65). These experiments, however, are complicated by the fact that T cells can expand in an antigen-independent manner in T cell–deficient hosts (66). Although the H-Y-specific receptor itself does not show such expansion on its own, the possibility that endogenous TCR-α receptors were coexpressed on the same cell (because the TCR transgenic was not placed on a Rag−/− background) still makes an expansion scenario conceivable. Thus, the reversal of anergy could have been a consequence of the entry into the cell cycle driven by this process and not a consequence of the removal of antigen. The reversal question was also addressed by Jenkins and colleagues upon transfer of CD4+ TCR transgenic T cells into a normal unirradiated syngeneic mouse, followed by challenge i.v. with soluble peptide antigen (67). Under these conditions the specific T cells showed limited peripheral expansion and a significant amount of

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death, with a residual subset of cells remaining that were unresponsive to antigen restimulation both in vivo and in vitro. These cells failed to expand rapidly in the lymph nodes following a subcutaneous challenge with antigen in incomplete Freund’s adjuvant (IFA), and all the cells failed to make much IL-2 or TNF-α as assessed by intracellular staining. In vitro IL-2 production was 10- to 20-fold less than that produced by na¨ıve untreated cells. BrdU labeling studies showed that the anergic cells all eventually underwent cell division by 5 days, but their maximum expansion was three- to sixfold less than that of na¨ıve T cells. Mixing experiments demonstrated that the unresponsiveness was not due to bystander suppression. The anergic state was still detected at three weeks after initial challenge but was completely gone by seven weeks. It is interesting that the unresponsive state could be extended if soluble peptide antigen was reinjected every week. The loss of anergy was roughly correlated with a decline in antigen presentation measured in vivo, although the anergic cells recovered at a much slower rate. It is clear from these studies and others (68) that the continued presence of antigen is required to maintain this adaptive tolerant state. More recent studies with double transgenic models have confirmed many of these characteristics of in vivo anergized T cells and have provided several new insights. In the work from our laboratory (69), TCR transgenic T cells on a Rag2−/− background were transferred into a CD3ε−/− mouse expressing the antigen pigeon cytochrome c under the control of an MHC class I promoter and an Ig enhancer. In this T cell–deficient mouse the homogeneous CD4+ T cell population undergoes an enormous expansion, 50- to 100-fold, and only a small amount of loss (50%) (Figure 1). As a consequence, the remaining anergic T cell population consists of anywhere from 10 to 40 million cells, making retransfer experiments and biochemical analysis feasible. In this model the unresponsiveness begins to emerge at 3 to 4 days when the cells are finishing their expansion phase and is generally complete by 7 to 10 days. It is not only characterized by a 10-fold decrease in IL-2 production in vitro compared to na¨ıve cells, but also a similar reduction was observed for all cytokines tested, including IFN-γ , IL-4, IL-3, IL-10, IL-6, and TNF-α. The loss of response was primarily a decrease in the number of cells producing the cytokine as measured for IFN-γ and IL-4, rather than a decrease in the amount of cytokine produced per cell by the entire population. Re-expression of the activation markers CD69 and CD25 was also impaired, and this seemed to affect all the cells in the population. The cells persisted in this state for as long as 5 months, but if they were retransferred to a second CD3ε −/− mouse not expressing the antigen, the cells lost their adaptive tolerance. CFSE-labeling showed that much of this reversal occurred within 7 days, prior to the onset of division in vivo driven by the T cell–deficient environment. These observations again argue that the T cells must continue to encounter antigen in order to maintain their anergic state. Finally, CFSE-labeled na¨ıve transgenic T cells transferred into an adaptively tolerant mouse filled with anergic T cells, proliferated at the same rate and to the same degree as na¨ıve T cells did in the original unmanipulated CD3ε −/− host. This result shows that antigen presentation is not affected by the

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Figure 1 Both panels display adoptive transfer experiments in which three million CD4+ lymph node T cells specific for pigeon cytochrome c (PCC) and I-Ea obtained from a Rag-2−/− TCR transgenic mouse were transferred into a 600R irradiated syngeneic host expressing the antigen (right panel) or a syngeneic CD3ε−/− unirradiated host (left panel) either expressing the mPCC antigen (closed circles) or not (open circles) (68). In the unpublished data (C. Tanchot & R.H. Schwartz) shown in the right panel, the open squares display the recovery of the endogenous CD4+ T cells after irradiation.

tolerant T cells and that they do not suppress or compete with the na¨ıve T cells. The latter is somewhat surprising but may reflect different homing properties of na¨ıve and anergic T cells or a lower effective avidity for peptide/MHC complexes by the anergic cells. Similar but less extensive observations have been made in irradiated and T cell replete antigen transgenic hosts, although the size of the population studied was much smaller because the initial expansion was less and the deletion component was somewhat larger (Figure 1). Nonetheless, some other important characteristics of the adaptive tolerant state were described in these experiments. One is that the proliferation defect observed in vitro using stimulation with either antigen or antiidiotypic antibody could not be reversed by the addition of IL-2 (70). Second, the half-life of the in vivo generated anergic cells was affected by the presence of other T cells. As shown in Figure 1, transfer of cytochrome c-specific TCR transgenic cells into a 600R irradiated antigen-bearing host induced a 10-fold smaller expansion but the same anergic state (between days 7 and 14) as seen for transfer into a T cell-deficient, antigen-expressing host. Beyond day 21, however, the anergic T cells slowly disappear. This correlates with the repopulation of the periphery by na¨ıve CD4+ T cells following the recovery of T cell development in the thymus; however, thymectomy experiments are still required to prove that it is these new T cells that are responsible for the disappearance of the anergic T cells. Third, the effect of antigen dose was examined in two hemagglutinin (HA) transgenics differing by 1000-fold in transgene-regulated antigen levels (71). Both conditions

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induced the adaptive tolerant state, even though the initial kinetics and magnitude of the expansion phases were different. More recently, we have also looked at two different levels of antigen presentation in the cytochrome c model and confirmed that both induced a tolerant state in which the threshold for reactivation was significantly heightened; however, these thresholds were subtly different in the two cases, which suggests that the T cells may have adapted differently to the two antigen environments (N. Singh & R.H. Schwartz, unpublished data). These observations lend some support to the Grossman & Paul (72) theory of tunable activation thresholds. Also possibly consistent with this model are our studies on the retransfer of anergic T cells into a second CD3ε−/− host expressing the antigen (69). The cells not only showed a greatly reduced in vivo proliferative response, as expected from the in vitro data, but also they slowly entered into an even deeper state of unresponsiveness, both in terms of cytokine production on restimulation in vitro and proliferative responsiveness following a third transfer into a CD3ε−/− antigen-bearing host. Because the final density of the anergic T cells in the second host was significantly lower than that in the first host, the competition for peptide/MHC complexes was less, resulting in an environment with an effectively greater concentration of antigen. That tolerant states in vivo can exist at different levels for the same antigen depending on the dose was first shown in the experiments of Ferber et al. (73).

Developmental Models In addition to anergy being described as a peripheral tolerance mechanism, this state has also been found in certain experimental models of T cell development. The first experiments of this nature were performed by Ramsdell & Fowlkes (74) with radiation-induced bone marrow chimeras. Vβ17+ and Vβ6+ T cells were only partially deleted during development in chimeras, in which both the I-E molecules and the mouse mammary tumor virus (MMTV) superantigens the T cells recognize were only expressed in host tissues (including the thymic epithelium) and not in bone marrow–derived cells. About 70%–90% of both CD4 and CD8 thymocytes and peripheral T cells carrying these Vβ receptors were not clonally deleted, but they were functionally unresponsive in mixed leukocyte reactions (MLRs) in vitro as well as in a graft-versus-host response following adoptive transfer into an IE+, MMTV+ host. In addition, direct stimulation of these cells with plate-bound, specific anti-Vβ antibodies could not elicit a proliferative response, and no IL-2 production was detected. Furthermore, addition of IL-2 only partially restored the proliferative response, which suggests that there was also a block in responsiveness to IL-2. Finally, the anergic state could be reversed by either culturing the cells in vitro in IL-7 for a week or by parking the cells in an irradiated host lacking the I-E molecule (75). If the cells were parked in a host expressing both the I-E molecule and the superantigen, then the unresponsiveness was not reversed, which suggests that persistent antigen exposure was required to maintain the anergic state. These last few results suggest that the state is more akin to adaptive tolerance than to clonal anergy.

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Around the same time, Burkly et al. (76) were experimenting with transgenic mice expressing MHC class II molecules under the control of tissue-specific promoters for models of peripheral tolerance. They made the puzzling observation that both thymus and lymph node Vβ5+ and Vβ17+ T cells were anergic in transgenic mice expressing the I-E molecule under the control of the rat insulin promoter. Although at first they could only detect I-E mRNA expression in the pancreas and kidney, subsequent studies using reverse transcriptase-polymerase chain reaction (RT-PCR) showed that tiny amounts of message could also be found in the thymus. To examine whether this expression was responsible for the tolerant phenotype, they thymectomized the transgenic mouse and then reconstituted it with an I-E negative thymus graft followed by lethal irradiation and I-E negative bone marrow reconstitution. They reported that the lymph node T cells from this manipulated mouse were also anergic. I have always felt that this lack of responsiveness was due to the early time point (6–8 weeks) at which they looked for function in the periphery. MLRs across an I-E only disparity are quite weak, and full reconstitution of the peripheral lymphoid pool after lethal irradiation can take up to three months. The critical experiment would have been to look at thymocyte responsiveness at late time points, but this was never done. Subsequent experiments by many laboratories have now shown that such ectopic expression of transgenic mRNA in the thymus can have tolerogenic consequences (77). Furthermore, there seems to be a normal mechanism for expressing peripheral tissue-specific antigens in a subpopulation of thymic medullary epithelial cells, which can also have functional consequences (78). Finally, direct targeting of transgenes to the thymic medulla induces tolerance, although the mechanism may be deletional (79). Thus, it seems reasonable to conclude that the anergic state observed by Burkly et al. (76) was likely induced during late thymic development. Unresponsive states have also been described in TCR transgenic mice under known negative selection conditions. The first of these described was for the H-Y specific transgene expressed in male mice (80, 81). Massive deletion occurs in the thymus at the double-negative to double-positive transition, but a few cells escape this process and appear in the periphery. They are unresponsive to stimulation with male spleen cells, but they can be activated by anti-TCR antibodies. They express lower levels of CD8, and this seems to be critical for their unresponsiveness to antigen. Constitutive expression of a CD8 transgene along with the TCR transgene resulted in the complete deletion of these cells in the thymus (82), and based on their phenotype some investigators feel that the cells are actually in the γ δT cell lineage (83). No matter what their lineage, however, their ability to proliferate to anti-TCR antibodies suggests that they are not really anergic. In contrast, more recently developed double transgenic models, in which a TCR transgenic mouse is crossed to a second transgenic mouse expressing the antigen, has given rise to undeleted T cell populations that appear to be anergic. Sarukhan & Von Boehmer (70) crossed a TCR transgenic line producing CD4+ T cells specific for an influenza hemagglutinin (HA) peptide and I-Ed to a line expressing HA under control of an Ig-κ promoter. Although a significant number of T cells were deleted in the thymus, a cohort escaped to the periphery. These cells expressed activation

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markers such as CD69 but failed to proliferate either to antigen and APCs or to an anticlonotypic antibody. Addition of IL-2 did not restore responsiveness. This anergic state resembles the in vivo anergy seen with superantigens expressed in the thymus and periphery, although it is not clear in this model in which location the anergy is induced. In a similar fashion, the αβ DN T cell lineage in the 2C TCR transgenic mouse was also made anergic, when this strain was crossed to one expressing the alloantigen (H-2Ld) recognized by the TCR (84). The resulting cells were CD44hi and failed to proliferate or make much IL-2 upon stimulation with alloantigen-bearing APC. It is interesting that they produced almost normal amounts of IFN-γ compared to αβ DN T cells from 2C H-2b hosts. This property seems to vary among different models of adaptive tolerance (69, 85). Why does the phenomenon of adaptive tolerance exist? As mentioned earlier (62, 85) these cells have not lost all effector functions but have in some cases merely downregulated their responses 10-fold (69). Proliferative expansion potential is also downregulated. Thus, it appears that after having made a vigorous response, the T cell immune system slows down to reassess the situation, under circumstances in which the antigen is still persisting in the host environment at a constant level. Such a reaction might give the system an opportunity to determine whether or not it has made a mistake, i.e., is responding to a self antigen, and thus prevent further damage. If the antigen concentration increases and the costimulatory milieu persists, as it might do in a chronic infection, then the cells can continue to respond, although with a somewhat reduced capacity, because they require a higher antigen dose to respond. If these amplifying conditions are not met, however, then the cells would slowly disappear. In the case of an overwhelming viral infection in which the viral antigens persist at a high level, the anergic response could also protect against severe immunopathology and death (59).

Comparison Between Adaptive Tolerance and Clonal Anergy At this point a summary of the findings in all the in vivo anergy models would suggest that adaptive tolerance has a number of characteristics that distinguish this state from clonal anergy (Table 1). Although both exhibit a block in IL-2 production and proliferation, only adaptive tolerance is typically associated with a limitation in the production of all cytokines. The only exception is IL-10, which is produced by anergic cells in several models. I return to this issue later on. Second, adaptive tolerance appears to require the persistence of antigen in order to maintain the unresponsive state. In clonal anergy, unresponsiveness remains for weeks after antigen and APCs are removed from the culture. The record in our lab is three months. The third difference is that in most adaptive tolerance models the proliferative block cannot be reversed by adding IL-2. This block in IL-2 receptor signaling can be achieved in vitro and seems to involve CTLA-4 signaling (see above) (19). The same may be true in vivo. Abbas and colleagues (7) were the first to show that blocking CTLA-4 in vivo prevented adaptive tolerance in the Jenkins’ model. Blocking antibodies against CD28 did not prevent tolerance,

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TABLE 1 Comparison of different anergic states Characteristic traits

Clonal anergy

Adaptive tolerance

Tr1 regulatory cell

CD25 regulatory cell

T-T cell presentation

Cell type initially affected

CD4/CD8

CD4/CD8

CD4

CD4

CD4

Preactivated versus na¨ıve

Preactivated

Na¨ıve

Na¨ıve

Na¨ıve

Preactivated

Induction signaling pathway

Ca/NFAT

?

IL-10R

?

?

Proliferation with induction

No

Yes

Yes

No

Yes

CTLA-4 involvement

No

Yes

?

Dispute

?

Inhibition of proliferation

Yes

Yes

Yes

Yes

Yes

Inhibition of IL-2 production

Yes

Yes

Yes

Yes

Yes

Inhibition of IFN-γ

No

Yes

No

?

Yes

Inhibition of IL-4

No

Yes

Yes

?

Yes

Production of IL-10

No

Some

Yes

Yes

?

Block in IL-2R signaling

No

Yes

Yes

No

No

Major signaling pathways blocked

Ras/MAP kinase

Ca/Tyr kinase

? Tyr kinase

?

Ca/NFAT

Reversal by IL-2

Yes

No

No

No

Yes

Reversal by Anti-Ox40

Yes

Yes

?

?

?

Antigen persistence required

No

Yes

No

? Yes

No

although such treatment did prevent priming. This topic was also examined using the CTLA-4-deficient mouse crossed onto a TCR transgenic background (86). Cells from such animals showed a two- to threefold enhancement of cytokine production (IL-2, IL-4, and IFN-γ ) and an enhancement of cell-cycle progression compared to wild-type T cells. This seemed to prevent the loss of IL-2 production and proliferation seen following the injection of soluble peptide. In contrast to these results, however, clonal anergy was readily inducible in CTLA-4-deficient T cells from TCR transgenic mice stimulated in vitro with plate-bound anti-CD3 antibody (5). Although the knock-out cells showed greater proliferation and IL-2 production in these experiments, the relative decrease following anergy induction was the same as that for the wild-type cells. It is interesting that these authors subsequently obtained similar results in an adaptive tolerance model (87). These results suggest that the primary effects of CTLA-4 are on CD4+ T cell expansion and survival and not on the anergy induction process per se. Nonetheless, there does appear to be a CTLA-4 effect on the cell’s responsiveness to IL-2 (19). This could indirectly affect anergy induction in vivo by influencing cell-cycle progression (88), or more likely be the only block contributed by the CTLA-4 signaling. Finally, one property that clonal anergy and adaptive tolerance share in common is the ability to be reversed by anti-OX40 antibody treatment. The primary effects of this mAb are to enhance T cell proliferation and diminish cell death following activation of na¨ıve T cells (89). OX-40 expression peaks at 48 h, and administration of the mAb at that time can prevent adaptive tolerance in the Jenkins model (51). More impressively, it could reverse adaptive tolerance once established. Tolerant

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mice (day 10) were immunized with peptide in complete Freund’s adjuvant (CFA) and given anti-OX40 at 48 h. This enhanced their expansion by seven- to eightfold and completely reversed the block in their ability to proliferate and make IL-2 following subsequent restimulation in vitro. Transfer experiments with CFSElabeled tolerant cells showed that expansion to anti-OX40 and antigen occurred in most of the cells in the population and not just in a minor fraction that may not have been tolerized. The reversal could be achieved up to 25 days after tolerance induction, but beyond that point the tolerant state waned naturally because of the disappearance of the antigen. Thus, it will be interesting to determine eventually the effect of anti-OX40 in double transgenic models where the antigen and adaptive tolerance persist.

Biochemical Alterations in Adaptive Tolerance Given the differences in the biological characteristics of adaptive tolerance and clonal anergy it should not be surprising that the biochemical blocks in signal transduction have turned out to be different (Table 1). Although many of the systems have not been well studied because of the difficulties in getting enough anergic cells to analyze, one can get sufficient information by pooling all the data from the various models to obtain a pretty clear picture of what is going on. The first attempt to examine TCR signaling in an Mls superantigen-induced adaptive tolerance model was carried out by Bhandoola & Quill (90). One of the things they observed was a decrease in the phosphorylation of a 38kD protein, which we now presume to be the linker for activation of T cells (LAT) adaptor molecule. This suggests a more proximal block in signal transduction than seen in T cell clonal anergy and similar to what has been reported for B cell clonal anergy (91). This was soon established by Ochi and colleagues (92) in an SEB superantigen model. They showed that in vivo anergy inhibited activation-induced TCR zeta chain (p23) and ZAP-70 phosphorylation. The consequences of this, as seen in all the adaptive tolerance models studied to date, is a profound inhibition in the mobilization of intracellular calcium (84, 93). This has been shown for both CD4 and CD8 T cells and in both developmental and peripheral tolerance models. In our cytochrome c double transgenic this effect is correlated with a block in Phospholipase C γ 1 phosphorylation (L. Chiodetti & R.H. Schwartz, unpublished data), which is required for the generation of the IP3 that mediates calcium release from intracellular stores. In contrast, activation of the Ras/MAP kinase pathway is only slightly inhibited, if at all. This is the exact opposite of the inhibtion seen in clonal anergy, in which calcium mobilization is normal and the Ras/MAP kinase pathway is blocked. It is not fully understood why a reduction in proximal tyrosine phosphorylation would preferentially affect calcium signaling and not the Ras/MAP kinase pathway. One model proposed by Madrenas and colleagues (94) is that the Ras pathway can be activated independently of LAT via the alternative adaptor Slp-76. It is interesting that in this regard, both Slp-76 and ERK phosphorylation were shown to be normal in the αβ DN T cell anergy model of the 2C TCR transgenic mouse (84). Finally, in a few of the models, attempts

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to bypass the block in proliferation with PMA and ionomycin did not work after prolonged exposure to antigen, even in the presence of IL-2 (93). This suggests that in adaptive tolerance the biochemical blockade may also eventually involve additional downstream components. The other biochemical block found in adaptive tolerance is in signal transduction through the IL-2 receptor. The upregulation of the CD25 α chain of the receptor is variable and is decreased in some models and increased in others (69, 84, 93). In the in vitro division arrest anergy, the activation of Stat 5 by IL-2 signaling was reported to be normal (19). Thus, the target of the block is still unknown. Some data exist to support a role for the src family kinase fyn. The original studies on clonal anergy showed that fyn kinase activity was increased (95). Fyn was then found to constitutively associate (coprecipitate) with phosphorylated Cbl and the adaptor molecule CrkL in clonally anergic human T cell lines (42). Although this complex was postulated to play a role in Rap-1 activation by binding the guanine nucleotideexchange protein C3G, gene targeting of fyn turned out to have no inhibitory effect on the induction of IL-2 in adaptive tolerance (96). Instead, it prevented the block in IL-2 receptor signaling. IL-2 receptor β chain expression was enhanced, and cell survival was increased in the presence of either IL-2 or IL-15. Possibly relevant to these findings, Taniguchi and colleagues (97) have shown that src family kinases bind to the IL-2 receptor β chain following IL-2 stimulation and that this interaction leads to phosphorylation of the kinase followed by activation of the Ras pathway. In clonal anergy this pathway is blocked (40). It is interesting that recent experiments of DeFranco and colleagues (98) in lyn-deficient mice have shown that a B cell autoimmune disease can develop as a consequence of decreased negative regulation stemming from a failure to phosphorylate inhibitory receptors such as CD22 and Fcγ RIIB. If fyn played a similar negative feedback role for the IL-2 receptor in T cells, this could explain how its premature elevation in anergic states interferes with IL-2 receptor signaling. Fyn elevation alone, however, is not sufficient because it is also elevated in clonal anergy, where IL-2R signaling is still intact. At this point there is not enough information to be sure that all adaptive tolerance models have the same biochemical alterations. My effort is simply to show that there are some common threads. There clearly seem to be several pathways affected, such as IL-2 production and IL-2R signaling, which can be independently regulated. As a consequence, subtle differences in phenotype could result from quantitative differences in how much each pathway is inhibited. On the other hand the differences among models could indicate still other pathways that have been affected in some states and not in others. This same caveat applies to the other models I now discuss.

ANERGY INDUCED BY T CELL ANTIGEN PRESENTATION With this overview it is now possible to reexamine the original model of T cell anergy in vitro described by Lamb & Feldmann (14) and see that it is a hybrid of clonal anergy and adaptive tolerance. In this model, human T cell clones were stimulated with peptide/MHC complexes presented by activated T cells (in the

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presence or absence of APC). The unresponsive state induced (T-T Cell presentation in Table 1) had clonal anergy properties in that IL-2 production was inhibited, the state persisted in the absence of antigen, and it was reversible upon stimulation with IL-2. However, it was also characterized by a proliferative response prior to induction, an inhibition of most effector cytokine production, and a major biochemical block in the calcium/NF-AT pathway, although a block in the Ras/MAP kinase pathway was also present (99). IL-2 receptor signaling was not blocked. This hybrid state seems to be a consequence of the method of antigen presentation because it is possible to induce canonical clonal anergy in human T cell clones using DR-transfected APC in the absence of CD28/B7 costimulation (49, 50). A similar state has been induced in rat T cells using T cell antigen presentation (100). In this latter model the degree of reversal by IL-2 depended on the initial concentration of antigen used to induce the state, which suggests that a block in IL-2R signaling can also be induced by this method.

ANERGY AND SUPPRESSION The first report of a clear link between clonal anergy and T cell suppressive activity was published by Lechler and colleagues (101). They added irradiated anergic human T cell clones to nonanergic clonal populations at different cell ratios and observed an inhibition of proliferation of the latter. Inhibition was optimum if both cell populations recognized the same antigen or if two different antigens were presented on the same APC (linked suppression). They interpreted these results as a passive competition between T cells for access to the membrane on the same APC, a model originally suggested by Herman Waldmann. The possibility of a bystander form of suppression through the production of inhibitory cytokines was discounted because of the lack of an effect of mAbs against IL-10, IL-4, and TGF-β, although the last of these did have a small but significant effect. The case for bystander suppression was made much more forcefully by Roncarolo and colleagues (13). They discovered that na¨ıve human CD4+ T cells could be induced into an anergic state if they were stimulated in the presence of IL-10 (102). Subsequent work showed that a combination of IL-10 and interferon alpha was optimal. This state was also reported to be induced by IL-10-treated APC (103) and by IL-10 in the absence of APC when the cells were stimulated with anti-CD3 and anti-CD28 (102). It was characterized by a block in proliferation and IL-2 production, although production of effector cytokines such as IL-4, and to a lesser extent IFN-γ , was also inhibited (13). IL-5 production was unaffected, and the cells rapidly produced copious amounts of IL-10 and some TGF-β on restimulation. The cells failed to express CD25 on reactivation and thus could not be brought out of the state by IL-2 stimulation. However, the cells could respond to IL-15, and this has been used to propagate them without reversing the anergic state (13). PMA and ionomycin could also bypass the block in proliferation. The exact biochemical nature of the anergy is still unclear. Initial studies showed that

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activation of the calcium pathway was normal, which suggests some form of clonal anergy (102). Surprisingly, however, signaling for phosphorylation of the ERK MAP kinase pathway was also intact, and the cells upregulated CD69, CD40L, and HLA-DR normally (13). By contrast, in a mouse model in which anergy to alloantigen stimulation was induced with a combination of IL-10 and TGF-β, activation-induced phosphorylation of TCR-ζ , ZAP-70, SLP-76, and ERK1/2 were all inhibited (104). In addition, phosphorylated fyn was constitutively increased, and the cells were blocked in early G1. Although calcium responses were not measured, one would suppose that this pathway was also blocked. This would constitute the most profound anergic state yet reported. The difference between the human and mouse data has yet to be reconciled. The most important advance to come out of these experiments was the realization that these anergic T cells (now called Tr1 cells) could suppress the activation of na¨ıve and memory CD4+ and CD8+ T cells by a cytokine-mediated mechanism (105). This was shown by mixing experiments in vitro for the suppression of proliferative responses of alloreactive or antigen-specific T cells, as well as by mixing experiments in vivo in adoptive transfer models. The latter have included protection from Th-1-mediated colitis induced by transfer of na¨ıve CD45RBhi cells into severe combined immunodeficient (SCID) mice (105), Th-2-mediated pathology in immediate hypersensitivity (106), and bystander suppression of experimental allergic encephalomyelitis (EAE) using OVA-specific Tr1 cells (107). In all cases, specific antigen stimulation is required to elicit the suppression, and it can be blocked with antibodies against IL-10 or its receptor. A similar type of in vivo suppression and antibody blocking has been demonstrated for T cells expressing large amounts of TGF-β (Th-3 cells) (108). The mechanisms of action of both IL-10 and TGF-β are quite complex and are beyond the scope of this chapter. Suffice it to say that the ability of some anergic T cells to suppress new immune responses provides an important potential biological reason for keeping them around following an initial immune response. Finally it should be noted that in several of the examples of adaptive tolerance discussed earlier, the cells were also shown to make significant amounts of IL-10 following antigen stimulation in vitro (70, 93, 109). In one case the cells were capable of suppressing the immune response (and ensuing pathology) of na¨ıve transgenic CD4+ T cells mounted against antigen expressed in skeletal muscle using adenoviral vectors (110). Not all adaptively tolerized T cells express IL-10, however, and they do not all suppress na¨ıve T cell responses (69) or use their IL-10 for that purpose (110). Thus, cytokine suppression and suppression mediated by anergic cells are not always linked, which suggests that they are separately regulated biochemical phenomena. Furthermore, Tr1 cells have a stable suppressive phenotype in the absence of antigen, whereas adaptive tolerance decays under such conditions. Whether the level of IL-10 produced accounts for such differences (13, 93) or whether the properties of the anergic state in Tr1 cells are unique (Table 1) remains to be determined. It was also clear from the original experiments of Lechler that cytokine bystander suppression could not explain the bulk of the regulatory effect mediated

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by anergic T cells in vitro (101). They subsequently reported similar results in an in vivo model involving the prolongation of skin graft survival (9). Insight into the nature of this alternative mechanism for suppression was first published by Wauben and colleagues (111). They showed that the suppression required cell contact with the APC but was not caused by passive competition for ligands on the APC surface. Suppression was still observed if the responding cells were added 24 hours after mixing anergic T cells and the APCs. In addition, purified APCs recovered from cocultures with anergic cells could suppress the proliferative response of na¨ıve T cells to the same antigen (112). Thus, the anergic T cells appeared to negatively condition the APCs. The nature of the conditioning was either the downregulation of antigen presentation function by decreasing the expression of MHC and costimulatory molecules, or, in one model, predisposition to apoptosis by Fas/FasL (112–114). The mechanism was not cytokine mediated (even though IL-10 can have similar effects) because the suppression was not blocked by antiIL-10 antibody (or anti-IL-4 or anti-TGF-β antibodies either). Recently a novel mechanism has been suggested by Suciu-Foca and colleagues (115) for CD8 suppressor T cells in which the APC is induced to express a family of APC-specific, NK-like inhibitory receptors (ILT3 and ILT4). These prevent the upregulation of costimulatory molecules following CD40 engagement. This results in clonal anergy induction in responding na¨ıve CD4+ T cells. It remains to be determined whether anergic CD4+ T cells use a similar mechanism. Relatively recently, another well-studied population of suppressor T cells has been more fully analyzed and also was shown to exist in an anergic state (116, 117). This is the CD4+ CD25+ suppressor T cell originally described by Nishizuka (118) and Sakaguchi (119). These cells do not make IL-2 or proliferate when stimulated with antigen or anti-CD3, but they can be grown in IL-2 following activation (116, 117, 120). They thus appear to be clonally anergic, although few biochemical experiments have been performed to characterize their signal transduction pathways. Their mechanism of suppression is still somewhat controversal—cytokine mediated or not, through the APC or not, involving CTLA-4 or not—but some fairly good evidence suggests that a unique type of T cell/T cell contact is required, at least in vitro (116). This interaction is not antigen specific, but the CD25+ regulatory cell must normally be activated by specific antigen. These cells develop in the thymus (118, 121) where they acquire their specificity for recognizing self antigens, possibly including peripheral antigens ectopically expressed in the thymic medulla (78), and they are thought to play a role in tolerance to tissuespecific antigens because neonatal thymectomy between days 3 and 5 results in organ-specific autoimmunity (116). These cells have also been found in the transplantation model of infectious tolerance (122, 123), where antigen persistence has been shown necessary to maintain the tolerant state. It thus appears that most types of regulatory T cells are anergic in one way or another. The teleological question is why? My own thoughts on this are that anergy prevents the regulatory cells from totally dominating the initial immune response to foreign antigens and shutting down this process prematurely. For example, in

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the model of CD4+ CD25+ regulatory T cells specific for tissue-specific antigens, one has to worry (as Polly Matzinger does) about whether their response to activation by self antigens, draining from the site of an infection, will also suppress the response of na¨ıve T cells specific for the pathogen. If the stimulus is weak, this sort of buffering against a response is what would happen in order to reduce the likelihood of autoimmunity. If the antigenic stimulus is strong, however, as in the challenge from an infectious agent, then some of the na¨ıve T cells will become activated despite the regulatory cells and undergo a burst of proliferative expansion. The regulatory cells, in contrast, are anergic and therefore cannot expand. Hence they will not be able to keep up, which will allow the T cells specific for the pathogen to gain the upper hand. The existence of such regulatory T cells, however, presupposes the existence of another set of na¨ıve T cells—specific for self antigens—that they regulate. These naive T cells can also escape regulation if the infectious signals are strong enough to overcome the suppression. These will then have to be dealt with by other forms of peripheral tolerance, including the adaptive tolerance mechanism described earlier in which the persistence of antigenic stimulation following resolution of the infection leads to an adjustment in the activation threshold of the responding T cells. Clonal anergy has also been postulated to play a role in this process following antigen presentation by nonprofessional APCs, although this has not yet been documented experimentally in vivo. For sure, though, the CD25+ T regulatory cells appear to be clonally anergic (with the exception that the state is not reversed following a response to IL-2) and perhaps that is the major in vivo role for this form of anergy, along with its regulation of CD8+ T cell expansion (AINR).

EPILOGUE Only those who were around at the beginning of the work on clonal anergy can fully appreciate the irony of the current marriage between anergy and suppression. Suppressor T cells were the debutantes of the 1970s, but by the mid 1980s they were old maids, having suffered the rejection of molecular immunologists and their diatribes against the I-J story. Marc Jenkins and I were still searching at that time for a way to characterize suppressor T cells more precisely based on their supposed specificity for free antigen when we first discovered, along with Helen Quill, the phenomenon of T cell clonal anergy. Soon anergy became the darling of the tolerance world, while suppression all but disappeared from the immunological scene. Since the late 1990s, however, the tables have turned, with anergy on the wane as a prominent player in tolerance for lack of a full molecular definition, while suppressor T cells have undergone a rebirth. Now it would appear that the two phenomena can be different sides of the same coin, joined in harmony to achieve an effective negative feedback regulatory mechanism for the immune response. This new vista, however, does not preclude previous ideas for the biologic function of anergic T cells (8–10); it simply expands their repertoire.

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ACKNOWLEDGMENTS I wish to thank Drs. Ronald N. Germain, Nevil Singh, and Lynda Chiodetti for their very helpful comments, which greatly improved this manuscript. The Annual Review of Immunology is online at http://immunol.annualreviews.org


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TOLL-LIKE RECEPTORS Kiyoshi Takeda1,2, Tsuneyasu Kaisho1,2,3, and Shizuo Akira1,2 Annu. Rev. Immunol. 2003.21:335-376. Downloaded from arjournals.annualreviews.org by HINARI on 09/01/07. For personal use only.

1

Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, 2Solution Oriented Research for Science and Technology, Japan Science and Technology Corporation, 3RIKEN Research Center for Allergy and Immunology, Suita Osaka 565-0871, Japan; email: [email protected], [email protected], [email protected]

Key Words innate immunity, signal transduction, MyD88, microbial components, drosophila ■ Abstract The innate immune system in drosophila and mammals senses the invasion of microorganisms using the family of Toll receptors, stimulation of which initiates a range of host defense mechanisms. In drosophila antimicrobial responses rely on two signaling pathways: the Toll pathway and the IMD pathway. In mammals there are at least 10 members of the Toll-like receptor (TLR) family that recognize specific components conserved among microorganisms. Activation of the TLRs leads not only to the induction of inflammatory responses but also to the development of antigenspecific adaptive immunity. The TLR-induced inflammatory response is dependent on a common signaling pathway that is mediated by the adaptor molecule MyD88. However, there is evidence for additional pathways that mediate TLR ligand-specific biological responses.

INTRODUCTION The immune system detects and eliminates invading pathogenic microorganisms by discriminating between self and non-self. In mammals the immune system can be divided into two branches: “innate immunity” and “adaptive immunity.” Adaptive immunity detects non-self through recognition of peptide antigens using antigen receptors expressed on the surface of B and T cells. In order to respond to a wide range of potential antigens, B and T cells rearrange their immunoglobulin and T cell receptor genes to generate over 1011 different species of antigen receptors. Engagement of antigen receptors by the cognate antigen triggers clonal expansion of the lymphocyte and further production of antigen-specific antibodies. This highly sophisticated system is observed only in vertebrates and is a potent defense against microbial infection. In contrast, the innate immune system, which was first described over a century ago, is phylogenetically conserved and is present in almost all multicellular organisms (1). Whereas the system of adaptive immunity 0732-0582/03/0407-0335$14.00

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has been the subject of considerable study in the past century, innate immunity has been less well appreciated. Therefore, the mechanism by which innate immunity recognizes non-self remained unknown until quite recently. However, the recent identification of Toll-like receptors in mammals has made immunologists aware that innate immunity plays an important role in the detection of invading pathogens. Recent evidence shows that Toll-like receptors recognize specific patterns of microbial components, especially those from pathogens, and regulates the activation of both innate and adaptive immunity. In this review we focus on recent progress regarding the functions of Toll-like receptors and their signaling pathways.

TOLL IN DROSOPHILA The Toll Pathway The involvement of the Toll receptors in innate immunity was first described in drosophila. Drosophila Toll was originally identified as a transmembrane receptor required for the establishment of dorso-ventral polarity in the developing embryo (2). Stimulation of Toll by the secreted Spätzle factor, a ligand of Toll, activates the cytoplasmic serine/threonine kinase Pelle via the adaptor protein Tube. Activation of Pelle promotes degradation of the ankyrin-repeat protein Cactus, which associates with the Rel-type transcription factor Dorsal in the cytoplasm. Once Cactus is degraded in response to the Toll-mediated signal, Dorsal is free to translocate to the nucleus, where it regulates transcription of specific target genes (3). The signaling pathway of drosophila Toll shows remarkable similarity to the mammalian IL-1 pathway, which leads to activation of NF-κB, a transcription factor responsible for many aspects of inflammatory and immune responses. Indeed, the cytoplasmic domains of drosophila Toll and the mammalian IL-1 receptor are highly conserved and are referred to as the Toll/IL-1 receptor (TIR) domain. Based on this similarity, it was proposed that the Toll-mediated pathway might be involved in regulating immune responses (3). This was clearly demonstrated in a study of mutant flies lacking individual components of the Toll-mediated pathway, i.e., Toll, Spätzle, Tube, or Pelle (4). Each mutant fly was highly sensitive to fungal infection owing to a lack of expression of the antifungal peptide Drosomycin. In drosophila two additional Rel-type transcription factors, Dorsal-type immune factor, and Relish, have been identified in addition to Dorsal. Dorsal-type immune factor is mainly involved in the induction of antifungal peptide genes in adult flies, whereas Dorsal is involved in dorso-ventral patterning in the embryo (5, 6). Relish regulates the induction of peptides active against Gram-negative bacteria (7). A recent study indicated that the Toll pathway is required for resistance to Gram-positive bacterial infections in addition to fungal infections (8). Indeed, infection with either Gram-positive bacteria or fungi induced Toll-dependent expression of the antifungal peptide Drosomycin. Tube is an adaptor that functions downstream of Toll and upstream of Pelle. Although Tube and Pelle have been shown to interact via conserved death domains

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(9, 10), no direct interaction has been demonstrated between Tube and Toll. However, the protein DmMyD88 appears to function as an adaptor linking Toll and Pelle; the TIR domain of DmMyD88 associates with the TIR domain of Toll, and the death domain of DmMyD88 associates with the death domain of Pelle (11, 12). DmMyD88 mutant flies are highly sensitive to fungal infection, suggesting that DmMyD88 is an essential component of the Toll pathway in drosophila. However, the functional relation between Tube and DmMyD88 remains unclear. The protein Spätzle is secreted as a precursor form that is cleaved to its active form by a serine protease in response to immune challenge. The cleaved Spätzle then activates Toll. Mutant flies with a loss-of-function mutation in the gene encoding the serine protease inhibitor Spn43Ac exhibit constitutive expression of cleaved Spätzle and, consequently, constitutive expression of Drosomycin (13). These data demonstrate that Toll is indirectly activated by Spätzle, rather than directly by microbial components. The precise mechanism by which Toll is activated in response to microbial infection is not well understood, but a recent genetic study has provided some possible clues. An ethyl-methyl-sulfonate–induced mutation of the semmelweis (seml) gene was shown to cause impaired expression of Drosomycin in response to infection by Gram-positive bacteria but not fungi (14). The gene responsible for the mutation was analyzed and found to encode PGRP-SA, the peptidoglycan recognition protein. PGRP-SA recognizes peptidoglycans that are abundant in Gram-positive bacterial cell walls (15). Thus, infection by Grampositive bacteria is detected by PGRP-SA, which in turn activates the Toll-mediated pathway. A factor that is involved in the detection of fungal infection and activation of the Toll-mediated pathway has recently been identified. Ethyl-methyl-sulfonate mutagenesis of drosophila produced a mutant with impaired activation of the Toll pathway in response to fungal infection but not to Gram-positive bacterial infection (16). This mutation was localized to the Persephone gene, which encodes a serine protease but possesses no obvious microbial pattern recognition domain (Figure 1). Most likely, there is a molecule upstream of Persephone that detects fungal infection. Identification of this molecule may provide new insights into the mechanisms of microbial recognition in drosophila.

IMD Pathway The immune response against Gram-negative bacteria is mediated by a distinct pathway first identified by a mutation in the immune deficiency (imd ) gene of drosophila (17). Imd mutant flies are highly susceptible to infection by Gramnegative bacteria but not to fungi, whereas Toll mutants are highly susceptible to fungi but not to Gram-negative bacteria. The imd gene encodes an adaptor protein containing a death domain with similarity to the mammalian receptor interacting protein (18). Genetic studies have identified several molecules involved in the IMD pathway that are involved in the response against Gram-negative bacteria. These include DmIKKβ, DmIKKγ , dTAK1, and Relish (7, 19–22). Fruitflies with mutations in these genes are defective in expression of the antibacterial peptide

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Diptericin and highly susceptible to Gram-negative bacterial infection. No receptor involved in the IMD pathway has been identified. Mutant flies lacking 18-wheeler, a member of the Toll family, are susceptible to Gram-negative bacterial infection, but expression of Diptericin is normal in these mutants (23, 24). Moreover, there are nine Toll family members in drosophila, but none of them has been shown to induce expression of Diptericin (25). These results indicate the existence of a non-Toll-related receptor that initiates signaling in the IMD pathway. Indeed, a member of the PGRP family, PGRP-LC, has been implicated in the activation of the IMD pathway, because induction of Diptericin in response to Gram-negative bacterial infection was shown to be defective in PGRP-LC mutant flies (26–28). Unlike PGRP-SA, PGRP-LC is a transmembrane protein (15). Although it remains unclear whether the intracellular portion of PGRP-LC possesses a domain required for activation of signaling cascades, it is possible that this protein may act as a receptor linking the recognition of Gram-negative bacteria to the activation of the IMD pathway (Figure 1). PGRPs in drosophila consist of a large family containing 12 members (15) and thus could be involved in sensing a variety of different microbes in drosophila. One component of the IMD pathway, Relish, is activated by a cleavage into two domains: the DNA-binding Rel homology domain and the inhibitory ankyrin repeat domain. Although the mechanism by which Relish is cleaved is unclear, it has been suggested that Dredd, a homologue of mammalian caspase-8, may be somehow involved. Dredd mutant flies are defective in cleavage of Relish, and are very susceptible to infection by Gram-negative bacteria (29–31). Dredd associates with drosophila Fas associating death domain (dFADD), a homologue of mammalian FADD (12, 32). In mammals the FADD/caspase8-dependent pathway is activated as a result of signaling from the type I TNF receptor (TNF-RI) and mediates induction of apoptosis. Overexpression of IMD, a homologue of mammalian receptor interacting protein that associates with TNF-RI, induces apoptosis in drosophila, whereas imd mutant flies are resistant to UV-induced apoptosis (18). Thus, the IMD pathway is presumably involved in the induction of apoptosis as well as the response against Gram-negative bacteria. This also indicates that IMD may act upstream of dFADD-Dredd, similar to receptor interacting protein’s acting upstream of FADD-casapase-8 in the mammalian TNF-RI-mediated signal pathway.

TOLL-LIKE RECEPTORS IN MAMMALS A year after the discovery of the role of the drosophila Toll in the host defense against fungal infection, a mammalian homologue of the drosophila Toll was identified (33). Subsequently, a family of proteins structurally related to drosophila Toll was identified, collectively referred to as the Toll-like receptors (TLRs). The TLR family is known to consist of 10 members (TLR1-TLR10), and no doubt more will be found in the future (33–38). The chromosomal location of each human TLR

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gene has been determined. TLR1 and TLR6 map very close to 4p14 (34, 35); TLR2 and TLR3 map to 4q32 and 4q35, respectively; TLR4 and TLR5 map to 9q32-33 and 1q33.3, respectively (34). TLR7 and TLR8 are located in tandem in Xp22, whereas TLR9 maps to 3p21.3 (36, 38). TLR family members are characterized structurally by the presence of a leucinerich repeat (LRR) domain in their extracellular domain and a TIR domain in their intracellular domain. A comparison of the amino acid sequences of the human TLRs reveals that members of the TLR family can be divided into five subfamilies: the TLR3, TLR4, TLR5, TLR2 and TLR9 subfamilies (Figure 2). The TLR2 subfamily is composed of TLR1, TLR2, TLR6, and TLR10; the TLR9 subfamily is composed of TLR7, TLR8, and TLR9. In the TLR2 subfamily TLR1 and TLR6 are highly similar proteins and exhibit 69.3% identity in overall amino acid sequence, but the TIR domains of both receptors are highly conserved, with over 90% identity (35). Because TLR1 and TLR6 have similar genomic structures, consisting of one exon, and are located in tandem in the same chromosome, they may be the products of an evolutionary duplication. Division of TLRs into five subfamilies is also based on genomic structure. The TLR2 gene has two exons, but all of the coding sequences are contained within, exon 2. In contrast, the TLR9 subfamily members including TLR7, TLR8, and TLR9 are encoded by two exons (36, 38). The genes for TLR7 and TLR8 show 42.3% identity and 72.7% similarity in their amino acid sequences, have similar genomic structures, and are located close to each other on the X chromosome (36, 38). The TLR4 and TLR5 genes have four and five exons,

Figure 2 Phylogenetic tree of human Toll-like receptors (TLRs). The phylogenetic tree was derived from an alignment of the amino acid sequences for the human TLR members using the neighbor-joining method.

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respectively. TLR3 has a unique structure among the TLRs in that it has five exons and the protein is encoded by exons 2 through 5. This is in contrast to all of the other TLRs, which are encoded by only one or two exons.


ROLES OF TLRS IN RECOGNITION OF MICROBIAL COMPONENTS Ectopic overexpression of TLR4, the first mammalian TLR identified, was shown to cause induction of the genes for several inflammatory cytokines and costimulatory molecules (33). Therefore, it was anticipated that the TLRs might be involved in immune responses, especially in the activation of innate immunity. In 1998 TLR4 was shown to be involved in the recognition of lipopolysaccharide (LPS), a major cell wall component of Gram-negative bacteria. Subsequently, other members of the TLR family have been shown to be essential for the recognition of a range of microbial components (Table 1). The structural similarity of TLRs seems to reflect their common function in the recognition of microbial components.

TLR4 TLR4 RECOGNIZES LIPOPOLYSACCHARIDE Two mouse strains, C3H/HeJ and C57BL10/ScCr, have long been known to be hypo-responsive to LPS. Two independent groups searching for the genes responsible for this hyporesponsiveness identified mutations in Tlr4 (39, 40). The C3H/HeJ mouse strain has a point mutation in the intracellular region of the Tlr4 gene leading to the replacement of a highly conserved proline with histidine. This mutation results in the generation of a dominant negative allele, defects in TLR4-mediated signaling, and consequent suppression of the response to LPS (41). Another LPS hypo-responsive strain, C57BL10/ScCr, has a null mutation in the Tlr4 gene (39, 40). TLR4-deficient mice generated by gene targeting are also hypo-responsive to LPS, confirming that TLR4 is an essential receptor for the recognition of LPS (41). Recognition of LPS requires other molecules in addition to TLR4. LPS binds to LPS-binding protein, present in the serum, and this LPS–LPS-binding protein complex is subsequently recognized by CD14, a glycosylphosphatidylinositolanchored molecule preferentially expressed in monocytes/macrophages and neutrophils. LPS stimulation is followed by increased physical proximity between CD14 and TLR4, suggesting that CD14 and TLR4 may interact in LPS signaling (42, 43). MD-2 was identified as a molecule that associates with the extracellular portion of TLR4 and enhances LPS responsiveness (44, 45). Chinese hamster ovary cell lines that are hypo-responsive to LPS have mutations in the MD-2 gene (46). Generation of MD-2-deficient mice demonstrated its essential role in the response to LPS. Macrophages, dendritic cells, and B cells from MD-2-deficient mice display severely impaired responses to LPS. Furthermore, MD-2-deficient mice are resistant to LPS-induced endotoxin shock, similar to TLR4-deficient mice (47). MD-2 associates with TLR4 in the endoplasmic reticulum/cis Golgi and then the

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TABLE 1 Toll-like receptors and their ligands TLR family

Ligands (origin)

TLR1

Tri-acyl lipopeptides (bacteria, mycobacteria) Soluble factors (Neisseria meningitides)

TLR2

Lipoprotein/lipopeptides (a variety of pathogens) Peptidoglycan (Gram-positive bacteria) Lipoteichoic acid (Gram-positive bacteria) Lipoarabinomannan (mycobacteria) A phenol-soluble modulin (Staphylococcus epidermidis) Glycoinositolphospholipids (Trypanosoma Cruzi) Glycolipids (Treponema maltophilum) Porins (Neisseria) Zymosan (fungi) Atypical LPS (Leptospira interrogans) Atypical LPS (Porphyromonas gingivalis) HSP70 (host)

TLR3

Double-stranded RNA (virus)

TLR4

LPS (Gram-negative bacteria) Taxol (plant) Fusion protein (RSV) Envelope proteins (MMTV) HSP60 (Chlamydia pneumoniae) HSP60 (host) HSP70 (host) Type III repeat extra domain A of fibronectin (host) Oligosaccharides of hyaluronic acid (host) Polysaccharide fragments of heparan sulfate (host) Fibrinogen (host)

TLR5

Flagellin (bacteria)

TLR6

Di-acyl lipopeptides (mycoplasma)

TLR7

Imidazoquinoline (synthetic compounds) Loxoribine (synthetic compounds) Bropirimine (synthetic compounds)

TLR8

?

TLR9

CpG DNA (bacteria)

TLR10

?

TLR4/MD-2 complex moves to the cell surface, where excess MD-2 is secreted (48). Whereas TLR4 normally resides on the cell surface in wild-type cells, it is found in the Golgi apparatus in cells deficient for MD-2, indicating that MD-2 is essential for the intracellular distribution of TLR4 (47). Another cell-surface protein, RP105, is also involved in the recognition of LPS. RP105 contains an LRR domain that is structurally related to those found in the extracellular portion of the TLRs

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and is preferentially expressed on the surface of B cells (49). B cells from RP105– deficient mice show a severely reduced response to LPS. RP105 functionally associates with TLR4 to recognize LPS (50). Thus, several components are implicated in the recognition of LPS, indicating that the functional LPS receptor forms a large complex. OTHER TLR4 LIGANDS In addition to LPS, TLR4 recognizes several other ligands. Taxol is a product of the Pacific yew (Taxus brevifolia) and exhibits potent antitumor activity in humans. The antimitotic action of Taxol is due to its ability to bind and stabilize microtubules, which prevents proper cell division during mitosis. Taxol possesses similar immunostimulatory activities to those of LPS in mice but not in humans. Mouse TLR4 and MD-2 mediate the LPS-mimetic activity of Taxol (51–53). TLR4 and CD14 recognize the fusion protein of respiratory syncytical virus (54, 55). Accordingly, C3H/HeJ and C57BL/10ScNCr mice, which are mutated for TLR4, exhibited a reduced inflammatory response against and impaired clearance of respiratory syncytical virus. Activation of B cells by murine retroviruses such as mouse mammary tumor virus is dependent on TLR4. The envelope proteins of mouse mammary tumor virus and Moloney murine leukemia virus were reported to co-immunoprecipitate with TLR4 (56). Thus, TLR4 is presumably involved in the recognition of a certain group of viruses. TLR4 seems to recognize some endogenous ligands as well. Heat shock proteins are highly conserved among organisms ranging from bacteria to mammals. A wide variety of stressful conditions such as heat shock, ultraviolet radiation, and viral and bacterial infection induce the increased synthesis of heat shock proteins. The primary functions of heat shock proteins are to chaperone nascent or aberrantly folded proteins. In addition, heat shock proteins activate macrophages and dendritic cells to secrete proinflammatory cytokines and to express costimulatory molecules. Thus, heat shock proteins may be representative of a type of endogenous “danger signal,” i.e., molecules or molecular structures that are released or produced by cells undergoing stress or abnormal cell death (necrosis). These signals are recognized by macrophages and dendritic cells and thereby initiate immune responses (57). The ability of heat shock protein to activate the immune cells is best documented for the heat shock protein HSP60. The immuno-stimulatory activity of HSP60 is mediated by TLR4 (58, 59). For example, HSP60 has been implicated in inflammation accompanying atherosclerosis, development of which is associated with chronic infection by Chlamydia pneumoniae. HSP60 derived from Chlamydia pneumoniae (cHSP60) colocalizes with macrophages in the atheromatous lesion and induces an inflammatory response. Therefore, cHSP60 is thought to be one of the factors causing atherosclerosis in chronic Chlamydial infection. cHSP60 also activates vascular smooth muscle cells and macrophages through TLR4 (60, 61). Mice defective for TLR4 show defective production of inflammatory cytokines in response to HSP70 as well as HSP60 (62–64). Thus, TLR4 seems to be responsible for the inflammatory responses elicited by heat shock proteins. However,

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both TLR2 and TLR4 are required for recognition of HSP70 (63, 64). CD91 (α-macroglobulin receptor) has been identified as a receptor for several heat shock proteins, including HSP70 (65). Furthermore, HSP60 binds to macrophages from TLR4-deficient C57BL/10ScCr mice, despite the fact that no HSP60-induced production of inflammatory cytokines is observed (66). These data suggest that TLR4 is not directly involved in the recognition of heat shock proteins. Extracellular matrix components, including fibronectin, hyaluronic acid, and heparan sulfate, are produced in response to tissue injury and play important roles in tissue remodeling, such as containing the agent of injury, closing the wound, and completing the healing. The type III repeat extra domain A of fibronectin has immuno-stimulatory activities similar to those provoked by LPS. This response to extra domain A of fibronectin is mediated by TLR4 (67). In addition, low molecular weight oligosaccharides of hyaluronic acid have been reported to be potent activators of dendritic cells, and activation of dendritic cells by hyaluronic acid is mediated by TLR4 (68). Furthermore, polysaccharide fragments of heparan sulfate have been reported to induce maturation of dendritic cells via TLR4 (69). Extravascular fibrin deposits are an early and persistent hallmark of inflammation accompanying injury, infection, and immune disorders. Fibrin is generated from plasma-derived fibrinogen, which escapes the vasculature in response to endothelial cell retraction at sites of inflammation. The capacity of fibrinogen to induce the production of chemokines from macrophages is elicited through recognition by TLR4 (70). Thus, TLR4 is presumably involved in several aspects of the inflammatory response by recognizing endogenous ligands produced during inflammation, even in the absence of infection. However, it should be noted that all of these endogenous TLR4 ligands activate immune cells only at very high concentrations, which is in sharp contrast to the low concentrations required for lipopolysaccharide (LPS). Therefore, there remains the possibility that these endogenous ligands might be contaminated with a true TLR4 ligand such as LPS. One intriguing question is whether TLR4 recognizes its ligands directly or not. Some groups have proposed that recognition of LPS by TLR4 involves direct binding, while others have suggested that LPS binds to MD-2, and this complex somehow stimulates TLR4 (71–74). Species-specific recognition of different ligands provides one kind of genetic evidence for direct interaction. For example, mouse but not human cells recognize Taxol, and this species-specific recognition is conferred by MD-2 (53). Another group showed that human but not murine TLR4 recognizes the highly acylated LPS from Pseudomonas aeruginosa (75). TLR4-INDEPENDENT RECOGNITION OF LIPOPOLYSACCHARIDE TLR4 has now been established as an essential component in the recognition of LPS. However, several reports have indicated that LPS can also be recognized independently of TLR4. A study using affinity chromatography, peptide mass fingerprinting, and fluorescence resonance energy transfer identified four molecules on the cell surface that bind LPS. These are HSP70, HSP90, chemokine receptor 4 (CXCR4), and growth differentiation factor 5 (76).

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LPS is rapidly delivered into the cytoplasm after binding to the cell surface. This intracellular movement appears to be necessary for certain cellular responses, since agents that block vesicular transport such as wortmannin or cytochalasin D block the integrin-mediated adhesion of neutrophils in response to LPS (77). This suggests that LPS may be recognized in the cytoplasm as well as on the cell surface. A candidate molecule that confers the intracellular recognition of LPS is Nod1. Nod1 was originally identified as a molecule that is structurally related to the apoptosis regulator, Apaf-1, which contains the caspase-recruitment domain and the nucleotide-binding oligomerization domain. Nod1 possesses an N-terminal caspase-recruitment domain linked to a nucleotide-binding domain and a C-terminal LRR domain. Unlike Apaf-1, Nod1 induces activation of NF-κB (78). Nod1 mediates activation of NF-κB in response to LPS and cell-invasive Shigella flexneri, indicating that Nod1 is a cytoplasmic receptor for LPS (79, 80). These findings suggest that the Nod family of proteins is involved in inflammatory responses, possibly through the recognition of LPS in the cytoplasm. Nod2, a molecule in the same family as Nod1 and Apaf-1, also confers LPS-induced activation of NF-κB. Furthermore, frameshift and missense mutations in NOD2 are associated with susceptibility to Crohn’s disease (81, 82). However, the mutations found in these patients are restricted to the LRR domain, which presumably recognizes LPS, and the mutant NOD2 protein is defective in LPS-induced NF-κB activation (81, 82). Therefore, it remains unclear exactly how mutation of NOD2 may be associated with susceptibility to Crohn’s disease.

TLR2, TLR1, and TLR6 TLR2 RECOGNIZES A VARIETY OF MICROBIAL COMPONENTS TLR2 recognizes components from a variety of microorganisms. These include lipoproteins from pathogens such as Gram-negative bacteria, Mycoplasma and spirochetes (83–87), peptidoglycan and lipoteichoic acid from Gram-positive bacteria (88–91), lipoarabinomannan from mycobacteria (90–93), glycoinositolphospholipids from Trypanosoma Cruzi (94), a phenol-soluble modulin from Staphylococcus epidermidis (95), zymosan from fungi (96), glycolipids from Treponema maltophilum (97), and porins that constitute the outer membrane of Neisseria (98). Analysis of TLR2deficient mice showed that TLR2 is critical to the recognition of peptidoglycan and lipoproteins (99, 100). Accordingly, TLR2-deficient mice showed higher susceptibility to infection by the Gram-positive bacteria S. aureus than wild-type mice (101). Another TLR2-deficient mouse strain showed defective clearance of spirochetes after infection by Borrelia burgdorferi and unresponsiveness to B. burgdorferi lipoproteins (102). Furthermore, TLR2 recognizes several atypical types of LPS from Leptospira interrogans and Porphyromonas gingivalis, in contrast to TLR4, which recognizes LPSs from enterobacteria such as Escherichia coli and Salmonella spp. (103, 104). The properties of the atypical LPSs recognized by TLR2 differ structurally and functionally from the enterobacteria LPS recognized by TLR4. In particular, the two types of LPS differ structurally in the number of

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acyl chains in the lipid A component (105). TLR2 and TLR4 may differentially recognize these structural variations in LPS. However, as with many of these studies, it remains possible that very small amounts of contaminating TLR2 ligand in the LPS preparations might obscure some of these results. TLR2 COOPERATES WITH TLR1 AND TLR6 One aspect of TLR2 ligand recognition involves cooperation with other TLR family members, in particular TLR6 and TLR1, which confer discrimination among different microbial components. The role of TLR6 was analyzed by introducing a dominant negative form into the RAW264.7 macrophage cell line. Peptidoglycan and secreted modulin from S. aureus are TLR2 ligands that induce TNF-α production in RAW264.7 cells, but these responses are suppressed by expression of dominant negative TLR6 (95, 106). TLR2 and TLR6 co-immunoprecipitate, suggesting that they physically interact in the cell (106). Analysis of TLR6-deficient mice further demonstrated that TLR6 functionally cooperates with TLR2 to recognize microbial lipopeptides (107). For example, bacterial lipopeptides have a NH2-terminal cysteine residue that is triacylated, in contrast to mycoplasmal macrophage-activating lipopeptides 2 (MALP-2) which are only diacylated. Macrophages from TLR6-deficient mice did not show any inflammatory response to MALP-2, whereas these cells responded normally to bacterial lipopeptides. Macrophages from TLR2-deficient mice showed no response to either type of lipopeptide. Reconstitution experiments in TLR2/TLR6 doubly deficient embryonic fibroblasts demonstrated that both TLR2 and TLR6 are required for the response to MALP-2. Thus, TLR6 functionally associates with TLR2 to confer specific recognition of the subtle differences between triacyl and diacyl lipopeptides. TLR1 has also been reported to functionally associate with TLR2. Cotransfection of TLR1 and TLR2 into HeLa cells confers responsiveness to soluble factors released from Neisseria meningitidis (108). Analysis of TLR1-deficient mice has demonstrated the importance of TLR1 in the recognition of triacyl lipopeptides (109). Macrophages from TLR1-deficient mice showed impaired production of inflammatory cytokines in response to several kinds of triacyl lipopeptides and lipoproteins from mycobacteria. When a range of triacyl lipopeptides with different lengths of fatty acid chains at their N-terminal cysteines was tested on cells from TLR1-deficient mice, the response to lipopeptides with an N-palmytoyl-S-dilauryl cysteine residue was found to be the most impaired. Although this impairment was not complete, this study suggests that TLR1 is responsible for recognizing subtle differences among the lipid moieties of lipopeptides. TLR1 is highly homologous to TLR6. Therefore, TLR6 may in some cases compensate for a deficiency in TLR1, or other TLRs, in the recognition of triacyl lipopeptides. Involvement of TLR1 in the recognition of the outer surface lipoprotein of B. burgdorferi was also shown (110). Thus, TLR2 has been shown to functionally associate with several TLRs, at least TLR1 and TLR6, and it recognizes a wide variety of microbial components. It is unknown whether dimerization of TLR2 with other TLRs occurs constitutively

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or if it is induced in response to ligand stimulation. It is also unknown whether TLR2 forms a large complex containing TLR1, TLR6, and other TLRs.


TLR5 Chinese hamster ovary cells expressing human TLR5 are responsive to the culture supernatants of Listeria monocytogenes. Purification of the culture supernatants containing TLR5-stimulating activity led to the identification of flagellin as the active component (111). Flagellin is the primary protein component of flagellar, a highly complex structure that extends out from the outer membrane of Gramnegative bacteria. Flagella serve as the propellers that move the bacteria through their aqueous environment. They also aid in the attachment of the bacteria to the host cells, assisting in bacterial invasion and thereby contributing to the virulence of pathogenic bacteria. The flagellin genes from a variety of Gram-negative bacteria share highly conserved regions at their amino- and carboxy-termini, and these regions are responsible for the immunostimulatory activity of flagellin (112). Flagellin elicits a potent immune response not only in mammals but also in plants. The flagellin-induced immune response in plants is dependent on a MAP kinase signaling cascade (113). A screen for flagellin-insensitive arabidopsis mutants led to the isolation of a single genetic locus, FLS2. This gene encodes a transmembrane receptor–like kinase with a leucine-rich repeat (LRR) domain, which shows a structure similar to the extracellular portion of the mammalian TLR family (114). Thus, flagellin represents an evolutionarily conserved pathogenic molecular pattern that is recognized by conserved host receptors containing an LRR domain. In addition to the FLS2 gene product, plants have several other gene products that are responsible for resistance to pathogens. Many of these products also possess an LRR domain, and some also possess a Toll/IL-1 receptor (TIR) domain, indicating that the LRR and TIR domains are important for the host defense against pathogens in many multicellular organisms (115).

TLR3 Double-stranded (ds) RNA is produced by many viruses during their replicative cycle, either as an essential intermediate in RNA synthesis or as a byproduct generated by symmetrical transcription of DNA virus genomes. dsRNA is a potent inducer of type I interferons (IFN-α and -β), which exert various physiological effects including antiviral and immuno-stimulatory activities. dsRNA also induces transcription of some IFN-inducible genes and promotes maturation of dendritic cells. Some synthetic dsRNAs, such as polyinosinic-polycytidylic acid [poly(I:C)], have similar activity to that of dsRNA. Some of the immunostimulatory activity of dsRNA is believed to be elicited by activation of dsRNA-dependent protein kinase (PKR). Embryonic fibroblasts from PKR-deficient mice showed impaired responses to dsRNA and poly(I:C), although some responses remained (116, 117). PKR-deficient mice were further shown to be susceptible to respiratory infection by vesicular stomatitis virus, but the responses to infections by other routes such

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as intravenous and intraperitoneal routes were apparently normal. This indicates that additional molecules other than PKR might be responsible for the recognition of dsRNA and viruses (118, 119). Expression of human TLR3 in the dsRNA-nonresponsive cell line 293 conferred enhanced activation of NF-κB in response to dsRNA and poly(I:C). Furthermore, TLR3-deficient mice showed impaired responses to dsRNA and poly(I:C), indicating that TLR3 is a receptor for dsRNA (120). However, further studies will be required to clarify the mechanisms by which PKR is linked with TLR3 in the dsRNA-mediated pathway. In addition, the more fundamental question of whether TLR3 is actually involved in the recognition of viruses remains an intriguing but unanswered one. The principal cell in human and mouse blood that produces type I interferon in response to viral challenge is plasmacytoid dendritic cell the (121–123); however, TLR3 is not expressed in this cell type (124). Therefore, other receptors in addition to TLR3 might be responsible for the recognition of viral infection leading to production of IFN-α/β. TLR3 has unique structural features among the TLRs. For example, TLR3 lacks the proline residue that is conserved among other TLRs. This proline is mutated in the Tlr4 gene of C3H/HeJ mice and is responsible for the LPS-hyposensitive phenotype of this strain. The genomic organization of TLR3 is also different from the other TLRs. TLR3 is also unique in that it is preferentially expressed in mature dendritic cells (125). Therefore, TLR3 may have a unique function in addition to the recognition of dsRNA.

TLR9 and TLR7 TLR9 IS ESSENTIAL FOR RECOGNITION OF CPG DNA Bacterial DNA is a potent activator of immune cells. The critical involvement of TLR9 in the recognition of bacterial DNA was demonstrated using TLR9-deficient mice (126). The immunostimulatory activity of bacterial DNA is attributed to the presence of unmethylated CpG motifs, which are relatively infrequent in the vertebrate genome and when they occur are typically methylated on their cytosine residues and lack any immunostimulatory activity. Thus, CpG DNA is another prototypic molecular pattern by which the immune system recognizes pathogens. Synthetic oligodeoxynucleotides containing unmethylated CpG motifs also activate immune cells. Administration of CpG DNA is sufficient to protect against infections by intracellular pathogens such as Leishmania major and Listeria monocytogenes in mice (127–129). Furthermore, CpG DNA activates dendritic cells to produce the Th1-polarizing cytokine IL-12, leading to the development of Th1-like immune responses. Therefore, CpG DNA has promising therapeutic value as an adjuvant and antiinfectious agent (130, 131). Human and mouse immune cells are optimally activated by slightly different CpG motifs (132). This specificity can be explained by species differences among TLR9s. When mouse or human TLR9 was expressed in the CpG DNAunresponsive cell line 293, these cells gained the ability to respond to the optimal mouse or human CpG sequence, respectively (133). These findings also indicate

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that TLR9 directly recognizes CpG DNA. Thus, the identification of optimal CpG motifs or other synthetic agonists of TLR9 from humans and other diverse animals may lead to the establishment of effective adjuvants for each species. Several studies have reported that CpG DNA is recognized in the endosome following nonspecific uptake into the cells (130, 131). This suggests that recognition of CpG DNA by TLR9 occurs in the endosome. Indeed, CpG DNA-induced activation of signaling cascades such as c-Jun N-terminal kinase (JNK) and NF-κB is delayed compared with LPS-induced activation in normal macrophages (126). Recently, a monoclonal antibody against TLR9 has been established, and staining with this antibody indicated the intracellular localization of endogenous TLR9 in a mouse macrophage cell line (134). This is in sharp contrast to TLR1, TLR2, and TLR4, which are expressed on the cell surface (44, 75, 135, 136). TLR2 is recruited to the phagosomes after stimulation with zymosan (96, 106). Thus, internalization of TLR ligands may be required for full activation of immune cells by TLRs, or signaling pathways via TLR9 may have some distinct characteristics from other TLRs. TLR7 RECOGNITION OF SYNTHETIC AGONISTS TLR7 and TLR8 are highly homologous to TLR9, as mentioned above. Although the natural ligands of TLR7 and TLR8 remain unclear, the TLR9 subfamily including TLR7, TLR8, and TLR9 may participate in the discrimination of nucleic acid-like structures in microorganisms. This parallels the situation in the TLR2 subfamily, which discriminates between differences in lipoproteins. One such example was demonstrated in TLR7-deficient mice. Several synthetic imidazoquinolines have demonstrated potent antiviral and antitumor properties, owing to their ability to induce inflammatory cytokines, especially IFN-α. One of these imidazoquinoline compounds, Imiquimod, has been approved for the treatment of genital warts caused by infection of human papillomavirus. Recently, it was shown that TLR7-deficient mice do not respond to synthetic imidazoquinolines (137). These compounds have structures similar to nucleic acids, and TLR7 may sense viral infection by recognizing a similar, as yet undetermined viral component or product, or a host compound induced in response to virus. In addition, our unpublished data (S Akira) indicate that two other immunomodulators, loxoribine and bropirimine, also activate immune cells through TLR7 (Figure 3). Loxoribine (7-allyl-8-oxoguanosine) enhances natural killer (NK) cell activity and induces production of cytokines including IFNs; it is anticipated to be useful for the clinical treatment of cancer (138). Bropirimine (2-amin-5-bromo-6-phenyl-4(3)-pyrimidinone) is an orally active immunomodulator that induces production of cytokines including IFN-α and is in clinical use against renal cell carcinoma (139). Thus, the TLR family recognizes not only microbial components but also clinically useful synthetic compounds, suggesting that a screen for TLR-activating agents will be useful for clinical applications. We anticipate that new therapies utilizing the TLR-mediated innate immune activation will be developed to treat several disorders such as infection, cancer, and allergy.

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Figure 3 Structures of synthetic compounds that activate TLR7. Analysis of TLR7-deficient mice revealed that TLR7 recognizes several synthetic compounds, which are structurally related to nucleic acids. These include imidazoquinoline (Imiquimod and R-848), loxoribine, and bropirimine.

EXPRESSION OF TLRs Distribution of TLRs The expression of TLR family members has been elucidated in several studies. Monocytes/macrophages express mRNA for most TLRs except TLR3 (125). Expression of TLRs in dendritic cells differs among their subsets (124, 140). In humans blood dendritic cells contain two subsets, myeloid dendritic cell (MDC) and plasmacytoid dendritic cell (PDC) (141–143). MDCs express TLR1, 2, 4, 5, and 8, and PDCs exclusively express TLR7 and TLR9, although there are some reports that TLR7 is also expressed in MDC (124, 140, 144, 145). Immature dendritic cells mature in response to microbial components (146–149), and the expression of different TLRs shows distinct patterns during maturation. Expression of TLR1, 2, 4, and 5 is observed in immature dendritic cells but decreases as the dendritic cells mature (136). TLR3 is expressed only in mature dendritic cells (125). Thus, TLRs are differentially expressed in different subsets and maturation stages of dendritic cells. Another study has examined expression of all the human TLR mRNAs in a range of tissues (150). This study indicated that most tissues express at least one TLR, and that phagocytes in particular show abundant expression of all known TLRs, although several TLRs are preferentially expressed in B cells. Further study will be required to clarify the tissue distribution of each TLR. Mast cells have been preserved throughout evolution and have the capacity to phagocytose pathogens, process antigens, and produce inflammatory cytokines, indicating their potential role in the innate immune response against infectious organisms as well as in allergic diseases (151). Mast cells express TLR2, 4, 6, and 8 but not TLR5 (152, 153). Furthermore, mast cells from TLR4-mutated mice

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showed defective production of inflammatory cytokines in response to LPS. When mice lacking mast cells were reconstituted with TLR4-mutated mast cells, it was observed that recruitment of neutrophils in the peritoneal cavity after enterobacteria infection was impaired (152). Intradermal injection of peptidoglycan (PGN) caused TLR2-mediated activation of mast cells in skin, which may be involved in the inflammatory lesions of atopic dermatitis (154). Thus, TLRs are expressed in mast cells and may play a role in their innate immune responses. In addition to innate immune cells, TLRs are expressed in several other types of cells that contribute to inflammatory responses. The mucosal surfaces of the respiratory and intestinal tract are covered by a single layer of epithelial cells, forming a protective barrier against pathogens. In the intestine the apical surfaces of epithelial cells are continually exposed to bacteria, but this does not result in exaggerated inflammation. These epithelial cells elicit inflammatory responses only against pathogenic bacteria that invade into the basolateral compartment from the apical side. For example, exposing the basolateral, but not apical, surface of model intestinal epithelia to the TLR5 ligand, flagellin, induces an inflammatory response. Furthermore, TLR5 is expressed exclusively on the basolateral surface of the intestinal epithelial cells (155). TLR4 is expressed at relatively low levels in intestinal epithelial cells, which may explain why lipopolysaccharide (LPS) does not elicit a strong inflammatory response in the intestine (156, 157). In contrast, intestinal epithelium from patients with inflammatory bowel diseases showed augmented expression of TLR4 (158). This is consistent with the idea that inflammatory bowel diseases may result from exaggerated inflammatory responses to intestinal bacterial flora. Thus, TLR expression is finely regulated in epithelial cells, perhaps explaining why pathogenic Gram-negative bacteria, but not commensal bacteria, induce inflammatory responses in the intestine. An epithelial cell line from the small intestine shows a peculiar type of LPS response: In response to LPS it does not produce inflammatory cytokines, but instead produces the chemokine MIP-1. TLR4 is not expressed on the cell surface of small intestine epithelial cells, but resides in the Golgi apparatus and is colocalized with LPS (159). LPS is internalized and delivered to the Golgi apparatus, thereby enabling LPS-induced cell activation (160). Therefore, the expression of TLR4 in the Golgi apparatus would be important for LPS-induced induction of chemokines by LPS in the small intestinal epithelia. Renal epithelial cells are important barriers to Gram-negative pyelonephritis. Expression of TLR2 and TLR4 in renal epithelial cells is induced by IFN-γ and TNF-α and contributes to the detection of bacterial invasion in the lumen of tubules and induction of the inflammatory response (161). TLR4-deficient mice are defective in the production of inflammatory cytokines after intrapulmonary administration of Haemophilus influenzae. This finding indicates that TLR4 plays an important role in sensing H. infulenzae infection in the pulmonary epithelia (162). TLR4 is also expressed on corneal epithelial cells and contributes to the inflammatory responses leading to river blindness following invasion of parasitic filarial nematodes (163). Microvascular endothelial cells are the first lines of defense against invading microorganisms. Human dermal

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endothelial cells express TLR4, indicating a possible role in detection of pathogens by endothelial cells (164).


Regulation of TLR Expression Expression of TLRs is modulated by a variety of factors such as microbial invasion, microbial components, and cytokines. Infection by Mycobacterium avium induces augmented TLR2 mRNA expression and decreased TLR4 mRNA expression in macrophages (165) and leads to increased TLR2 promoter activity accompanied by chromatin remodeling (166, 167). Nontypeable H. influenzae activates NF-κB through TLR2 and induces expression of TLR2 in epithelial cells in an autocrine manner (168, 169). Infection of mice with E. coli induces expression of TLR2 mRNA in γ δT cells, which is thought to represent a more primitive, early line of cellular defense, preprogrammed to recognize a limited set of antigens (170). Viral infection also induces expression of the TLR1, TLR2, TLR3, and TLR7 mRNAs in macrophages. Increased TLR expression is suppressed by treatment with anti-IFNα/β antibody, indicating that IFN-α/β mediates virus-induced activation of innate immunity via modulation of TLR expression (171). LPS enhances expression of TLR2 in macrophages and adipocytes (172, 173). In contrast, LPS stimulation of mouse macrophages causes a reduction in surface expression of the TLR4/MD-2 complex, and this may be one mechanism underlying the phenomenon of LPS tolerance (74, 174). Several cytokines regulate expression of the TLRs. Colony-stimulating factor 1 is induced in vivo after infection or challenge with LPS and can prime macrophages to respond to further LPS stimulation with enhanced inflammatory cytokine production. Colony-stimulating factor 1 can downregulate TLR9 expression in macrophages and strongly suppresses CpG DNA-induced production of inflammatory cytokines (175). Macrophage migration inhibitory factor (MIF) is an important cytokine that mediates inflammation and sepsis (176). MIF-deficient mice are defective in their responses to LPS. Recently, this defect was shown to be the result of decreased expression of TLR4. Introduction of antisense MIF mRNA into normal cells resulted in reduced TLR4 promoter activity and a reduced LPS response, indicating that MIF regulates TLR4 expression (177). IFN-γ , which primes phagocytes to respond to LPS, enhances surface expression of TLR4 in human monocytes and macrophages (178). Expression of the Tlr2 gene in macrophages is induced by LPS and inflammatory cytokines such as IL-2, IL-15, IL-1β, IFN-γ , and TNF-α (172). IL-15, a cytokine that promotes extrathymic development and survival of T cells, especially CD8+ T cells and NK cells, induces expression of the Tlr2 gene in T cell lines through the activation of Stat5 (179). T1/ST2, a member of the IL-1 receptor (IL-1R) family, is expressed by fibroblasts, mast cells, and Th2 cells, but not Th1 cells, and exists in both membrane-bound and soluble forms. Blocking ligand activation of T1/ST2 causes downregulation of TLR4. For example, incubation of macrophage cultures with the soluble form of T1/ST2 downregulates TLR4 mRNA expression, and administration of

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anti-T1/ST2 antibody to mice reduces the mortality of LPS-induced endotoxin shock (180).


TLR-MEDIATED SIGNALING PATHWAYS The pathways that transduce TLR signals in mammals have both similar and dissimilar characteristics from those in drosophila. In drosophila the Toll- and IMDpathways are essential for antifungal and anti–Gram negative bacterial responses, respectively. In mammals the host defense against microorganisms mainly relies on pathways that originate from the common TIR domain of TLRs. The TLR family signaling pathway is highly homologous to that of the IL-1R family. Both TLR and IL-1R interact with an adaptor protein MyD88, which has a TIR domain in its C-terminal portion but a death domain in its N-terminal portion instead of the transmembrane domain found in TLRs. MyD88 associates with both the TLRs and the IL-1R via interaction between the respective TIR domains. Upon stimulation, MyD88 recruits a death domain–containing serine/threonine kinase, the IL-1R-associated kinase (IRAK). IRAK is activated by phosphorylation and then associates with TRAF6, leading to activation of two distinct signaling pathways, JNK and NF-κB (181–185).

MyD88-Dependent Signaling Pathway Studies of MyD88-deficient mice revealed that this protein plays a critical role in the response to IL-1 and LPS (186, 187). Macrophages from MyD88-deficient mice do not produce any inflammatory cytokines in response to peptidoglycan, lipoproteins, CpG DNA, dsRNA, or the imidazoquinolines (100, 120, 137, 188– 190). MyD88-deficient mice are also unable to produce any detectable level of IL-6 in response to flagellin (111). These results demonstrate that MyD88 is critical to the production of inflammatory cytokines induced by the TLR family. Indeed, no activation of NF-κB or JNK was observed in MyD88-deficient macrophages in response to peptidoglycan, lipoprotein, CpG DNA, or the imidazoquinolines. Accordingly, MyD88-deficient mice were found to be highly susceptible to infection by S. aureus (101). Similarly, TRAF6-deficient mice exhibit impaired responses to both IL-1 and LPS, indicating that TRAF6 is a critical component of both the IL-1R- and TLR4-mediated signaling pathways at a level downstream of MyD88 (191, 192). The IRAK family is comprised of four members that contain a conserved death domain and kinase domain: IRAK-1, IRAK-2, IRAK-M, and IRAK-4 (193). IRAK-1-deficient mice show partial defects in their responses to IL-1 and LPS (194–196). In contrast, IRAK-4-deficient mice show almost no inflammatory responses to either IL-1 or LPS (197). Among the IRAK homologs, IRAK-4 is most structurally related to its drosophila counterpart, Pelle (197). These findings indicate that IRAK-4 is an essential component in IL-1- and TLR4-dependent signaling pathways. The MyD88-dependent pathway signals via MyD88, IRAK, and TRAF6 and leads to NF-κB activation. The activity of NF-κB is regulated by association with

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IκB, which sequesters NF-κB in the cytoplasm until phosphorylated on serine residues by the IκB kinase (IKK) complex. This phosphorylation leads to the dissociation and nuclear translocation of NF-κB. The IKK complex contains two catalytic subunits, IKKα and IKKβ, as well as a scaffold protein, IKKγ . LPS stimulation enhances the activity of IKK in a human monocytic cell line (198, 199). Although IKKα is dispensable for IL-1- and LPS-induced NF-κB activation, cells from mice deficient in IKKβ or IKKγ show impaired NF-κB activation and IL-6 production in response to IL-1 and LPS (117, 200). This shows that these IKK components are critical to the TLR-mediated signaling pathway. In drosophila dTAK1 acts upstream of dIKKβ and dIKKγ . Studies using dTAK1-mutant flies showed that dTAK1 plays an essential role in Gram-negative bacteria-induced activation of Relish, an NF-κB-like transcription factor in the IMD pathway (21). In vitro over-expression studies showed that both IL-1 and LPS activate mammalian TAK1, which in turn activates NF-κB (201–203). However, the physiological role of TAK1 remains to be elucidated. Recent studies have suggested a unique mechanism by which TRAF6 is linked to the IKK complex. A mammalian protein complex that activates IKK was purified and analyzed and found to be composed of two subunits: TAK1 and a ubiquitin conjugating enzyme complex composed of Ubc13 and Uev1A. TRAF6 functions together with Ubc13/Uev1A to catalyze the Lys 63 (K63)–linked polyubiquitination of TRAF6 itself (204). TAK1 is consequently activated via its association with the ubiquitinated TRAF6. Once activated, TAK1 mediates phosphorylation of the IKK complex (205). Ubiquitination is thought to be a step that directs modified target proteins to the proteasome, where they are degraded. However, ubiquitination of TRAF6 mediates activation of NF-κB through a process that does not require protein degradation. A candidate molecule that links TRAF6 and NF-κB was identified in a screen of TRAF6-interacting molecules. This molecule is designated ECSIT (evolutionarily conserved signaling intermediate in Toll pathways) (206) and it interacts with TRAF6 and MEKK1, a MAP kinase kinase kinase family member that mediates the activation of NF-κB. However, the biological function of ECSIT remains to be elucidated. An additional molecule that mediates TLR-induced signaling has been reported. Receptor interacting protein-2 (RIP2) contains a C-terminal caspase-recruitment domain and was originally identified as a serine/threonine kinase that associates with the TRAFs and with TNF receptor family members such as the type I TNF receptor and CD40 to induce NF-κB activation and apoptosis (207, 208). Mice deficient in RIP2 exhibit partial impairment in their response to LPS, peptidoglycan, and dsRNA (209, 210). Furthermore, RIP2 associates with TLR2, indicating that RIP2 is somehow involved in TLR signaling pathways.

MyD88-Independent Signaling Pathway LPS-INDUCED RESPONSE IN THE ABSENCE OF MYD88 MyD88 is essential for the production of inflammatory cytokines in response to a variety of microbial

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components. However, LPS is still able to induce activation of NF-κB and JNK in MyD88-deficient macrophages, but with delayed kinetics (187). This indicates that although MyD88 is important for LPS-induced production of inflammatory cytokines, there exists an MyD88-independent component in the LPS signaling pathway. Evidence is accumulating that MyD88-independent activation of the LPS-TLR4 signaling pathway is of biological importance. Dendritic cells from MyD88-deficient, but not from TLR4-deficient mice showed enhanced expression of costimulatory molecules and increased T cell allo-stimulatory activity in response to LPS. This indicates that LPS-induced maturation of dendritic cells depends on the MyD88-independent pathway (211, 212). LPS stimulation induces caspase-1-dependent cleavage of the IL-18 precursor into its mature form in Kupffer cells from MyD88-deficient mice (213). Analysis of LPS-induced genes in MyD88-deficient macrophage showed that a number of IFN-regulated genes are upregulated, such as those encoding IP-10 and GARG16 (214). Thus, several LPSinduced responses occur in MyD88-deficient mice. In addition to LPS, dsRNA induced activation of NF-κB in MyD88-deficient mice, although no dsRNA-induced production of inflammatory cytokines was observed (120). It is not known whether activation of MyD88-independent signaling induced by dsRNA and LPS are equivalent or not. MOLECULES INVOLVED IN THE MYD88-INDEPENDENT PATHWAY In the course of analyzing the MyD88-independent activation of LPS signaling, a novel adaptor molecule named TIR domain–containing adaptor protein (TIRAP)/MyD88adaptor-like (Mal) was identified (215, 216). Similar to MyD88, TIRAP/Mal possesses a C-terminal TIR domain but lacks an N-terminal death domain. It specifically associates with TLR4 through interaction between their respective TIR domains. The dominant-negative form of TIRAP/Mal inhibited TLR4-mediated activation but not TLR9-mediated activation of NF-κB. Furthermore, LPS-induced maturation was abolished in both wild-type and MyD88-deficient dendritic cells treated with a cell-permeable TIRAP peptide that blocks TIRAP-mediated signaling. These in vitro findings indicate that TIRAP/Mal is a possible adaptor molecule involved in LPS-induced, MyD88-independent signaling (Figure 4). The generation and analysis of TIR domain–containing adaptor protein/MyD88-adaptor-like (TIRAP/Mal)–deficient mice will clarify its physiological role in TLR4-mediated signaling. LPS stimulation of MyD88-deficient macrophages also activates IRF-3 (214). LPS-induced activation of IRF-3 causes expression of several IFN-inducible genes (216). Activation of IRF-3 was observed when cells were stimulated with ligand for TLR4 but not TLR2 (214, 217). Viral infection and dsRNA, which also activate the MyD88-independent pathway, are also known to activate IRF-3, thereby inducing the IFN-α/β- and IFN-regulated genes (218–220). Therefore, IRF-3 may play an important role in the MyD88-independent pathway. Similar to TLR4-mediated signaling, each TLR seems to have its own signaling pathway in addition to the common MyD88-dependent pathway. In

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TLR2-mediated signaling, stimulation with heat-killed S. aureus results in the recruitment of active RacI and phosphatidyl-inositol-3 to the cytoplasmic portion of TLR2. This in turn causes activation of Akt, which is followed by the activation of the p65 subunit of NF-κB in a process that is independent of IκBα degradation (221). Stimulation of dendritic cells with TLR2 and TLR4 agonists induces mRNA expression for distinct types of cytokines and chemokines (222). The existence of individual pathways for each TLR may explain the distinct biological responses elicited by different TLR agonists. In addition to MyD88 and TIRAP/Mal, another adaptor molecule named Tollinteracting protein (Tollip) has been identified (223). Tollip was first identified in the context of IL-1 signaling and was shown to be present in a complex with IRAK. Upon stimulation with IL-1, the Tollip-IRAK complex is recruited to the IL-1R complex through the association of Tollip with IL-1RAcP. Interaction with MyD88, which is also recruited to the signaling complex, then triggers IRAK autophosphorylation, which in turn leads to the rapid dissociation of IRAK from Tollip. A subsequent study showed that Tollip negatively regulates the TLR-mediated signaling pathway (224, 225). Overexpression of Tollip blocked activation of NFκB in response to IL-1, TLR2, and TLR4 agonists. However, it remains unclear what physiological roles Tollip plays in TLR signaling.

Transcription Factors Activated in the TLR-Mediated Signaling Pathway NF-κB NF-κB is a transcription factor that was originally identified as a nuclear factor necessary for the transcription of immunoglobulin light chain in B cells. Subsequently, NF-κB was shown to be expressed in a variety of cell types. The NF-κB family of transcription factors is evolutionarily conserved. In drosophila, three members have been identified, as mentioned earlier: Dorsal, Dorsal-type immune factor, and Relish. In mammals five family members have been identified: RelB, c-Rel, p65 (RelA), p100/p52, and p105/p50 (226). Each member of the NF-κB family plays an important role in LPS-mediated responses. For example, B cells from mice deficient in p50, RelA, c-Rel, or RelB displayed an impaired growth response to LPS (226). Mice lacking individual NF-κB subunits were very susceptible to microbial infections (227–229). The critical involvement of NF-κB in the development and function of dendritic cells has also been shown. RelBdeficient mice showed defective development of a dendritic cells subset (230–232). In mice doubly deficient for p50 and p65, the development of dendritic cells was also impaired. In contrast, the development of dendritic cells was normal, but IL12 production was severely impaired in mice doubly deficient for p50 and c-Rel (233). This indicates that the function and development of dendritic cells is finely regulated by distinct NF-κB subunits. Necrotic cells, but not apoptotic cells, induce inflammatory responses (57). Necrotic cells induce TLR2-dependent activation of NF-κB, and embryonic fibroblast cells from mice deficient in the p65 subunit of NF-κB are defective in the necrotic cell–induced expression of chemokines (234).

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AP-1 The AP-1 (activating protein-1) family of transcription factors consists of homodimers and heterodimers of the Jun and Fos family, which bind to the 12-O-tetradecanoylphorbol-13-acetate response element (235). Jun proteins form not only homo- and hetero-dimers within the AP-1 family but also form heterodimers with members of the CREB/ATF family of transcription factors, such as ATF, and therefore are able to bind to the cAMP response element (CRE). The activity of AP-1 is upregulated through phosphorylation by the MAP kinases JNK and ERK (236). LPS and peptidoglycan enhance the transcriptional activity of AP-1 and the CREB/ATF family of transcription factors (237–239). In addition, viral infection and dsRNA activate AP-1 through induction of JNK (117). NF-IL6 NF-IL6 is a member of the C/EBP family of transcription factors, which contain basic and leucine zipper domains (240). NF-IL6 was originally identified as a nuclear factor that specifically binds to an IL-1 responsive element in the IL-6 gene promoter (241). NF-IL6 was subsequently shown to be activated by phosphorylation in response to inflammatory stimuli and to play an important role in macrophage responses (242). Indeed, macrophages from NF-IL6-deficient mice display defective killing activity against Listeria monocytogenes (243). NF-IL6 is critical for LPS-induced gene expression in macrophages. Macrophages from NF-IL6-deficient mice show defective expression of LPS-inducible genes such as Cox-2, a C-type lectin Mincle, and membrane-bound glutathione-dependent prostaglandin E2 synthase (244–246). Thus, NF-IL6 is a nuclear target in the TLR-mediated signaling pathway. IRF The IRF family of transcription factors is composed of nine members that are critical regulators of innate immune responses (247). Among these, IRF-3 is presumably involved in the MyD88-independent signaling pathway, as described above. The expression of IRF-1 is markedly induced by viral infection. Macrophages from IRF-1-deficient mice show defective induction of IL-12 and iNOS in response to LPS (248, 249). IRF-7 is also induced by viral infection and critically involved in the biphasic system of IFNα/β gene induction in conjunction with IRF-3 (220). IRF-8/ICSBP is critical for induction of the IL-12 gene. As a result, IRF-8/ICSBP-deficient mice are highly susceptible to infection with Toxoplasma gondii and Leishmania major owing to defective Th1 responses (250, 251).

OTHER TRANSCRIPTION FACTORS LPS stimulation induces activation of the STAT family of transcription factors (252). Bacterial infection or LPS stimulation of macrophages leads to the rapid phosphorylation of a serine residue in Stat1 (253). In macrophages from Stat1deficient mice, LPS-induced expression of IFN-regulated genes such as IP-10, IRF-1, and iNOS was reduced. These findings indicate that Stat1 may be involved

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in the response to LPS (254). The STAT family of transcription factors has been established as critical molecules in cytokine signaling pathways (255). Indeed, other studies showed that LPS stimulation of macrophages induced expression of IFN-β through activation of the MyD88-independent pathway, and IFN-β in turn induced IFN-regulated gene expression through activation of Stat1 (256, 257). Therefore, Stat1 seems to be indirectly involved in the LPS-induced expression of IFN-regulated genes. The Sp1 transcription factor is also involved in LPS-induced gene expression and plays a prominent role in the induction of IL-10 gene expression in both human and mouse macrophages (258, 259).

MODULATION OF IMMUNE RESPONSES BY TLRs Regulation of Adaptive Immunity by TLRs Recognition of microbial components by TLRs triggers activation of not only innate immunity but also adaptive immunity. The signals for activation of adaptive immunity are largely provided by dendritic cells. Immature dendritic cells residing in the periphery have a high capacity for endocytosis, which facilitates antigen uptake. They are activated by various microbial components to undergo maturation and express many of the TLRs, such as TLR1, 2, 4, and 5 (136). Furthermore, maturation of dendritic cells by a variety of microbial components is elicited through TLRs; this includes LPS, CpG DNA, peptidoglycan, lipoprotein, and the cell wall skeleton of Mycobacteria (126, 147–149, 212). TLR-mediated recognition of microbial components by dendritic cells induces the expression of costimulatory molecules such as CD80/CD86 and production of inflammatory cytokines such as IL-12 (260). Once matured, dendritic cells lose their capacity for endocytosis and migrate into the draining lymph nodes. Here they present microorganism-derived peptide antigens expressed on the cell surface with MHC class II antigen to naive T cells, thereby initiating an antigen-specific adaptive immune response (261, 262). The involvement of TLRs in the regulation of the adaptive immune response was demonstrated in vivo using MyD88-deficient mice. MyD88-deficient mice immunized with Ag mixed with complete Freund’s adjuvant (CFA) exhibited defective production of both IFN-γ from CD4+ T cells and Ag-specific IgG2a (263, 264). Furthermore, the Th1 immune response provoked by a protozoan parasite was abolished in MyD88-deficient mice (265). Thus, the Th1 immune response is regulated by the MyD88-dependent signaling pathway. It has been proposed that distinct types of dendritic cell subsets differentially induce Th1 and Th2 responses (141–143). However, the functions of these dendritic cell subsets are rather flexible, and their ability to steer a particular type of Th cell development can depend on the microbial microenvironment (162, 266). Activation of TLR4 or TLR9 in dendritic cells induces production of IL-12, thereby skewing Th cell differentiation toward the Th1 type. LPSs from E. coli (TLR4

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ligand) and Porphylomonas gingivalis (a putative TLR2 ligand) induce Th1-type and Th2-type responses, respectively, in vivo (267). This differential outcome was attributed to the ability of E. coli LPS but not P. gingivalis LPS to induce production of IL-12 from CD8+ dendritic cells. Thus, TLR signaling in dendritic cells is critically involved in determining the Th1/Th2 balance. MyD88-deficient mice exhibit a skewed Th2 response against Ag administered along with CFA or Th1-inducing microbial stimuli (263–265). The skewed Th2 response in MyD88deficient mice does not seem to be caused by a default pathway active in the absence of IL-12 production, because IL-12-deficient mice do not show a Th2 response (265). Furthermore, TLR4 signaling stimulates wild-type and MyD88deficient dendritic cells to support Th1 and Th2 cell differentiation, respectively (264). Although this finding indicates that activation of the MyD88-independent pathway downstream of TLR4 leads to differentiation of dendritic cells into Th2supporting dendritic cells, there is little evidence to show that TLRs are involved in the helminth-induced Th2 response (268). It remains unclear whether the Th2 response is TLR-independent or not. Analysis of the in vivo antigen-specific responses in MyD88-deficient mice suggested that the immuno-stimulatory activity of adjuvants such as CFA is elicited through the TLRs (263). Indeed, CFA contains a complex mixture of mycobacterial components. In addition to CFA, several microbial components have potent immuno-stimulatory activity as adjuvants. CpG DNA, which is recognized by TLR9, is a potent adjuvant that elicits a skewed Th1 response (269, 270). The outer membrane proteins of Neisseria, porins, have potent immunogenicity and are used as adjuvants in various vaccine formulations. The Neisserial porins have been shown to be recognized by TLR2 (98). Similar to CFA, the cell-wall skeletal fraction from Mycobacterium bovis BCG strain (BCG-CWS) has potent immunogenicity and is used as an adjuvant for immunotherapy in cancer (271). Recognition of BCG-CWS is dependent on TLR2 and TLR4 (147). Thus, several pathogen-derived adjuvants are recognized by TLRs, which may explain the molecular mechanism of their adjuvanticity.

Crosstalk Between Type I IFNs and TLRs Activation of TLRs in dendritic cells leads to production of type I IFNs (IFNα/β), which promote dendritic cell maturation and induce some Th1-type chemokine genes (257, 272). Type I IFNs induce production of antigen-specific immunoglobulins with all isotypes in a dendritic cell–dependent manner (273). CFA-induced immune responses are abolished in IFN-α/βR-deficient mice (273). Thus, type I IFNs are critical to the link between innate and adaptive immunity. Patients with systemic lupus erythematosus manifest elevated levels of serum IFN-α, which induces dendritic cell differentiation, indicating that disregulation of type I IFN production can lead to immunological disorders (274). Dendritic cell subsets, such as myeloid dendritic cells (MDC) and plasmacytoid dendritic cells (PDC), respond to different repertoires of pathogenic stimuli. In humans

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MDCs produce IL-12 in response to a variety of stimuli including LPS, whereas PDCs preferentially produce IFN-α upon viral infection and in response to CpG DNA (121, 275). Different TLRs are expressed between MDC and PDC, as described above. However, the pattern of TLR expression alone does not determine how dendritic cell subsets differentially respond to pathogenic stimuli. It has recently been shown that the same TLR7 ligand induces production of IL-12 in MDC, but IFN-α in PDC, indicating that distinct patterns of response are determined not only by TLR expression but also by the dendritic cell lineage (145). A murine counterpart of human PDCs has been identified as an IFN-α producing cell population (MIPC) (122, 123). MIPCs are CD11cdullB220+Gr-1+ and reside in the spleen or bone marrow. Similar to human PDCs, MIPCs express TLR7 and TLR9 and produce IL-12 in response to CpG DNA (122, 123, 276). MIPCs play crucial roles in the production of type I IFN and IL-12 during MCMV infection (277). It remains unknown how TLRs on MIPC are involved in antiviral immune responses.

Involvement of the TLRs in Microbial Killing In addition to controlling the development of adaptive immunity, activation of TLRs appears to be directly involved in induction of antimicrobial activity. TLR2 activation leads to nitric oxide–dependent and –independent killing of intracellular Mycobacterium tuberculosis in mouse and human macrophages, respectively (278). In drosophila activation of the Toll and IMD pathways by microbial invasion leads to the synthesis of antimicrobial peptides. Expression of a single antimicrobial peptide is sufficient to rescue the susceptibility of Spätzle/IMD double mutant flies to microbial infection, indicating that antimicrobial peptides play an essential role in the host defense in drosophila (279). These antimicrobial peptides are evolutionarily ancient and conserved between humans and plants and have been shown to directly kill microbes (280). In mammals antimicrobial peptides such as β-defensins are produced in several kinds of epithelial cells residing in the gastrointestinal tracts, respiratory tracts, and skin (280). Paneth cells in the base of the crypts of gastrointestinal tracts secrete α-defensins in response to LPS or bacterial challenge (281). Thus, mammalian antimicrobial peptides are produced in response to microbial stimuli at the epithelial surface, the front line of defense between pathogen and host. Strong expression of TLR4 occurs in the crypts of the small intestine (159). LPS induces expression of mouse β-defensin-2, -3, and -6 (282). Stimulation of the human lung epithelial cell line A549 with lipoprotein led to TLR2-mediated induction of β-defensin-2 (283). These findings indicate that TLRs are likely to mediate the secretion of antimicrobial peptides, thereby regulating the direct killing of microbes at the epithelial surface. This potential involvement of TLRs in induction of mammalian antimicrobial peptides needs to be analyzed more precisely. Macrophages infected with invasive bacteria undergo apoptosis (284). Although the implications of this phenomenon remain elusive, the induction of apoptosis

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may limit the spread of pathogens by localizing cell death at the site of pathogen invasion. Apoptosis of macrophages and endothelial cells is triggered by several microbial components such as LPS and lipoprotein. TLR2 confers lipoproteininduced apoptosis of macrophages, indicating the possible involvement of TLRs in infection-induced cell death (83). TLR2-mediated apoptosis involves MyD88 and an apoptotic pathway involving FADD and caspase 8. MyD88 associates with FADD via their respective death domains (84). LPS-induced apoptosis in endothelial cells is mediated by MyD88, IRAK-1, and FADD (285, 286). Thus, TLRs are presumably involved in apoptosis induced by microbial components. MyD88 and IRAK-1, both of which possess death domains, may induce apoptosis via interaction with FADD and consequent activation of the FADD–caspase 8 apoptotic signaling pathway. In addition to the induction of apoptosis, the FADD-dependent pathway mediates the activation of NF-κB and induction of inflammatory gene expression, indicating the possible involvement of FADD in TLR-mediated pathways (287, 288). However, there is a report showing that FADD suppresses activation of NF-κB by LPS (289). Thus, more experiments are required to clarify the role of FADD in TLR signaling.

FUTURE PROSPECTS Since the discovery of the TLRs a few years ago, much progress has been made in our understanding of the mechanisms of innate immune recognition. The innate immune system detects the invasion of microorganisms through the TLRs, which recognize microbial components and trigger inflammatory responses. The TLRs also play a role in instructing the adaptive immune response. However, many questions remain to be answered. There are some TLRs that have unknown microbial ligands. It remains unclear whether TLR recognizes microbial components by direct binding or by some indirect mechanism. It is also unclear where each TLR recognizes these components—on the cell surface or in some intracellular compartment such as the phagosome or endosome. Many questions also remain to be answered with regard to TLR signaling pathways: How does activation of individual TLRs lead to differential gene expression and biological responses, and what kinds of signaling cascades do individual TLRs activate in addition to the common MyD88-dependent pathway? Finally, the fact that activation of TLRs leads to the induction not only of innate immunity but also of adaptive immunity suggests that the TLRs could be involved in some immune disorders as well as infectious diseases. Indeed, several autoimmune diseases have been shown to be associated with infection and dysregulation of innate immune activation (290). Furthermore, autoreactive B cells specific for self-IgG have been shown to be activated by an IgG/chromatin immune complex that synergistically activates the antigen receptor–and MyD88-dependent signaling pathways (291). This strongly suggests that autoimmune disorders are induced by the cross talk between adaptive and innate immune signaling pathways. By elucidation of these issues, we should

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be able to increase our understanding of the complex nature of both the innate and adaptive immune systems.


ACKNOWLEDGMENTS We thank E. Horita for secretarial assistance, M. Lamphier for critical reading of the manuscript, and K. Hoshino for preparing figures. This work was supported by grants from Special Coordination Funds, the Ministry of Education, Culture, Sports, Science and Technology; the Virtual Research Institute of Aging of Nippon Boehringer Ingelheim; and Japan Research Foundation for Clinical Pharmacology. The Annual Review of Immunology is online at http://immunol.annualreviews.org

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switching by stimulating dendritic cells in vivo. Immunity 14:461–70 Blanco P, Palucka AK, Gill M, Pascual V, Banchereau J. 2001. Induction of dendritic cell differentiation by IFN-α in systemic lupus erythematosus. Science 294:1540–43 Cella M, Jarrossay D, Facchetti F, Alebardi O, Nakajima H, et al. 1999. Plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type I interferon. Nat. Med. 5:919– 23 Bruno L, Seidl T, Lanzavecchia A. 2001. Mouse pre-immunocytes as nonproliferating multipotent precursors of macrophages, interferon-producing cells, CD8α + and CD8α − dendritic cells. Eur. J. Immunol. 31:3403–12 Dalod M, Salazar-Mather TP, Malmgaard L, Lewis C, Asselin-Paturel C, et al. 2002. Interferon α/β and interleukin 12 responses to viral infections: pathways regulating dendritic cell cytokine expression in vivo. J. Exp. Med. 195:517–28 Thoma-Uszynski S, Stenger S, Takeuchi O, Ochoa MT, Engele M, et al. 2001. Induction of direct antimicrobial activity through mammalian toll-like receptors. Science 291:1544–47 Tzou P, Reichhart JM, Lemaitre B. 2002. Constitutive expression of a single antimicrobial peptide can restore wild-type resistance to infection in immunodeficient Drosophila mutants. Proc. Natl. Acad. Sci. USA 99:2152–57 Zasloff M. 2002. Antimicrobial peptides of multicellular organisms. Nature 415:389–95 Ayabe T, Satchell DP, Wilson CL, Parks WC, Selsted ME, et al. 2000. Secretion of microbicidal a-defensins by intestinal Paneth cells in response to bacteria. Nat. Immunol. 1:113–18 Lehrer RI, Ganz T. 2002. Defensins of

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vertebrate animals. Curr. Opin. Immunol. 14:96–102 Birchler T, Seibl R, Buchner K, Loeliger S, Seger R, et al. 2001. Human Toll-like receptor 2 mediates induction of the antimicrobial peptide human β-defensin 2 in response to bacterial lipoprotein. Eur. J. Immunol. 31:3131–37 Zychlinsky A, Prevost MC, Sansonetti PJ. 1992. Shigella flexneri induces apoptosis in infected macrophages. Nature 358:167–68 Choi K-B, Wong F, Harlan JM, Chaudhary PM, Hood L, et al. 1998. Lipopolysaccharide mediates endothelial apoptosis by a FADD-dependent pathway. J. Biol. Chem. 273:20185–88 Bannerman DD, Tupper JC, Erwert RD, Winn RK, Harlan JM. 2002. Divergence of bacterial lipopolysaccharide proapoptotic signaling downstream of IRAK1. J. Biol. Chem. 277:8048–53 Hu WH, Johnson H, Shu HB. 2000. Activation of NF-κB by FADD, Casper, and caspase-8. J. Biol. Chem. 275:10838–44 Schaub FJ, Han DK, Liles WC, Adams LD, Coats SA, et al. 2000. Fas/FADDmediated activation of a specific program of inflammatory gene expression in vascular smooth muscle cells. Nat. Med. 6:790– 96 Bannerman DD, Tupper JC, Kelly JD, Winn RK, Harlan J. 2002. The Fasassociated death domain protein suppresses activation of NF-κB by LPS and IL-1β. J. Clin. Invest. 109:419–25 Bachmann MF, Kopf M. 2001. On the role of the innate immunity in autoimmune disease. J. Exp. Med. 193:F47–50 Leadbetter EA, Rifkin IR, Hohlbaum AM, Beaudette BC, Shlomchik MJ, et al. 2002. Chromatin/IgG complexes activate autoreactive B cells by dual engagement of IgM and Toll-like receptors. Nature 416:603–7


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Figure 1 Host defense responses in drosophila. In drosophila, the Toll and IMD pathways confer host defense against pathogen invasion. The Toll pathway regulates production of antimicrobial peptides against fungi and Gram-positive bacteria. PGRPSA is essential for activation of the Toll pathway in response to Gram-negative bacteria. Persephone is involved in activation of the Toll pathway in response to fungi. PGRP-LC recognizes the invasion of Gram-negative bacteria and is required for activation of the IMD pathway, which is essential for anti-Gram negative bacterial responses.


Figure 4 Toll-like receptor (TLR) signaling pathway. TLRs recognize specific patterns of microbial components. MyD88 is an essential adaptor for all TLRs and is critical to the inflammatory response. In the case of the TLR4-mediated pathway, lipopolysaccharide (LPS)induced activation of signaling molecules such as IRF-3, PKR, MAP kinase, and NF-kB has been reported, indicating the presence of the MyD88-independent pathway. TIRAP/Mal was identified as a component specifically involved in TLR4-mediated signaling.


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POXVIRUSES AND IMMUNE EVASION Bruce T. Seet,1 J.B. Johnston,2 Craig R. Brunetti,2 John W. Barrett,2 Helen Everett,2 Cheryl Cameron,1 Joanna Sypula,1 Steven H. Nazarian,1 Alexandra Lucas,2,3 and Grant McFadden1,2 1

Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 5C1, Canada 2 Biotherapeutics and 3Vascular Biology Research Groups, Robarts Research Institute, London, Ontario N6G 2V4, Canada; email: [email protected], [email protected], [email protected], [email protected], [email protected], [email protected], [email protected], [email protected], [email protected], [email protected]

Let it . . . be borne in mind how infinitely complex and close-fitting are the mutual relations of all organic beings to each other and to their physical conditions of life; and consequently what infinitely varied diversities of structure might be of use to each being under changing conditions of life. Charles Darwin

Key Words viroceptor, virokine, apoptosis, inflammation, cell-mediated immunity ■ Abstract Large DNA viruses defend against hostile assault executed by the host immune system by producing an array of gene products that systematically sabotage key components of the inflammatory response. Poxviruses target many of the primary mediators of innate immunity including interferons, tumor necrosis factors, interleukins, complement, and chemokines. Poxviruses also manipulate a variety of intracellular signal transduction pathways such as the apoptotic response. Many of the poxvirus genes that disrupt these pathways have been hijacked directly from the host immune system, while others have demonstrated no clear resemblance to any known host genes. Nonetheless, the immunological targets and the diversity of strategies used by poxviruses to disrupt these host pathways have provided important insights into diverse aspects of immunology, virology, and inflammation. Furthermore, because of their anti-inflammatory nature, many of these poxvirus proteins hold promise as potential therapeutic agents for acute or chronic inflammatory conditions.

INTRODUCTION The coevolution of viruses and their hosts has had a significant impact on how each has evolved, and the consequences of this ancient and dynamic battle are manifested in both host and virus genomes. Viruses, and other invading pathogens, have exerted unrelenting selection pressure upon the mammalian host, necessitating 0732-0582/03/0407-0377$14.00

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the development of a complex and adaptable immune system. The successful propagation of many viruses within the mammalian host requires the evasion or manipulation of the host’s immune defenses. Large DNA viruses such as herpesviruses and poxviruses provide some of the most extensive inventories of gene products that serve to defend these viruses against the aggressive assault executed by the host immune response. Here, we highlight the many strategies that poxviruses utilize to systematically modulate key components of the innate and acquired immune responses. The observation that viruses deploy specifically targeted proteins to modulate the normal functioning of diverse immune pathways underscores the significant influence that these host responses possess in mediating the antiviral response. This review focuses primarily on recent advances, and the reader is referred to other excellent reviews for a more comprehensive accounting of this subject (1–10). Not covered in this review are the immune evasion strategies of the insect poxviruses (Entomopoxvirinae) or any other large DNA virus families that infect chordates, such as the Herpesviridae and the Asfarviridae (African swine fever virus).

GENOMIC ORGANIZATION OF POXVIRUS IMMUNE EVASION GENES Viruses of the Poxviridae are divided into either the Chordopoxvirinae or the Entomopoxvirinae. There are presently 20 complete poxvirus genomic sequences deposited in sequence databases, including 18 Chordopoxvirinae and 2 Entomopoxvirinae. Three other complete genomes that have not yet been made publicly available belong to Yaba monkey tumor virus, rabbitpox virus, and orf virus. Some characteristics of representative genomic sequences for seven of the eight Chordopoxvirinae genera are shown in Supplemental Table 1 available online (follow the Supplemental Material link on the Annual Reviews homepage at http://www.annualreviews.org/). Only the Parapoxvirus genus lacks a member whose genome has been entirely sequenced and made available. Poxviruses contain linear double-stranded DNA genomes with termini that form covalently closed hairpin loops (11). Chordopoxvirinae genomes range in size from 135,000 (Yaba monkey tumor virus; C.R. Brunetti, H. Amano, Y. Ueda, T. Miyamura, T. Suzuki, X. Li, J. Barrett, G. McFadden, unpublished data) to 289,000 (fowlpox virus) base pairs (bps) (12) and encode between 136 and 260 open reading frames (ORFs) respectively. Poxvirus ORFs are usually defined as being in excess of 50 amino acids and are generally nonoverlapping. In addition, the noncoding regions between ORFs are often very small and in some cases just a few nucleotides. Genes that are centrally located in the genome are mostly conserved among all poxviruses and tend to be involved with common molecular functions such as replication or virion assembly, whereas terminally located genes tend to be more variable and are often involved in host range restriction or immune subversion (11). The genome of each poxvirus is flanked by terminal inverted repeat

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(TIR) regions that typically contain a small number of genes whose positions and orientations are mirrored at the opposing ends of the genome. TIRs range in length from 58 bp in variola virus (variola virus, strain India 1967) (13) to 12,400 bp in Shope fibroma virus (Shope fibroma virus, strain Kasza) (14). The number of ORFs that map within the TIR range from zero in variola virus (13, 15, 16) to 12 ORFs in myxoma virus (17). Sequence analysis of ORFs within the variable regions from Chordopoxvirinae has revealed a wide array of viral proteins that modulate components of the host immune response. In fact, the viral proteins that interact with host components are so diverse that no single common immunomodulatory gene can be found in all poxvirus genomes (see Tables 1 and 2). Nevertheless, two host immune pathways that are consistently targeted by all poxviruses include those mediated by the interferons (IFNs) and by the chemokine superfamily. The absence of immunomodulatory gene conservation between poxvirus family members may reflect differences in host and tissue tropism and may help explain the phenotypic differences observed during clinical manifestations of diverse poxviral diseases. However, it is possible to group the Chordopoxvirinae genera based on the common presence or absence of particular host interaction proteins. For example, the Capripoxvirus, Leporipoxvirus, Suipoxvirus, and Yatapoxvirus genera all share the same set of host interaction proteins not found in other Chordopoxvirinae genera, such as an OX-2 homolog (17); a mitochondrial protein that inhibits apoptosis (M11L of myxoma virus is the prototypical member) (18); and a gene responsible for downregulation of major histocompatibility complex (MHC) molecules (19). In addition, swinepox virus and Yaba-like disease virus share a secreted homolog of the class I MHC heavy chain (20, 21). In contrast, the Orthopoxvirus genera have a distinct set of host interaction proteins that often include multiple copies of tumor necrosis factor (TNF) receptor homologs, an interleukin1β (IL-1β) receptor homolog, and an apparent absence of predicted proteins that modulate the antigen presentation/MHC pathway. Finally, several immunomodulatory homologs exist only within the Orthopoxvirus and Leporipoxvirus genera, such as Toll-like receptor inhibitors and secreted CC-chemokine-binding proteins. Fowlpox virus, a representative member of the Avipoxvirus genus, contains the largest Chordopoxvirinae genome yet sequenced. Despite sharing several immunomodulatory genes present in other Chordopoxvirinae genera, fowlpox virus contains a number of unique genes not found in other poxvirus genera, such as putative homologs of Bcl-2, β-NGF, and TGF-β (12). Finally, Molluscipoxviruses appear to be unique when compared to other Chordopoxvirinae genera with respect to host immunomodulatory proteins (22).

POXVIRUS EVASION OF CELL-MEDIATED IMMUNITY Although both the humoral and cell-mediated immunity (CMI) components of the vertebrate acquired immune system jointly participate in the host response to infection, CMI is particularly critical for the clearance of poxvirus-infected cells (23, 24). An effective CMI response requires both innate effector cells, such as

CD30 eIF2α Type II interferon receptor Type I interferon receptor

vCD30

eIF2α homolog

IFN-γ receptor

IFNα/β binding proteins

IL-10 OX-2

IL-18-binding protein

Viral IL-10

OX-2 homolog

MIP-1β ? G protein coupled chemokine receptor

Chemokine homolog

Chemokine-binding protein

CC chemokine receptor

MC148R

MC054

(Continued )

J1L (J3R)

D6L

A47R

B14R

F3L

B16R

B9R

K1R

J2R (J2L)

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Cell surface, function unknown

Secreted chemokine-binding protein

Secreted, antagonizes chemokines

Negative signaling of lymphocytes?

Immune suppressive

Secreted, inhibits IL-18

Signaling inhibition

Secreted, blocks IL-1β

Inhibitor of PKR and 2,5A synthetase

Secreted

Secreted

Inhibition of translation

TNF receptor family member, binds CD153

Secreted, inhibits TNF

Secreted, inhibits TNF

Secreted, inhibits LTα

Secreted, inhibits TNF and LTα

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Chemokine pathway

? IL-18-binding protein

Toll-like receptor inhibitor

?

TNF receptor

TNF receptor, CrmD

IL-1β receptor

TNF receptor

TNF receptor, CrmE

IL-1β receptor

TNF receptor

TNF receptor, CrmC

MPV

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dsRNA-binding protein

TNF receptor

TNF receptor, CrmB

MCV

AR

Cytokine pathway

Function

380

Cellular homolog

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TABLE 1 Poxvirus modulation of cytokines, chemokines, and various signaling pathwaysa


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G2R, [G2R]

Cytokine pathway TNF receptor, CrmB

C3L, [E3L] D4R, [B15R, B16R] A56R, [A52R] B6L, [D5L]


IL-1β receptor



C23L (B29R)

A46R, A52R

B16R

E3L

B19R

CMLV001, 211

CMLV166

CMLV193, 194, 196

CMLV055

CMLV201

D1L (H5R)

C8L

A49R

B14R

F3L

B17R

E1 {K2R}

E19

E168, E174

E191 {C9R}

E60

E194 {C12R}

E185 {C4R}


(Continued )

FPV060, 061, 116, 121

FPV073

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Chemokine binding protein

Chemokine homolog

Chemokine pathway

OX-2 homolog

G3R, [G3R]

D9R, [B20R]


E6 {I1R} E13

FPV

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Viral IL-10

M3L B7R

IFN-γ receptor

CMLV032

H9R, [B9R]

eIF2α homolog CMLV184

P3L, [C3L]

vCD30

ECT

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B8R

K2R C5L

TNF receptor, CrmD K3L

A56R

D2L (H3R)

CPV

K3R

CMLV007

CMLV002, 210

CMLV

TNF receptor, CrmE

A53R

C22L (B28R)

VAC-COP

AR

TNF receptor, CrmC

VAR-GAR, [VAR-IND]

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TABLE 1 (Continued )


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M002R/L

Cytokine pathway TNF receptor, CrmB

LSDV008


Chemokine-binding protein

Chemokine homolog M001R/L, M007R/L

S001R/L LSDV011

LSDV138

OX-2 homolog

SPV005, 145

SPV012

SPV032

SPV132

SPV008

SPV010

7L, 145R

141R

134R

14L

34L

136R

12L

(Continued )

ORF-IL-10

ORF 20.0L

ORF

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LSDV005

Viral IL-10

LSDV034

LSDV135

LSDV015 S141R

S029L

S007R/L

LSDV014



M141R

M029L


IL-1β receptor

M007R/L M135R


S008.2R/L

YLD

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IFN-γ receptor

M156R

eIF2α homolog

vCD30

TNF receptor, CrmD

SPV

AR

TNF receptor, CrmE

LSDV

382

S002R/L

SFV

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MYX

Viral protein



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MHC class Iα chain-like

EGF/TGFα CD-46 ? CD-47 β-NGF Lymphocytic-activating molecule TGF-β VEGF

Viral growth factor

Complement inhibition

GM-CSF/IL-2 inhibitor

CD47-like protein

β-NGF

Lymphocytic-activating molecule

TGF-β

VEGF

Semaphorin

Semaphorins

dehydrogenase

3β-hydroxysteroid

Hydroxysteroid dehydrogenase

MHC class I heavy chain

LAP domain

Vascular growth factor

Immune modulation

Cell surface

?

Cell surface, integrin associated protein

D14L

D3R

A45L

(Continued )

MC002L

MC080R

MC033L

MPV

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Secreted, blocks inflammatory cell migration

Secreted, blocks inflammation

Secreted

MHC downregulation

Cell surface

Cell surface

MCV

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Other poxvirus immunomodulators

MHC I protein ?

MHC class 1 heavy chain homolog

Class I-MHC

Class I-MHC

Function

AR

Antigen presentation/MHC pathway

Cellular homolog

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Viral protein

TABLE 1 (Continued)


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chain homolog

A45R, [A42R] B3R, [D2R] B18L, [D12L]

Semaphorins

Viral growth factor


TGF-β VEGF


β-NGF

CD47-like protein

A38L

C3L

C11R

A39R

A44L

CMLV158

CMLV023

CMLV010

CMLV159

CMLV164

C17L

C5R

A41R

A47L

CPV

E160

E28

E16

E161

E166

ECT

FPV080

FPV072, 076

FPV211

FPV047

FPV046

FPV

(Continued )

M128L

M144R

M010L

M153R

MYX

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A54L, [A50L]

3β-hydroxysteroid dehydrogenase


CMLV

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LAP domain

VAC-COP

AR

Antigen presentation/MHC pathway Class I-MHC MHC class 1 heavy

VAR-GAR, [VAR-IND]

384

Viral protein

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TABLE 1 (Continued)


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SPV009L

SPV125

SPV139

SPV128

SPV003L

5L

128L

144R

15L

133L

2L

vVEGF

GIF

ORF

Abbreviations: molluscum contagiosum virus (MCV) (22), monkeypox (MPV) (110, 276), variola-Garcia (VAR-GAR), variola-India 1967 (VAR-IND) (277), vaccinia virus Copenhagen (VAC-COP) (130), cowpox (CPV) (275), ectromelia virus (ECT), fowlpox (FPV) (12), myxoma virus (MYX) (17), Shope fibroma virus (SFV) (14), lumpy skin disease virus (LSDV) (111), swinepox (SPV) (20), Yaba-like disease (YLD) (21), orf virus (ORF ). The genomes of CPV and ORF virus have not been completely sequenced, so other immunomodulatory genes may be identified in these species. Genes in square brackets [ ] are the gene product from VAR-IND strain. A duplicate copy of a gene present in the terminal inverted repeats is designated by parentheses ( ). Alternate names for a gene are listed in braces { }.

a

VEGF

TGF-β


LSDV128

LSDV141

LSDV016

LSDV010

YLD

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CD47-like protein

S128L

S144R



S010L

Viral growth factor

Semaphorins

3β-hydroxysteroid dehydrogenase

S153R

SPV

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MHC class 1 heavy chain homolog

Class I-MHC

LSDV

AR

Antigen presentation/MHC pathway

SFV

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TABLE 1 (Continued)


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eIF2α homolog

None

Inhibition of apoptosis

Serpin

SPI-3

and IL-18 processing Fusion, anti-inflammatory

(Continued)

C2L

B12R

B19R

Anti-apoptotic, blocks IL-1β

Serpin

SPI-2/CrmA

Host range

Serpin

SPI-1

MC066L

F3L

D5R

Intracellular, inhibitor apoptosis

Mitochondrial checkpoint

Endoplasmic reticulum

MC159R, MC160R

MPV

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RING finger

M-T2

Glutathione peroxidase


Blocks interferon-induced apoptosis

Blocks interferon-induced apoptosis

Intracellular, blocks death signals

MCV

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M-T4

? eIF2α

DED domains (vFLIP)

Prevents apoptosis

Bcl-2 FLIP

Bcl-2 gene family

Prevents apoptosis

Function

Ankyrin repeat

Anti-apoptotic molecules

Cellular homolog

386

AR

Viral protein

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E3L

CMLV055

F3L

K2L

CMLV031

M2L

FPV040 FPV044

E189 {C7R} E36 {H14-B}

(Continued)

M008.1L

M151R

M152R

[C2L]

FPV010, 204, 251

E197 {C14R}

SPI-3

B12R

B20R

D2R, [B13R]

SPI-2/CrmA

CMLV191

D14R, [B25R]

SPI-1 B13R/B14R

M143R

FPV157, 150

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P28

D6R

M004L/R

M156R

M029L

M005L/R

MYX

RING finger

E60

FPV039

FPV

M002L/R D7R

M3L

ECT

M011L

CMLV205

CMLV032

CPV

M-T2

C12L

K3L

CMLV



M-T4

C3L, [E3L] P3L, [C3L]

eIF2α homolog

VAC-COP

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Bcl-2 gene family

Ankyrin repeat

Anti-apoptotic molecules

VAR-GAR, [VAR-IND]

AR

Viral protein

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S152R S151R

SPI-1

SPI-2/CrmA

SPV145

SPV138

SPV012

SPV010

10L

149R

143R

16L

12L

34L

YLD

ORF 20.0L

ORF

Abbreviations: molluscum contagiosum virus (MCV) (22), monkeypox (MPV) (110, 276), variola-Garcia (VAR-GAR), variola-India 1967 (VAR-IND) (277), vaccinia virus Copenhagen (VAC-COP) (130), cowpox (CPV) (275), ectromelia virus (ECT), fowlpox (FPV) (12), myxoma virus (MYX) (17), Shope fibroma virus (SFV) (14), lumpy skin disease virus (LSDV) (111), swinepox (SPV) (20), Yaba-like disease (YLD) (21), orf virus (ORF). The genomes of CPV and ORF virus have not been completely sequenced, so other immunomodulatory genes may be identified in these species. Genes in square brackets [ ] are the gene product from VAR-IND strain. A duplicate copy of a gene present in the terminal inverted repeats is designated by parentheses ( ). Alternate names for a gene are listed in braces { }.

a

LSDV149

LSDV140

LSDV017

LSDV014

SPV032

SPV

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SPI-3

gp143R

RING finger

M-T2


Glutathione peroxidase S011L

S008.2R/L

eIF2α homolog

LSDV034

LSDV

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M-T4

S029L


DED domains (vFLIP)

Bcl-2 gene family

Ankyrin repeat

SFV

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AR

Viral protein

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natural killer (NK) cells, and educated effector cells, such as cytotoxic T lymphocytes (CTLs), to rapidly identify and eliminate infected cells before the virus can replicate and spread. Consequently, viruses have evolved mechanisms to reduce the efficiency of the CMI response, including the masking of outward signs of infection, a strategy that can be considered as virostealth. One such mechanism employed by poxviruses involves downregulation of cell surface receptors such as those that participate in antigen presentation and immune cell recognition. Although poxvirus-induced downregulation of class II MHC has not been reported, decreased expression of class I MHC molecules, which present endogenous antigens to circulating CD8+ CTLs, has been detected following infection by several poxviruses [reviewed in (4, 25)]. The extent of class I MHC depletion is quite variable between poxviruses. Infection by myxoma and malignant rabbit fibroma viruses, both of which severely compromise the CMI response of the host, is associated with a specific, rapid, and profound loss (>90%) of class I MHC on infected cells (26). In contrast, Orthopoxviruses such as vaccinia virus, which do not induce comparable systemic immunosuppression, cause only moderate downregulation of cell surface class I MHC. The ability of poxviruses to inhibit pro-inflammatory cytokines that regulate MHC expression, such as TNF and IFN (discussed in later sections), provides one indirect mechanism by which MHC upregulation may fail to occur. Additionally, a more direct mechanism of proactive downregulation has been recently proposed for myxoma virus in which the virus directly interferes in the antigen presentation pathway using the product of the M153R gene (19). Deletion of M153R abrogates the loss of class I MHC observed during myxoma virus infection in vitro and renders cells infected with the deletant virus more susceptible to CTL-mediated cytolysis than cells infected with wild-type virus (19). Furthermore, in vivo studies in infected rabbits demonstrate that the M153R-deletion mutant of myxoma virus exhibits decreased virulence and an infection profile characterized by increased mononuclear infiltrates at the primary site of infection (19). The mechanism by which the M153R protein reduces class I MHC levels is thought to be linked to the MHC trafficking pathway (19). M153R is predicted to encode an early protein possessing an atypical N-terminal zinc finger motif (C4HC3). This motif, known as a PHD (plant homeodomain) or LAP (leukemiaassociated protein), is found in proteins from other poxviruses, including Shope fibroma virus, swinepox virus, Yaba-like disease virus, and lumpy skin disease virus, as well as in the K3 and K5 proteins of the gammaherpesvirus, human herpesvirus-8 (HHV-8) (27, 28). The C-terminal portion of M153R contains two predicted transmembrane domains that serve to localize the protein to the endoplasmic reticulum (ER), a pattern of expression that is essential for class I MHC downregulation. Similar to the ER-based mechanism proposed for HHV-8 K3 and K5 (27, 28), M153R may promote the preferential loss of β2-microglobulinassociated class I MHC molecules, both at the cell surface and in an intracellular post-Golgi compartment, by somehow targeting them for retention and degradation via the late endosomal/lysosomal pathway (29).

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Although class I MHC downregulation has the potential to protect infected cells from CTL-mediated cytolysis, it should also reduce the capacity for these cells to generate the class I MHC–dependent inhibitory signal required to prevent killing by NK cells. Genes encoding putative decoy class I MHC homologs, which in theory might avert this increased susceptibility to NK cells, have been identified in the genomes of a few poxviruses such as molluscum contagiosum virus and swinepox virus (20, 30). To date, little is known about the mechanism of action of these class I MHC homologs, but the molluscum contagiosum version (MC80R) was detected in stable intracellular complexes with β2-microglobulin (31). Poxviruses also alter the expression of other cell surface molecules that play roles in immune regulation. For example, downregulation of CD4 expression has been demonstrated following myxoma virus infection of T lymphocytes, providing a mechanism to alter CD4+ lymphocyte function (32). Although the specific gene product responsible has not been identified, CD4 depletion involves a protein kinase C–independent increase in the internalization of the receptor and targeting to lysosomal vesicles for degradation, as well as an uncoupling of the T cell activation pathway (32). In the event that infected cells are recognized and targeted for clearance, poxviruses have developed diverse strategies to circumvent effector killing mechanisms and regulatory controls exerted upon CTLs and NK cells. As detailed in the following sections, poxviruses employ an impressive array of strategies to disrupt both extracellular cytokine networks and intracellular signaling cascades that transduce death signals associated with functional CMI responses.

VIROKINES AND VIROCEPTORS: POXVIRUS MODULATION OF THE EXTRACELLULAR ENVIRONMENT The damage incurred by cells and tissues following viral infection stimulates a series of nonspecific events that collectively make up the early inflammatory response. The net effect of inflammation is to promote an influx of leukocytes into the site of infection and create a localized microenvironment that impedes viral spread until specific acquired immunity can be generated. Given the integrated nature of inflammation, it is unlikely that any single viral protein would be sufficient to circumvent this response; rather, the cooperative actions of multiple secreted and cell-associated viral immune modulators are expected to protect diverse cell types and tissues that can support virus replication. Representative poxvirus proteins that modulate the extracellular circuitry comprising the complement, cytokine, and chemokine networks are listed in Table 1.

Modulation of Complement The complement system is composed of numerous cell surface–anchored effector proteins and soluble regulatory proteins (33). By interacting with such regulatory

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proteins, poxviruses inhibit, modulate, and exploit various levels of the complement system (34, 35). COMPLEMENT BINDING PROTEINS Vaccinia virus encodes a secreted complement control protein (VCP) that possesses four tandem copies of a short consensus repeat (SCR), a motif that is commonly found among host complement regulators (36). VCP inhibits both the alternative and classical pathways of complement activation by binding and inhibiting C3b and C4b (37). VCP binds C3b and C4b with higher affinity than the human C4b binding protein, and accelerates the decay of the classical and alternative C3 convertases (38). Moreover, VCP can serve as a cofactor for the cleavage and inhibition of C3b and C4b by factor I (38, 39). VCP is not only necessary to inhibit complement-mediated antibody-dependent neutralization of intracellular mature virus (IMV) particles, but contributes to pathogenesis such that infection of rabbits with vaccinia virus deficient for VCP expression results in smaller skin lesions compared to wild-type virus (40). In addition to inhibiting complement, VCP has been shown to have distinct functions endowed by its interaction with glycosaminoglycans (41–43). VCP is an extended asymmetrical molecule that possesses putative heparin-binding domains within SCR 1 and 4 (41, 43–47). Binding glycosaminoglycans may enable VCP to localize to cell surfaces, allowing the uptake of VCP by mast cells and endothelial cells as well as the ability to inhibit chemokine-mediated migration of leukocytes (42). The anti-inflammatory nature of VCP is further underscored by observations that VCP may be capable of functioning therapeutically to prevent xenorejection by inhibiting complement and preventing cytotoxic cell–mediated death (48, 49). Orthologs of VCP are also present in cowpox virus, monkeypox virus, and variola virus (50–52). The cowpox virus complement control protein, termed the inflammation modulatory protein (IMP), has been shown to play a role in limiting mononuclear cell infiltration, reducing tissue destruction and formation of nodular lesions during viral infection (51–53). Overall, VCP/IMP appears to aid in preserving host tissue within the local virus-infected tissue microenvironment (35). In the case of variola virus, the smallpox inhibitor of complement enzymes (SPICE) protein is considerably more potent than VCP at inactivating human C3b and C4b, reinforcing the close relationship between this virus and the human immune system (54). OTHER POXVIRUS COMPLEMENT-RELATED PROTEINS The B5R protein is a 42-kDa extracellular enveloped virus (EEV) glycoprotein with type I membrane topology that contains four SCRs (55, 56). Although no complement binding activity has yet been ascribed to B5R, it is required for the formation of intracellular enveloped virus (IEV), externalization of the virus, actin tail formation, development of normal plaque size, and virus virulence (57–64). Because of the homology of the extracellular domain to complement control proteins, it is predicted that B5R may function in some aspect of viral immune evasion, but its only documented role to date is as a target for EEV neutralization (65, 66).

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Poxviruses may also circumvent complement by retaining components of the host cell membrane, including host regulators of complement on the EEV outer membrane. In support of this notion, IMV of vaccinia virus, but not EEV, was sensitive to complement inhibition, and EEV grown from cells of one species was more susceptible to complement from another species. Indeed, EEV derived from rat cells expressing human regulators of complement, CD55 and CD59, were more resistant to human complement than EEV derived from control rat cells that did not express either CD55 or CD59 (67).

Inhibition of Interferon IFN constitutes one of the most potent first-line host defenses against virus infection and can induce direct antiviral effects as well as promote T helper cell type 1 (Th1) responses [recently reviewed in (68–70)]. Signaling through interferon receptors (IFN-Rs) requires the binding of an IFN ligand that mediates the heterodimerization of IFN-R subunits. The recruitment and activation of the Janus kinases (JAKs) results in the phosphorylation of specific members of the signal transducers and activators of transcription (STATs) that translocate to the nucleus and activate an array of genes involved in establishing, among other things, an antiviral state. As an integral component of the antiviral host response, the IFN system is strategically targeted by all poxviruses for disruption at a variety of extracellular and intracellular levels (Figure 1). IFNγ RECEPTOR HOMOLOGS The critical role of IFNγ in limiting poxvirus infection is supported by findings that administration of IFNγ in mice infected with ectromelia virus and vaccinia virus can increase resistance and limit infection (71–75). Furthermore, transgenic mice with disrupted IFNγ or IFNγ -Rs show increased susceptibility to vaccinia virus infection (76–78). To circumvent the effects of IFNs, many poxviruses encode IFNγ -R homologs (Table 1). All known poxvirus-encoded IFNγ -Rs function as competitive antagonists of IFNγ (Figure 1), although the ligand specificity varies according to the poxvirus species (79–81). Similar to mammalian IFNγ -Rs, the viral inhibitors from Leporipoxviruses typically exhibit restricted species-specific binding to IFNγ , and thus the myxoma virus M-T7 protein binds and inhibits only rabbit IFNγ (82). However, orthopoxvirus IFNγ -Rs often possess the ability to bind IFNγ from a broader variety of species (80, 81, 83, 85). Cross-species recognition of host ligands by other Orthopoxvirus cytokine-binding proteins is also observed among the Orthopoxvirus TNF and IFNα/β inhibitors and likely reflects the diverse host range and evolutionary history of the Orthopoxvirus genus (81, 83, 86). Although the myxoma virus IFNγ -R (M-T7) has been shown to exist as a stable trimer in solution (87), the vaccinia virus, camelpox virus, and cowpox virus IFNγ -Rs exist naturally as homodimers (88). The oligomeric nature of poxvirus IFNγ -Rs likely assists in the binding of the homodimeric IFNγ molecules.

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The role of poxvirus IFNγ -R homologs in dampening the effects of host IFNγ during viral infection has been examined using recombinant poxviruses with disrupted IFNγ -R homologs. The results of these knockout studies are complicated by the discovery of secondary interactions with other molecules (e.g., M-T7 with chemokines) or lack of species-specific interactions of the viral protein with IFNγ in the model host animal (e.g., vaccinia virus B8R in mice). European rabbits infected with a recombinant myxoma virus lacking expression of its IFNγ -R homolog (M-T7) showed a dramatic attenuation in lethality, disease symptoms, and viral dissemination to secondary sites (89). Moreover, the infiltrating leukocyte profile was altered at sites of infection with the knockout virus, but the extent to which this phenotype can be attributed solely to IFNγ inhibition by M-T7 was confounded by the subsequent observation that M-T7 is also a chemokine-binding protein (discussed below) (87). Disruption of the IFNγ -R homolog in vaccinia virus (B8R) originally showed no difference in virulence compared to wild-type viruses in a mouse model, but the knockout virus was attenuated in rabbits (85, 90). This was anticipated given that B8R does not bind or inhibit mouse IFNγ with high affinity but does inhibit the rabbit ligand (80, 81). However, contrasting results recently reported from another group demonstrated that disruption of B8R in vaccinia virus results in a significant decrease in weight loss and increased mortality in normal mice following intranasal infection with vaccinia virus (91). This suggests that B8R may recognize additional ligands or possess other activities not related to its binding of IFNγ . A promising model to help shed light on this issue may lie in the study of ectromelia virus, for which the natural host is the mouse and whose IFNγ receptor homolog specifically binds to and inhibits the activity of mouse IFNγ (92).

INTERFERON-α/β BINDING PROTEINS Many poxviruses encode a protein with some limited sequence similarity to the cellular IFNα/β receptor (Figure 1). The bestcharacterized poxvirus inhibitor of IFNα/β was identified in vaccinia virus (strain Western Reserve) (93). The vaccinia virus (strain Western Reserve) B18R gene (designated B19R in strain Copenhagen) encodes a secreted 60- to 65-kDa glycoprotein that exhibits a modest amino acid similarity to the α subunits of mouse, human, and bovine type I IFN receptors (94). In fact, the closest host sequence similarity of B18R is to members of the immunoglobulin superfamily and hence the term IFNα/β-binding protein is preferred over IFNα/β receptor homolog (93, 95). Composed of three immunoglobulin domains, B18R binds and competitively inhibits IFNα, β, δ, and ω from various mammalian species (93, 96, 97). The deletion of B18R from vaccinia virus (strain Western Reserve) produced a knockout virus that was attenuated in both intranasally and intracranially infected mice, emphasizing the importance of the type I IFNs in controlling vaccinia virus infection (93, 94). B18R has also been shown to bind cell surfaces from uninfected and infected cells, an attribute that likely aids in limiting an IFN-mediated establishment of an antiviral state (93, 98, 99). Ectromelia virus also encodes an IFNα/β-binding

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protein that was recently shown to potently inhibit human and mouse IFNα in addition to human, but not mouse, IFNβ (92). INHIBITORS OF INTERFERON-INDUCING CYTOKINES: INTERLEUKIN-18 BINDING PRO-

Interleukin-18 (IL-18), first known as the IFNγ -inducing factor, is a potent, pleiotropic cytokine that can stimulate the synthesis of various cytokines and chemokines, regulate Th1 and Th2 cell responses, and activate NK and cytotoxic T cells (100). IL-18 is a member of the IL-1 family and bears a structural resemblance to IL-1β. Similar to IL-1β, IL-18 is cleaved from a precursor form into its active, secreted form by the IL-1β-converting enzyme (ICE) and other caspases (101), and this cleavage presumably renders IL-18 susceptible to the inhibitory effects of the poxvirus caspase inhibitors CrmA/SPI-2 (discussed in the section Prevention of Apoptosis by Caspase Inhibition). One mechanism that modulates the effects of IL-18 is the mammalian IL-18binding protein (IL-18BP), a naturally occurring antagonist that blocks IL-18 from binding its receptor (Figure 1) (102, 103). The identification of the human IL-18BP prompted the immediate identification of poxvirus-encoded IL-18BPs that possess sequence similarities to the immunoglobulin domain of the human IL-18BP (102). Molluscum contagiosum virus encodes three gene products (MC51L, 53L, and 54L) that exhibit sequence similarity to IL-18BP, of which only one, MC54L, has so far been shown to bind IL-18 (104, 105). The high affinity interaction of MC54L with human and murine IL-18 is mediated by moderate association rates and slow dissociation rates, similar to other poxvirus IL-18BPs and comparable to the human and mouse IL-18 interactions with the human IL-18BP (106). MC54L binding to IL-18 requires a set of conserved MC54L residues (105) that correspond to those used by the human IL-18BP (107). The related MC53L and MC51L, which do not bind IL-18, lack these conserved residues (105). Interestingly, restoration of key residues in MC53L or generating hybrid proteins of MC53L could not bestow binding for IL-18 [Y. Xiang, B. Moss, unpublished data, but noted in (105)], which suggests that these proteins may interact with other ligands or may represent new members of the IL-1-R family (105). The in vivo contribution of the virus-encoded IL-18BP during infection has been assessed in mice infected with ectromelia virus containing a disrupted IL-18BP (ectromelia virus-p13−) (108). Following peritoneal infection with ectromelia virusp13−, mice exhibited an increase in cytotoxic activity mediated primarily by NK cells that correlated with an increase in the levels of IFNγ . Overall, the role of the poxvirus IL-18BPs appears to be to dampen the Th1 response and inhibit the early induction of IFNγ by IL-18, particularly in the epidermis, a site where IL-18 is highly expressed and where ectromelia virus and molluscum contagiosum virus primarily infect. Other poxviruses containing functional IL-18BPs include vaccinia virus (109), and cowpox virus (106, 109). Genomic sequencing of swinepox virus (20), Yabalike disease virus (21), monkeypox virus (110), and lumpy skin disease virus


TEINS

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(111) revealed that these viruses also encode putative IL-18BPs, but none have yet been characterized.


Modulation of the TNF Family TNF is a potent pro-inflammatory cytokine secreted primarily by macrophages and activated T cells (112). Three classes of TNFs have been identified: TNF, lymphotoxin-α (LT-α), and LT-β, all of which form functional trimers that recognize two cellular members of the TNF receptor (TNFR) superfamily, TNFRI (p55) and TNFRII (p75). The pleiotropic effects of TNF on the host response to infection, which include promoting an antiviral state and mediating the cytolysis of infected cells (113), has provided powerful selection pressure for viruses to evolve strategies to circumvent the TNF-mediated responses to infection. The best-characterized anti-TNF strategy employed by poxviruses involves encoded homologs of TNFR, termed vTNFRs, that function by binding and sequestering extracellular TNF prior to cellular TNFR engagement (114, 115). Secreted vTNFRs share sequence similarity to regions of the extracellular domains of p55 and p75 that include up to four cysteine-rich domains (CRDs). However, the C-terminal transmembrane domain of cellular TNFRs is always absent from the viral homologs. vTNFRs are secreted in a variety of oligomeric states that can influence their ability to inhibit TNF (116, 117). The best-studied vTNFRs are the T2-like family members encoded by Leporipoxviruses and the cytokine response modifier (Crm)-like orthologs encoded by Orthopoxviruses. The T2-like genes encode early, glycosylated proteins that bind rabbit TNF with an affinity comparable to that of the cellular rabbit TNFR (118, 119). M-T2 is an important determinant of myxoma virus pathogenesis, and virus constructs lacking this gene exhibit marked decrease in pathogenicity (119). M-T2 also functions as an intracellular inhibitor of apoptosis in virus-infected lymphocytes (120, 121). These anti-apoptotic properties have been ascribed to the first two N-terminal CRDs and are independent of the ability of M-T2 to inhibit TNF, a property that requires the first three CRDs (122). Cowpox virus encodes four vTNFRs, termed CrmB (cytokine response modifier B) (123), CrmC (124), CrmD (117), and CrmE (125), that vary in ligand specificity and patterns of expression. CrmA is a distinct intracellular serpin-like modulator considered later in this review. A fifth related family member, designated vCD30, appears to be more closely related to CD30 and binds the appropriate cellular target, namely CD153 (126). CrmB and CrmD are expressed early and late postinfection, respectively, and possess sequence similarity to T2-like vTNFRs. These two genes express proteins that bind and inhibit both TNF and LT-α (117). CrmD is absent in most cowpox virus strains and is usually encoded by Orthopoxviruses that lack CrmB and CrmC (117, 125). CrmC, a late viral protein, selectively inhibits TNF and prevents the cytolysis of target cells mediated by this cytokine (124). Unlike CrmB and CrmD, CrmC lacks the C-terminal domain conserved among other

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vTNFRs (124). CrmE, a recently identified member of the Crm-like vTNFRs, has been shown to bind rat, murine, and human TNF, but it protects cells only from cytolysis by human TNF (125). Although CrmE genes have been identified in several Orthopoxviruses, only the cowpox and vaccinia (strain USSR) versions have been so far shown to encode functional TNF inhibitors (125, 127). Among other Orthopoxviruses, the nature of secreted vTNFRs varies according to the individual virus. Variola and camelpox viruses possess a single predicted CrmB-like protein (15, 16, 125, 126), whereas ectromelia virus encodes a functional CrmD variant that binds rodent and human TNF (117, 129). The genomes of some vaccinia virus strains contain multiple discontinuous and nonfunctional TNFR homologs, including the CrmC-like A53R and the CrmB-like B28R/C22 (130, 131). However, three strains of vaccinia virus (Lister, USSR, and Evans) have been shown to possess both soluble and cell-associated vTNFR activity (132). These activities have been mapped to an intact A53R gene in the Lister and USSR strains of vaccinia virus that encodes a functional, soluble vTNFR (127, 132) and to the CrmE gene in the USSR strain that is associated with soluble and membranebound TNF-binding activity (127). The cell-surface form of CrmE from vaccinia virus (strain USSR) also protects cells from TNF-mediated apoptosis (127). Another TNF-binding protein, identified in tanapox virus, is a unique multi-cytokine-binding protein, termed gp38 (133). No gene has yet been identified, but this viral protein has been reported to bind TNF, IL-2, IL-5, and IFNγ and to inhibit the interaction of these cytokines with their receptors. In addition to poxviral strategies that target the TNF molecule itself, indirect strategies that abrogate TNF-mediated intracellular signaling pathways include the molluscum contagiosum virus MC159L gene, which encodes a viral FLICE inhibitory protein (vFLIP) homolog, that blocks the activation of NF-κB, a critical molecule in the TNF pathway (134). Other poxviral regulators of NF-κB such as the product of the cowpox virus CPV016 gene (135), have been reported.

Manipulation of IL-1β The interleukin-1 (IL-1) family comprises potent pro-inflammatory cytokines that control diverse early inflammatory processes. Poxviruses inhibit IL-1 effector functions at the level of intracellular precursor processing of the ligand (e.g., by CrmA-mediated inhibition of caspase 1, discussed later) as well as by preventing ligand/receptor engagement and downstream intracellular mechanisms of signaling. Several species of the Orthopoxviruses encode a secreted decoy IL-1 receptor (136, 137). Vaccinia virus (strain Western Reserve) B15R and cowpox virus B14R express secreted proteins that bind IL-1β, but not IL-1α or the host IL-1 receptor antagonist (129, 136, 137). Animal studies with knockout vaccinia virus constructs demonstrated that B15R suppresses the febrile response of infected mice mediated by IL-1β (138). In fact, this was the first demonstration that IL-1β functions as the major endogenous pyrogen during poxvirus infection (138). The inactivation of B15R decreases virus virulence in mice after intracranial injection (137) but

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enhances virulence when administered intranasally (136). More recently, an IL-1R homolog in ectromelia virus (E191) was found to bind soluble IL-1β and prevent signaling downstream of the IL-1 receptor (129).


Modulation of the Chemokines Chemokines are produced in response to virus infection and coordinate the activation and migration of leukocytes to sites of infection. Poxviruses utilize a variety of methods to modulate chemokines by encoding chemokine-binding proteins (CBPs), homologs of chemokine receptors, and chemokine ligand mimics [reviewed in (139–145)]. Although there is not one single class of chemokinemodulating protein that is shared by all poxviruses, the fact that all poxviruses modulate the chemokine network in some fashion demonstrates the central importance of this host pathway in poxvirus pathogenesis. LOW AFFINITY CHEMOKINE-BINDING PROTEIN (TYPE I CBP) The myxoma virus interferon-γ receptor (IFNγ -R) homolog, termed M-T7 (79, 82), binds not only IFNγ but also a broad spectrum of CXC, CC, and C-chemokines with no apparent species restriction (87). Designated the type I CBP (CBP-I), M-T7 binds chemokines through a low affinity interaction with the C-terminal heparin-binding domain present on many chemokines. The binding of rabbit IFNγ and chemokines by M-T7 is mutually exclusive, suggesting that the chemokine-binding domain and the IFNγ -binding domain on M-T7 may overlap (87). Functionally, it is postulated that M-T7 disrupts the glycosaminoglycan (GAG)-bound chemokine gradients present on the surface of endothelial cells or in the extracellular matrices (144, 145). Rabbits infected with a recombinant myxoma virus disrupted for M-T7 expression exhibited both an increase in the number of infiltrating leukocytes into sites of infection as well as a more focalized taxis toward infected cells (89), but it is not formally proven whether this phenotype is a result of the inhibition of IFNγ or of chemokines by M-T7. Nonetheless, purified M-T7 protein can reduce the migration of inflammatory cells in a rodent model of atheroma development following balloon angioplasty (146). Since M-T7 cannot inhibit murine IFNγ (80), the reduction of inflammatory cells was attributed to the species-nonspecific inhibition of chemokines by M-T7 (146). Unexpectedly, the IFNγ -R family members found in the orthopoxviruses (e.g., B8R of vaccinia virus) do not interact with chemokines. HIGH AFFINITY CC-CHEMOKINE-BINDING PROTEINS (TYPE II CBPS) Members of the Leporipoxvirus and Orthopoxvirus genera produce a mechanistically different class of secreted CBP (CBP-II; also called vCCI) that binds to CC-chemokines with high affinity and prevents the binding of CC-chemokines, but not C, CXC, or CX3C chemokines, to their cognate high affinity G protein–coupled receptors (GPCRs) (129, 147–149). Remarkably, these related poxvirus proteins bear no resemblance to any known chemokine GPCR or other known mammalian protein.

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The CBPs of the Leporipoxvirus and Orthopoxvirus genera possess very similar binding affinities toward CC-chemokines (147–150). A recent affinity analysis with over 80 chemokines binding to the vaccinia virus CBP-II demonstrated that this protein can bind most, but not all, CC-chemokines, reflecting some level of selectivity in recognition (151). Mechanistically, the CBP-II proteins bind and block CC-chemokines from interacting with native host GPCRs (129, 147–149). CBP-II members occlude the receptor-binding domain on CC-chemokines, a site that is independent of the heparin-binding domain found on many chemokines (148, 152, 153). The residues required for the high affinity interaction between vaccinia virus CBP-II and the CC-chemokine MCP-1 are largely conserved among most CC-chemokines, thus explaining how the viral CBP-II proteins are capable of promiscuously interacting with many CC-chemokines with high affinity (152, 153). Disruption of the CBP-II/M-T1 gene in myxoma virus or the disruption of the CBP-II/35-kDa gene in rabbitpox virus results in an increase in early leukocyte infiltration into tissue sites of virus infection (147, 154, 155). However, unlike CBP-I, no effect on virus lethality was observed in either of the deletion viruses. The myxoma virus CBP-II/M-T1 protein, but not the vaccinia virus CBP-II/35kDa protein, has been shown to bind glycosaminoglycans, enabling this protein to simultaneously bind cell surfaces and CC-chemokines, an adaptation that may allow M-T1 to inhibit locally secreted chemokines at the site of infection (156). The crystal structure of the cowpox virus CBP-II protein (vCCI) reveals two parallel β-sheets (β-sheet I and II) that form a β-sandwich of novel topology distantly resembling the collagen-binding domain of Staphylococcus aureus adhesin (157). A negatively charged surface within β-sheet II was predicted to be a potential chemokine-binding site since it contains a region of conserved residues present among other members of the poxvirus CBP-II proteins (157). The unique structure of CBP-II proteins and their ability to bind many CC-chemokines suggests that these novel poxvirus proteins have recapitulated elements of the binding surface provided by cellular CC-chemokine receptors despite the substantial difference between the structures. CHEMOKINE HOMOLOGS Only two poxviruses have been observed to possess chemokine-mimics: molluscum contagiosum virus and fowlpox virus (12, 30). To date, only the molluscum contagiosum virus chemokine has been characterized (158). Following the discovery of the molluscum contagiosum virus chemokine mimic, termed MC148R, the host homolog of the MC148R was discovered and termed ILC, for IL-11 receptor α-locus chemokine (159). Notably, ILC is expressed selectively in the skin, coinciding with the location of molluscum contagiosum virus infection. MC148R is a 104-amino-acid protein that is structurally related to the CC-chemokine family of chemokines but possesses a notable deletion in the Nterminal region preceding the dicysteine motif, a region necessary for activation of receptors. MC148R is detected as a secreted protein (158) and was initially shown to potently inhibit binding, signaling, and chemotaxis of various leukocytes in

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response to a number of CC and CXC chemokines (160, 161). Moreover, MC148R could inhibit colony formation of myeloid, erythroid, and multipotential progenitor cells, although the physiological relevance of this activity is unclear (160). More recent reports argue that MC148R is a highly selective antagonist of human, but not mouse, CCR8 and is unable to bind a number of chemokine receptors or orphan GPCRs (162–164). The role of MC148R in controlling the inflammatory response is unknown since a suitable animal model of molluscum contagiosum virus pathogenesis is lacking. However, the idea that MC148R possesses anti-inflammatory properties is supported by the observation that MC148R can inhibit allograft rejection in transgenic mice (165). This is unexpected since MC148R is a selective antagonist of human, but not mouse, CCR8. Nonetheless, these results suggest that MC148R may be able to bind other still-undefined chemokine receptors or may have novel anti-inflammatory properties that facilitate graft survival. Several poxvirus encode putative chemokine receptors (21, 141), but these proteins remain to be functionally characterized.

Proteins That Bind Multiple Cytokines TANAPOX VIRUS 38-kDa MULTIPLE-CYTOKINE-BINDING PROTEIN Tanapox virus is a poxvirus of the Yatapoxvirus genus that causes a mild, self-limiting disease in humans (166). Tanapox virus–infected cells express a secreted 38-kDa protein that can bind and inhibit human IL-2, human IL-5, and human IFNγ (167). A subsequent study revealed that supernatants from tanapox virus–infected cells could also inhibit TNF-mediated induction of NK-κB and upregulation of cell adhesion molecule expression (133). Binding of TNF was shown to be mediated by a 38-kDa protein present in the supernatants of cells infected with tanapox virus, but not mock-infected cells. A complete analysis of these phenomena awaits identification of the protein(s) that mediate these activities. ORF VIRUS GM-CSF/IL-2 INHIBITORY FACTOR Orf virus is a member of the Parapoxvirus genus that causes an acute contagious skin condition, termed contagious ecthyma, in sheep, goats, and humans (10). Orf virus encodes a secreted protein that binds and inhibits the function of ovine, but not human or murine, GM-CSF and IL-2 (168). This protein, termed GM-CSF/IL-2 inhibitory factor (GIF), forms dimers and tetramers that bind with high affinity to both ovine GM-CSF and ovine IL-2 (Kd = ∼369 pM and ∼1.04 nM, respectively), effectively allowing GIF to competitively bind these cytokines away from their host receptors (168). The binding specificity of GIF for ovine, but not human, cytokines is consistent with the idea that orf virus is evolutionarily adapted for sheep as its primary host. GIF has no sequence similarity to any known mammalian gene although it appears to be related to the vaccinia virus A41L protein and may be a distant member of the poxvirus T1/35-kDa chemokine-binding protein (CBP-II) family (169). However, GIF has no demonstrable interaction with various tested chemokines (MCP-1, RANTES, IL-8, MIP-1α), cytokines (IL-3, IL-4, IL-5, IFNγ , TNF-α),

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or heparin (168). In addition to inhibiting the biological activities of ovine GMCSF and ovine IL-2 in assays in vitro, GIF inhibitory activity of GM-CSF was observed in sheep infected with orf virus (168). It is predicted that GIF affects the Th1 arm of the effector immune response by inhibiting IL-2, and it may function to obstruct GM-CSF-mediated neutrophil and macrophage activation and/or antigen presentation by dendritic cells.


Semaphorin Homolog Semaphorins are a highly conserved family of regulatory molecules found in animals ranging from invertebrates to mammals. The defining characteristic of semaphorins is the presence of a SEMA domain, an approximately 500-aminoacid region within the extracellular component that mediates receptor-binding specificity. Semaphorins may be secreted, anchored to the membrane by glycosylphosphatidylinositol (GPI), or occur as transmembrane molecules (170, 171). Although semaphorins were originally identified by their ability to induce axon steering and growth cone collapse, several semaphorins have now been shown to have immunological roles (170–172). Virus-encoded semaphorins have been identified among the Poxviridae and the Herpesviridae (172–174). Of interest here are the versions that have been identified in members from two of the seven Chordopoxvirinae genera that contain a semaphorin homolog (12, 172, 173, 175). Vaccinia virus A39R is a 403-aminoacid ORF that encodes a 50- to 55-kDa product with a modest but significant identity (25%) to cellular semaphorins (172, 173). Related members have been identified in 8 of 15 vaccinia virus strains tested (175). In addition to vaccinia virus, ectromelia virus and cowpox virus also encode secreted orthologs of A39R that resemble the extracellular region of SemaA7A (173, 175). In contrast, FPV047 was identified from the fowlpox virus genome based on the presence of a SEMA domain and sequence similarity to vaccinia virus A39R (12). Functionally, the ectromelia virus version of A39R can induce monocyte aggregation due to activation of CD54 (ICAM-1) on the cell surface (173). Through an interaction with a virus-encoded semaphorin protein receptor (VESPR; also called CD232 or plexin C1), A39R can also induce the production of IL-6 and IL-8, suggesting that A39R may play a role in mediating inflammation (173). Deletion of A39R from vaccinia virus (strain Copenhagen) has no effect on virus titer or its ability to replicate in vitro (175). Mice intranasally infected with vaccinia virus exhibit no difference in virulence, irrespective of whether A39R was expressed, although when the Copenhagen A39R gene was expressed from vaccinia virus strain WR, the lesion size was increased after intradermal infection (175).

Growth Factor Homologs Most poxviruses encode a growth factor homolog; the exceptions are molluscum contagiosum virus and swinepox virus. Poxvirus growth factor homologs exploit the machinery connected to growth factor receptors to tap into cellular signaling

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pathways that induce cell cycle progression, proliferation, wound healing, and/or angiogenesis (176). Consequently, these viral proteins have an impact on various aspects of virus virulence and spread. EPIDERMAL GROWTH FACTOR HOMOLOGS Epidermal growth factor (EGF) regulates cell differentiation and proliferation in a number of cell types by binding to the ErbB family of receptors (177). Upon ligand binding, these receptor tyrosine kinases initiate a number of signaling pathways, including the MAPK pathway, which results in cell growth and differentiation (178). All poxvirus homologs of EGF have the characteristic spacing of six key cysteine residues, which appears to be important for the folding of the domains and binding to the ErbB receptor. Poxvirus EGFs are expressed from early promoters, secreted as highly glycosylated proteins, and bind the ErbB family of receptors to induce proliferation of infected and noninfected cells. The first discovered poxvirus-encoded growth factor was the vaccinia virus growth factor (VGF), a 19-kDa early protein with binding specificity for the ErbB-1/ErbB-1 homodimer as well as the ErbB-1/ErbB-2 and ErbB-1/ErbB-3 heterodimers (176, 178). Upon receptor binding, VGF stimulates quiescent cells to enter unscheduled cell division and induces cell proliferation (176, 179). Other poxviruses that contain an EGF homolog include cowpox virus, myxoma virus, Shope fibroma virus, variola virus, and tanapox virus. The Shope fibroma virus EGF homolog binds ErbB-1/ErbB-1 homodimers and several of the ErbB heterodimers, whereas the EGF homolog of myxoma virus is a narrow-specificity ligand that binds only ErbB-2/ErbB-3 heterodimers (178). These poxvirus growth factors possess a 10- to 1000-fold lower binding affinity to their receptors but have a similar, if not higher, capacity to stimulate proliferation in cells compared to host growth factors (178). This function of the viral proteins can be explained by their unique ability to reduce receptor downregulation and subsequent degradation, which prolongs signal transduction. VASCULAR ENDOTHELIAL GROWTH FACTOR HOMOLOG The vascular endothelial growth factor (VEGF) family of growth factors induce vascular permeability and play a crucial role in the formation of new blood vessels during vasculogenesis and angiogenesis (180, 181). So far, only orf virus, which infects sheep, goats, and humans, has been found to encode a functional VEGF homolog. This protein is expressed early, contains the characteristic cysteine spacing motif, binds to specific members of the mammalian tyrosine kinase receptors, namely the VEGF receptors (182), and may provide a molecular basis for the characteristic lesions caused by orf virus, which exhibit extensive vascular proliferation and dilation (183). Orf virus VEGF (ORFV-VEGF) binds to VEGFR-2 and neuropilin-1, but not VEGFR-1 or VEGFR-3 (184). ORFV-VEGF shows very similar activities to VEGF-A in that both stimulate the proliferation of endothelial cells in vitro and angiogenesis in vivo (180). Infection of sheep with a recombinant orf virus containing a disrupted ORFVVEGF gene results in lesions with reduced pustule formation, vascularization,

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inflammatory cell influx, and epidermal hyperplasia (183). Moreover, virus titers are reduced in lesions at late times of infection and characteristic scabs fail to form with the ORFV-VEGF deletion virus. Thus, the VEGF homolog enhances wound healing and promotes the formation of orf virus–containing scabs, which may contribute to virus transmission and protection from environmental inactivation following the sloughing process.


Cytokine Homologs IL-10 is a multifunctional cytokine that can exert either immunostimulatory or immunosuppressive effects on many cell types (185). To date, a number of viral IL-10 homologs have been identified among the herpesviruses and more recently among a number of poxviruses: orf virus, Yaba-like disease virus, and lumpy skin disease virus (21, 111, 186). The first identified, and so far the only characterized, poxvirus IL-10 homolog is the orf virus IL-10 (ORFV-IL-10). The ORFV-IL-10 gene shows high amino acid identity to host IL-10 from sheep, cattle, humans, and mice, as well as to other viral IL-10-like proteins (186). ORFV-IL-10 is transcribed early during infection and exhibits the same biological activity as ovine IL-10 in a murine thymocyte proliferation assay (186). ORFV-IL-10 also costimulates the growth of murine mast cells in culture, producing stimulatory effects equivalent to ovine IL-10 (185). ORFV-IL-10 suppresses macrophage activation and is thought to play a role in immune evasion by exerting the immunosuppressive effects of IL-10 and therefore protecting virus-infected cells from Th1-mediated immune responses (187).

Anti-Inflammatory Serpins The myxoma virus SERP-1 protein is a late 55- to 60-kDa glycoprotein that is so far the only identified viral serpin shown to be secreted, and is the first viral protein shown to be sialylated by a virus-encoded glycosyltransferase (188, 189). Disruption of both copies of the SERP-1 gene results in an increased inflammatory cell response, attenuated virulence, and a more rapid clearance of the infection (188, 190). Although the in vivo targets of SERP-1 are unknown, in vitro binding studies have shown that the protein effectively inhibits pro-inflammatory serine proteinase substrates, including plasmin, tissue plasminogen activator, urokinase, and thrombin (191, 192). Viral proteins with proposed functions similar to that of SERP-1 have been reported in other poxviruses, including the product of the Yaba-like disease virus 10L gene and the SPI-3 serpin of the Orthopoxviruses, such as vaccinia virus (K2L) and cowpox virus (M2L). SPI-3 was originally shown to inhibit cell-cell fusion following infection (193–195), but recent experiments have demonstrated that SPI-3 exhibits a profile of substrate inhibition similar to that of SERP-1 (196), and that this inhibition is mediated by different key residues than those required to block fusion (197). SPI-3 is an early protein that is not secreted from wild-type cowpox virus–infected cells, is not essential for virulence, and shares only 29%

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identity with SERP-1 (197). Moreover, neither SERP-1 nor SPI-3 are functionally interchangeable (198) despite their capacity to inhibit similar proteinases (196).

POXVIRUS INHIBITION OF THE INTRACELLULAR ENVIRONMENT


Inhibition of Apoptosis Apoptosis is a regulated form of cell death that is designed to eliminate defective or unwanted cells, including those that are virus-infected (199–201). Indeed, apoptosis is so effective a mechanism for counteracting virus infections that it can be viewed as a form of innate immunity (202). Poxviruses quench this host response by producing viral proteins that are rapidly expressed during the early stages of replication (see Table 2) (200, 203–205). These anti-apoptotic effectors have different modes of action. They can be secreted and neutralize signals emanating from the extracellular environment (for example, the TNF decoy proteins described previously) or they can act to manipulate transduction of cell death pathways within the cell, as described below. PREVENTION OF APOPTOSIS BY CASPASE INHIBITION A key event in the induction of most cell death signals is the activation of a family of pro-apoptotic proteases termed caspases (201). Consequently, these apoptotic proteases are frequently targeted for inactivation by different poxviruses. For example, the strategy employed by molluscum contagiosum virus is directed toward preventing activation of the initiator caspase, caspase-8 (206–208). Activation of caspase-8 is triggered by the ligation of cell surface Fas, TNF, or TRAIL death receptors and the formation of intracellular molecular scaffolds that incorporate Fas-associated death domain (FADD) adapter molecules. Each FADD molecule contains an important death effector domain (DED) motif, the same motif that is present in the pro-domain of inactive pro-caspase-8. Clustering of FADD DED motifs allows recruitment of pro-caspase-8 molecules to receptor complexes and proximity-mediated transactivation (201). Molluscum contagiosum virus prevents caspase-8 activation by producing two DED-containing proteins, MC159 and MC160. MC159 and MC160 fall into the general category of viral FLICE/caspase-8 inhibitory proteins (vFLIPs) that bind to FADD and procaspase-8, thereby inhibiting transduction of death receptor– mediated apoptotic signals (206–208). MC159 is thought to be the primary functional vFLIP. MC160 has an unclear role but undergoes caspase-mediated degradation when expressed independently of MC159, which suggests that these proteins could have a specialized combined functional relationship (209). Recent evidence also indicates that the mechanism of MC159 action may be more complex than originally anticipated (210). As expected, mutation of the FADD and procaspase8 binding regions within the DED motifs of MC159 abolishes the anti-apoptotic

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properties of this protein. However, mutation of adjacent hydrophobic regions within the DED motifs, which do not impair binding of MC159 to FADD or procaspase-8, renders this protein unable to inhibit Fas-, TNF-, or TRAIL-mediated apoptosis. This suggests that MC159 may interact with additional cellular proteins within the death receptor complex that may be required to prevent death signals. Other poxviruses inhibit caspases by producing proteins that act as suicide substrates. The most extensively studied protein in this group is CrmA produced by cowpox virus. CrmA was first identified as an inhibitor of the pro-inflammatory IL1β converting enzyme (caspase-1) (211). Viruses with a targeted disruption in this gene have a modestly attenuated disease phenotype (212). CrmA is a member of the serine protease inhibitor (serpin) superfamily and, at least in vitro, CrmA can inhibit the serine protease granzyme B that is delivered to target cells by cytotoxic T cells and natural killer (NK) cells to initiate perforin-dependent apoptosis (213, 214). CrmA therefore at least has the potential to protect infected cells against apoptosis induced by both cytotoxic T cells and NK cells (214, 215). Furthermore, CrmA is able to inhibit caspase-8, and possibly caspase-10, and can block apoptosis by several pathways. These pathways can be initiated by diverse stimuli including serum deprivation (216), removal of nerve growth factor (217), detachment from extracellular matrix (218), hypoxic conditions (219), and TNF and Fas ligation (220–223). The novel cross-class inhibition of caspases by CrmA (224) can be rationalized on the basis of its unique structural features (225). The versatility of CrmA allows this protein to disarm granzyme B–mediated apoptosis as well as caspase-8-mediated cell death pathways. The SPI-2 family of poxvirus serpins, such as B13R from vaccinia virus (strain Copenhagen), can protect cells from Fas- and TNF-mediated apoptosis (226, 227) but, in comparison to CrmA of cowpox virus, are generally less potent apoptosis inhibitors. In addition, viruses with a targeted disruption of this gene are not noticeably attenuated (227, 228), although a recent report indicates an increased lesion size in mice inoculated dermally with B13R-deficient vaccinia virus (229). SERP-2, a SPI-2-like serpin expressed by myxoma virus that was first described as a caspase-1 inhibitor, plays an important role in pathogenesis by preventing apoptosis of lymphoid cells in infected rabbits (230, 231). SERP-2, however, is considerably less effective at inhibiting caspase-1 and granzyme B than CrmA. In addition, SERP-2 cannot substitute for CrmA in preventing apoptosis in the context of cowpox virus infection (232), indicating that SERP-2 may have distinct cellular targets. INHIBITION OF PKR-INDUCED APOPTOSIS Activation of RNA-dependent protein kinase (PKR) results in the interruption of translation and, frequently, the induction of apoptosis (233). Vaccinia virus encodes two PKR inhibitors (E3L and K3L) (as discussed in the section Intracellular Mechanisms of Interferon Inhibition). Given their target pathway, both can be categorized as anti-apoptotic proteins (234). However, E3L has been found to be more important for prevention of apoptosis during infection (235). In addition, recent evidence suggests that PKR triggers

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apoptosis by activating caspase-8. Consequently, the poxvirus caspase-8 inhibitors, MC159 and CrmA, have also been shown to counteract PKR-mediated apoptosis (134, 236). ANTI-APOPTOSIS STRATEGY OF PREVENTING OXIDATIVE STRESS Another poxvirus protein that prevents cell death triggered by stress signals is the molluscum contagiosum virus protein MC066. This protein incorporates an unusual selenocysteine and functions as a glutathione peroxidase by catalyzing the conversion of toxic reactive oxygen species, such as H2O2, to water (237). H2O2 can arise as a result of the effector functions of macrophages or neutrophils or it can be produced following UV irradiation. MC066 could therefore be important for virus replication at the primary site of infection in basal keratinocytes of the skin. Two unrelated poxvirus RING finger proteins, p28 of ectromelia virus and N1R of Shope fibroma virus, are able to counteract UV- but not TNF- or Fas-induced apoptosis and may target the same pathway (238, 239). OTHER ANTI-APOPTOTIC PROTEINS Sensors of cellular stress are also situated within specific organelles. Mitochondria are important coordinating centers for apoptotic signaling (240). Myxoma virus expresses a 166-amino-acid protein, termed M11L, that is able to inhibit transduction of death signals via the mitochondrial checkpoint. M11L was first identified as a virulence factor and the knockout virus also displayed a pro-apoptotic phenotype following infection of a rabbit lymphocyte cell line (120, 241). M11L is targeted to mitochondria and prevents mitochondrial changes associated with apoptosis including the loss of inner mitochondrial membrane potential (242). Vaccinia virus infection also protects mitochondria from apoptosis mediated by Fas, staurosporine, and granzyme B, although the viral protein(s) involved remain to be reported (243). Therefore, a need to preserve mitochondrial function and inhibit apoptotic signal transduction via this checkpoint appears to be a common theme among poxviruses. Other apoptotic modulators from myxoma virus have been identified based on the apoptotic phenotypes of specific knockout viruses. These include the M-T4 protein, an endoplasmic reticulum (ER)-resident protein that may inhibit an ER stress response to infection (244, 245), and the M-T5 protein, an ankyrin repeat protein with homology to the Chinese hamster ovary host range (CHOhr) gene of cowpox virus (246). Although the functional mechanisms of M-T5 and CHOhr are unknown, both proteins can prevent apoptosis associated with virus infection (246, 247). M-T2, the myxoma virus TNFR homolog, also functions as an intracellular inhibitor of apoptosis (121). Collectively, the properties of these viral proteins suggest that inhibiting apoptosis at multiple points is an important survival strategy for poxviruses.

Intracellular Mechanisms of Interferon Inhibition In addition to the extracellular mechanisms of IFN inhibition (discussed previously), many poxviruses also target intracellular signaling elements of the IFN

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response, including two IFN-inducible enzymatic pathways: PKR and 20 ,50 -oligoadenylate synthetase (OAS). Activation of these enzymes by dsRNA produced during poxviral transcription inhibits the translation and synthesis of viral proteins in infected cells, and frequently leads to the induction of apoptosis (Figure 1) (69, 70, 233, 248, 249). Vaccinia virus E3L is a dsRNA-binding protein that binds and sequesters dsRNA to prevent the activation of PKR and OAS (234, 250). E3L can bind directly to PKR and inhibits its activity, resulting in reduced phosphorylation of eukaryotic initiation factor 2α (eIF2α) (251), IRF-3, and IRF-7 (252). Recently, E3L was reported to reduce adenosine deaminase editing activity (253) and to bind to SUMO-1 (254). E3L also blocks IRF-3 activation and prevents upregulation of the host cell IFNβ gene (255). A second vaccinia virus gene, termed K3L, encodes a homolog of the eIF2α subunit that acts as a nonphosphorylatable pseudosubstrate of PKR and competitively inhibits phosphorylation of eIF2α (256, 257). Deletion of E3L and K3L in vaccinia virus renders the virus sensitive to IFN and severely limits host range and disease progression (258–260). To date, E3L and K3L orthologs have been identified in myxoma virus, Yaba-like disease virus, variola virus, Shope fibroma virus, swinepox virus, and orf virus (14, 17, 261–263). Ectromelia virus encodes an E3L ortholog and ectromelia virus–infected cells are highly resistant to mouse IFN, but surprisingly, its K3L gene product is nonfunctional (92). Expression of the vaccinia virus E3L and K3L genes in canarypox-based vaccine vectors improves epitope expression levels and inhibits apoptosis (264). In addition to directly inhibiting the PKR and OAS pathways, some poxviruses may also act indirectly to alleviate the antiviral state induced by IFN, such as by targeting the transcription factors that transduce the biological effects of IFNinducible genes. For example, the vaccinia virus H1L gene encodes a phosphatase that prevents IFN-induced activation of STAT-1, a vital transcription factor in the intracellular signaling pathways employed by IFNs (265). In a related fashion, molluscum contagiosum virus, which lacks an ortholog of either E3L or K3L, may use MC159L to inhibit IFN-mediated PKR-induced apoptosis and activation of NF-κB (134).

Intracellular Inhibition of IL-1βR Signaling Poxviruses disrupt intracellular IL-1 receptor signaling by producing proteins that have acquired the cytoplasmic signaling components of Toll-like receptors (TLRs). TLRs play an important role in innate immunity and function as pattern recognition receptors on cells of the innate immune system (266). Vaccinia virus A46R and A52R contain a Toll/IL-1 receptor (TLR) domain motif within the cytoplasmic domain that permits interactions with adapter molecules and ultimately prevents intracellular signaling downstream of the IL-1 receptor (267). Although the level of sequence conservation between the viral proteins and the IL-1R/TLR family members is low, convincing in vitro data suggests that these two proteins are members of the IL-1R/TLR family.

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The ectopic expression of A46R and A52R demonstrated that A46R had a minor inhibitory effect on IL-1-induced NF-κB activation, and A52R had a greater inhibitory effect on NF-κB activation (267). Moreover, the effect of A52R on IL1, TLR4, and IL-18 signaling mimicked a dominant-negative, truncated version of MyD88, a TIR- and death domain–containing adapter protein. Overall, A52R likely provides vaccinia virus the ability to simultaneously inhibit the signaling pathways emanating from multiple cell surface receptors with TIR domains, such as the TLRs and IL-1R.

A NEW TYPE OF DRUG: POXVIRUS IMMUNE EVASION PROTEINS AS ANTI-INFLAMMATORY AGENTS Viruses in many ways have been consummate drug researchers and developers. Components of the host immune system that are modulated by viruses illuminate potential key targets that uniquely regulate the early inflammatory responses. However, the concept of exploiting viral anti-inflammatory proteins directly as therapeutic agents has emerged only in the past few years. Purified SERP-1 protein, the secreted myxoma virus serpin described previously, was found to have considerable potency for the inhibition of inflammatory responses to arterial injury following balloon angioplasty with an associated reduction in early inflammatory cell invasion in a rabbit model (268). Subsequent work demonstrated that SERP-1 was effective in other models at preventing chronic transplant rejection by reducing mononuclear cell invasion and subsequent intimal hyperplasia in a rat model of aortic transplant (269), by reducing vasculopathy in rat models of heterotopic heart transplant and renal transplant (270), and by reducing joint inflammation in rabbit and rat models of collagen-induced arthritis (271). Two other classes of poxvirus inhibitors that target chemokines and complement have been tested in animal models of inflammatory-based diseases (49, 145). It was reported that the CBP-II of vaccinia virus could mediate effective reductions of eotaxin-induced eosinophilia in guinea pig skin (148), and that CBP-II of cowpox virus inhibited bronchospasm and cellular infiltration in a murine asthma model (272). The myxoma virus CBP-I, M-T7, was also shown to possess potent anti-inflammatory activity in rat and rabbit models of angioplasty injury and transplant vasculopathy and was highly effective at inhibiting early mononuclear cell infiltration and reducing transplanted organ scarring and vasculopathy development after arterial injury resulting from either surgery or balloon angioplasty (146). The poxvirus chemokine homolog MC148R, which functions as a competitive antagonist for CCR8, has also been shown to be effective at preventing transplant rejection using plasmid-mediated gene transfer technology (273). In each case, the viral proteins studied have demonstrated anti-inflammatory properties even when administered systemically at very low concentrations. The dose range for these agents likely reflects the low dosage of these inhibitors when secreted by the infecting virus in situ. The list of virus-derived anti-inflammatory agents that are exploitable as potential therapeutic agents is likely to continue

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to grow as the cellular targets for more of these remarkable virus-encoded immunomodulators become identified and better defined.


CONCLUSIONS The field of poxvirus-encoded immunomodulators is now over 15 years old, and yet fundamental discoveries in this area continue to occur with undiminished frequency. Although no one virus member utilizes all of the collective strategies described in this review, all seem to have targeted host pathways that regulate the earliest aspects of immune responses, particularly the IFNs, the chemokines, the pro-inflammatory cytokines (IL-1, TNF, IL-18), complement, and the regulatory components that orchestrate cellular immunity and apoptosis (see Figure 2). As more of these viral anti-immune regulators are defined and investigated, our appreciation of the selective pressures that drive the coevolution between virus and host seems destined to increase. ACKNOWLEDGMENTS This work was funded by the Canadian Institutes of Health Research (CIHR) and the National Cancer Institute (Canada). G.M. holds a Canada Research Chair in Molecular Virology. B.T.S. is funded by an Ontario Graduate Scholarship (O.G.S) and an O.G.S. for Science and Technology (O.G.S.S.T.). J.S. and S.N. are funded by a Special University Scholarship. C.C. is funded by a CIHR doctoral award. We wish to thank Antonio Alcami, Michele Barry, Richard Moyer, and Geoffrey Smith for critically reviewing the manuscript. Because of space restrictions, we were able to cite only a fraction of the relevant literature on this subject, and we apologize to any colleagues whose contributions may not be appropriately represented in this review. The Annual Review of Immunology is online at http://immunol.annualreviews.org

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SEET ET AL. Vaccinia virus-encoded eIF-2α homolog abrogates the antiviral effect of interferon. Virology 183:419–22 Carroll K, Elroystein O, Moss B, Jagus R. 1993. Recombinant vaccinia virus K3L gene product prevents activation of double-stranded RNA-dependent, initiation factor-2-alpha-specific protein kinase. J. Biol. Chem. 268:12837–42 Beattie E, Denzler KL, Tartaglia J, Perkus ME, Paoletti E, et al. 1995. Reversal of the interferon-sensitive phenotype of a vaccinia virus lacking E3L by expression of the reovirus S4 gene. J. Virol. 69:499–505 Beattie E, Kauffman EB, Martinez H, Perkus ME, Jacobs BL, et al. 1996. Hostrange restriction of vaccinia virus E3Lspecific deletion mutants. Virus Genes 12:89–94 Brandt TA, Jacobs BL. 2001. Both carboxy- and amino-terminal domains of the vaccinia virus interferon resistance gene, E3L, are required for pathogenesis in a mouse model. J. Virol. 75:850– 56 McInnes CJ, Wood AR, Nettleton PF, Gilray JA. 2001. Genomic comparison of an avirulent strain of orf virus with that of a virulent wild type isolate reveals that the orf virus G2L gene is non-essential for replication. Virus Genes 22:141–50 McInnes CJ, Wood AR, Mercer AA. 1998. Orf virus encodes a homolog of the vaccinia virus interferon resistance gene E3L. Virus Genes 17:107–15 Kawagishi-Kobayashi M, Cao CN, Lu JM, Ozato K, Dever TE. 2000. Pseudosubstrate inhibition of protein kinase PKR by swine pox virus C8L gene product. Virology 276:424–34 Fang Z-Y, Limbach K, Tartaglia J, Hammonds J, Chen X, et al. 2001. Expression of vaccinia E3L and K3L genes by a novel recombinant canarypox HIV vaccine vector enhances HIV-1 pseudovirion production and inhibits apoptosis in human cells. Virology 291:272–84 Najarro P, Traktman P, Lewis JA. 2001.

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Vaccinia virus blocks gamma interferon signal transduction: Viral VH1 phosphatase reverses Stat1 activation. J. Virol. 75:3185–96 O’Neill L. 2000. The Toll/interleukin-1 receptor domain: a molecular switch for inflammation and host defence. Biochem. Soc. 28:557–63 Bowie A, Kiss-Toth E, Symons JA, Smith GL, Dower SK, et al. 2000. A46R and A52R from vaccinia virus are antagonists of host IL-1 and toll-like receptor signaling. Proc. Natl. Acad. Sci. USA 97:10162– 67 Lucas A, Liu L, Macen J, Nash P, Dai E, et al. 1996. Virus-encoded serine proteinase inhibitor SERP-1 inhibits atherosclerotic plaque development after balloon angioplasty. Circulation 94:2890–900 Miller L, Dai E, Nash P, Liu L, Icton C, et al. 2000. Inhibition of transplant vasculopathy in a rat aortic model after infusion of an anti-inflammatory viral serpin. Circulation 101:1598–605 Hausen B, Boeke K, Berry GJ, Morris RE. 2001. Viral serine proteinase inhibitor (Serp-1) effectively decreases the incidence of graft vasculopathy in heterotopic heart allografts. Transplantation 72:364– 68 Maksymowych WP, Nation N, Nash PD, Macen J, Lucas A, et al. 1996. Amelioration of antigen-induced arthritis in rabbits treated with a secreted viral serine proteinase inhibitor. J. Rheumatol. 23:878– 82 Dabbagh K, Xiao Y, Smith C, StepickBiek P, Kim SG, et al. 2000. Local blockade of allergic airway hyperreactivity and inflammation by the poxvirus-derived pan-CC-chemokine inhibitor vCCI. J. Immunol. 165:3418–22 DeBruyne LA, Li K, Bishop DK, Bromberg JS. 2000. Gene transfer of virally encoded chemokine antagonists vMIP-II and MC148 prolongs cardiac allograft survival and inhibits

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POXVIRUSES AND IMMUNE EVASION donor-specific immunity. Gene Ther. 7: 575–82 274. Afonso CL, Tulman ER, Lu Z, Zsak L, Sandybaev NT, et al. 2002. The genome of camelpox virus. Virology 295:1– 9 275. Shchelkunov SN, Safronov PF, Totmenin AV, Petrov NA, Ryazankina OI, et al. 1998. The genomic sequence analysis of the left and right species-specific terminal region of a cowpox virus strain reveals unique sequences and a cluster of

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Figure 1 Inhibition of IFNs and IFN-induced mechanisms by poxviruses. Binding of IFNγ and IFNα/β to cellular receptors is inhibited by poxvirus IFNγ receptor homologs or IFNα/β-binding proteins, respectively. The IL-18BP from several poxviruses can sequester IL-18 and inhibit the induction of IFNγ by IL-18. Intracellular inhibition of IFN signaling is achieved by the vaccinia virus VH1 (H1L) phosphatase (shown to dephosphorylate IFNγ -induced STAT1), E3L [binds double-stranded RNA (dsRNA) and prevents activation of RNA-dependent protein kinase (PKR) and/or 20 ,50 oligoadenylate synthase activation], and K3L (inhibits PKR activation). Proteins shown in red represent poxvirus proteins; host proteins are shown in black and gray.


Figure 2 Schematic representation of selected poxvirus immunomodulators. Secreted viral proteins are shown above the membrane whereas intracellular proteins are shown below the membrane. Poxvirus viroceptors function as soluble or cell surface decoy receptors that bind host cytokines or chemokines. Poxvirus virokines are also secreted but function as agonistic or antagonistic ligands for host cellular receptors. A number of intracellular poxvirus proteins function to modulate apoptosis, cytokine processing, and host range. Proteins shown in red represent poxvirus proteins; host proteins are shown in black and gray.


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Annu. Rev. Immunol. 2003. 21:425–56 doi: 10.1146/annurev.immunol.21.120601.141142

IL-13 EFFECTOR FUNCTIONS∗ Thomas A. Wynn Annu. Rev. Immunol. 2003.21:425-456. Downloaded from arjournals.annualreviews.org by HINARI on 09/01/07. For personal use only.

Immunopathogenesis Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892; email: [email protected]

Key Words IL-4, parasite, cancer, asthma, fibrosis ■ Abstract IL-13 was first recognized for its effects on B cells and monocytes, where it upregulated class II expression, promoted IgE class switching and inhibited inflammatory cytokine production. It was also thought to be functionally redundant with IL-4. However, studies conducted with knockout mice, neutralizing antibodies, and novel antagonists demonstrate that IL-13 possesses several unique effector functions that distinguish it from IL-4. Resistance to most gastrointestinal nematodes is mediated by type-2 cytokine responses, in which IL-13 plays a dominant role. By regulating cell-mediated immunity, IL-13 modulates resistance to intracellular organisms including Leishmania major, Leishmania mexicana, and Listeria monocytogenes. In the lung, IL-13 is the central mediator of allergic asthma, where it regulates eosinophilic inflammation, mucus secretion, and airway hyperresponsiveness. Manipulation of IL-13 effector function may also prove useful in the treatment of some cancers like B-cell chronic lymphocytic leukemia and Hodgkin’s disease, where IL-13 modulates apoptosis or tumor cell growth. IL-13 can also inhibit tumor immunosurveillance. As such, inhibitors of IL-13 might be effective as cancer immunotherapeutics by boosting type1-associated anti-tumor defenses. Finally, IL-13 was revealed as a potent mediator of tissue fibrosis in both schistosomiasis and asthma, which indicates that it is a key regulator of the extracellular matrix. The mechanisms that regulate IL-13 production and/or function have also been investigated, and IL-4, IL-12, IL-18, IFN-γ , IL-10, TGF-β, TNF-α, and the IL-4/IL-13 receptor complex play important roles. This review highlights the effector functions of IL-13 and describes multiple pathways for modulating its activity in vivo.

INTRODUCTION IL-13 was originally described as a T cell–derived cytokine that inhibits inflammatory cytokine production (1, 2, 3). Though this original description remains accurate, the known effector functions of IL-13 have expanded dramatically over the past few years. This increase in knowledge is primarily attributable to the ∗ The U.S. Government has the right to retain a nonexclusive, royalty-free license in and to any copyright covering this paper.

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development of several unique experimental animal models that either promote or block the activity of IL-13. Early studies comparing IL-4-, IL-4R-, and Stat6deficient mice were the first to suggest a nonredundant role for IL-13 in host immunity (4–8). Since then, at least two IL-13 transgenic mice have been developed that overexpress IL-13 in the lung (9) or in lymphoid tissues (10). Two distinct IL-13-deficient animals have also been generated. One has a disruption in exon 1 (11) and the second a neocassette inserted into exon 3 (12). Both knockout animals were instrumental in demonstrating nonredundant roles for IL-13. Mice simultaneously deficient in IL-4 and IL-13 were also valuable tools and highlighted the cooperative and distinct functional activities of IL-4 and IL-13 (13). In addition to transgenic and knockout mice, novel IL-13 antagonists and neutralizing antibodies have also been generated and offer the advantage of blocking IL-13 at various time points and in other transgenic mice (14–16). Moreover, the utility of IL-13-deficient mice alone to study IL-13 effector function may be limited because the mice appear to manifest a partial defect in the closely linked IL-4 gene (17). This must be taken into consideration when comparing findings generated from knockout and IL-13-blocking studies and may explain some of the published functional discrepancies. The list of important effector functions mediated by IL-13 continues to grow and includes a diverse array of biological activities including regulation of gastrointestinal parasite expulsion, airway hyperresponsiveness (AHR), allergic inflammation, tissue eosinophilia, mastocytosis, IgE Ab production, goblet cell hyperplasia, tumor cell growth, intracellular parasitism, tissue remodeling, and fibrosis. Although IL-4 and IL-13 are functionally related, it is surprising to note in many situations IL-13 appears to play a more important role than IL-4. The general availability of both ligands and the particular receptor combinations expressed on responding cells likely dictates the overall importance of IL-4 versus IL-13 in specific disease settings. This review focuses on the effector functions of IL-13, revealed primarily through knockout, blocking, and transgenic mouse studies. The various mechanisms that regulate the production and/or effector activities of IL-13 are also discussed. The findings generated over the past few years illustrate a complex and pleiotropic nature for IL-13 in host immunity. More importantly, they emphasize that IL-13 is an important target for therapeutic intervention.

THE CENTRAL ROLE OF IL-13 IN DISEASE SUSCEPTIBILITY AND RESISTANCE Resistance to Gastrointestinal Nematodes Helminth parasites induce strong Th2 responses that contribute to the mast cell, eosinophil, giant cell, IgE/IgA, and mucus responses, which are hallmarks of these infections. For intestinal helminths in particular it is clear that elements of the type 2 response are critical for host resistance to infection (18). The mechanisms involved have been investigated intensively and experimental studies with Nippostrongylus

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brasiliensis were the first to reveal a unique and nonredundant role for IL-13 in host immunity (8, 11). Since then, several worms have been studied in detail in this regard: Trichuris muris, a natural parasite of the mouse and closely related to human whipworm, Heligomosoides polygyrus, and Trichinella spiralis, as well as N. brasiliensis, the rat hookworm. In N. brasiliensis infection, parasites infect mice through the skin, migrate to the lungs, are coughed up, swallowed, develop into adults in the gut lumen, produce eggs that are excreted in the feces, and are themselves expelled approximately 10 days postinfection (18). Although studies show the expulsion mechanism requires a CD4+ Th2 cell response (19), IL-4 itself is not critical because anti-IL-4 mAbtreated and IL-4−/− mice are as resistant to infection as WT animals (20, 21). As such, these studies were the first to suggest that another Th2 cell-derived mediator might be more important for worm expulsion. IL-13 was a likely candidate because IL-4Rα-deficient, Stat6-deficient, and IL-4/IL-13-deficient mice are all much more susceptible than animals deficient in IL-4 alone (7, 8, 13). Studies conducted with a soluble IL-13 antagonist (8) and in IL-13-deficient mice (11) confirmed the critical and nonredundant role of IL-13 in immunity to N. brasiliensis. Thus, although exogenous rIL-4 (22) and rIL-13 (7, 11) are both capable of stimulating resistance in susceptible hosts, the blocking and knockout studies conducted in resistant animals suggested IL-13 is the more important endogenous mediator of resistance. Moreover, the fact that IL-13 is capable of stimulating parasite expulsion even in immunodeficient RAG2−/− mice (7) suggests IL-13 mediates parasite expulsion by directly activating a yet undefined nonlymphoid cell within the gut. In support of this conclusion, recent evidence suggests expression of IL-4Rα by non–bone marrow–derived gastrointestinal cells is sufficient for parasite elimination (23). In contrast to N. brasiliensis infection, where IL-13 clearly plays a superior role to IL-4, both cytokines appear to be necessary for optimal immunity to T. muris. T. muris and H. polygyrus are both transmitted by the oral-fecal route independently of an intermediate host, and, in some strains of mice, they cause chronic infections. In the case of T. muris, susceptibility depends on the mouse strain, where some animals reject the parasite shortly after exposure and others develop long-lived infections (24). In this system, resistant mice express type 2 responses, susceptible mice mount type 1 responses, and IL-12 can induce a switch from a protective type 2 to an infection-permissive type 1 response. Similarly, in normally susceptible strains, in vivo depletion of IFN-γ or IL-12 allows the expansion of a type-2 response and effective clearance of infection (24). In contrast to T. muris, most strains of mice are susceptible to a primary H. polygyrus infection, but following drug clearance animals exhibit a strong type 2 response and are highly resistant to secondary infections. Similar to N. brasiliensis infection, CD4+ T cells are required for the induction and/or expression of immunity to both H. polygyrus and T. muris (24). Exogenous IL-4 can cure primary infections with T. muris and H. polygyrus, anti-IL-4R mAb blocks resistance to both, and IL4−/− mice are susceptible to a challenge infection with H. polygyrus or a primary infection with T. muris (24, 25). The relative role of IL-4, however, depends on

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the background of the mouse because C57BL/6 IL-4 KO mice develop chronic T. muris infections, whereas some BALB/c IL-4 KO mice clear their infections (26–28). This latter finding suggested an IL-4-independent role for IL-13 in resistance, which was confirmed by treating BALB/c IL-4-deficient mice with an IL-13 antagonist (27). A recent study suggested differences in IFN-γ production in C57BL/6 versus BALB/c mice likely dictate the relative importance of IL-13 and/or IL-4 in T. muris immunity (29). Indeed, chronic infections are also established in IL-13-deficient mice despite the fact that they develop relatively normal IL-4 responses (26), which thus further emphasizes the cooperative and additive roles of IL-4 and IL-13 in immunity to T. muris. Similar to what is observed during T. muris and N. brasiliensis infection, IFN-γ also antagonizes the actions of IL-4 and IL-13 in T. spiralis infection, effectively suppressing parasite expulsion. In contrast to T. muris infection, however, expulsion of T. spiralis proceeds normally in the absence of IL-4 or IL-13. Thus, expulsion is prevented only in the combined absence of both cytokines (30). Spontaneous T. spiralis expulsion also requires expression of IL-4Rα and Stat6 (30, 31). As such, many observations made with T. spiralis closely resemble findings with N. brasiliensis, except IL-13 is clearly more important than IL-4 for expulsion of the latter. In contrast to N. brasiliensis, however, where IL-13 appears to have direct effects on the gut, T. spiralis expulsion appears to be mediated indirectly by IL-13, IL-4Rα, and Stat6 by enhancing IL-13 and IL-4 and suppressing IFN-γ production; this, in turn, enhances the intestinal mast cell response required for T. spiralis expulsion (30). Extrapolation of the findings from these various experimental systems to state that resistance to intestinal nematodes requires Th2-mediated responses is supported by recent studies that provide new insight as to how IL-4 and IL-13 actually function to mediate protection. In uninfected mice, IL-4 has dramatic effects on intestinal physiology causing increased mucosal permeability and reduced sodiumlinked glucose absorption (18, 32). Muscle hypercontractility and goblet-cell hyperplasia are also regulated in the gut by IL-4 and IL-13 and are attenuated by IL-12 treatment, possibly implicating them in the resistance mechanism (31–35). For the most part, these protective responses appear to be T cell and mast cell dependent (18). Their net effect is to trap parasites in mucus within the gut lumen, to increase intestinal fluid content, and to increase contractility, all of which facilitate expulsion of parasites via a “weep and sweep” mechanism (18, 32). Recent studies indicate that many of these properties are shared by IL-13 (36). Indeed, IL-13 has a greater effect on intestinal smooth muscle contractility than IL-4 (37). The hypercontractility induced by IL-13 is mediated by direct effects on the smooth muscle cell as well as by an action on enteric nerves.

Regulation of Intracellular Parasitism Although control of many extracellular parasitic infections requires a Th2-polarized immune response, resistance to most intracellular parasites requires a strong cellmediated response, where CD4+ Th1 effector cells play a prominent role. The

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immune response to Leishmania parasites is no exception and is arguably one of the most well-studied systems in the field of immunoparasitology. Infection with Leishmania major in susceptible BALB/c mice results in a Th2-dominant immune response, progressive disease, and eventual death (38). Most other strains of mice develop strong Th1 responses, and consequently, they kill the majority of parasites within the inoculation site and fail to develop significant lesions. Thus, low-grade infection without significant disease is the most common outcome following infection with L. major (39). Early studies suggested IL-4 was likely the key factor influencing resistance versus susceptibility (38); however, a plethora of studies now indicate several host genes are involved (40–42), and the parasite substrain and/or Leishmania species also play a significant role (43, 44). Localized cutaneous leishmaniasis lesions express high levels of IL-13 (45), and over 50% of patients with visceral leishmaniasis have detectable levels of IL13 in their serum, whereas many fewer have a detectable IL-4 response (46). These observations provided, from infected humans, evidence of a possible regulatory role for IL-13 in disease progression, which was recently investigated in a series of experimental studies where L. major infection was compared in BALB/c IL-4-, IL-4Rα-, Stat6-, and IL-13-deficient mice (43, 44, 47–50). In one study, genetically pure BALB/c IL-4−/− and IL-4Rα −/− mice were fully susceptible to LV39, whereas IL-4−/− mice were partially resistant and IL-4Rα −/− mice were highly resistant to the IR173 substrain (43). This finding provided the first experimental evidence linking IL-13 with nonhealing infections in BALB/c mice. Subsequent studies conducted in BALB/c IL-13- and IL-4/IL-13-deficient mice confirmed the role of IL-13, which is important because they showed that there is an additive protective effect of deleting IL-4 and IL-13 (49). However, it remains unclear how ubiquitous the contribution of IL-13 will be because growth of the LV39 substrain was little affected by IL-4Rα deficiency (43). Moreover, studies conducted with a second cutaneous leishmaniasis strain, L. mexicana, demonstrated no role for IL-13 in susceptibility (44), although this finding is disputed in a more recent study (51). These findings suggest other susceptibility factors, such as IL-10 and TGF-beta (39, 52), may also play significant roles during Leishmania infection, as suggested by genetic linkage studies. In contrast to the findings of Noben-Trauth et al. (43), a protective role for IL-13 was postulated in a second study comparing L. major infection in BALB/c IL-4−/− and IL-4Rα −/− mice (48). However, in this study, the authors’ interesting findings were revealed in the chronic phase of infection, when additional factors are believed to be needed for the complete healing and elimination of parasites. In these studies, IL-4Rα −/−, but not IL-4−/−, mice infected with the LV39 strain showed signs of progressive and disseminating lethal disease approximately 80 days postinfection. Prior to this point in time, however, both strains of mice appeared to be fully resistant and effectively restricted parasite growth. The different outcomes displayed between IL-4−/− and IL-4Rα −/− mice suggest that IL-13-mediated effector functions are involved in controlling dissemination of the parasite in chronic leishmaniasis. It remains unclear, however, why IL-13 displays an exacerbative role in

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the early phases of some leishmanial infections (43, 51) and protective activity in the chronic phase (48), but it will likely prove an exciting area of future research. It is interesting that a similar protective role for IL-13 was also reported in mice infected with the intracellular pathogen, Listeria monocytogenes (53). In this case, rIL-13 increased IL-12 p40 and p70 production by infected bone marrow–derived macrophages, implicating IL-13 in the initiation and/or maintenance of protective cell-mediated immunity.


Inflammatory Diseases of the Lung One of the areas where the in vivo biological activity of IL-13 has been investigated intensively is the lung; many important findings have come out of this research in the past few years alone. Several distinct disease models have been studied in this regard including models of pathogen-induced pulmonary inflammation (54, 55), asthma (9, 56, 57), anaphylaxis (58), hyperoxic acute lung injury (59), emphysema, and chronic obstructive pulmonary disease (COPD) (60). When viewed together, it is clear that IL-13 plays a critical and nonredundant role in the pathophysiology of the lung. One of the first studies to report pulmonary expression of IL-13 was an experimental model of Th2-mediated granulomatous disease (61). In this model, eggs of the helminth parasite Schistosoma mansoni are injected intravenously into na¨ıve or egg-sensitized mice, and the animals mount a vigorous granulomatous response in the lung. Consistent with many helminthic diseases, Th2-type cytokines dominate the response, and studies conducted with exogenous IL-12 showed the Th2-type response is critical for normal egg-induced lesion formation (61). Since then, at least three separate groups using different approaches have attempted to dissect the individual contributions of IL-4 and IL-13 in pulmonary granuloma formation (13, 16, 54). In one study, sIL-13Rα2-Fc treatment reduced peak granuloma size in both na¨ıve and egg-sensitized mice by ∼50% (54). However, when similar experiments were conducted in IL-4-deficient mice, granuloma formation and tissue eosinophilia were almost completely ablated, which demonstrated that IL-4 and IL-13 play additive roles in pulmonary lesion formation. Similar conclusions were generated in studies using IL-4-, IL-13-, and IL-4/IL-13-deficient mice (13) or anti-IL-13 mAbs (16). The individual roles of IL-13 and IL-4 in the pathogenesis of asthma have also been the subject of several research programs in the past few years. Previous studies showed that although IL-4 drives Th2 cell development, it does not appear to be necessary for the expression of allergic asthma, which suggests a more important role for other Th2 family members (62). To elucidate the various effector functions of IL-13, Zhu and colleagues engineered a mouse that specifically overexpresses IL-13 in the lung (9). These mice, in contrast to their transgene negative littermates, developed several characteristics of asthma including pulmonary eosinophilia, airway epithelial cell hyperplasia, mucus cell metaplasia, subepithelial fibrosis, Charcot-Leyden-like crystals, airways obstruction, and nonspecific

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airways hyperresponsiveness to cholinergic stimulation. IL-13 blockade inhibited many of the same characteristics in allergen-immunized wild-type mice, which is important because it reveals for the first time the critical and nonredundant role of IL-13 in allergen-induced AHR (56, 57). Thus, although exogenous IL-4 and IL-13 are both capable of inducing an asthma-like phenotype (56, 57, 63), as reported with some of the gastrointestinal parasites (8, 11), the effector activity IL-13 appears to be superior to that of IL-4. Given the overall importance of these findings, the mechanism underlying the IL-13-dependent expression of AHR has been a major research focus. Because IL-4 and IL-13 both utilize the IL-4Rα chain for signaling, it was probably not unexpected that the activity of IL-13, at least in these acute asthma models, is abrogated in IL-4Rα- and Stat6-deficient animals (57, 64–67). One of the more important early findings demonstrated that exogenous IL-13 can also induce AHR in the absence of T and B cells (57). This finding was one of the first to suggest a direct role for IL-13, which has been substantiated in several other studies (68–71). Eosinophils have also been implicated as primary effector cells in asthma and asthmatic AHR; however, allergen-induced pulmonary eosinophilia was not significantly affected in one of the studies using sIL-13Rα2-Fc treatment (56), potentially lessening their overall importance to AHR. Antigen-specific IgE was also unchanged in IL-13 antagonist-treated mice (56), indicating that AHR is not dependent on IgE production; these findings are consistent with studies conducted in IgE-deficient mice (72). Subsequent AHR studies conducted in IL-13-deficient mice have suggested that IL-13 might instead play more of an additive role with IL-4 (73); however, these findings remain somewhat controversial (74). It may also prove inappropriate to directly compare results generated with IL-13 antagonists and IL-13-deficient mice because the latter animals may display a partial defect in the closely linked IL-4 gene (17). Nevertheless, the findings from IL-13-deficient mice have confirmed the importance of the IL-13-, IL-4Rα-, Stat6-dependent pathway in the induction of AHR. In one study, AHR failed to develop in Ovalbumin (OVA)-sensitized and challenged IL-13−/− mice, despite the presence of a strong Th2-biased pulmonary eosinophil response (74). Administration of rIL-13, however, completely restored AHR in the knockout animals. Moreover, OVA-specific Th2 cells derived from TCR-transgenic IL-13−/− mice failed to induce AHR in recipient SCID mice even though they produced high levels of IL-4 and IL-5 and had a significant eosinophilic infiltrate. These studies suggested that IL-13 was, by itself, necessary and sufficient to induce AHR. Nevertheless, a second study using IL-13−/− mice showed that it is possible for AHR and pulmonary eosinophilia to develop in the absence of IL-13 (73). Here, AHR was reduced in IL-13−/− mice only when they were treated with either anti-IL-4 or anti-IL-5 mAbs, which suggests additive roles for IL-13, IL-4, IL-5, and tissue eosinophilia in the development of AHR. Consistent with findings that suggest a direct role for IL-13 in AHR, a recent study showed that IL-13-induced AHR and mucus secretion can occur independently of IL-5 and/or the eosinophil chemoattractant eotaxin (67). It is important

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to note this study also showed AHR-inducing activity for IL-13 in the early preinflammatory phase, which is characterized by an absence of inflammation (eosinophilia) and mucus hypersecretion. Thus, although IL-13-induced recruitment of eosinophils to lung requires IL-5 and eotaxin (75), the eosinophil regulatory circuit does not appear to be necessary for the development of AHR or mucus hypersecretion when IL-13 is abundant. Nevertheless, subsequent studies have suggested that IL-5 and eotaxin may both be required to maintain IL-13 production in CD4+ Th2 cells (76). Indeed, the ability of Th2 cells to produce IL-13 was significantly impaired in mice deficient in IL-5/eotaxin. However, this T cell IL-13 defect was overcome when eosinophils were transferred to the double knockout mice. Thus, the IL-5, eotaxin, eosinophil regulatory circuit is not necessarily a dispensable effector pathway in allergic disease because it appears to play a central role in regulating production of the key effector cytokine, IL-13. It is surprising that, although many features of allergic airway disease are IL4Rα chain- and Stat6-dependent, at least two recent papers have shown that IL13 may also function in the absence of these downstream mediators (77, 78), which possibly suggests a novel pathway for IL-13 signaling. In one study, IL13-sufficient and IL-13-deficient OVA-specific T cells were adoptively transferred to na¨ıve BALB/c WT and IL-4Rα −/− mice concurrently with OVA delivery to the airways (77). The IL-13-expressing T cells were found unexpectedly to induce several features of asthma independently of the IL-4Rα chain. The IL-13+/+ cells were, however, completely ineffective in Stat6-deficient mice. Together, these observations suggest a novel IL-13 receptor signaling system must exist to modulate allergic disease, which does not require the IL-4Rα chain but utilizes the Stat6-signaling cascade. This is consistent with a recent study that suggested Stat6-deficient mice are protected from all pulmonary effects of IL-13. However, reconstitution of Stat6 only in epithelial cells was sufficient for IL-13-induced AHR and mucus production (79). These findings argued for direct IL-13/Stat6-induced effects on epithelial cells. Nevertheless, a separate study of chronic fungal asthma suggested that a Stat6-independent but IL-13-dependent pathway might also exist (78). Thus, though nearly every published study points to IL-13 as a key effector cytokine in asthma, the downstream events and cellular targets of IL-13 remain somewhat unclear and will certainly be an exciting research focus in the coming years.

Cancer Another important area of research is in the field of cancer where depending on the tumor cell type in question, targeting IL-13 or IL-13 receptors may either inhibit or promote tumor cell growth. Early in vivo studies showed potent antitumor activity for both IL-4 and IL-13 in mice. IL-4 inhibited the proliferation of several human cancer cell lines, including various hematologic malignancies as well as non–small cell lung cancer, colon cancer, head and neck carcinomas, and glioblastomas (80). Similar antiproliferative effects on human breast cancer cells, renal cell carcinoma, and B-lineage acute lymphoblastic leukemia were also shown for IL-13 (81–84). In one study, the growth inhibition mediated by IL-13 was completely reversible

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by anti-IL-4 receptor antibody treatment (84), which demonstrated that a classical type II IL-4 receptor and a Stat6-signaling cascade were involved. In another study, mice injected with transfected P815 mastocytoma cells secreting large amounts of IL-13 rejected their tumors and developed systemic specific antitumor immunity leading to long-lasting specific antitumor protection. In vivo, overexpression of the IL-13Rα2 chain in the tumor inhibits the tumorigenicity of some breast and pancreatic cancers in immunodeficient mice (85). In each case, activation of specific and/or nonspecific antitumor defenses by IL-13 or IL-13Rα2 was postulated as the primary explanation for the observed antitumor response (86). Nevertheless, subversion of host antitumor defenses has also been demonstrated for IL-13. Type 1 T cell–mediated host defenses are widely believed to mediate optimal tumor rejection in vivo. Therefore, deviation toward a more dominant Th2type response has been postulated as a mechanism that might block tumor rejection and/or promote tumor recurrence (87). In support of this hypothesis, three recent studies using transplantable tumor cell lines demonstrated that Stat6, IL-4, and IL-13 were capable of inhibiting tumor rejection (88–90). In contrast to wild-type mice that showed progressive growth of their tumors, Stat6-deficient mice were highly resistant and spontaneously rejected the transplanted tumor cells. Enhancement of tumor-specific IFN-γ production and CTL activity in the absence of Stat6 was proposed as the primary explanation for the potent antitumor activity. In one of the studies, the authors were investigating the mechanisms regulating tumor immunosurveillance (88). In this model, tumors spontaneously regress after initial growth; however, within 20 to 40 days, they recur. Here, the mechanism of tumor regression is mediated by CD8+ cytotoxic T lymphocytes specific for a model tumor antigen HIV gp160. These authors showed that IL-4Rα- and Stat6-, but not IL-4-deficient, animals were highly resistant to tumor recurrence, which implicated IL-13 as the primary mediator. When susceptible WT or IL-4-deficient mice were treated with IL-13 inhibitor, tumor recurrence was almost completely blocked (88). It is interesting that loss of natural killer T cells (NKT cells) in CD1-deficient mice resulted in decreased IL-13 production and resistance to recurrence. Thus, IL-13, produced in part by NKT cells, appears to antagonize tumor immunosurveillance. As such, these findings suggest IL-13 inhibitors or novel IL-13 antagonists may prove highly effective as cancer immunotherapeutics. In addition to boosting type-1-associated antitumor defenses, in some situations IL-13 inhibitors may also block tumor cell growth more directly. Such is the case with B chronic lymphocytic leukemia (B-CLL) and Hodgkin’s disease, where IL-13 either blocks apoptosis or promotes tumor cell proliferation (91, 92). In the case of B-CLL, IL-13 does not exhibit direct growth factor activity but instead protects B-CLL cells from in vitro spontaneous apoptosis (2, 91, 93). Thus, IL-13 may be an important factor in the pathogenesis of CLL by preventing the death of neoplastic cells. In Hodgkin’s disease, a condition that mostly affects young adults, tumors are typically detected in lymph nodes in the neck and chest, with eventual spread throughout the lymphatic system. The cancer is characterized by the presence of Hodgkin/Reed-Sternberg cells (H/RS), which are large multinucleated cells that rarely occur throughout the lymph tissue. In a large majority of cases, the

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malignant cell population arises from B cells. Several Hodgkin’s disease cell lines overexpress IL-13 and IL-13 receptors. Lymph node tissues taken from Hodgkin’s disease patients also exhibit high levels of the cytokine (92, 94–96). Constitutive activation of Stat6 was also reported in Hodgkin’s lymphoma cell lines (97). More importantly, however, neutralizing anti-IL-13 mAbs or IL-13 antagonists inhibited H/RS cell proliferation in a dose-dependent manner (92, 96). Increased apoptosis was also observed following IL-13 blockade. Thus, the available evidence suggests IL-13 stimulates the proliferation of H/RS cells in an autocrine fashion (92, 98). As such, neutralization of IL-13 may represent an attractive and highly effective treatment for Hodgkin’s lymphoma and other B cell–associated cancers by inhibiting tumor cell growth and simultaneously enhancing antitumor defenses (88–90). Given that many tumor cell types overexpress IL-4/IL-13 receptors, the receptors themselves have also been exploited as possible targets for cancer therapy. By linking human IL-13 with a mutated form of Pseudomonas exotoxin (99), Puri and colleagues have developed a novel fusion protein that is highly cytotoxic to several IL-13 receptor-positive tumor cells, including glial, renal, AIDS-associated Kaposi’s tumors, squamous cell carcinoma of head and neck and prostate cancer cells, as well as several human epithelial carcinomas such as adenocarcinoma of the stomach, colon, and skin (14, 100–105). In this system, the toxin kills tumor cells but only after they internalize the ligand-receptor complex. The cytotoxic action of the IL-13-toxin is blocked when excess human IL-13 is included, which further confirms the high specificity of the reaction. With some tumor cell types, the IL-13toxin appears to be more effective than a similarly engineered IL-4 chimeric protein because the tumors preferentially express high levels of IL-13Rα2 (82, 106–108). A mutation was also recently engineered in the IL-13 portion of the fusion protein to alter its interaction with the IL-13 receptor, which decreased its toxicity on normal tissues and simultaneously enhanced its antitumor activity (109, 110). Thus, targeting the IL-13/IL-13 receptor system may prove highly effective in the treatment of several malignancies. Nevertheless, given that IL-13 can exhibit both proand antitumorigenic activity, the exact role of IL-13 must be carefully dissected in each case so that the most appropriate IL-13-based strategy is implemented.

Tissue Remodeling and Fibrosis In the helminth infection, Schistosoma mansoni, adult parasites reside in the mesenteric veins where they lay hundreds of eggs per day. Some of the eggs become entrapped in the microvasculature of the liver, and once there, they induce a granulomatous response (111). Subsequently, fibrosis and portal hypertension may develop, which is the primary cause of morbidity in infected individuals and in some cases may be lethal (112). Consequently, much of the symptomatology of schistosomiasis is attributed to the egg-induced inflammatory response and associated fibrotic liver pathology. CD4+ Th cells are essential for granuloma formation, and early studies examining the respective roles of Th1- and Th2-associated cytokines showed that the granulomatous response evolves from early Th1 to sustained and dominant

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Th2-type cytokine response (113, 114). The importance of Th2 cells to this pathologic process was shown by experiments in which mice vaccinated with egg antigen plus IL-12, to induce an egg-specific Th1 response upon subsequent infection, developed smaller granulomas and less severe fibrosis than did nonvaccinated infected controls (61, 115). Decreased fibrosis was associated with a diminished IL-4/IL-13 response and increased type-1 cytokine production. Although Th2mediated pathology is ultimately detrimental to the host, it is also clear that granulomas serve an important host-protective function during infection. In chronically infected hosts, schistosome eggs provide a continuous antigenic stimulus for the immune response. If these antigens are not sequestered or neutralized effectively, they may damage host tissues, the liver being particularly sensitive. In support of this conclusion, T cell–deprived, nude, SCID, egg-tolerized, and some IL-4−/− and IL-10−/− mice die earlier than comparably infected, immunologically intact control mice because they are unable to satisfactorily mount a normal Th2-dominant response (116–119). Widespread microvesicular hepatic damage induced by toxic egg products contributes to the poorer survival of the infected immunosuppressed mice. Presumably, the chronic detrimental effects associated with egg granulomas (e.g., fibrosis and portal hypertension) represent a compromise solution for the host and parasite because the parasite will only survive when the host is effectively protected from these hepatotoxins. Nevertheless, although the Th2 response plays a host-protective role, persistent expression of these mediators will also scar the liver, which eventually contributes to the development of hepatosplenic disease. The Th2 response, therefore, represents a double-edged sword by exhibiting protective and host-damaging activities during acute and chronic infection, respectively. It is surprising that early IL-4 ablation experiments and studies with IL-4-deficient mice failed to demonstrate a significant role for IL-4 in the development of fibrosis (111, 120). In contrast, granuloma size and fibrosis markedly decreased in Stat6- and IL-4Rα −/− animals (121, 122), implicating IL-13 (54). Microscopic, biochemical, and molecular techniques all indicated that IL-13, but not IL-4, plays the major role in the development of egg-induced fibrosis (123, 124). Earlier studies showed that perturbations in the Th1/Th2 cytokine balance can also significantly affect the extent of tissue fibrosis in S. mansoni–infected mice (115). However, the IL-13 blocking studies suggested the effects of IL-13 might be more direct. Similar conclusions were drawn from studies conducted in S. mansoni– infected IL-13 and IL-4/IL-13-deficient mice (125). Mortality was delayed significantly in infected IL-13-deficient animals, further highlighting the major contribution of IL-13 to the pathogenesis of schistosomiasis. In vitro studies showed that IL-13 is a potent activator of collagen production in fibroblasts (123). Thus, the effects of IL-13 on fibrosis may indeed be direct and not dependent upon induction of other profibrotic mediators or perturbations in the Th1/Th2 cytokine response. Studies conducted with IL-13 on normal human skin and keloid fibroblasts also suggested a direct role for IL-13 in collagen production (126). Additional studies are needed, however, to determine whether other mediators are involved, such as TGF-β, as suggested in a murine asthma model (127), and whether granulomaderived fibroblasts exposed to IL-13 exhibit similar collagen-producing activity.

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Although activated fibroblasts are the primary producers of collagen, recent studies suggest macrophages and dendritic cells may be equally important mediators of IL-13-induced fibrosis (128) and Th2-mediated inflammation in general (129). Macrophages possess two cytokine-inducible enzymes, NOS-2 and arginase (Arg-1), which share L-arginine as a substrate. Modolell and colleagues showed that Th1-type cytokines activate NOS-2 in macrophages designated “classically activated,” whereas the Th2-type cytokines IL-4 and IL-13 stimulate Arg-1 activity in macrophages termed alternatively activated (130). Similar observations were generated with dendritic cells (131). Arginase uses L-arginine as a substrate to make L-ornithine, which is converted to proline by ornithine-aminotransferase and to polyamines by ornithine decarboxylase. Proline is an essential amino acid involved in collagen production and, therefore, is necessary for the development of fibrosis. Fibrosis is inhibited in mice sensitized with egg antigens plus IL-12 (115), which appears to be owing to the induction of NOS-2 rather than arginase. NOS-2 uses arginine to make nitric oxide (NO) and citrulline. An intermediate product in this pathway, L-hydroxyarginine is also a potent inhibitor of arginase. Thus, in Th1-polarizing conditions, consumption of arginine by NOS-2 is increased, which blocks arginase, effectively reducing the amount of proline that is available for collagen synthesis (128). The IL-12 sensitization protocol is completely ineffective in NOS-2-deficient mice despite induction of a strong Th1 response (132). Here, competition for arginine by NOS-2 and production of the arginase inhibitor Lhydroxyarginine is eliminated, thus allowing for unobstructed arginase activity and a vigorous fibrotic response. These findings are particularly exciting because they provide a mechanistic explanation for the induction of fibrosis by IL-13 by showing a direct connection between the immune response (Th1 versus Th2), fibroblasts, and the arginine metabolic pathway in macrophages and dendritic cells (Figure 1). The link between IL-13 and fibrosis revealed in the murine schistosomiasis model may also extend to other Th2-associated diseases. Indeed, tissue −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→ Figure 1 IL-13 effector functions.Type-1 cytokines stimulate nitric oxide production in classically activated macrophages, whereas IL-13 produced by multiple cell types preferentially activates Arginase-1 and production of L-ornithine in alternatively activated cells. L-ornithine serves as substrate for ornithine-decarboxylase (ODC) and ornithine-amino transferase (OAT), which generate polyamines and proline, respectively. Proline is an essential amino acid used in collagen production. Fibroblasts are another significant target of IL-13 activity. IL-13 may act directly on fibroblasts or indirectly by stimulating and activating TGF-β production in macrophages. In addition, smooth muscle, mucus-producing cells, B cells, endothelium, and epithelium are also important target cells that ultimately regulate a wide-variety of IL-13-associated effector functions. The type-2 cytokine response also upregulates IL-13Rα2 expression (IL-13 decoy receptor), which dampens IL-13 effector activity. In contrast, the type-1 response appears to antagonize IL-13Rα2 expression in vivo, which may increase IL-13 effector activity when IL-13 levels are low or when mixed Th1/Th2 responses prevail.


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remodeling and fibrosis are important pathological features in a variety of disorders where type-2 cytokines dominate, including asthma (9, 55), idiopathic pulmonary fibrosis (133), chronic graft rejection (134), bleomycin-induced pulmonary fibrosis (135), progressive systemic sclerosis (136), radiation-induced pulmonary fibrosis (137), and hepatic fibrosis (123, 125). Therefore, IL-13 antagonists may prove highly effective in a variety of situations where chronic exposure to IL-13 triggers excessive healing, tissue remodeling, or the formation of destructive tissue pathology.

REGULATION AND SUPPRESSION OF IL-13-MEDIATED EFFECTOR FUNCTION The studies discussed above illustrate the pivotal role of IL-13 in the regulation of type-2 cytokine-mediated immune responses. In some pathological conditions, IL-13 is either under- or overexpressed resulting in decreased resistance to infection, uncontrolled tumor cell growth, severe asthma, or destructive inflammation and tissue scarring. Thus, understanding the molecular mechanisms that govern the expression and/or functional activity of IL-13 is of critical importance. Several inhibitory pathways have been described, which may offer novel therapeutic strategies to treat infectious, malignant, and inflammatory diseases. The following sections outline a few of the important endogenous IL-13 regulatory systems.

IL-4 IL-4 is the central differentiation factor for Th2 response development (138). In the absence of IL-4, Th2 responses are significantly impaired, although, in some studies, low yet significant production of IL-5 and IL-13 has been reported, even in the absence of the IL-4 receptor and Stat6 signaling molecule (6, 139, 140). In contrast, IL-13 is not thought to play a significant role primarily because T cells lack the appropriate receptors (141, 142). Nevertheless, IL-13 may participate indirectly because impaired Th2 response generation was also reported in some but not all studies utilizing IL-13 knockout mice (12, 26, 125). The discovery of IL-4-independent IL-13 production, however, was significant because it provided one of the first clues that IL-13 might be the critical Th2 effector cytokine in vivo. It also helped explain some of the divergent findings reported with IL-4- and IL-4Rα-deficient mice. Indeed, although IL-13 production is markedly decreased in the absence of IL-4, the residual IL-13 response remains highly active as explained in many of the disease models discussed above (8, 43, 48, 56, 88, 123). Even a small amount of IL-13 is sufficient to mediate many of its in vivo effector functions. In the murine schistosomiasis model, for example, IL-13 production decreases over 80% in the absence of IL-4 (123). Nevertheless, granuloma formation proceeds normally in these IL-4-deficient animals unless IL-13 is also neutralized. Thus, although targeting IL-4 will likely yield a lower IL-13 response, in many

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situations both cytokines must be neutralized to effectively block effector activity completely. This may account for the efficacy of soluble IL-4 receptor for the treatment of moderate persistent asthma (143). IL-13 is produced at much greater levels than IL-4, which may also explain why IL-13 operates as the key effector cytokine in vivo (16, 45, 46, 54, 123).


IL-12, IL-18, IFN-γ , and Immunostimulatory CpG Oligodeoxynucleotides Many of the cytokines and mediators that drive Th1 cell development are potent inhibitors of IL-13 production and/or activity. This was perhaps exemplified best in early studies examining the mechanisms of nematode expulsion, asthma, and schistosome egg-induced granuloma formation. In schistosomiasis, the pulmonary egg-induced granulomatous response evolves from an early Th0-like phase to a strong Th2 response, which peaks approximately 7 to 10 days post-primary iv egg challenge (113, 114). In the early phase, high levels of IFN-γ and IL-12 are detectable; both cytokines play important regulatory roles. IL-12 is a potent stimulus for T cell– and NK cell–derived IFN-γ and inhibits T cell production of IL-4 (144). In support of these observations, mAb depletion of IFN-γ or IL-12 augmented IL-4/IL-13 levels and increased pulmonary granuloma formation (61). A similar enhancement was observed when antiasialo GM1 antibodies were administered, which suggested the early IFN-γ response was derived from an NK cell source. In contrast, exogenous treatment with rIL-12 profoundly inhibited primary granuloma formation and IL-4/IL-13 production, while increasing IFN-γ and IL-12 levels. IFN-γ was of critical importance because similar IL-12 treatments in IFN-γ -deficient mice not only failed to inhibit granuloma formation but also actually caused a marked exacerbation in the response (145). Similar results were generated with the N. brasiliensis model, where exogenous IL-12 inhibited parasite expulsion and anti-IFN-γ mAb treatment blocked IL-12 activity (146). Exogenous IL-12 also reduced secondary granuloma formation and IL-13 production in schistosome egg-sensitized mice, which demonstrated a role for the cytokine in reversing established Th2-type responses (61). Mice immunized with egg antigens plus IL-12, to establish a memory Th1 response, were also protected from secondary granuloma formation (61), and, more importantly, displayed a marked reduction in IL-13-induced fibrosis when subsequently infected (115). These findings suggested that IL-12 provides an effective strategy to prophylactically inhibit IL-13-driven pathologies. In agreement with the parasite models, IL-12 inhibits antigen-induced airway hyperresponsiveness, inflammation, and Th2 cytokine expression in a mouse model of asthma (147). Here again, the effects of IL-12 were dependent on IFN-γ because concurrent treatment with IL-12 and anti-IFN-γ mAb partially reversed the inhibition of airway hyperresponsiveness and eosinophilia by IL-12. There was also evidence that IL-12 was active during ongoing secondary responses, which provides further proof that IL-12 and immune-deviation might be highly effective

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for the treatment of allergic disease. Similar changes were observed when IL-12 was substituted with CpG oligodeoxynucleotides (CpG ODN) (148, 149), which are potent inducers of Th1-associated cytokines (150). In each case, the protective effects were highly dependent on the induction of IFN-γ and simultaneous suppression of IL-13 activity (124, 151). Given that IL-18 was originally described as an interferon-gamma-inducing factor (IGIF) (152), it was probably not unexpected that IL-18 showed potent Th2-inhibiting activity (153–155). Nevertheless, the role of IL-18 in the regulation of Th2 responses remains somewhat controversial (156). IL-18 is secreted by activated macrophages and can act synergistically with IL-12 to induce high levels of IFN-γ production in T cells (152). Consistent with its role as a Th1-inducing cytokine, treatment of OVA-sensitized mice with exogenous IL-18 or with an IL-18-expressing adenovirus reduced allergen-specific airway eosinophilia (154), IL-4 production, and mucus hypersecretion; increased IFN-γ ; and prevented the development of AHR (155). In contrast, mice deficient in IL-18 showed an enhancement in allergen-induced eosinophilia (154). The effects of IL-18 were also highly dependent on IFN-γ and IL-12 (155), which provides evidence of a regulatory loop in which IL-12 and IL-18 act in concert with IFN-γ to antagonize the allergy-inducing activities of IL-13. Nevertheless, this inhibitory activity may depend on the cytokine milieu (156). Indeed, other studies suggest that IL-18 may exhibit Th2-promoting activities (157–160), particularly when produced in the absence of IL-12. In one study, basophils and mast cells expressed IL-18 receptors and produced large amounts of IL-4 and IL-13 when stimulated with IL-3 and IL-18 (157). A similar response was also observed when IL-18 was injected in vivo. The addition of IL-12, however, blocked the type-2 cytokine promoting activity of IL-18 and in an IFN-γ -dependent fashion. Consistent with these findings, administration of IL-18 in conjunction with allergic sensitization and challenge increased serum levels of IgE and splenocyte production of Th2-type cytokines (159). Moreover, while eosinophil recruitment decreased early after IL-18 treatment, as the response matured, lung eosinophilia increased dramatically, which demonstrated again that IL-18 was promoting a Th2-type response in vivo. The IL-18-induced IgE response was also CD4+ T cell-, IL-4-, and Stat6-dependent (158), and in a related study, eosinophil recruitment triggered by IL-18 was at least partially dependent on TNF-α (160). Nevertheless, it should be emphasized again that in some settings, IL-18 exhibits potent Th2-suppressing activity. In the T. muris model, resistance clearly depends on IL-4 and IL-13, and mice treated with IL-18 displayed decreased IL-4/IL13 levels (161). It is surprising, however, that the IL-18-treated animals failed to develop an upregulated IFN-γ response, and IFN-γ -deficient mice remained susceptible to the Th2-suppressing activities of IL-18, which suggested IL-18 might directly inhibit IL-4 and IL-13 (161). Thus, when viewed together, the results from all of these studies clearly indicate that, under the right circumstances, IL-18 may either inhibit or promote IL-13 activity. The factors that influence this decision, however, remain unclear, although IL-12 may be a key player.

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TNF-α Like IL-18, the role of TNF-α may be context dependent in that it has been shown to both promote and antagonize IL-13-driven responses. In the T. muris model, in vivo blockade of TNF-α in mice that normally mount a protective IL-4/IL-13 response significantly delayed worm expulsion for the duration of the treatment (28). IL-13mediated worm expulsion in IL-4-deficient mice was also TNF-α dependent and enhanced by exogenous treatment with TNF-α. TNF receptor-deficient mice were also more susceptible, and there was evidence that the protective Th2 response was being skewed to a susceptible Th1 phenotype in the absence of TNF-α signaling (28). The results from these studies were the first to demonstrate a role for TNF-α in regulating Th2-type cytokine responses at mucosal sites. They were also the first to directly implicate TNF-α as an important protective cytokine during helminth infection. Nevertheless, the protection mediated by TNF-α appears to be entirely attributable to its Th2-promoting activity because high levels of TNF-α are produced in Th1-deviated mice; and yet, these mice remain highly susceptible to T. muris infection (33). In schistosomiasis, granuloma formation and fibrosis are also IL-4/IL-13-dependent and reduced in mice pretreated with schistosome eggs and IL-12 (115). However, if TNF-α is neutralized at the time of egg laying, IL4/IL-13 production, granuloma formation, and fibrosis are rapidly restored, which suggests TNF-α is necessary to maintain the protective Th1 response (162). Nevertheless, TNF-α is also expressed in mice exhibiting highly skewed Th2-type responses (119), and studies of human schistosomiasis suggest it may be contributing to the development of periportal fibrosis (163). Thus, under the right circumstances, TNF-α can exhibit either Th1- or Th2-promoting activities. The contribution of TNF-α to the regulation of IL-13-driven responses, therefore, appears to be influenced greatly by the cytokine milieu.

IL-10 IL-10 was initially described as a Th2-type cytokine that antagonizes Th1 cell development by suppressing IL-12 and IFN-γ production (164). Nevertheless, it has become increasingly clear that IL-10 exhibits potent immunosuppressive activity during both Th1- and Th2-dominant immune responses (165, 166). In the absence of IL-10, mice infected with S. mansoni displayed a mixed Th1/Th2-type cytokine response (167). However, mice deficient in both IL-10 and IL-12 (119) or IL-10 and IFN-γ (168) developed highly polarized and exaggerated type-2 cytokine responses, with IL-13 levels approaching 10-fold higher than WT. Consequently, fibrogenesis also increased dramatically in these double cytokine-deficient animals (119, 168). These studies showed that IL-10 and the type-1-inducing cytokines IL-12 and IFN-γ cooperate to suppress type-2 cytokine-driven inflammation. Thus, mice displaying high IL-13 and low IL-10/IFN-γ responses tend to exhibit the greatest IL-13-mediated effector activity (124). Similar conclusions were drawn in studies investigating gastrointestinal parasite expulsion and asthma (33, 151). In fact, in the experimental asthma model, IL-10, rather than the counter-regulatory

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type-1 cytokine response, was hypothesized to provide the primary mechanism by which allergic inflammatory processes in the lungs are inhibited (169, 170). This is consistent with the observation that few Th1-effector cells are found in the lungs of healthy people, whereas IL-10 levels are lower in patients with asthma (170). Thus, normal production of IL-10 in nonasthmatic people, rather than a protective type-1 response, may be the key factor limiting IL-4/IL-13-mediated inflammatory processes. IL-10 may also help explain the hygiene hypothesis, which suggests the increase in allergic inflammation and asthma observed in westernized countries is due to a cleaner environment and decreased incidence of childhood infections (171, 172). Indeed, decreased atopy was reported in children infected with S. haematobium, and this was associated with higher concentrations of antigenspecific IL-10 (173). Thus, antiinflammatory cytokines induced during long-term chronic infections appear to provide protection from allergic inflammation (174). Recent studies suggest regulatory T cells producing IL-10 and/or TGF-β may also play an essential inhibitory role (175, 176). Dendritic cells exposed to respiratory antigen transiently produced IL-10 and stimulated the development of IL-10-producing CD4+ T regulatory cells (175). Thus, induction of tolerance by IL-10 may provide an effective means to reduce AHR and asthma. Nevertheless, it is important to note other studies examining the function of IL-10 found that AHR (177, 178), eosinophilic inflammation, and Th2 effector function can also be increased by IL-10 (179). Similar findings were reported in the T. muris model, where IL-10 promoted IL-4/IL-13-mediated parasite expulsion by suppressing the counter-regulatory type-1 cytokine response (33). Therefore, additional study is needed before IL-10 or regulatory T cells can be advocated as a therapy for asthma or other IL-4/IL-13-driven disorders.

TGF-β Like IL-10, TGF-β both antagonizes and promotes IL-13 effector function. As indicated above, regulatory T cells producing TGF-β and/or IL-10 provide significant protection from IL-13-dependent allergic inflammation (175, 176). Nevertheless, recent studies indicate TGF-β is also an important downstream mediator of IL13-induced activity. One of the more significant effector functions of IL-13 is the promotion of wound healing and fibrosis (9, 123, 180, 181). A recent study showed IL-13 induces fibrosis by selectively stimulating and activating the fibrogenic cytokine TGF-β (127). In this study, macrophages were the major site of TGF-β production (127), although a second study suggested bronchial epithelial cells may be another important source (182). Activation of TGF-β was mediated by an IL-13, a plasmin/serine protease, and an MMP-9-dependent mechanism. TGF-β antagonists were also highly effective at ameliorating the IL-13-induced pulmonary fibrosis (127). As such, these observations suggest an indirect TGF-β-dependent role for IL-13 in the development of fibrosis. Nevertheless, IL-13 directly stimulates collagen deposition in fibroblasts cultured in vitro (123, 181). Moreover, recent unpublished work in the schistosomiasis model showed that liver fibrosis can develop in the absence of MMP-9, TGF-β, and Smad-3 signaling pathways (M. Kaviratne, M. Hesse, A.W. Cheever, J. Letterio, M. Leusink, A. Roberts,

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S. Davies, L. Wakefield, T.A. Wynn, unpublished observations). These latter observations suggest a TGF-β-independent but IL-13-dependent pathway may also exist. Therefore, further study is needed to elucidate the common and distinct fibrogenic pathways triggered by IL-13 and TGF-β.


The Decoy IL-13 Receptor Alpha 2 In many of the disease models discussed above, IL-13 is produced at much higher levels than IL-4, which may, in part, explain why it acts as the central Th2-effector cytokine. Nevertheless, the receptors that bind these cytokines may also have a significant impact on their functional activity in vivo. The receptor-binding chains for IL-13 include IL-13Rα1 and IL-13Rα2 (15, 183–185). When expressed alone, IL-13Rα1 binds IL-13 with low affinity. However, when IL-13Rα1 is coexpressed with IL-4Rα, a high-affinity receptor-signaling complex is formed (183, 185). This receptor complex is expressed widely on both lymphoid and nonlymphoid cells and can also be activated by IL-4, thus accounting for the functional overlap between IL-4 and IL-13 (186). The second IL-13 binding chain, IL-13Rα2, binds IL-13 with high affinity and, in contrast to IL-13Rα1, is also found as a soluble receptor in mouse serum and urine (15, 187). Structural differences between the cytoplasmic domains of the IL-13 receptors, however, suggest they are functionally distinct. The cytoplasmic region of murine IL-13Rα2 does not possess an obvious signaling motif or JAK/STAT binding sequence (15), which raises the possibility that it is a dominant negative inhibitor or decoy receptor, as originally described for the IL-1 receptor type II (188). To understand the role of IL-13Rα2 in regulating the biological activity of IL-13, mice with targeted deletion of IL-13Rα2 were recently generated (N. Wood, M. Whitters, B.A. Jacobson, J. Witek, J.P. Sypek, M. Kasaian, M. Eppihimer, M. Unger, S. Goldman, M. Collins, D.D. Donaldson, M. Grusby, submitted manuscript). Basal serum IgE levels were elevated in IL-13Rα2−/− mice despite the fact that serum IL-13 was absent and IFN-γ production increased compared to WT mice. The frequency of bone marrow macrophage progenitors also increased in the knockout animals, whereas the ability of tissue macrophages to produce NO and IL-12 in response to LPS decreased substantially (N. Wood, M. Whitters, B.A. Jacobson, J. Witek, J.P. Sypek, M. Kasaian, M. Eppihimer, M. Unger, S. Goldman, M. Collins, D.D. Donaldson, M. Grusby, submitted manuscript). From these studies, the authors concluded that the primary function of the IL13Rα2 is to limit IL-13 effector function in vivo (Figure 1). IL-13Rα2 deficiency was also studied in the murine schistosomiasis model, where chronic exposure to IL-13 is the principle cause of liver fibrosis (M.G. Chiaramonte, M. Mentink-Kane, B.A. Jacobson, A.W. Cheever, M.J. Whitters, submitted manuscript). The mechanisms regulating expression of IL-13Rα2 in vivo were also examined. Basal expression of IL-13Rα2 mRNA was nearly undetectable in the livers of uninfected mice. However, shortly after the onset of egg-laying, a marked induction of IL-13Rα2 expression was observed, which persisted throughout the chronic stage of infection and correlated almost perfectly with the development of liver fibrosis. Receptor expression was also highly

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dependent on IL-10, IL-13, and Stat6 and inhibited by the Th1-inducing adjuvant IL-12, thus showing a strong correlation with the egg-induced Th2 response (M.G. Chiaramonte, M. Mentink-Kane, B.A. Jacobson, A.W. Cheever, M.J. Whitters, submitted manuscript). Schistosome-infected IL-13Rα2-deficient mice showed a marked exacerbation in hepatic fibrosis, despite displaying no change in granulomatous inflammation, tissue eosinophilia, or mastocytosis. Fibrosis increased despite the fact that IL-13 levels decreased significantly, thus supporting findings from unchallenged mice (N. Wood, M. Whitters, B.A. Jacobson, J. Witek, J.P. Sypek, M. Kasaian, M. Eppihimer, M. Unger, S. Goldman, M. Collins, D.D. Donaldson, M. Grusby, submitted manuscript). Pathology was prevented when IL13Rα2-deficient mice were treated with a soluble IL-13Rα2-Fc construct (M.G. Chiaramonte, M. Mentink-Kane, B.A. Jacobson, A.W. Cheever, M.J. Whitters, submitted manuscript), formally demonstrating that their exacerbated fibrotic response was due to heightened IL-13 activity. As such, these studies illustrate a novel and previously unappreciated mechanism for limiting pathology associated with chronic IL-13-mediated inflammatory responses. Indeed, effective exploitation of this endogenous IL-13 dampening system has already been achieved in several experimental models (54, 56, 57, 63, 88, 123, 124).

SUMMARY IL-13 is produced by and either directly or indirectly affects the function of multiple cell types, including T cells, eosinophils, mast cells, basophils, epithelial cells, smooth muscle cells, fibroblasts, and macrophages. The published literature on this area alone was much too broad to adequately cover in one review. However, this more focused area of research on IL-13 is extremely important and will certainly remain an exciting topic in the coming years. What cell types produce IL-13 and under what conditions? Who are the responding cells? What regulates IL4/IL-13 receptor expression, and how does this affect the functional activity of IL-13? What role does the IL-13 decoy receptor play in other diseases, and how is the functional activity of IL-4 and IL-13 regulated? These are just a few of the important questions that remain unclear. The type-2 response was originally thought of as a counter-regulatory system for the type-1 immune response (189). Nevertheless, the recent explosion in research on IL-13 clearly demonstrates this is an extreme oversimplification. Given its central status in the Th2 response, a complete understanding of the many effector functions of IL-13 is needed so that it can be effectively exploited for therapeutic intervention. ACKNOWLEDGMENTS I would like to thank Terez Shea-Donohue, Joe Urban, Frank Brombacher, David Sacks, Marsha Wills-Karp, Raj Puri, Jay Berzofsky, Masaki Terabe, Mallika Kaviratne, and Deb Donaldson for their helpful comments and unpublished observations. I would also like to sincerely thank the past and present members of my laboratory for their invaluable contributions.

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Annu. Rev. Immunol. 2003. 21:457–81 doi: 10.1146/annurev.immunol.21.120601.141021

LOCATION IS EVERYTHING: Lipid Rafts


and Immune Cell Signaling∗

Michelle Dykstra, Anu Cherukuri, Hae Won Sohn, Shiang-Jong Tzeng, and Susan K. Pierce Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland 20852; email: [email protected], [email protected], [email protected], [email protected], [email protected]

Key Words B cells, T cells, mast cells, antigen receptors, coreceptors ■ Abstract The cells of both the adaptive and innate immune systems express a dizzying array of receptors that transduce and integrate an enormous amount of information about the environment that allows the cells to mount effective immune responses. Over the past several years, significant advances have been made in elucidating the molecular details of signal cascades initiated by the engagement of immune cell receptors by their ligands. Recent evidence indicates that immune receptors and components of their signaling cascades are spatially organized and that this spatial organization plays a central role in the initiation and regulation of signaling. A key organizing element for signaling receptors appears to be cholesterol- and sphingolipidrich plasma membrane microdomains termed lipid rafts. Research into the molecular basis of the spatial segregation and organization of signaling receptors provided by rafts is adding fundamentally to our understanding of the initiation and prolongation of signals in the immune system.

INTRODUCTION Over the past several years, a detailed picture of the biochemical cascades triggered by ligand-engagement of immune cell signaling receptors has emerged. It has become apparent that the immune receptors and the biochemical cascades that emanate from these are spatially organized in cells and that such organization is a critical element of the signaling process [reviewed in (1, 2)]. Most extensively studied to date in this regard are members of the multichain immune recognition receptor (MIRR) family, which includes the T and B cell receptors for antigen (the TCR and BCR) and the high-affinity receptor for IgE (FcεR1) expressed by mast cells and basophils (3). All MIRRs contain ligand-binding chains with short intracellular tails that are not directly involved in the transduction of biochemical ∗

The U.S. Government has the right to retain a nonexclusive, royalty-free license in and to any copyright covering this paper.

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signals. The ligand-binding chains are coupled to signaling cascades by association with transmembrane proteins with long intracellular domains containing immunoreceptor tyrosine-based activation motifs (ITAMs). Significantly, the MIRRs have no intrinsic kinase activity but when engaged by their multivalent ligands are phosphorylated by Src-family kinases on tyrosine residues contained within their ITAMs. Here we review the evidence that the initiation of MIRR signaling is facilitated by the concentration of Src-family kinases and other key signaling components in lipid rafts and that lipid rafts play a key role in the regulation of immune cell activation. To provide a context in which to evaluate the current evidence, we begin with a brief description of lipid rafts and a model for how lipid rafts function in immune cell signaling.

What is a Lipid Raft? In recent years evidence has accumulated that the plasma membrane is not a uniform lipid bilayer but rather that it contains within it sphingolipid- and cholesterolrich microdomains, termed lipid rafts (Figure 1) (1). Lipid rafts are evolutionarily conserved structures that play a role in a number of signaling processes involving receptors expressed by a variety of cell types, including the EGF receptor,

Figure 1 A schematic depiction of a lipid raft.

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the insulin receptor, and integrins. Several excellent reviews have been published recently describing the biochemistry and function of rafts (1, 4–7). Briefly, the outer leaflet of lipid rafts is composed of both sphingolipids with highly saturated acyl chains that pack tightly into gel-like microdomains and cholesterol, which by binding to the sphingolipids promotes formation of a liquid ordered phase. The sphingolipids and cholesterol partition out of the glycerophospholipid bilayer, which exists in a liquid disordered phase due to the unsaturated, kinked acyl chains of glycerophospholipids. Thus, lipid rafts are relatively ordered domains that float in the disordered glycerophospholipid bilayer. The partitioning of sphingolipids into rafts allows the lipids themselves to be used as markers for rafts. For example, the glycosphingolipid GM1 that binds to the β subunit of cholera toxin is a commonly used raft marker. The inner leaflet of lipid rafts is less well characterized but is probably composed of saturated phospholipids. The inner and outer leaflets are coupled, although the nature of the coupling is not known. A central feature of lipid rafts is that they allow for the lateral segregation of proteins within the plasma membrane (1). The ability to segregate provides a mechanism for the compartmentalization of signaling components within the membrane, concentrating certain components in lipid rafts and excluding others. At present, the exact lipid and protein composition of rafts has not been determined, and this information is essential to understand the principles of raft assembly and function. Although the rules that govern the constitutive or induced association of proteins with lipid rafts are not fully understood, some generalizations can be made at this point (Figure 1). Proteins are associated with the outer leaflet of lipid rafts through glycosylphosphatidylinositol (GPI)-linkage, in which case the lipid tail of the GPI-linked proteins preferentially partitions into rafts (8). Examples of raft-associated GPI-linked immune receptors include CD14, the receptor for the bacterial mitogen LPS; CD16, an Fc receptor; and CD48 and CD58, adhesion/costimulatory molecules. Although they lack transmembrane or cytoplasmic domains, many GPI-linked proteins including these have been shown to transduce signals when cross-linked. It has been postulated that the raft association of GPI-linked proteins is critical to their ability to signal (8), although the mechanism is not completely understood. Cytoplasmic proteins associate with the inner leaflet of lipid rafts through acylation. Proteins that are dually acylated by saturated fatty acids (N-myristoylation and S-palmitoylation) partition into rafts while proteins modified by unsaturated fatty acids or prenyl groups are excluded (9, 10). Significantly, most of the Src-family kinases are dually acylated and raft associated (11–13). The GTPase H-ras, which is palmitoylated and farnesylated, is targeted to lipid rafts; in contrast K-ras, which is farnesylated but not palmitoylated, associates with the inner leaflet of the plasma membrane but is excluded from rafts (14). Thus, a posttranslational addition of palmitate effectively segregates these two GTPases in the plasma membrane. Because palmitoylation is posttranslational and labile, the process is reversible, giving cells the potential to control the modification and thus to control raft association. In addition, S-acylation of proteins with heterogeneous unsaturated fatty acids

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appears to be a widespread mechanism by which cells regulate signal transduction by altering the association of proteins with rafts (15, 16) The vast majority of transmembrane proteins are excluded from rafts constitutively and cannot be induced to partition into rafts upon cross-linking or oligomerization (1). A small number of transmembrane proteins constitutively partition into rafts, and for these acylation, usually S-palmitoylation, is generally required (9). Important examples of raft-resident proteins in terms of lymphocyte signaling are the linker for activation in T cells (LAT) (17) and the T cell coreceptors CD4 and CD8 (18, 19). Lastly, some integral membrane proteins reside constitutively outside of rafts but when activated become raft associated (1). The MIRRs are important examples of this group (3). The ability to associate with rafts following cross-linking appears to be a selective feature of only a small number of proteins, and many proteins, for example, CD45 and the type-1 IL-1 receptor, when cross-linked do not associate with rafts (20, 21). The characteristics of integral membrane proteins that allow raft association are not known, although evidence from the studies of chimeric proteins implicates the transmembrane domains as critical (21, 22). Lipid rafts are most commonly isolated from cells based on their differential solubility in nonionic detergents. In certain nonionic detergents at low temperatures, the cholesterol- and sphingolipid-rich membrane domains are insoluble and can be separated from the soluble membranes based on their buoyant density (1, 9). Thus, the identification of a protein in a raft is operational and dependent on the particular detergent concentration and temperature selected (1, 23, 24). A number of issues arise from the use of detergent solubility to define the microenvironment of a protein on the plasma membrane, including the concern that the detergent itself induces the formation of rafts. However, evidence is rapidly accumulating that supports the existence of rafts in the membranes of living cells. These studies have taken advantage of a variety of techniques, including chemical cross-linking (25) and fluorescence resonance energy transfer (FRET) (26) to detect the proximity of two proteins in the membrane, photonic force microscopy (27) to measure the local diffusion of single membrane proteins, and single fluorophore tracking microscopy (28) to monitor the diffusion and dynamics of individual proteins and lipids in the plasma membrane. The identification by such techniques of proteins in the microdomains of living cells has correlated well with their detergent solubility. Of particular relevance to the future study of immune cell receptors and lipid rafts is the recent demonstration of FRET in living cells between Aequorea fluorescent proteins that contained the Src-family kinase N-terminal acylation sequence and were dually acylated (29). In contrast, prenylated fluorescent proteins showed no FRET. Furthermore, the dually acylated proteins but not the prenylated fluorescent proteins were present in detergent insoluble membranes providing a link between the function of rafts in living cells and detergent solubility. Lipid rafts are estimated to represent a significant portion of immune cell membranes, greater than 40% by measurements of fluorescence anisotropy of the lipid order in plasma membranes and lipid rafts (30). This proportion is in agreement

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with estimates based on the quantification of sphingolipids recovered after detergent solubilization (31). In resting cells rafts appear to be highly dynamic, submicroscopic structures (50 nm in diameter) containing only thousands of lipids and a small number of proteins (1, 26, 27). These have been referred to as elemental rafts. Upon cross-linking of signaling receptors associated with rafts, lipid rafts become larger, microscopic (100s of nm to µm in diameter), and more stable structures, often attached to the actin cytoskeleton (1, 32, 33). These structures are referred to as clustered rafts. It is not known if elemental and clustered rafts are equally insoluble in detergents, an important issue in the interpretation of results of raft isolations. Lastly, the observation that lipid rafts segregate proteins in the plasma membrane, including some and excluding others, raises the questions: Are all rafts the same, and is there heterogeneity among the rafts present in the membrane of any given cell? At present there is little evidence addressing this important issue.

Lipid Rafts and Signal Transduction in Immune Cells: A Model The following model for the mechanism by which rafts function in MIRR signaling is provided as a framework in which to discuss the existing data concerning the role of lipid rafts in immune cell signaling (Figure 2). The primary tenet of the model is that the rafts serve to spatially segregate signaling components in the plasma membrane, and by doing so they regulate the initiation and prolongation of signaling. In this model, in resting cells the MIRRs are excluded from lipid

Figure 2 A model for a role for lipid rafts in MIRR signaling depicting the progression from resting cells, to ligand binding and initiation of signaling cascade (∗ ), to raft clustering, to formation of an immunological synapse.

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rafts that concentrate the Src-family kinases and additional signaling components indispensable for cell activation, for example LAT in T cells (17). Significantly, rafts exclude negative regulators of receptor signaling such as CD45 (34–36). The exclusion of the monomeric MIRRs from elemental rafts is not absolute but rather reflects an equilibrium of the receptor with the rafts that is heavily skewed toward exclusion. Thus, monomeric MIRRs transiently associate with rafts; however, the affinity of the monomeric receptor for rafts is too weak to allow a sufficiently long residency time in rafts to fully propagate signals. The transient association of receptors with rafts may be significant and play a role in tonic signaling for cell survival. The multivalent binding of ligands to the MIRRs induces the oligomerization of the receptors, and in this model, the oligomer has a higher affinity for lipid rafts, shifting the equilibrium toward residence of the receptors in the rafts. If the oligomer is sufficiently stable, which would depend on both the affinity and valency of the ligand, the receptor would remain in the rafts for a sufficient length of time to assemble a “signalosome” composed of adaptors and other signaling components that contribute to raft clustering. A second important repercussion of signaling is the attachment of the receptor complex to the actin cytoskeleton, further stabilizing the receptors in clustered rafts. If the receptor oligomer is not stable owing to a weak association with its ligand, the receptor monomers would diffuse out of rafts and signaling would cease. It is not critical to the model that the ligand-induced oligomers form outside the rafts (as shown in Figure 2) versus within rafts from receptors transiently associated with the rafts. Similarly, the model is also consistent with the weak association of monomeric immune receptors and the Src-family kinases with separate small elemental rafts in resting cells that coalesce upon receptor oligomerization, as has been proposed for the FcεRI receptor (37). Lastly, the model makes no prediction about the number of ligated receptors required to initiate and sustain signaling in rafts and does not preclude the possibility that signaling is a multistep process involving rounds of raft association and signal initiation followed by raft clustering and stabilization as has been proposed for the TCR (38, 39). It follows from this model that any factor affecting the affinity of the receptor for rafts or the nature of the rafts themselves would have a significant impact on signaling. As is described below, current evidence indicates that both the association of receptors with rafts and the composition of rafts may function to regulate signaling in immune cells. Lastly, for T cells the engagement of an antigen presenting cell leads to the dramatic reorientation of the TCR, adhesion molecules, and associated signaling molecules into a structure, termed an immunological synapse, microns in diameter, at the interface of the T cell and the antigen presenting cell (40). An analogous structure recently has been described for B cells (41). Immunological synapses appear to be essential to establish stable, persistent signaling necessary for full activation. In the model presented, it is assumed that formation of clustered rafts precedes the formation of the immunological synapse. However, whether the lipid rafts play a role in the formation of the immunological synapse or are key organizing

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elements within the synapse remains to be determined. The immunological synapse and its relationship to lipid rafts have been the subject of several recent informative reviews (40, 42, 43) and are only briefly discussed here.

A ROLE FOR LIPID RAFTS IN IMMUNE CELL SIGNALING The MIRR Family The current evidence from studies relying on detergent solubility to isolate rafts or on confocal microscopy to colocalize receptors with raft markers indicates that rafts play a significant role in TCR signaling. In resting mature T cells, the TCR is excluded from lipid rafts that concentrate several key components of the TCR signaling pathway, including the Src-family kinase, Lck, and LAT (Table 1) (35, 36). In addition, a portion of CD4 and CD8, which is palmitoylated, are constitutively present in lipid rafts where they interact with Lck through specific protein-protein interactions (18, 19). Significantly, the T cell rafts also compartmentalize key regulators of Src-family kinases, namely, the C-terminal Src kinase, Csk, and the phosphatase, CD45. Csk is a soluble protein recruited to rafts by binding to phosphorylated Cbp (Csk-binding protein), a palmitoylated raft resident protein (44, 45). CD45 is an integral membrane protein excluded from rafts (34–36). Upon engagement of the TCR, either by CD3-specific antibodies or antigen presenting cells, the TCR associates with rafts where the ζ chain of the CD3 complex becomes phosphorylated as does ZAP-70 (35, 36). A number of the components of the T cell “signalosome” including the adapter protein Slp-76 and kinase PKCθ are recruited to lipid rafts following TCR activation (46, 47) (Table 1). In addition, the TCR in rafts becomes associated with the actin cytoskeleton (48). The proteins that are constitutively raft associated or are induced to associate with rafts following TCR engagement have been identified by specific antibodies; they likely represent only a subset of all associated proteins. Indeed, recent application of protein identification techniques, termed proteomics, has identified over 70 different raft-associated proteins in resting T cells including those listed in Table 1 (49). Significantly, the vast majority of these proteins are either elements of the cytoskeleton or components of signaling pathways. Recent evidence suggests that T cell rafts may be heterogeneous. T cell polarization during chemotaxis results in the asymmetric distribution of rafts containing the glycosphingolipids GM1 and GM3, suggesting that membrane components required for chemotaxis partition initially into rafts of different compositions (50). In addition, rafts containing Lck and LAT in resting cells were shown to be differentially soluble, which suggests differences in composition (51). It is possible that the composition and heterogeneity of rafts play a role in the regulation of signaling in T cells. In addition to a role for lipid rafts in mature T cell signaling, recent evidence indicates that lipid rafts are essential for positive selection of immature T cells in the thymus. T cells that express mutant TCR α chains or that fail to express CD3δ


THE TCR

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TABLE 1 Lipid raft–associated signaling components in resting and activated immune cells1,2 MIRR

Resting

Activated

BCR

Lyn (74, 76) c-Abl (143) Cbp (44, 45)

Syk (107) Btk (107) Vav (107) SHIP (75) PLC-γ 2 (76, 107) PI3-K (75, 107) BLNK (107)

TCR

Lck (11, 35, 36) Fyn (35, 36) ∗ Itk (24, 144) Rlk (145) Syk (35) ∗ LAT (17, 36) Cbp (44, 45) Csk (44, 45) Cbl (35) ∗ PI3-K (some isoforms) (35) Ras (24, 35) Grb-2 (35) PKA (148) PIP2 (24, 131)

∗

ZAP-70 (35, 36) Vav (17, 35) Slp-76 (46) Shc (35) ∗ PLC-γ 1 (17, 24, 35) SHP-1 (58) Hpk-1 (146) Gads (46) ∗ PKC α/θ (18, 47, 147) ∗ PI3-K (some isoforms) (35) IKK (147) ∗ Grb-2 (17, 24)

∗

∗

FcεR

Lyn (68, 69) LAT (68) Rac1 (68)

∗

Syk (67, 69) PLC-γ 1 (67) Plc-γ 2 (149) Vav1 (68) Grb2 (68) Slp-76 (68) PI3K (149) Gab-2 (149)

1

Only those molecules that have been investigated thus far and demonstrated to be included or recruited to lipid rafts in cells expressing the BCR, TCR, or FcεR are listed here.

2

An asterisk indicates that the molecule is found phosphorylated in the rafts upon activation.

chains are deficient in positive selection and fail to localize activated Lck, ZAP70, phosphorylated LAT and CD3ζ in rafts (52, 53). These observations may be of particular significance in suggesting similarities in mechanisms between positive selection and activation of mature peripheral T cells. Evidence that lipid rafts are both necessary for T cell signaling and play an important role in maintaining T cells in a resting state comes from several observations. Disruption of rafts by cholesterol depletion from T cell membranes using drugs such as methyl-β-cyclodextrin, filipin, and nystatin profoundly inhibits T cell

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signaling through phospholipase C–dependent pathways (35) yet activates other signaling pathways (54). T cells from mice deficient in acid sphingomyelinase, which affects the formation of lipid rafts, are defective in T cell signaling (55). Mutant versions of Lck and LAT that are not palmitoylated and consequently do not localize in rafts do not function in T cell activation (17, 56, 57). Conversely, the targeting of the phosphatase SHP-1 to lipid rafts inhibits TCR-mediated T cell activation (58). In addition, raft-associated Lck appears to be more catalytically active than Lck present in soluble membranes (59). Lastly, Slp-76, which normally associates with phosphorylated LAT and links the TCR to several downstream signaling pathways, when artificially targeted to rafts negates the requirement for active LAT (46). Although rafts appear to play a key role in the spatial organization of TCR signaling, the exact location of the initial phosphorylation of the TCR by Lck is not certain. Based on the inability to detect phosphorylated CD3ζ in detergent insoluble microdomains immediately following TCR engagement (36, 60), the proposal has been made that the TCR is initially phosphorylated outside of lipid rafts (61). In addition, raft association of the TCR has been reported to be dependent on an intact cytoskeleton, which suggests that actin cytoskeleton attachment precedes raft association (62). However, it is possible that the elemental rafts with which the TCR initially associates are not as detergent insoluble as the clustered rafts that subsequently form. Thus, the biochemical events attributed to non-raft regions of the membrane may actually occur in elemental rafts. Indeed, recent evidence using a solubilization protocol designed to preserve elemental rafts at 37◦ C revealed that a fraction of the TCR and ZAP-70 are constitutively associated with rafts, and it is within this subset that signaling is initiated (63). The role of rafts in the very initial events in TCR signaling is clearly important to establish. The technologies described above that allow the detection of raft associations in living cells should help address this issue. THE FCεRI Mast cells express a high-affinity receptor for IgE, the FcεRI, which when cross-linked by antigen results in the tyrosine phosphorylation of the ITAMs of the receptor complex by the Src-family kinase, Lyn, triggering a signal transduction cascade that leads to degranulation and cytokine synthesis (64). The relationship between the FcεRI and lipid rafts has been studied by a number of experimental techniques, including confocal imaging of both fixed and live cells (65–68), detergent solubilization (67, 69, 70), and electron microscopy (EM) (71). The results of these studies indicate that lipid rafts play a key role in mast cell activation. In resting cells the FcεRI is excluded from detergent insoluble membranes, and upon cross-linking, the FcεRI associates with Lyn-containing rafts where it is phosphorylated and recruits several components of the FcεRI signalsome to the rafts (Table 1). The initial association of the FcεRI with rafts does not require the actin cytoskeleton (65). The treatment of cells with methyl-β-cyclodextrin to remove cholesterol blocks FcεRI phosphorylation and signaling (72). Recent analyses suggest that the segregation of the FcεRI and signaling components is

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highly dynamic. By electron microscopy, a portion of FcεRI and Lyn (∼20%) appears clustered in small submicroscopic domains (71). As judged by detergent solubility, the association of FcεRI with Lyn-containing microdomains is weak, and the small clusters observed by EM may represent the equilibrium distribution of the monomeric FcεRI with elemental rafts. EM reveals that following FcεRI cross-linking large patches of FcεRI are formed that concentrate Syk and exclude Lyn, which is present at the periphery of the patches (71, 73). Similarly, by detergent solubilization, cross-linked FcεRI is first detected in association with Lyn. Subsequently, in an actin cytoskeleton-dependent event, Lyn is excluded, and the FcεRI rafts become less buoyant (65). Thus, microdomains containing FcεRI and Syk may represent the relevant prolonged signaling domains in mast cells. It might be anticipated that when examined more closely the association of other immune receptors with rafts will appear similarly dynamic.

THE BCR The relationship of the BCR with lipid rafts has been studied primarily by detergent solubility. In resting cells the BCR is excluded from lipid rafts. Following cross-linking either by Ig-specific antibodies or antigen, the BCR associates with lipid rafts, and a number of components of the BCR signaling pathways are recruited to rafts (74–76) (Table 1). The association of the BCR with lipid rafts is dependent on membrane cholesterol, but it does not require a signaling competent receptor or active Src-kinases and is not dependent on the actin cytoskeleton (77, 78). Disruption of rafts by cholesterol sequestration blocks BCR redistribution but enhances BCR-mediated calcium mobilization, which suggests that rafts play a role in both enhancing and suppressing B cell responses (75, 79). Indeed the phosphatase SHIP that inhibits BCR signaling was shown to be transiently recruited to lipid rafts following BCR cross-linking (75). The association of the BCR with rafts is transient; by 15 to 30 min after cross-linking, the BCR is no longer isolated in rafts (74). The association of the BCR with rafts is even less stable and more transient when the BCR is unable to initiate signaling or to attach to the actin cytoskeleton. This fact suggests that signaling and raft clustering are necessary to stabilize the BCR in rafts (77).

Immune Receptors Other Than the MIRRs NK cells express an array of activating and inhibitory receptors that in concert allow NK cells to distinguish target cells for lysis from normal healthy cells (80). Recent studies have shown that conjugate formation of NK cells with sensitive tumor cells results in the actin-dependent redistribution of the raft marker GM1 to the contact site in a Src- and Syk-dependent fashion (81, 82). Significantly, both the redistribution of rafts and cytotoxicity were blocked by the engagement of an inhibitory receptor in a SHP-1-dependent fashion (81). As the integration of signals from multiple receptors is essential for NK cell activation, the role of lipid rafts in NK cell signaling is of interest. NK CELL RECEPTORS

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MHC CLASS II MOLECULES The demonstration that lipid rafts play a role in the organization of signaling receptors in T cells raised the question of whether lipid rafts also are involved in the spatial organization of peptide-MHC class II complexes on antigen-presenting cell (APC) surfaces. At present the nature of the organization of class II molecules on APC is not fully understood, although lipid rafts have been implicated. Following cross-linking with specific antibodies, class II molecules colocalize with GM1 and partition into insoluble membranes (83). Other studies demonstrated a constitutive association of class II molecules with lipid rafts on APC that, when disrupted by cholesterol depletion, diminished antigen presentation to T cells (84). Recently, evidence was provided that MHC class II molecules are organized into functional clusters containing the tetraspanins CD9, CD81, and CD82 in a raft-independent fashion (85). Thus, the organization of class II molecules on APC is of interest but requires further study.

CD40 is a tumor necrosis factor family member that interacts with its ligand CD154 on activated T cells and contributes to B cell proliferation, differentiation, isotype switching, and memory development (86). When oligomerized by the binding of CD154, CD40 associates with lipid rafts where signaling is initiated through recruitment of TRAF molecules to the cytoplasmic domain of CD40 (87). CD40 ligation also alters the kinetics of BCR association with lipid rafts (88). The physiological relevance of the association of CD40 with lipid rafts was underscored by the observation that a CD40 signaling complex anchored in lipid rafts in nonHodgkin’s lymphomas leads to the constitutive activation of NF-κB and neoplastic cell growth (89). The engagement of CD154 by CD40 in dendritic cells (DC) results not only in the association of CD40 with lipid rafts and the recruitment of TRAFs but also in the phosphorylation of intracellular substrates by the Srcfamily kinases, which suggests that, by associating with lipid rafts, CD40 triggers Src-kinase dependent signaling pathways (90).

CD40

Sphingolipid metabolites such as ceramide and sphingosine-1-phosphate have been implicated as mediators in signaling cascades for apoptosis (91). Because sphingolipids are concentrated in lipid rafts, it is likely that rafts play a role in signaling for apoptosis. Recently, CD95 or Fas, which signals for apoptosis, was shown to associate with and cluster lipid rafts upon Fas-ligand binding, following hydrolysis of sphingomyelin to ceramide (92). In this case the lipid rafts appear to concentrate essential biochemical substrates for the apoptosis signal transduction pathway.

CD95

The Relationship of Coreceptors with Lipid Rafts Both T cells and B cells express coreceptors that, when engaged, serve to enhance or dampen signals transduced by the antigen-specific TCR and BCR. Recent evidence suggests novel mechanisms by which coreceptors function, namely, by facilitating or impeding the association of antigen receptors with lipid rafts.

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DYKSTRA ET AL. CD28/CTLA-4 The ultimate outcome of T cell activation is critically dependent on the positive and negative regulatory functions of the coreceptors CD28 and CTLA-4 (reviewed in 93). The coreceptor CD28 plays a central role in lowering the threshold for TCR activation, allowing T cells to respond to significantly lower levels of TCR occupancy as compared with activation through the TCR alone. Na¨ıve resting T cells appear to have only low levels of GM1 and Lck on the cell surface (94, 95). Cross-linking the TCR alone results in increased GM1 synthesis and the transport of intracellular GM1- and Lck-containing membranes to the T cell surface (94, 95). Coengagement of CD28 results in both an increase in the concentration of lipid rafts on the surface (95) and a redistribution of these to the immunological synapse (96), presumably facilitating the association of the engaged TCR with lipid rafts. The mechanism by which TCR engagement triggers transport of intracellular rafts to the plasma membrane is not known but is of considerable interest. CTLA-4, a potent negative regulator of TCR activation, when engaged, was shown to block the CD28-induced transport of intracellular rafts to the T cell surface (95). The CTLA-4-mediated block in raft transport correlates with the reduction in T cell activation. Thus, it has been proposed that CD28 and CTLA-4 mediate their enhancing and inhibitory effects on T cell activation in part through common mechanisms that control the cell surface expression of lipid rafts, thereby controlling the availability of raft-associated signal mediators to the TCR (97).

CD2 and its ligands, CD58 in humans and CD48 in mice, promote T cell adhesion and signal transduction. Following cross-linking, a significant fraction of human CD2 is recruited to lipid rafts where it induces the accumulation of tyrosine-phosphorylated proteins in a Src-family kinase-dependent fashion (98). Thus, CD2 in human cells may function to create active signaling platforms for the TCR prior to TCR engagement of peptide-MHC complexes. Coengagement of CD48 and the TCR on mouse T cells was shown to increase the level of raftdependent TCR ζ phosphorylation, association of ζ with the actin cytoskeleton, and actin reorganization, which together suggest that raft clustering through CD48 cross-linking facilitates TCR signaling (39, 48). In addition, other T cell molecules that have been described to provide costimulation, including CD5, CD9, and CD44, also enhance the association of the TCR with lipid rafts (99). CD2/CD58/CD48

CD19/CD21 The coreceptor CD19/CD21 complex plays a key role in B cells by setting the threshold for activation. The CD19/CD21 complex is coligated to the BCR by the binding of C3d-tagged antigens by CD21 resulting in a dramatic decrease in the number of ligand-engaged BCR required to activate B cells as compared with activation through the BCR alone (100). Recently, it was shown that the CD19/CD21 complex functions to prolong BCR residency in and signaling from lipid rafts (101). BCR cross-linking alone results in the relatively transient association of the BCR with lipid rafts. However, when coligated by C3d-tagged antigens, the BCR and the CD19/CD21 complex associate with lipid rafts where the BCR, CD19, Vav, and PLCγ 2 are phosphorylated for prolonged periods of time.

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Recent investigations into the mechanisms by which the CD19/CD21 complex prolongs raft association demonstrated the importance of CD81, a tetraspanin family member that associates with the CD19/CD21 complex. Both chimeric CD19 proteins that fail to associate with CD81 and CD19 expressed on CD81-deficient B cells failed both to associate with rafts and to promote signaling from rafts upon coligation with the BCR (A. Cherukuri, unpublished observation). The tetraspanins are a ubiquitously expressed, highly conserved family whose members associate with a number of adhesion and signaling molecules and have been implicated in a variety of normal and pathological cellular processes (102). The finding that CD81 is required for the stable association of CD19/CD21 with lipid rafts raises the possibility that a general feature of tetraspanins is their ability to facilitate raft association. Recently, an analysis of three prototypical tetraspanin complexes (CD9 and CD81 with the α3β1 integrin and CD63 with the phosphatidylinositol kinase Ptd Ins4-K) with lipid rafts provided evidence that these complexes associate with rafts but can be solubilized as discrete units (103). FCγ RIIB1 The Fcγ RIIB1 on mature B cells is a potent negative regulator of BCR signaling when ligated to the BCR through the binding of immune complexes (reviewed in 104). Recent studies have shown that when coligated in mature B cells both the BCR and the Fcγ RIIB1 associate with lipid rafts, where Fcγ RIIB1 recruits the inositol phosphatase SHIP and blocks BCR signaling (105). Thus, the Fcγ RIIB1 appears to associate with lipid rafts to block signals initiated by the BCR. Blocking the BCR signals results in a more transient association of the BCR with rafts, which may reflect the failure of the BCR, blocked in signaling, to promote raft clustering and stabilization. In immature B cells the cross-linking of the Fcγ RIIB1 alone signals for apoptosis (106). This function of the Fcγ RIIB1 is proposed to play a role in the elimination of germinal-center B cells whose somatically mutated BCRs lose affinity for the immunizing antigen. We have recently shown that the Fcγ RIIB1 when cross-linked becomes raft associated in the immature DT40 B cells and signals for apoptosis (S-J. Tzeng, unpublished observations). The cross-linked Fcγ RIIB1 also destabilizes the association of independently cross-linked BCR, which may enhance apoptotic signaling by blocking survival signals from the BCR.

Lipid Rafts in Immune Cell Development and Differentiation The process of generating an antigen-specific repertoire of T cells and B cells devoid of self-reactivity requires that the TCR and BCR transduce qualitatively different signals at different stages of development. Current evidence suggests that the association of antigen receptors with lipid rafts changes during development and differentiation, presumably to regulate the outcome of signaling. Initially, the first rearranged chains of the B cell and T cell receptors are expressed on the cell surface of pre-B and pre-T cells and signal for cell survival. Significantly, a large portion of these pre-BCR and pre-TCR constitutively reside in lipid rafts, unlike mature BCR and TCR, which are excluded from rafts (107, 108).

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In pre-T cells the surrogate α chain expressed as a component of the pre-TCR is palmitoylated, providing a biochemical basis for the increased association of the pre-TCR with lipid rafts (108). The constitutive presence of the pre-BCR and preTCR in lipid rafts presumably allows signaling for survival, although this has not been shown directly. In contrast, in immature B and T cells at the stage in development in which negative selection occurs, the BCR and TCR are excluded from rafts in the resting state and do not stably associate with rafts following receptor cross-linking (109–111). Whether the receptors transiently associate with rafts after cross-linking is not known, but the rafts do not appear to be sites of signal propagation, at least in B cells, as evidenced by the absence of phosphotyrosine-containing proteins within the rafts (109). A similar phenomenon was observed with tolerant B cells in which case the BCR did not become associated with rafts upon BCR cross-linking (78). Differences in raft composition and abundance have been implicated in regulating responses in the T cell subsets Th1 and Th2 cells and in effector T cells. A number of differences in the regulation of cell signaling and activation have been described for Th1 and Th2 cells, and recently these have been related to distinct patterns of membrane compartmentalization of the TCR mediated by lipid rafts (112, 113). Activation of primary Th1 cells through the TCR was shown to be highly dependent on CD4-containing lipid rafts and to result in raft clustering at the interface of the T cell and the antigen presenting cell. In contrast, Th2 cell activation showed little raft dependence, and Th2 cells did not recruit rafts to the activated receptor. The failure of the Th2 TCRs to partition into and to cluster rafts correlated with the poor ability of Th2 cells to respond to low-affinity peptide stimulation. The raft compositions of na¨ıve and effector T cell surfaces also appear to be different (38). Effector T cells are more responsive than na¨ıve T cells to ligand and less dependent on costimulatory signals for activation. As commented upon above, in na¨ıve T cells a significant portion of Lck and GM1 appear to be present in intracellular, endocytic membranes (94). In contrast, in effector T cells nearly all the Lck is expressed at the plasma membrane in rafts containing the coreceptors CD4 and CD8 (94). Thus, na¨ıve and effector T cells appear to regulate the quantity of rafts at the plasma membrane in order to control the reactivity of the TCR. Taken together these studies indicate that the nature of the BCR and TCR association with microdomains is altered during development and differentiation, correlating with the outcome of signaling. An analysis of the lipid and protein composition of rafts from lymphocytes at different stages of development and differentiation will be important to elucidate the molecular basis of the raft behavior.

Raft Clustering and Regulation of Immune Cell Signaling As described above, raft clustering appears to be an important mechanism by which immune cell signaling is controlled. Clustered rafts presumably provide more stable, efficient signaling platforms for ligand-engaged immune receptors, thus allowing the activation of cells by fewer and lower affinity ligands. Clustered rafts could also shift the equilibrium of unligated immune receptors toward

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residency in rafts, which provides a means of amplifying the signaling of a very small number of receptors engaged with high-affinity ligand by recruiting unligated or weakly engaged receptors. Cross-linking the GPI-anchored protein CD59 on T cells clusters rafts, as shown by the patching of the raft marker GM1, and so induces the accumulation of phosphorylated proteins and actin in rafts by mechanisms involving Src-family kinase activity (114). Inducing the clustering of lipid rafts by cross-linking GM1 using cholera toxin results in signaling in T cells and the redistribution of several membrane proteins including β1 integrins, CD59, and CD43 to clustered rafts that exclude LFA3 (115). The physiological significance of raft clustering was recently underscored by the description of an endogenous inducer of raft aggregation in T cells. Agrin, a neuronal aggregating factor was shown to be secreted by activated T cells and to induce the clustering of lipid rafts that coclustered with T cell signaling molecules (116). Significantly, agrinmediated raft clustering resulted in an increased reactivity of the TCR to peptideMHC complexes. It was suggested that agrin-induced raft clustering reflects a conserved mechanism to facilitate receptor signaling in the immune and neuronal systems. If endogenous promoters of raft clustering exist, it is likely that there are also endogenous mechanisms for restricting or limiting raft clustering. Galectin-1, an endogenous T cell lectin that binds to a number of lactosamine-containing receptors on T cells, including CD45 and CD43, was recently reported to block the raft clustering and TCR recruitment to rafts induced by engagement of the coreceptors CD28 and CD48 (117).

The Relationship of Lipid Rafts and Pathogens Recent evidence indicates that lipid rafts play key roles in the life cycle of a variety of intracellular pathogenic viruses, bacteria, and parasites [reviewed in (7)]. The general theme emerging is that lipid rafts facilitate the access of pathogens to cells by concentrating cellular receptors for the pathogens, influencing pathogen trafficking to appropriate subcellular sites for replication and playing critical roles in the assembly of enveloped viruses during replication. HIV-1 infection of T cells illustrates these functions of rafts. HIV-1 binding to T cells occurs through sequential interactions of its envelope glycoproteins gp120-gp41 with CD4 and raft-associated coreceptors such as CCR5 and CXCR4, which leads to raft clustering (118–120). Lipid rafts also may induce a conformation of the CD4gp120-coreceptor complex required for viral fusion through interactions with raft glycosphingolipids (121). In any event, disruption of rafts by cholesterol depletion or by blocking sphingolipid synthesis inhibits HIV-1 entry into host cells (119, 120, 122). Rafts also play a role in HIV-1 assembly and budding. The viral protein Gag that mediates multiple steps in viral assembly at the T cell plasma membrane localizes in raft-like domains upon multerimization (123, 124). Another important envelope protein, Env is palmitoylated (125), and it along with Gag colocalizes with T cell lipid raft markers during virus assembly (126). T cell raft markers, but not raft-excluded molecules such as CD45, become incorporated into newly formed virus particles, indicating that viral budding also occurs in lipid

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rafts (126). As with viral entry, HIV-1 release and infectivity are inhibited if rafts are disrupted by cholesterol depletion (124). In addition to a role of lipid rafts in pathogen entry and assembly, pathogens encode gene products that coopt the functions of lipid rafts in infected cells. During latent infection of B cells, EBV encodes two such integral membrane proteins, the latent membrane proteins 1 and 2A (LMP1 and LMP2A). LMP1 promotes growth transformation of infected cells and is constitutively present in rafts where it generates signals that mimic those of ligand-bound CD40 (127–129). LMP2A appears to mediate EBV latency both by blocking BCR signaling and by generating its own signals for B cell development and survival (130). LMP2A accomplishes both these functions from lipid rafts where it is constitutively present and phosphorylated and blocks BCR raft association and subsequent BCR signaling and internalization (20, 128).

THE ROLE OF LIPID RAFTS IN MEMBRANE TRAFFICKING Lipid rafts were first identified in studies of the selective transport of membrane proteins to the cell surface in polarized epithelial cells (1). In T cells, trafficking of an intracellular store of rafts to the plasma membrane also occurs upon T cell activation, as mentioned above (94). In addition to regulating membrane transport to the cell surface, rafts may also play a role in endocytosis. In B cells, BCR cross-linking directs the internalization of the lipid raft marker GM1, but not of a raft-associated GPI-linked protein, to class II loading compartments, indicating that BCR internalization is initiated in rafts, although sorting of raft components occurs during endocytosis (74). Recent evidence suggests that rafts organize regulators of the actin cytoskeleton network and endocytosis, potentially allowing the coordination of receptor signaling with receptor internalization. The phosphoinositide lipid PI(4, 5)P2 accumulates in rafts and recruits PH-domain-containing proteins, many of which are involved in cytoskeletal organization and membrane trafficking (131, 132). In addition, Cbl and Nedd4, ubiquitin ligases that can target receptors for internalization by ubiquitination, are recruited to rafts upon FcεRI signaling (133). Although clathrin-dependent endocytosis has been traditionally assumed distinct from caveolae- and raft-mediated internalization, recent evidence indicates that raft signaling and clathrin-dependent endocytosis may be linked. By electron microscopy, clathrin coated pits were shown to bud from areas immediately adjacent to FcεRI-containing rafts (71). In addition, BCR cross-linking leads to the phosphorylation of clathrin in rafts (133a). Finally, members of the flotillin/reggie protein family have been implicated as organizing centers for signal transduction in rafts (134, 135). The localization of flotillin family members in endosomes, lysosomes, and phagosomes, as well as the involvement of flotillins in the uptake of insulin, suggest that these proteins may provide a link between raft-associated signaling and raft-mediated trafficking (134, 136, 137).

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RAFTS AS POTENTIAL TARGETS OF THERAPIES The observations that lipid rafts play roles in both immune cell signaling and in the process of infection for a variety of pathogens suggest that rafts may provide new targets for therapeutic strategies. Indeed, at present there are emerging links between existing clinical therapies and their effects on lipid rafts that suggest that new classes of drugs may be developed to modify immune responsiveness based on their ability to modulate the function of lipid rafts. Glucocorticoids (GCS) are a class of cholesterol-derived steroids produced by the hypothalamic-pituitary-adrenal axis that have profound immunosuppressive and anti-inflammatory effects in the immune system (138). Consequently, GCS are widely used to prevent graft rejection and to treat autoimmunity, allergies, and inflammatory diseases. GCs are small lipophilic compounds that diffuse across membranes and mediate their effects by binding to intracellular receptors that alter nuclear gene transcription. However, recent evidence indicates that in T cells GCs also modify the lipid composition of the inner leaflet of lipid rafts and palmitoylation of cellular proteins, which results in the failure of treated cells to compartmentalize the normally raft-associated proteins LAT, Cbp, Lck, and Fyn (139). The mechanism by which GCs alter membrane composition is not known; however, the observation that in addition to their effects on gene transcription GCs alter lipid raft function suggests a mechanism for immunosuppression that can be further exploited. Polyunsaturated fatty acids (PUFA) such as those abundant in marine fish oils modulate immune responses and consequently have been used clinically as immunosuppressants and in the treatment of inflammatory diseases (140). Recently PUFA have been shown to inhibit T cell activation by modifying the inner leaflet of rafts and by disrupting raft localization by incorporating directly into proteins, including Src-family kinases, through S-acylation (15, 16, 141). The understanding that dietary PUFA modulate lipid raft function may provide new targets for therapeutic uses of PUFA in the treatment of autoimmune diseases. Lastly, the cholesterol reducing drugs, the statins, already in widespread clinical use, have been shown to modulate T cell responses (142). It is interesting to speculate that the statins may affect immune T cell activation by influencing membrane cholesterol levels necessary for the function of rafts.

CONCLUSIONS The segregation and compartmentalization of signaling receptors and key components of signal cascades by lipid rafts represent a previously unappreciated level of organization and regulation of signal transduction in immune cells. Investigations into the role of lipid rafts in immune cell activation thus far have been largely descriptive. Nonetheless, results of these studies have provided important new evidence that lipid rafts function in the initiation of signaling in a variety of immune cells and have implicated rafts in the control of signaling by coreceptors,

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during development and differentiation, and in infection. A future challenge is to understand the molecular mechanisms underlying the ability of lipid rafts to segregate signaling receptors and components of their signal cascades in the plane of the membrane. Important new technologies that allow the detection of the interactions of rafts, cellular receptors, and components of signaling pathways in living cells and the identification of proteins and lipid contents of rafts should provide powerful tools for this effort. The recognition of a role for lipid rafts in immune cell activation provides a new context in which to phrase longstanding questions concerning the mechanisms by which immune cell signaling is initiated and prolonged. In this new context, in which location is everything, interesting new answers to these questions may be expected. The Annual Review of Immunology is online at http://immunol.annualreviews.org

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123. Lindwasser OW, Resh MD. 2001. Multimerization of human immunodeficiency virus type 1 Gag promotes its localization to barges, raft-like membrane microdomains. J. Virol. 75:7913–24 124. Ono A, Freed EO. 2001. Plasma membrane rafts play a critical role in HIV-1 assembly and release. Proc. Natl. Acad. Sci. USA 98:13925–30 125. Rousso I, Mixon MB, Chen BK, Kim PS. 2000. Palmitoylation of the HIV-1 envelope glycoprotein is critical for viral infectivity. Proc. Natl. Acad. Sci. USA 97:13523–25 126. Nguyen DH, Hildreth JE. 2000. Evidence for budding of human immunodeficiency virus type 1 selectively from glycolipidenriched membrane lipid rafts. J. Virol. 74:3264–72 127. Clausse B, Fizazi K, Walczak V, Tetaud C, Wiels J, et al. 1997. High concentration of EBV latent membrane protein 1 in glycosphingolipid-rich complexes from both epithelial and lymphoid cells. Virology 228:285–93 128. Higuchi M, Izumi KM, Kieff E. 2001. Epstein-Barr virus latent-infection membrane proteins are palmitoylated and raft-associated: protein 1 binds to the cytoskeleton through TNF receptor cytoplasmic factors. Proc. Natl. Acad. Sci. USA 98:4675–80 129. Kaykas A, Worringer K, Sugden B. 2001. CD40 and LMP-1 both signal from lipid rafts but LMP-1 assembles a distinct, more efficient signaling complex. EMBO J. 20:2641–54 130. Merchant M, Swart R, Katzman RB, Ikeda M, Ikeda A, et al. 2001. The effects of the Epstein-Barr virus latent membrane protein 2A on B cell function. Int. Rev. Immunol. 20:805–35 131. Liu Y, Casey L, Pike LJ. 1998. Compartmentalization of phosphatidylinositol 4,5bisphosphate in low-density membrane domains in the absence of caveolin. Biochem. Biophys. Res. Commun. 245: 684–90

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THE REGULATORY ROLE OF Vα14 NKT CELLS IN INNATE AND ACQUIRED IMMUNE RESPONSE Masaru Taniguchi, Michishige Harada, Satoshi Kojo, Toshinori Nakayama, and Hiroshi Wakao Laboratory of Immune Regulation, RIKEN Research Center for Allergy and Immunology, and Department of Molecular Immunology, Graduate School of Medicine, Chiba University, Chuo-ku, Chiba 260-8670, Japan; email: [email protected], [email protected], [email protected], [email protected], [email protected]

Key Words α-galactosylceramide, Vα14 NKT cell–deficient mice, GM-CSF receptor, gene rearrangement, regulation of immune response, innate immunity ■ Abstract A novel lymphocyte lineage, Vα14 natural killer T (NKT) cells, is now well established as distinct from conventional αβ T cells. Vα14 NKT cells express a single invariant Vα14 antigen receptor that is essential for their development. Successful identification of a specific ligand, α-galactosylceramide(α-GalCer), and the establishment of gene-manipulated mice with selective loss of Vα14 NKT cells helped elucidate the remarkable functional diversity of Vα14 NKT cells in various immune responses such as host defense by mediating anti-nonself innate immune reaction, homeostatic regulation of anti-self responses, and antitumor immunity.

DISCOVERY OF Vα14 NKT CELLS Vα14 natural killer T (NKT) cells have been identified as a novel lymphocyte lineage, and are characterized by the expression of a single invariant Vα14 antigen receptor and the NK1.1 marker (1, 2). Three independent lines of study contributed to the identification of Vα14 NKT cells. First, in 1986 we isolated complementary DNA (cDNA) encoding Vα14 from a suppressor T cell hybridoma (34S-281), and found that it was utilized by most (12/13: more than 90%) independently established suppressor T cell hybridomas from numerous sources (3, 4). In fact, Vα14 cDNAs isolated from all of these hybridomas consisted of the Vα14 and Jα281 gene segments with a single-nucleotide N-region. Since the N-region is composed of the third base of a glycine residue (GGX) at position 93, all nucleotide insertions give rise to only a glycine residue. Thus, all N-regions result in the generation of a Vα14 receptor that is invariant at the amino acid level. Although the Vα14 and Jα281 genes are located in the T cell receptor (TCR) gene cluster on chromosome 14, the invariant Vα14 receptor is used only by Vα14 NKT cells and 0732-0582/03/0407-0483$14.00

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not by conventional T cells (5). Moreover, the Vα14 receptor has an extra cysteine residue at position 19 in the variable region, a characteristic feature distinct from other TCRs (3, 6). Since the invariant Vα14 receptor appeared to be a unique marker of this cell type, the Vα14 cDNA was used in our initial studies as a probe for the detection and analysis of Vα14+ cells. For example, RNase protection assays using the invariant Vα14 cDNA probe revealed Vα14+ cells to constitute a relatively large population in peripheral lymphoid organs [1–2% of spleen lymphocytes, 10–20% of liver lymphocytes, and 40% of CD3+ cells in bone marrow (BM)] rather than the thymus (0.4%) (2, 7). The numbers of Vα and Jα gene segments are about 100 each, and the diversity of the TCRα repertoire is calculated to be about 108. In other words, the frequency of any one particular expressed TCR is expected to be about 1/108. Therefore, the degree of expression of the invariant Vα14 seen in peripheral tissues is estimated to be 104–106 times higher than expected. Thus, we considered this cell type to be a unique population and coined the term Vα14+ cells. All laboratory inbred mouse strains tested carrying different major histocompatibility complex (MHC) haplotypes possessed considerable numbers of Vα14+ cells, and Vα14+ cells were diminished in numbers in β2-microglubulin (β2M)deficient mice (8, 9). These data suggest that Vα14+ cells are selected by a monomorphic MHC-like molecule, rather than by the polymorphic MHC essential for the selection of conventional T cells with a diverse repertoire. Moreover, Vα14+ cells were selected by BM-derived cells, including thymocytes, but not by thymic epithelial cells as shown by BM chimera experiments (9). Thus, it is suggested that Vα14+ cells develop through unique selection processes distinct from those of conventional T cells. In addition, the most important conclusion from these studies is that Vα14+ cells are able to develop in extrathymic lymphoid tissues, including BM and liver but not spleen (10). This is based on the observation that circular DNA copies created by Vα14-Jα281 gene-mediated rearrangement events were abundantly detected in some extrathymic tissues. Since the copy number of circular DNA reflects the frequency of TCR rearrangement events, these studies strongly suggest the existence of a unique Vα14+ cell population distinct from conventional αβ T cells. The second line of studies on the identification of Vα14 NKT cells came in 1987 when Fowlkes et al. and Budd et al. reported that a small population (0.4%) of CD4−CD8− double negative (DN) thymocytes (hitherto believed to be TCRαβnegative immature thymocytes) exclusively expressed TCR Vβ8 on their cell surface, suggesting a unique population distinguishable from conventional T cells by its DN phenotype and a restricted TCR Vβ repertoire (11, 12). They were later found to express CD44, CD5, and NK1.1 (13, 14). This novel cell type was also found among mature T cells having either CD4+ or DN phenotypes, and it produced both Th1 and Th2 cytokines (15, 16). Interestingly, no CD8 positive population was detected in the thymus, probably due to the negative selection of Vα14 NKT cells when they express CD8 (17).

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The third study, which provided a link between the above two sets of studies, was carried out by Lantz & Bendelac in 1994 (1). They succeeded in establishing Vβ8+, CD5high, CD44+ thymocyte hybridomas that had either a CD4+ or double negative phenotype. These hybridomas were also found to express messenger RNA (mRNA) for an invariant Vα14 receptor, and thus are identical to the Vα14+ cells described by us (3, 4) and the DN Vβ8+ thymocytes demonstrated by Fowlkes et al. and Budd et al. (11, 12). We also demonstrated that the Vα14+ cells in peripheral tissues expressed an invariant Vα14/Vβ8.2 receptor and NK1.1 (2). These observations strongly suggest that Vα14+ cells, DN Vβ8+ thymocytes, and Vβ8+ CD44+ thymocyte hybridomas are the same cell type. Thus, this population was designated as Vα14 NKT cells. Another important finding by Bendelac and associates is that their hybridomas recognize CD1d, a class Ib molecule with a monomorphic nature (18, 19). This finding provided new insight into the immune system, in particular the existence of a CD1d-dependent immune system. In any event, the majority of NK1.1+ CD3+ cells in mice are Vα14+ NKT cells that belong to the CD4+/DN CD62L− CD69+ DX5dull population in the spleen, liver, thymus, and BM.

A Minor Subset of NKT Cells Another category of unusual lymphocytes somewhat similar to Vα14 NKT cells in terms of their NK1.1 expression has also been described as a minor population. These cells include CD1d-dependent and CD1d-independent non-Vα14 NKT cells expressing a diverse repertoire of TCR, as well as T cells expressing intermediate levels of TCR (called intermediate T cells) (20–24). CD1d-DEPENDENT NON-Vα14 NKT CELLS CD1d-dependent non-Vα14NKT cells express diverse receptors and constitute a minor population expressing CD8+ or DN DX5dull Ly49A− CD62L− CD69+ phenotypes, detected mainly in the spleen and BM. They were increased in number in Vα14 NKT cell–deficient mice, particularly with age, but were diminished in CD1d-deficient mice (20–23). CD1d-INDEPENDENT NKT CELLS The majority of CD1d-independent NKT cells appeared to be conventional T cells that expressed NK cell markers upon activation, and were mainly DX5+ Ly49A+ CD62L+ CD69−. They consisted of less than one fifth of the total NK+CD3+ cell population present mainly in BM of CD1-deficient mice (20–23). In fact, more than 90% of antigen-specific CD8+ and CD4+ T cells coexpress one or more NK cell markers, such as NK1.1, DX5, or asialo GM1, after infection with lymphocytic choriomeningitis virus (26). Therefore, it is apparent that the current nomenclature defining NKT cells includes conventional antigenspecific CD8+ and CD4+ T cells. DX5+ CD1d-independent CD8+ NK1.1+CD3+ cells, expanded in vitro with interferon γ (IFNγ ), interleukin-2 (IL-2), and anti-CD3 antibody, produced Th1biased cytokines, and suppressed graft-versus-host disease (GVHD) (27). IFNγ

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derived from CD8+ CD1d-independent NK1.1+ T cells mediates the suppression of GVHD, since CD8+ CD1d-independent NK1.1+ CD3+ cells from mice deficient in IFNγ could not suppress acute GVHD. Interestingly, induction of GVHD requires Fas/Fas L and perforin/granzyme B, as the cells from mice deficient for Fas, Fas ligand (Fas L), and perforin did not cause acute GVHD (27). Similar findings were also reported in the human system where CD1d-reactive nonVα24 NKT cells with diverse TCR repertoire bearing CD161 were derived from BM, produced Th2-biased cytokines, and suppressed mixed lymphocyte reaction (MLR) (28). Abo and his colleagues described “intermediate T cells” that contain NK1.1+ and NK1.1− populations but are characterized by the expression of IL-2Rβ with high intensity, which are distinct from conventional T cells. These intermediate T cells are detected in nude mice and in adult-thymectomized irradiated mice reconstituted with syngeneic BM cells that give rise to CD8αα + NKT cells, which suggests their extrathymic development (24). Despite their unique characteristics, intermediate T cells seem to consist of various types of cells, including DN αβ T cells with a CD3dull B220+ CD44+ NK1.1− phenotype found in lpr mice, prone to a lupus-like disease, and in normal mice (29). Thus, these cells contain heterogeneous cell populations in terms of their phenotypic expression, antigen receptor expression, and selection processes. INTERMEDIATE T CELLS

CHARACTERISTIC FEATURES OF Vα14 NKT CELLS Since Vα14 NKT cells express both NK1.1 and invariant Vα14 receptor, they are recognized as a subpopulation of αβ T cells that express NK1.1. However, at least four facts indicate that Vα14 NKT cells are not a subgroup of conventional αβ T cells or activated T cells expressing NK1.1 marker, but rather are an independent lineage distinct from other lymphocytes. First, the invariant Vα14 receptor is expressed only by Vα14 NKT cells but not by conventional αβ T cells (5). Second, unlike conventional T cells, the precursor of Vα14 NKT cells expresses the granulocyte macrophage colony stimulating factor receptor (GM-CSFR) and receives GM-CSF signals to allow a critical maturation process to induce Vα gene rearrangement (30). Third, the invariant Vα14 expression is essential for Vα14 NKT cell development (5, 31). Fourth, the transcripts of Vα14/Vβ8 and CD3ε, and circular DNA mediated by Vα14-Jα281 and Vβ8 gene rearrangements, were detected at an early stage of embryogenesis before thymus formation (32).

Exclusive Usage of Invariant Vα14 Antigen Receptor by Vα14 NKT Cells ESTABLISHMENT OF Vα14 NKT CELL–DEFICIENT MICE The exclusive expression of invariant Vα14/Vβ8.2 receptor on Vα14 NKT cells and the essential requirement

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Figure 1 Fluorescence activated cell sorter (FACS) patterns of spleen cells derived from wild-type (Jα281+/+), Vα14 NKT cell–deficient (Jα281−/−), RAG-1-deficient (RAG-1−/−), and Vα14 NKT (RAG-1−/− Vα14/Vβ8.2 tg) mice. The squares indicate the position of NKT cells. tg: transgenic.

of Vα14 expression for the development of Vα14 NKT cells were demonstrated in Vα14 NKT cell–deficient (Jα281−/−) mice (5). As expected, the deletion of Jα281 gene expression caused a complete failure to develop Vα14 NKT cells, leaving other lymphoid lineages intact (Figure 1). This observation strongly suggests that the invariant Vα14/Vβ8.2 segment is indispensable for the generation of Vα14 NKT cells. A similar decrease in the number of Vα14 NKT cells has also been shown in β2M-deficient and CD1d-deficient mice (8, 9, 33–35), suggesting that positive selection of Vα14 NKT cells through specific interaction of Vα14/Vβ8.2 receptors with CD1d is required for differentiation. GENERATION OF Vα14 NKT MICE Further evidence for the exclusive usage of the invariant Vα14 receptor on Vα14 NKT cells is that preferential generation of Vα14 NKT cells with severe impairment in the development of conventional αβ T cells was observed in Vα14 transgenic mice crossed with either Cα-deficient or RAG-1-deficient mice (31). As shown in Figure 1, no NK1.1-negative conventional T cells were detected in the spleen of Vα14/Vβ8.2 double transgenic mice with a RAG-1-deficient background. If conventional T cells could utilize the invariant Vα14/Vβ8.2 receptor chains, they should have developed in Vα14 NKT mice. Thus, we refer to these mice as Vα14 NKT mice. These results indicate that the invariant Vα14 is exclusively used by Vα14 NKT cells, but not by conventional T cells, and is required for Vα14 NKT cell development (5). Also, no conventional NK cells were detected in Vα14 NKT mice. Since NK cells are present in RAG-1-deficient mice, the introduction of the invariant Vα14/Vβ8.2 genes may inhibit NK cell development and induce the preferential generation of Vα14 NKT cells (Figure 1). It is thus possible that the expression of transgenic Vα14 and Vβ8.2 in common precursors leads to a preferential development of Vα14 NKT cells.

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Identification of ImmatureVα14 NKT Cells and Their Development


Vα14 NKT PRECURSOR CELLS IDENTIFIED IN THE SPLEEN OF Vβ8.2 TRANSGENIC MICE The selective usage of the invariant Vα14 by Vα14 NKT cells encouraged us to clarify their unique developmental processes, such as the existence of a specific precursor population and particular maturation steps. We found the spleen of Vβ8.2 transgenic mice to contain an NK1.1+ TCRβ + cell fraction consisting of two distinct populations, surface CD3high and CD3dim cells (31). Only the CD3high fraction expressed Vα14 mRNA, whereas the CD3dim fraction showed no Vα14 mRNA but did express transcripts relevant to the immature developing T-lineage cells, such as RAG-1, RAG-2, and pTα, indicating precursor phenotypes (30). However, the phenotype of Vβ8.2+ pTα + NKT cell precursors detected is unusual in terms of CD3 expression because it is reported that pTα + immature T cells are generally CD3+ (36, 37). Unlike Vβ8.2 transgenic mice, in Vα14/Vβ8.2 double transgenic mice the CD3dim fraction disappeared and became the CD3high population (31). This may suggest that the transgenic expression of Vα14 accelerates the development of mature Vα14 NKT cells, and thus the CD3dim fraction is likely to be a precursor of mature Vα14 NKT cells. In addition, Vα14 NKT cells have been reported absent in pTα-deficient mice (38). Thus, pre-TCR-dependent selection (β-selection) and the following proliferation process may be required for Vα14 NKT cell development.

INDUCTION OF Vα14 GENE REARRANGEMENTS IN THE PRECURSOR CELLS BY GMCSF We succeeded in the development of pre-Vα14 NKT cells (CD3ε dim, RAG+,

Vα14−) into mature Vα14+ NKT cells when culturing them in combination with IL-15 and GM-CSF in the presence of stroma cells in vitro (30). Distinct from conventional T cells, these precursor cells expressed GM-CSFR at a level comparable to that in myeloid cells, even though GM-CSFR is reported to be expressed on myeloid cells but not lymphoid lineage cells (39). Moreover, when the pre-Vα14 NKT cells were cultured with GM-CSF alone without feeder cells, Vα14 gene rearrangement events, such as the Vα14-Jα281 gene-mediated signal sequence and the rearranged Vα14-Jα281 genomic DNA, were detected in the precursor cells (30). It is worth mentioning that preferential and programmed Vα14-Jα281 rearrangements do not give rise to the canonical Vα14 sequence, since a variety of Vα gene rearrangements were detected after GM-CSF treatment. Therefore, the most intriguing and reasonable possibility for GM-CSF-mediated rearrangements is that GM-CSF allows the recombination machinery to gain access to the TCRα locus. This possibility is now under investigation. Similar mechanisms have been suggested in other systems, such as Igκ gene rearrangement in pre-B-cell lines (40, 41) and the induction of the germ line transcripts of TCRγ after stimulation with IL-7 signaling (42). Consistent with the finding that GM-CSF is critical for Vα14-Jα281 gene rearrangement, GM-CSFR (βc)-deficient mice showed a significantly reduced frequency of Vα14-Jα281 gene rearrangement and diminished

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numbers of Vα14 NKT cell precursors, while no significant reduction in T cell numbers was observed (30). This suggests that GM-CSFR is expressed on the precursor cells for Vα14 NKT cells but not on conventional T cell precursors. In addition to the above evidence for the existence of Vα14 NKT precursor cells in extrathymic tissues and for extrathymic development, Vα14 NKT cells do develop in the thymus (43–45). Lineage expansion followed by Vα14-Jα281 gene rearrangement and positive selection occurred prior to NK1.1 receptor expression at the double positive (DP) stage. Precursor cells in the thymus initially do not express NK1.1, and they secrete only IL-4 until they express CD44, when they are then seen to secrete IL-4 and IFNγ . After expression of NK1.1 at a later stage, they change their cytokine secretion pattern to produce mainly IFNγ and only a little IL-4 (44, 45). Thus, Vα14 NKT cells do develop both in the thymus and in peripheral tissues via unique differentiation processes. DETECTION OF Vα14 TRANSCRIPTS AND CIRCULAR DNA MEDIATED BY Vα14-Jα281 GENE REARRANGEMENTS AT AN EARLY STAGE OF EMBRYOGENESIS Vα14 NKT

cells, unlike conventional T cells, seem to develop starting in early embryogenesis. To study the ontogeny of Vα14 NKT cells, we analyzed F1 progeny generated by crossing RAG-1-deficient females with BALB/c males (RAG-1−/− × BALB/c). Since RAG-1-deficient mothers are immunologically defective, all TCR transcripts detected must be derived from the embryos rather than by maternal contamination (32). Vα14, Vβ8.2, and CD3ε transcripts were detected in embryo bodies but not in yolk sac at an early stage of embryogenesis, gestation day 9.5 (i.e., before thymus formation), indicating the early development of Vα14 NKT cells. Also, only the paternal polymorphisms in Vα14 transcrips were detected in (RAG-1−/− × BALB/c) F1 embryos (32). The above results were confirmed by experiments showing paternally inherited polymorphisms in the Vα14 cDNA, detected in both (BALB/c × C57BL/6) F1 and (C57BL/6 × BALB/c) F1 fetuses. The genetic polymorphisms are at positions 50– 52 of Vα14, as well as the 18-nucleotide insertion in the 30 -UTR of the Cα region of C57BL/6 mice compared to that of BALB/c mice. The paternal polymorphisms were detected in the Vα14 cDNA isolated from day 11 and day 14 embryo bodies. In addition, Vα14-Jα281-mediated signal sequences were also detected in the circular DNA, indicating that Vα14-Jα281 gene rearrangements detected in the fetal tissues are derived from the fetus. Therefore, it is likely that Vα14 NKT cells develop at an early stage of gestation before thymus formation.

EVOLUTION OF THE V α14 GENE FAMILY IN MICE V α14 Gene Family Laboratory strains of mice can be divided into three groups based on the analysis of Vα14-hybridizing Pst I restriction fragment length polymorphisms [26 strains tested: A/J, AKR, BALB/c, CBA/J, CE/J, C3H, I/LnJ, 129/J, NZB, PL/J, RIIIs/J,

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RFM/MsNrs, SJL, SM/J, SWM, WB/ReJ, DBA/1, DBA/2, C57BL/6, C57BL/10, C57BR, B10A, B10A (3R), B10A (4R), and B10A (5R)] (46). Group I includes C57 mice (i.e., C57BL/6) with 3.0-kb (Vα14.1) and 1.8-kb (Vα14.3; pseudo gene) DNA fragments. Group II includes the majority of strains with 2.4-kb fragments (Vα14.2), such as BALB/c, C3H, A/J, 129, SJL and others, except C57 and DBA mice. Group III includes DBA (DBA/1 and DBA/2) mice with 1.9-kb (Vα14.4), 2.2-kb (Vα14.5), and 3-kb (Vα14.1) DNA fragments (see Figure 2). These Vα14 subfamily genes are all preferentially associated with the Jα281 gene fragment with a single-nucleotide N-region in Vα14 NKT cells, and the invariant Vα14+ cells dominate in the peripheral lymphoid population at a level of 2–3% of total TCRα used in these laboratory strains. Wild-type mice, including Mus musculus subspecies and other Mus species, also possess Vα14 gene homologues with the same subfamily groups of Vα14 genes found in laboratory strains. Among four different wild subspecies, three of them, M. m. domesticus, M. m. musculus, and M. m. molossinus, possessed Vα14.1, whereas M. m. castaneous carried Vα14.4 of DBA mice. However, there are also wild-type-specific Vα14 subfamily genes; these include M. m. dom-1 (similar to Vα14.1), M. m. dom-2 (similar to Vα14.5), M. m. dom-3 (unique), M. m. mus-1 (similar to Vα14.1), M. m. mol (equivalent to Vα14.1), M. m. cas-1 (similar to Vα14.4), and M. m. cas-2 (unique). Concerning Vα14 genes in Musculus species in Europe, M. spretoides and M. spicilegus have a single Vα14 gene similar to that of Vα14.2. However, M. spretus carries three unique Vα14 genes different from M. spretoides and M. spicilegus. However, M. caroli, which mainly inhabits India and Southeast Asia, possess a single Vα14 gene similar to Vα14.5 of DBA or M. m. dom-2, which suggests that Vα14.5 is conserved among members of the genus Mus despite their genetic distance. M. platythrix is genetically different from the rest of genus Mus but similar in morphology. The order of evolutionary dichotomy is suggested to start with Rattus around 8–11 million years ago, to continue with Apodemus speciosus or M. platythrix at intermediate times, to see the ancestors of modern species of the genus Mus split off from M. leggada between 1.5 and 3.2 million years ago, and finally to arrive at M. musculus subspecies between 0.5 and 1.0 million years ago. The Vα14 gene in M. platythrix is a single member and is positioned between A. speciosus and M. leggada (Figure 2).

Positive Darwinian Selection of the V α14 Gene Family The frequency of nucleotide substitutions in TCR V regions as a whole is generally calculated to be 0.58 in the first position, 0.47 in the second position, and 0.89 in the third position of the triplet code (47, 48). This pattern of nucleotide substitution is similar to that of most eukaryotic and prokaryotic genes (49), suggesting that amino acid substitutions in most TCR V regions are evolutionarily neutral. Surprisingly, Vα14 but not other Vα genes have more substitutions at the first and second nucleotide positions of codons over their entire length, suggesting a high proportion of the nonsynonymous mutation (amino acid altering) ratios for

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Figure 2 Dendrogram of the genetic relationship of Vα14 genes in Mus and Rattus. The data are based on the nucleotide sequences of Vα14 genes and their homologues. See text for details.

pairwise comparison among Vα14 sequences. Particularly, the rates of nonsynonymous nucleotide substitutions are higher than those of synonymous substitutions at the time of divergence of a certain species, such as A. speciosus versus Mus subspecies and Rattus versus hamster (Table 1). For example, the nonsynonymous/ synonymous ratios are much higher when A. speciosus is compared with the genus Mus (Vα14.2, M. platythrix, M. leggada), suggesting the positive selection occurred at the time the genus Mus diverged from A. speciosus. Similar Darwinian positive selection is observed between Rattus and hamster (0.953). This positive selection in Vα14 genes does not seem to have occurred during the evolution of the genus Mus or in other Vα genes (i.e., Vα1) because the ratios of nonsynonymous/ synonymous mutations among the genus Mus (i.e., Vα14.2 versus M. caroli) or Vα1 gene and their homologues are not high (generally 0.1–0.5). Therefore, the Vα14 gene family has been affected by selection mechanisms in its evolution. This is probably because Vα14 NKT cells are necessary for the survival of the species and also because the Vα14 receptor might undergo critical mutations at the time of divergence. This assumption is likely to be true since Vα14 NKT cells are essential for protection against various infectious diseases, such as granuloma formation in tuberculosis infection, and several bacterial, viral, and parasitic infections.

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TANIGUCHI ET AL. TABLE 1 Ratio of mutation types in the variable region of Vα14 and Vα1 related genesa


Ratio of nonsynonymous/ synonymous mutation Genes compared

Vα14 related

Vα1 related

Apodemus speciosus versus M. musculus M. platythrix M. leggada

1.117 1.442 1.063

0.408 0.408 0.348

M. musculus versus M. caroli

0.220

NDb

Rattus versus Hamster Human

0.953 0.189

ND ND

a

The full length of the variable region is included.

b

ND, not done

DISCOVERY OF A SPECIFIC LIGAND FOR Vα14 NKT CELLS α-Galactosylceramide as a Specific Ligand for Vα14 NKT Cells The ligand for Vα14 NKT cells has been identified as a glycolipid, α-galactosylceramide (α-GalCer), which is presented by CD1d. The glycolipid nature of the ligand was suggested by our earlier work demonstrating that the development of Vα14 NKT cells is apparently normal in transporter-associated protein (TAP)deficient mice, but severely disturbed in β2M-deficient mice (9). TAP is essential for peptide presentation to the MHC class I, and Vα14 NKT cells do not require TAP, indicating that the ligand is likely to be a nonpeptidic antigen. Thus, we screened synthetic glycolipids using splenocytes from Vα14 NKT mice, which have only Vα14 NKT cells. α-GalCer or α-glucosylceramide (α-GluCer) but not β-GalCer was identified as a specific ligand, suggesting that the α-anomeric conformation of the inner sugar is important for stimulation of Vα14 NKT cells (50).

Molecular Interaction of α-GalCer with CD1d Necessary for Vα14 NKT Cell Activation α-GalCer is a glycolipid comprising a hydrophilic carbohydrate moiety with an α-linkage to the hydrophobic ceramide portion, which consists of a long fatty acyl chain (C26) and sphingosine base (C18). It is thus easy to speculate that the ceramide portion binds to the floor of the hydrophobic cleft of CD1d, while the hydrophilic

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sugar moiety is likely to interact with the Vα14/Vβ8.2 receptor and/or α-helix of CD1d. Functional analyses using various analogues of α-GalCer revealed that the length of the carbon chains on the ceramide is important, because a shorter length of either the fatty acyl chains or the sphingosine base reduced its ability to cause Vα14 NKT cell proliferation (50). Concerning the sugar moiety, the configuration of the 20 -hydroxyl (OH) group is critical because α-mannosylceramide (α-ManCer), having a different configuration of the 20 -OH from α-GalCer, failed to stimulate Vα14 NKT cells. In a similar manner, the 3-OH on the sphingosine is important, because α-GalCer lacking 3-OH sphingosine had no effect. Based on the functional data, a docking model of α-GalCer with the crystal structure of CD1d was constructed (Figure 3) (51). The model clearly shows that the 20 -OH and 30 -OH on the sugar moiety and the 3-OH and the amide nitrogen on the ceramide portion are crucial for stable binding with CD1d. Both Arg79 and Asp80 on CD1d seem to interact with the 20 -OH group, while both Asp80 and Glu83 interact with the 30 -OH group of the carbohydrate moiety. In addition, the amide nitrogen on the fatty acyl chain and the 3-OH on the sphingosine seem to interact with Asp153 and Val149, respectively. The functional assay using a series of transfectants expressing mutant CD1d with an alanine substitution also supports the docking model, demonstrating that mutations at positions Arg79, Asp80, Glu83, and Asp153 clearly affect the activity that stimulates Vα14 NKT cells. It is, however, still possible that some of these positions are the binding sites for the Vα14/Vβ8 receptor. In fact, three different Vα14 NKT cell hybridomas bearing Vβ8 with distinct CDR3 sequences failed to react with CD1d mutated to aspartic acid at position Arg79, while two other Vα14 hybridomas with either Vβ7 or Vβ10 reacted fully with α-GalCer (52). These findings indicate that the 20 -OH on the galactose interacts mainly with Asp80 on CD1d, whereas Arg79 is likely to bind with the Vβ8 chain of Vα14 NKT cells. Studies using surface plasmon resonance have demonstrated that the affinity of CD1d for α-GalCer binding is at the 0.01–1 µM range (53). Interestingly, nonfunctional glycolipids, such as β-GalCer, α-GalCer lacking 3-OH on the sphingosine, or N-dipalmitoyl-L-α-phosphatidylethanolamine, can bind to CD1d with similar affinity to the functional glycolipids (53). Thus, CD1d appears to have the ability to bind a variety of lipid-containing antigens regardless of their stimulatory activities, but only α-GalCer and α-GluCer are able to stimulate Vα14 NKT cells.

THE CONTRIBUTION OF Vα14 NKT CELL-DEFICIENT MICE TO THE IDENTIFICATION OF THE PHYSIOLOGICAL ROLES OF Vα14 NKT CELLS Vα14 NKT cells play critical physiological roles in various immune responses. So far, two important areas of functional activities have been identified by using Vα14 NKT cell–deficient mice. Specifically, Vα14 NKT cells are important in infectious diseases and in the regulation of immune responses, such as immune suppression

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by peripheral tolerance, maintenance of transplantation tolerance, inhibition of tumor development, and protection against autoimmune disease development.


Peripheral Tolerance Peripheral tolerance is attributed to a variety of mechanisms, including low expression of MHC/TCR/CD8 molecules (54), production of immunosuppressive cytokines such as IL-10 and TGFβ (55), constitutive expression of Fas L (56), and induction of regulatory cells (57–59) and Vα14 NKT cells with regulatory function (60–62). In the model of immune privilege in the eye, known as anterior chamber– associated immune deviation (ACAID), antigen-activated intraocular BM-derived dendritic cells (DC) lacking CD40 expression showed upregulated production of MIP-2, a functional murine homologue of human IL-8 (63). The production of MIP-2 appeared to recruit Vα14 NKT cells to the spleen, where they induced regulatory T cells conveying peripheral tolerance and suppression of delayed-type hypersensitivity (DTH) responses. Vα14 NKT cells and their IL-10/TGFβ are essential for the generation of regulatory T cells and induction of tolerance in this model (60–62). In fact, CD1d-deficient or Vα14 NKT cell–deficient mice as well as IL-10-deficient mice failed to develop systemic tolerance, ACAID. As IL-10/TGFβ has a capacity to downmodulate costimulatory molecules including CD40 on DC (64), the lack of CD40 expression on DC may be due to the effects of IL-10, and by these interactions, DC may acquire tolerogenic functions. Although the requirement of IL-10/TGFβ in the final step of suppression is uncertain, it is possible that IL-10/TGFβ or tolerogenic DC induce regulatory T cells that suppress effector T cell responses (61, 64, 65). Thus, Vα14 NKT cells may serve as an inducer for the development of regulatory T cells in the ACAID system. It is well documented that CD25+ CD4+ regulatory T cells contribute to the maintenance of self- or non-self-tolerance, because mice lacking CD25+ regulatory T cells by neonatal thymectomy show a breakdown in self-tolerance and suffer autoimmunity (58, 59, 66). On the contrary, augmentation of the regulatory T cell activity by antigen activation suppressed allograft rejection and induced allograft tolerance (58). However, it is unclear whether Vα14 NKT cells have interactions with CD25+ T cells or whether they operate totally independently of each other in their functions. It is therefore important to elucidate the relationship between Vα14 NKT cells and CD25+ regulatory T cells in the maintenance of tolerance and regulation of various immune responses.

Transplantation Tolerance Rat islet cells transplanted into the liver of wild-type mice together with injection of anti-CD4 (50 µg/mouse) survived for more than 200 days (67). The same treatment failed to maintain xenograft survival in Vα14 NKT cell–deficient mice. However, xenograft survival was observed when Vα14 NKT cell–deficient mice received Vα14 NKT cells by cell transfer after transplantation, which suggests an

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essential role for Vα14 NKT cells in xenograft transplantation. Similarly, Vα14 NKT cells also play a critical role in allotransplantation tolerance; after tolerance induction against BALB/c cardiac grafts by anti-ICAM-1 and anti-LFA-1 or anti-B7-1/B7-2 antibodies, long-term acceptance of the grafts was observed in wild-type mice but not in Vα14 NKT cell–deficient mice (68). Adoptive transfer with Vα14 NKT cells restored long-term acceptance of allografts in Vα14 NKT cell–deficient mice. Therefore, Vα14 NKT cells obviously contribute to the maintenance of transplantation tolerance. However, the molecular mechanisms still remain unclear.

Immunological Surveillance of Tumors Vα14 NKT cells play a decisive immunosurveillance role in methylcholanthreneinduced fibrosarcoma development (69). When tumor incidence and the time of tumor development were compared between C57BL/6 and Vα14 NKT cell–deficient mice after injection of various doses of methylcholanthrene, tumor development occurred significantly earlier in Vα14 NKT cell–deficient mice, and the incidence of tumor development was always four- to sixfold higher than in C57BL/6 mice at any dose of methylcholanthrene. Since Vα14 NKT cell–deficient mice still possess NK cells that are also known to contribute to immunological surveillance (70), the higher incidence of tumor development might be explained if Vα14 NKT cells serve as a modulator that influences NK cell function to control tumor development and tumor immunity. This possibility is likely because IL-12 produced by activated DC primarily acts on Vα14 NKT cells to produce IFNγ that in turn activates NK and CD8 T cells to mediate tumor killing. In fact, NK and T cells in the absence of Vα14 NKT cells failed to produce IFNγ even after IL-12 activation and thus could not exert full functional activity. In some tumor models CD1d-dependent CD4+ NKT cells appear to have immunosuppressive regulatory function against CD8+ cytotoxic T lymphocytes (CTL), since elimination of CD1d-dependent CD4+ NKT cells enhanced CTL activity leading to tumor regression (25). The result suggests that CD1d-dependent NKT cells possess opposite function to protective feature of Vα14 NKT cells in tumor surveillance. The suppressive activity of CD1d-dependent CD4+ NKT cells was mediated by their producing IL-13, and several other cell types, including myeloid cells, appeared to be involved (25), suggesting an indirect immunosuppressive mechanism. It is also possible that the suppressive activity is mediated by CD1d-dependent but non-Vα14 NKT cells described by several groups (20–23, 27). In fact, human CD1d-dependent non-Vα24 NKT cells were reported to exert immunosuppressive function in MLR by producing Th2 cytokines (27). Therefore, tumor surveillance mechanisms may not function in Vα14 NKT cell–deficient mice due to the loss of the protective function of Vα14 NKT cells together with the increased numbers of CD1d-dependent non-Vα14 NKT cells with immunosuppressive, tumor enhancing activity.

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Autoimmune Diseases The association between the selective reduction in numbers of Vα14 NKT cells and autoimmune disease development was first found in the autoimmune-prone MRL/lpr mouse, a mouse model for human systemic lupus erythematosus (SLE) (71). Vα14 NKT cell numbers in these mice start to decrease at around 3–4 weeks of age before the onset of disease and completely disappear at around 10 weeks of age, after which autoimmune disease manifests itself. Introduction of transgenic Vα14 into MRL/lpr mice caused delayed reduction in Vα14 NKT cell numbers and delayed onset of the disease development. Similar findings on the pathogenesis of CD1d-reactive NKT cells of lupus were observed in NZB/NZW F1 mice (72). These results suggest a tight connection between the reduction of Vα14 NKT cell numbers and autoimmune disease development. It is intriguing that selective reduction in Vα24/Vβ11+ NKT cell numbers has also been shown in patients with various autoimmune diseases, such as systemic sclerosis (73), SLE (74), rheumatoid arthritis (74), type I diabetes (75–77), and multiple sclerosis (78, 79), indicating that reduction of Vα14/Vα24 NKT cell numbers is a general phenomenon associated with autoimmune disease development. TYPE I DIABETES Nonobese diabetic (NOD) mice develop spontaneous autoimmune Type I diabetes as a result of Th1-mediated destruction of pancreatic islet cells. A reduced number of Vα14 NKT cells and their abnormality in Th2 cytokine production have both been reported to be associated with disease development. In fact, Bach and his colleagues described a clear deficit in the number of Vα14 NKT cells at 3 weeks of age, and their ability to produce IL-4 was virtually absent at 4 weeks of age (80). Autoimmune-diabetes-prone BB rats also show severe defects in the number of NKT cells (81). These abnormalities in NOD Vα14 NKT cells can be corrected by transfer of thymic Vα14 NKT cells from nondiabetic (BALB/c × NOD) F1 mice, which protected against diabetes development induced by diabetic NOD spleen cells (82, 83). However, an overabundance of Vα14 NKT cells in Vα14 transgenic mice was only partially protective, which indicates that a functional rather than a quantitative defect of Vα14 NKT cells correlates with the pathogenesis in NOD mice (84, 85). Conversely, protection against diabetes development correlated with recovered production of Th2 cytokines, such as IL-4 and IL-10 (83). In fact, treatment of NOD mice with the Th1 cytokine IL-12 or anti-Th2 cytokine monoclonal antibodies (mAb), such as anti-IL-4, abolished protective effects by Vα14 NKT cells (85, 86). These results indicate that diabetes development is tightly associated with the defect in Th2 polarization, while disease protection is accompanied by recovery of Th2 cytokine production. Similar abnormalities were found in patients with Type I diabetes, including identical twin sets in which the diabetic siblings had fewer invariant Vα24 NKT cells than their nondiabetic twins (75). In addition, while Vα24 NKT cell clones from normal controls or nondiabetic siblings were able to produce both IFNγ and IL-4, those from diabetic patients produced only IFNγ but not IL-4, which

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indicates their abnormality in Th2 cytokine production. Similar to the murine diabetes model, Vα24 NKT cell abnormality is tightly associated with human Type I diabetes development. A causative link between the deficiency of Vα14 NKT cells and the induction of Type I diabetes has been directly demonstrated by CD1d-deficient NOD mice, clearly showing a protective role of Vα14 NKT cells (87, 88). It is surprising however that Vα14 NKT cell deficiency did not alter Th1/Th2 cytokine profiles in these mice. Moreover, dysfunction of Vα14 NKT cells is associated with accelerated accumulation of CCR4+ diabetogenic T cells in the islets, suggesting that Vα14 NKT cells may have another important role in the prevention of autoaggressive T cell recruitment to sites of inflammation (88). Furthermore, mature myeloid CD8α − DC accumulated in pancreatic lymph nodes only after treatment with αGalCer, which protected against diabetes development. Transfer of the myeloid DC into NOD mice completely prevented diabetes development (89). Therefore, an immunoregulatory role for Vα14 NKT cells in the recruitment of tolerogenic myeloid DC (CD8α − myeloid DC) to pancreatic lymph nodes, rather than Th1/Th2 cytokine imbalance, is suggested. It is, however, necessary to determine the molecular events operating in Vα14 NKT cell and tolerogenic DC interactions. CONCANAVALIN A–INDUCED HEPATITIS Concanavalin A (Con A)–induced hepatitis is considered to be a mouse model of autoimmune hepatitis mediated by T cells (90). However, recent evidence suggests the involvement of NK1.1+ cells in the pathogenesis of Con A–induced hepatitis as demonstrated by the administration of anti-NK1.1 antibody in vivo (91). Vα14 NKT cells are essential for Con A–induced hepatitis, because Vα14 NKT cell–deficient mice did not develop hepatocyte injury or mononuclear cell infiltration in the liver (92). Conversely, Vα14 NKT mice lacking other lymphocytes, including CD4 T cells and NK cells, do develop hepatitis and all mice die within 24 h. In addition, the failure to induce hepatitis in Vα14 NKT cell–deficient mice was rescued by transfer of freshly prepared or in vitro–activated Vα14 NKT cells (92). These results suggested that Vα14 NKT cells alone are able to function as effector cells in the absence of other lymphocytes. IL-4 produced by Con A–activated Vα14 NKT cells plays a crucial role in disease development by augmenting the cytotoxic activity of Vα14 NKT cells in an autocrine fashion. Indeed, the enhanced cytotoxic activity by IL-4 was accompanied by an increase in the expression levels of transcripts of perforin, granzyme B, and Fas L in Vα14 NKT cells. Moreover, Vα14 NKT cells obtained from IL4-deficient mice, perforin-deficient mice, or Fas L–mutant gld/gld mice failed to induce hepatitis, indicating that both perforin/granzyme B and Fas/Fas L–mediated killing are essential (92). Similar findings were also reported by others (93). The requirement of both perforin/granzyme B and Fas L for the induction of hepatocyte injury is intriguing, since Fas-mediated signaling induced by anti-Fas monoclonal antibody alone is enough to cause apoptotic cell death in hepatocytes (94).

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Vα14 NKT cells have been implicated in protective immune responses against several pathogens. In particular, Vα14 NKT cells interplay with both the innate immune system and acquired immune system, and these cells are essential for amplifying initial signals from the innate immune system and for promoting Th1/Th2 development in acquired immunity. Several areas of significance are detailed in the following sections. LOW-DOSE LIPOPOLYSACCHARIDE–INDUCED LETHAL SHOCK Vα14 NKT cells play an important role in various infectious diseases, although they do not express Toll-like receptors (TLRs) at a resting state, except for TLR-1. In fact, Vα14 NKT cells are essential for innate immune responses such as the generalized Shwartzman reaction, a lethal shock syndrome of mice caused after two consecutive injections of low doses of lipopolysaccharide (LPS) (95). IL-12, IFNγ , and tumor necrosis factor α (TNFα) are known to be involved in this pathogenesis , but IL-12 plays a pivotal role in the priming phase because it induces IFNγ production. IFNγ in turn induces TNFα, critical for liver injury (96). Interestingly, Vα14 NKT cell–deficient mice were resistant to this low-dose LPS shock. In Vα14 NKT cell–deficient mice, IFNγ as well as TNFα but not IL-12 production was greatly reduced compared to that in wild-type mice, and injection of recombinant IFNγ but not IL-12 restored LPSinduced mortality and TNFα production. These results indicate that LPS first acts on macrophages through TLR-4 to produce IL-12 that then acts directly on Vα14 NKT cells to produce large amounts of IFNγ and TNFα. IFNγ produced by IL-12activated Vα14 NKT cells induces production of TNFα that leads to hepatocyte injury. We do not know, however, whether it is TNFα produced by either Vα14 NKT cells or other cell types that is crucial for hepatocyte injury. In any event, Vα14 NKT cells clearly regulate innate immune responses just as they participate in the regulation of Th1 and Th2 differentiation in acquired immune responses. GRANULOMA FORMATION BY MYCOBACTERIUM TUBERCULOSIS Granuloma formation is an early immune response of the host during tuberculosis infection that is considered to hinder tuberculosis bacilli dissemination. Th1 cytokines, such as IFNγ , are known to be involved in granuloma formation (97). In a granuloma model using deproteinated bacterial cell walls [containing putative Vα14 NKT cell ligand, mycobacterial oligomannosylated glycosylphosphatidylinositol (GPI), particularly phosphatidylinositol mannoside (PIM)] in the absence of bacterial proliferation, the major cell type detected in early granulomas was the Vα14 NKT cell, and Vα14 NKT cell–deficient mice failed to form granulomas, which indicates their primary role in the processes of granuloma formation (98). Although Vα14 NKT cells are indispensable for granuloma formation, they are not primarily responsible for protection against mycobacterial infection. This is because the protective response against Mycobacterium tuberculosis infection in β2M-deficient, CD1d-deficient, and wild-type mice was indistinguishable (99, 100). Recently, Vγ 2Vδ2 T cells have been demonstrated to help protect against mycobacterial infections; clonal expansion of Mycobacterium bovis

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bacillus Calmette-Guérin (BCG)–specific Vγ 2Vδ2 T cells was associated with both the clearance of bacteremia and immunity to fetal tuberculosis in BCGvaccinated hosts (101). This may indicate that Vα14 NKT cells contribute to granuloma formation but γ δ T cells are responsible for the protective immunity against tuberculosis infection. CRYPTOCOCCUS Cryptococcus neoformans is a ubiquitous fungal pathogen that causes granulomatous lesions in the lung and disseminates to the central nervous system, frequently leading to lethal meningoencephalitis, particularly in AIDS patients. IFNγ produced by Vα14 NKT cells critically controls the Th1-dependent host defense against this pathogen (102). In particular, Vα14 NKT cell numbers were not increased in the lungs of monocyte-chemoattractant-protein-1 (MCP1)-deficient mice by Cryptococcus infection (unlike wild-type mice), suggesting that infection causes MCP-1 production that then recruits Vα14 NKT cells. Also, elimination of this fungal pathogen was drastically delayed in Vα14 NKT cell– deficient mice because of the limited IFNγ production and the failure to induce protective responses (103). TRYPANOSOMA Trypanosoma cruzi is a protozoan parasite that chronically infects mammalian species and causes Chagas’ disease (104). GPI from the parasite induces IL-12 production and activated Vα14 NKT cells to protect against parasitemia during acute Trypanosoma cruzi infection (105). In the chronic phase of infection, Vα14 NKT cells augmented Trypanosoma-specific antibodies that are considered to trigger self-damaging chronic inflammatory responses. In fact, Vα14 NKT cell–deficient mice showed greater parasitemia in the acute phase, and the antibody responses to a GPI-coupled protein were dramatically decreased in the chronic phase. Therefore, Vα14 NKT cells play an important role in the pathogenesis of both acute and chronic Trypanosoma cruzi infection.

THE CONTRIBUTION OF α-GalCer TO THE IDENTIFICATION OF Vα14 NKT CELL FUNCTIONS Despite significant early studies on Vα14 NKT cells, it was difficult to define their function because of their limited numbers in various tissues in vivo. However, the identification and use of a specific ligand, α-GalCer, revealed several important activities of Vα14 NKT cells in vivo and in vitro.

Polarized Th1 Cytokine Production and Disease Protection Vα14 NKT cells produce Th1 and Th2 cytokines as well as perforin/granzyme B and Fas L upon stimulation with α-GalCer, and such cells exert multifunctional activities in various immune responses (Figure 4). In fact, α-GalCer-activated Vα14 NKT cells induce expression of CD40 L (CD154) on their cell surface, which engages CD40 on DC and stimulates DC to produce IL-12 (106, 107). Then, activated Vα14 NKT cells exert two distinct functional activities

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at different times. As early as 90 min after α-GalCer injection in vivo, IFNγ produced by Vα14 NKT cells stimulated NK cells to enhance IFNγ production. B and T cells were activated at later times to express CD69 and to enhance immunoglobulin E (IgE) antibody production (108, 109). Thus, Vα14 NKT cells support Th1 differentiation at an early stage and then contribute to the development of Th2 cells at a later stage. IL-12/IFNγ CASCADE IN Vα14 NKT CELL–MEDIATED CYTOTOXICITY Vα14 NKT cells, in addition to their effector function, serve as inducers to activate other cytotoxic effector cells, such as NK cells or CD8 T cells (Figure 4). α-GalCerpulsed DCs were activated to produce IL-12 that acts primarily on Vα14 NKT cells but not NK and T cells (5, 107). This is because the mature form of IL-12 receptor is expressed only on Vα14 NKT cells but not on NK cells and CD8 T cells at the resting state. Although antitumor effects of IL-12 were thought to be associated with NK and/or CD8 killer cells, the IL-12-induced antitumor activity was not found in Vα14 NKT cell–deficient mice, indicating that Vα14 NKT cells are essential for IL-12-mediated antitumor activity (5, 110). In addition, the majority of IFNγ detected after IL-12 treatment is produced by Vα14 NKT cells but not by NK and T cells, because Vα14 NKT cell–deficient mice as well as RAG1-deficient mice (having only NK cells) produced only small amounts of IFNγ compared to wild-type mice (less than one fifth as much) (5). IFNγ produced by IL-12-activated Vα14 NKT cells in turn stimulates NK and CD8 T cells as a secondary effect, all of which mediate cytotoxic activity through perforin/granzyme B, tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), or Fas/Fas L mechanisms (111, 112). Therefore, IL-12 produced by DC is a key factor that preferentially activates the Vα14 NKT cell system. Like IL-12, IL-18 was originally identified as an IFNγ -inducing factor (IGIF). It induces IFNγ production, enhances NK-like cytotoxicity, drives Th1 responses, and mediates Fas/Fas L–dependent cytotoxicity (113). Vα14 NKT cells do not seem to be involved in IL-18-mediated IFNγ -production cascade interactions despite their expression of IL-18 receptor, because the enhanced cytotoxic activity induced by IL-18 was not seen in NK cell–depleted mice treated with anti-asialo GM1 antibody, but was still seen in Vα14 NKT cell–deficient mice (112, 114, 115). It is interesting, however, that IL-18 directly acts on Vα14 NKT cells and enhances IL-4 production only when Vα14 NKT cells are activated by anti-TCR or their ligand, α-GalCer (116). ANTITUMOR ACTIVITY The α-GalCer-activated Vα14 NKT cells also serve as cytotoxic effector cells mediating strong antitumor activities, because Vα14 NKT mice but not Vα14 NKT cell–deficient mice were protected against experimental liver metastasis of melanoma upon stimulation with α-GalCer in vivo (50). Thus, Vα14 NKT cells are activated by α-GalCer in vivo, in situ in the liver, and have the potential to serve as cytotoxic effector cells to directly kill tumor cells. NK and CD8 T cells also contributed to the cytotoxic effector mechanisms as a secondary effect by IFNγ produced by activated Vα14 NKT cells (108, 111, 112, 117).

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ERADICATION OF ONGOING TUMORS BY α-GalCer-PULSED DC THERAPY Although α-GalCer alone showed potent antitumor activity, it was no longer effective when α-GalCer-treatment started 3 days after melanoma cell injection (118, 119). Histological examination clearly revealed no significant tumor nest formation in the liver on day 5 after tumor injection. However, tumor cells quickly grew and tumor nodules became visible on day 7. Therefore, we speculate that in this experimental model, injection of α-GalCer alone does not provide sufficient effects compared to the tumor growth rate. To circumvent these problems, α-GalCer-pulsed DC (α-GalCer-DC) rather than α-GalCer injection alone was used. α-GalCer-DCs (3 million cells) were injected into tumor-bearing mice that had multiple small (0.5–1 mm) metastatic nodules in the liver. Seven days after α-GalCer-DC treatment, tumor nodules disappeared and metastatic tumors were completely cured. By this treatment, Vα14 NKT cell numbers in tissues (i.e., lung and liver) were increased five- to sixfold 72 h after α-GalCer-DC injection (as determined by α-GalCer-CD1d tetramer-staining), and they continued to be maintained up to 7 days after the treatment compared to normal control mice or mice treated with a single injection of α-GalCer alone (M. Taniguchi, unpublished observation). Thus, unlike injections of α-GalCer alone, α-GalCer-DC therapy is efficient for tumor eradication. In the last several years, three groups have independently reported that α-GalCer stimulates human Vα24/Vβ11 NKT cells equivalent to murine Vα14 NKT cells (120–123). Since α-GalCer binding sites (Arg79, Asp80, Glu83, Val149, Asp153) on CD1d are all conserved among species, CD1d in various mammalian species is able to present α-GalCer. It was shown that α-GalCer-activated Vα24 NKT cells mediated a potent perforin/granzyme B–dependent cytotoxic activity against a wide variety of human tumor cell lines (123). These observations suggest the possibility that α-GalCer-CD1d could be used as an effective new tool for cancer immunotherapy with great advantages. First, cytotoxicity against various tumors is very high, irrelevant of cell type and histological grade, and normal cells are not susceptible. Second, α-GalCer can be used in all patients, since the α-GalCer activation of Vα24 NKT cells is dependent on monomorphic CD1d. Finally, the functions of DC and Vα24 NKT cells in cancer patients can be evaluated before immunotherapy by assay systems using mouse Vα14 NKT cells and DC as stable indicators, because α-GalCer-pulsed mouse DC activates human Vα24 NKT cells and α-GalCer-pulsed human DC activates mouse Vα14 NKT cells (123). IgE SUPPRESSION BY Vα14 NKT CELL–DERIVED IFNγ Since activated Vα14 NKT cells produce large amounts of both IFNγ and IL-4, a regulatory role of Vα14 NKT cells in Th1/Th2 differentiation has been suggested (16, 50, 124–126). However, Vα14 NKT cells are not required for IgE responses because CD1d-deficient mice or Vα14 NKT cell–deficient mice showed normal IgE production (33–35, 127). On the contrary, activated Vα14 NKT cells suppressed Th2 differentiation and IgE production. IgE suppression was not induced in Vα14 NKT cell–deficient mice or IFNγ -deficient mice, indicating that the activated Vα14 NKT cells and their IFNγ suppress IgE responses after α-GalCer injection (127). Similarly, when na¨ıve CD4

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T cells were cultured in the presence of IL-4 with α-GalCer-activated Vα14 NKT cells, Th2 differentiation was severely suppressed, indicating that Vα14 NKT cell– derived IFNγ drives Th1 cell differentiation even in the presence of IL-4 (127). This may indeed be the case because IFNγ downregulates the expression of IL-4 receptor on Th2 cells, which are then not able to receive IL-4 signals important for Th2 development and IgE production. α-GalCer-activated Vα14 NKT cells have potent antimalaria activity, inhibiting the development of intrahepatic stages of the rodent malaria parasites Plasmodium yoelii and P. berghei. Antimalaria activity is stage specific and is effective for only a short time during the liver stage of the parasite. Thus the timing of α-GalCer administration in vivo was critically important for the antimalaria activity. The activity was highest when α-GalCer was administered 1 or 2 days prior to sporozoite challenge. Vα14 NKT cell–deficient, CD1d-deficient, and IFNγ -deficient mice failed to show α-GalCer-mediated protection against malaria infection (128). Only IFNγ produced by activated Vα14 NKT cells but not other cytotoxic molecules (Fas L, perforin, or TNFα) is responsible for malaria protection. The activity can be elicited in the absence of NK, B, and T cells, which suggests that Vα14 NKT cells serve as effector cells to control parasite replication in the liver. Most recently, it has been shown that Vα14 NKT cells not only exert a direct inhibitory activity against the liver stage of malaria but also play a role in enhancing memory responses elicited by malaria vaccines, including irradiated sporozoites and a recombinant circumsporozoite protein (129). The increased protective immunity is due to the increased level of malaria-specific CD8+ T cell responses by α-GalCer-activated Vα14 NKT cells. The effect of α-GalCer was abolished in mice lacking CD1d, IFNγ R, or Vα14 NKT cells, indicating that IFNγ produced by Vα14 NKT cells mediates the adjuvant activity of α-GalCer. Similar enhancing effects of activated Vα14 NKT cells on CD8+ effector T cell function specific for Toxioplasma gondii have also been reported in other intracytoplasmic parasitic infections (130).


ANTIMALARIA ACTIVITY

ANTI–HEPATITIS B VIRUS INFECTION It is well known that IFNγ and IFNα/β inhibit hepatitis B virus (HBV) replication (131). Intrahepatic Vα14 NKT cells were activated to produce IFNγ and IFNα/β within 24 h and inhibited HBV replication when administered with α-GalCer into HBV transgenic mice (132). Since the antiviral activity by α-GalCer was abolished in mice deficient for receptors of either IFNγ or IFNα/β, most of the antiviral activity was mediated by these cytokines derived from Vα14 NKT cells (132). Therefore, it is possible that Vα14 NKT cells are a good candidate for the control of viral replication during natural HBV infection, and thus represent a new strategy for the treatment of chronic HBV infection. α-GalCer -INDUCED ABORTION In the decidua is a unique subset of Vα14 NKT cells expressing the Vβ7 receptor that provokes abortion (almost 100%) after stimulation with α-GalCer (133). The Vβ7+ Vα14 NKT cells produced IFNγ and

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TNFα but not Th2 cytokines upon activation by α-GalCer. α-GalCer-mediated abortion appeared to result from selective degeneration of embryonic trophoblasts by apoptotic cell death, and was not observed in mice deficient in Vα14 NKT cells, IFNγ , TNFα, or perforin. Thus, the apoptotic process in Vα14 NKT cell–mediated abortion seems to be a multifactorial event in which both cytokines and direct cell-to-cell contact, followed by perforin/granzyme-dependent killing mediated by Vα14 NKT cells, are essential. The physiological role of decidual Vα14 NKT cells remains elusive, but they might be involved in feto-maternal immune responses against pathogens to protect the mother by inducing abortion of infected fetuses.

Polarized Th2 Cytokine Production and Disease Protection It was originally reported that Vα14 NKT cells favor the establishment of Th2 immunity through their selective ability to secrete IL-4 at an early stage after α-GalCer or anti-CD3 stimulation (15, 16, 50, 124–126). However, Vα14 NKT cells instead contribute to Th1 development by secreting IFNγ (15, 50, 108, 109, 126, 127).The number of α-GalCer administered to mice determines the cytokine profile of Vα14 NKT cells. Repeated exposure to α-GalCer polarized the cells to only IL-4 secretion and to the Th2 pathway (134, 135). Although the mechanism of Th2 shift of Vα14 NKT cells after repeated α-GalCer injection remains unclear, IL-7 or IL-18 seems to contribute to the enhanced IL-4 production and increased numbers of IL-4-producing Vα14 NKT cells. In fact, IL-7 enhanced IL-4 production by Vα14 NKT cells only after TCR engagement (136, 137). Moreover, IL-18 augmented IL-4 production only when Vα14 NKT cells were activated with α-GalCer (116). Since IL-18 receptor is expressed on Vα14 NKT cells (115) and no IL-18induced IL-4 enhancement was detected in Vα14 NKT cell–deficient mice, IL-18 acts directly on Vα14 NKT cells (116). Thus, the IL-7 or IL-18 pathway might explain the polarized Th2 switch of Vα14 NKT cells upon repeated exposure to α-GalCer. PREFERENTIAL Th2 CYTOKINE PRODUCTION BY REPEATED α-GalCer INJECTION AND PREVENTION OF TYPE I DIABETES Prevention of autoimmune diabetes develop-

ment in NOD mice seems to correlate with the ability of α-GalCer to enhance Th2 cytokines (IL-4/IL-10) and suppress IFNγ production, resulting in islet-specific protective Th2 cell generation (138, 139). Repeated injection (i.e., every day for 2 weeks, or twice per week for 30 weeks) of α-GalCer and IL-7 into NOD mice prevented the onset of diabetes. The mechanisms of Th2 switch of Vα14 NKT cells by α-GalCer and IL-7 in NOD mice remain unclear. PREFERENTIAL Th2 CYTOKINE PRODUCTION BY α-GalCer ANALOGUE AND PREVENTION OF EXPERIMENTAL ALLERGIC ENCEPHALITIS Vα14 NKT cell dysfunction

has been demonstrated to correlate with the pathogenesis of experimental allergic encephalitis (EAE), a murine model of Th1-mediated autoimmune disease of the central nervous system. The high susceptibility of SJL mice to EAE is associated with a functional defect of Vα14 NKT cells because of a lack of TCRVβ8 genes

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in the genome (140). The depletion of NK+ cells in non-autoimmune-prone mice rendered them extremely susceptible to the induction of EAE (141). Furthermore, EAE development in IL-4-deficient mice was enhanced by multiple α-GalCer treatments, while IFNγ deficiency suppressed EAE (142), which suggests that Th2 cytokines produced by the repeated α-GalCer-activation of Vα14 NKT cells prevents EAE. Protection from EAE conferred by α-GalCer also correlated with its ability to suppress myelin antigen–specific Th1 responses and/or to promote myelin-specific Th2 responses (143). It would be interesting to develop α-GalCer analogues that would activate Vα14 NKT cells to selectively secrete IL-4 but not IFNγ ; these cells would be expected to promote the selective development of Th2 cells. In fact, an α-GalCer analogue with a truncated sphingosine base induced preferential IL-4 production by Vα14 NKT cells (144). As expected, a single injection of the α-GalCer analogue consistently induced Th2 polarization, resulting in the suppression of EAE development. Although further investigation of this reagent is required, such a reagent would be ideal for controlling the production of protective cytokines and for efficient prevention of autoimmune diseases.

CONCLUSIONS Functional analyses of Vα14 NKT cells have provided new insights into the regulatory mechanisms operating in both protection against disease development and host defense. In particular, the discovery of the Vα14 NKT cell ligand, α-GalCer, and the establishment of gene-manipulated mice lacking only Vα14 NKT cells have helped to elucidate the functional diversity of Vα14 NKT cells in the regulation of innate immune responses in host defense, protection against autoimmune disease development caused by dysfunction of adaptive immune responses, and antitumor immunity (Figure 4). Innate immune responses are in the first instance triggered by DC, B cells, macrophages, or neutrophils through their innate immune receptors. The responses include acute and chronic inflammatory responses induced by bacterial, parasitic, or fungal infections, and viral replication. Cells of the innate immune system are activated by pathogens and their products through the innate immune receptors to produce cytokines such as IL-12, which primarily activates Vα14 NKT cells because of their preferential expression of IL-12 receptor. Vα14 NKT cells thus activated secrete cytokines such as IL-4 and/or IFNγ that augment their functional activities by autocrine mechanisms or by activation of other effector molecules and cells in the innate immune system, such as NK cells or macrophages. In fact, Vα14 NKT cell–deficient mice failed to induce innate immune responses against certain pathogens. Thus, Vα14 NKT cells appear to play pivotal regulatory roles in anti-nonself responses in innate immunity. Besides the above functional importance of Vα14 NKT cells in innate immunity, Vα14 NKT cells serve as regulatory cells to control anti-self and anti-nonself responses in adaptive immunity. Studies of Vα14 NKT cell–deficient mice, as

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well as molecular and cellular analyses of Vα14 NKT cell function after activation with a specific ligand, α-GalCer, have revealed that Vα14 NKT cells are essential to maintain homeostasis in the adaptive immune system by controlling Th1/Th2 differentiation via their cytokines. In addition, Vα14 NKT cells serve as regulatory T cells that mediate self-tolerance and suppression of various immune responses. Thus, dysfunction of Vα14 NKT cells leads to the development of various diseases, including autoimmune diseases, rejection of organ transplantation, and failure of immunological surveillance of tumors. Thus, Vα14 NKT cells control both innate and adaptive immune responses. Vα14 NKT cells, once activated, exert a potent antitumor activity through activation of NK cells and CD8 cytotoxic T lymphocytes. Moreover, activated Vα14 NKT cells also serve as bystander effector cells to kill various tumor target cells by NK-like cytotoxic activity through perforin/granzyme B, Fas/Fas L, TNFα, or TRAIL interactions. The use of α-GalCer provides us with a powerful tool to manipulate functions of activated Vα14 NKT cells. Since α-GalCer binding sites on CD1d are all conserved among various species, it might have utility for the treatment of patients. From a therapeutic point of view, the possibility of preventing disease development by the manipulation of Vα24 NKT cell functions with α-GalCer is of particular interest. Finally, the endogenous ligands of Vα14 NKT cells remain unknown. The identification of an endogenous ligand should lead to a better understanding of the physiological roles of Vα14 NKT cells in either homeostatic processes or in pathological situations, such as autoimmune disease development or surveillance of tumor development. ACKNOWLEDGMENTS We thank Dr. Pandelakis A. Koni (Medical College Georgia and RIKEN Research Center for Allergy and Immunology) for discussion and Hiroko Tanabe for her help throughout the preparation of this review. This work was in part supported by The Human Frontier Science Program Research Grant (#RG0168/2000-M), and Core Research for Evolutional Science and Technology (JST) and a Grant-in-Aid for Scientific Research A (#13307011) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The Annual Review of Immunology is online at http://immunol.annualreviews.org

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Bergmann CC, Wilson JM, Schmieg J, et al. 2002. Natural killer T cell ligand αgalactosylceramide enhances protective immunity induced by malaria vaccines. J. Exp. Med. 195:617–24 Denkers EY, Scharton-Kersten T, Barbieri S, Caspar P, Sher A. 1996. A role for CD4+ NK1.1+ T lymphocytes as major histocompatibility complex class II independent helper cells in the generation of CD8+ effector function against intracellular infection. J. Exp. Med. 184:131–39 McClary H, Koch R, Chisari FV, Guidotti LG. 2000. Relative sensitivity of hepatitis B virus and other hepatotropic viruses to the antiviral effects of cytokines. J. Virol. 74:2255–64 Kakimi K, Guidotti LG, Koezuka Y, Chisari FV. 2000. Natural killer T cell activation inhibits hepatitis B virus replication in vivo. J. Exp. Med. 192:921–30 Ito K, Karasawa M, Kawano T, Akasaka T, Koseki H, et al. 2000. Involvement of decidual Vα14 NKT cells in abortion. Proc. Natl. Acad. Sci. USA 97:740–44 Burdin N, Brossay L, Kronenberg M. 1999. Immunization with α-galactosylceramide polarizes CD1-reactive NK T cells towards Th2 cytokine synthesis. Eur. J. Immunol. 29:2014–25 Singh N, Hong S, Scherer DC, Serizawa I, Burdin N, et al. 1999. Cutting edge: activation of NK T cells by CD1d and α-galactosylceramide directs conventional T cells to the acquisition of a Th2 phenotype. J. Immunol. 163:2373– 77 Gombert JM, Tancrede-Bohin E, Hameg A, Leite-de-Moraes MC, Vicari A, et al. 1996. IL-7 reverses NK1+ T cell-defective IL-4 production in the non-obese diabetic mouse. Int. Immunol. 8:1751–58 Hameg A, Gouarin C, Gombert JM, Hong

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S, Van Kaer L, et al. 1999. IL-7 up-regulates IL-4 production by splenic NK1.1+ and NK1.1− MHC class I-like/CD1dependent CD4+ T cells. J. Immunol. 162: 7067–74 Hong S, Wilson MT, Serizawa I, Wu L, Singh N, et al. 2001. The natural killer T-cell ligand α-galactosylceramide prevents autoimmune diabetes in nonobese diabetic mice. Nat. Med. 7:1052– 56 Sharif S, Arreaza GA, Zucker P, Mi QS, Sondhi J, et al. 2001. Activation of natural killer T cells by α-galactosylceramide treatment prevents the onset and recurrence of autoimmune Type 1 diabetes. Nat. Med. 7:1057–62 Acha-Orbea H, Steinman L, McDevitt HO. 1989. T cell receptors in murine autoimmune diseases. Annu. Rev. Immunol. 7:371–405 Zhang B, Yamamura T, Kondo T, Fujiwara M, Tabira T. 1997. Regulation of experimental autoimmune encephalomyelitis by natural killer (NK) cells. J. Exp. Med. 186:1677–87 Pal E, Tabira T, Kawano T, Taniguchi M, Miyake S, Yamamura T. 2001. Costimulation-dependent modulation of experimental autoimmune encephalomyelitis by ligand stimulation of Vα14 NK T cells. J. Immunol. 166:662–68 Singh AK, Wilson MT, Hong S, OlivaresVillagomez D, Du C, et al. 2001. Natural killer T cell activation protects mice against experimental autoimmune encephalomyelitis. J. Exp. Med. 194:1801– 11 Miyamoto K, Miyake S, Yamamura T. 2001. A synthetic glycolipid prevents autoimmune encephalomyelitis by inducing TH2 bias of natural killer T cells. Nature 413:531–34


Figure 3 Docking model of α-GalCer with the crystal structure of CD1d. The hydrogen-bond network near the galactose and ceramide linkage is represented by dashed blue lines. The oxygen atoms on the side chains of Arg79, Asp80, Glu83, Val149, Asp153, and α-GalCer are shown in red and nitrogen atoms in violet. The α-helices and carbon atoms of CD1d and carbon atoms of α-GalCer are colored in yellow and white, respectively.


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Figure 4 Schematic diagram of Vα14 NKT cell activation and their interactions. Two types of activation pathways, through innate immune system/IL-12 receptor and through specific recognition of the α-GalCer ligand, are represented. Each activation pathway displays different functional activities of Vα14 NKT cells. TLR, Toll-like receptor; APC, antigen presenting cells; LPS, lipopolysaccharide; LAM, lipoarabinomannane; M, macrophage.


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Annu. Rev. Immunol. 2003. 21:515–46 doi: 10.1146/annurev.immunol.21.120601.141045 c 2003 by Annual Reviews. All rights reserved Copyright ° First published online as a Review in Advance on December 6, 2002

ON NATURAL AND ARTIFICIAL VACCINATIONS Rolf M. Zinkernagel Annu. Rev. Immunol. 2003.21:515-546. Downloaded from arjournals.annualreviews.org by HINARI on 09/01/07. For personal use only.

Institute for Experimental Immunology, University Hospital, Zurich CH-8091, Switzerland; email: [email protected]

Key Words immunological memory, immunity, maternal antibodies, autoimmunity, infections, virus, tumors ■ Abstract This review summarizes the general parameters of cell- and antibodymediated immune protection and the basic mechanisms responsible for what we call immunological memory. From this basis, the various successes and difficulties of vaccines are evaluated with respect to the role of antigen in maintaining protective immunity. Based on the fact that in humans during the first 12–48 months maternal antibodies from milk and serum protect against classical acute childhood and other infections, the concept is developed that maternal antibodies attenuate most infections of babies and infants and turn them into effective vaccines. If this “natural vaccination” under passive protective conditions does not occur, acute childhood diseases may be severe, unless infants are actively vaccinated with conventional vaccines early enough, i.e., in synchronization with the immune system’s maturation. Although vaccines are available against the classical childhood diseases, they are not available for many seemingly milder childhood infections such as gastrointestinal and respiratory infections; these may eventually trigger immunopathological diseases. These changing balances between humans and infections caused by changes in nursing habits but also in hygiene levels may well be involved in changing disease patterns including increased frequencies of certain autoimmune and degenerative diseases.

INTRODUCTION During the past 100 years the nature of immunological memory has been widely debated, not only by immunologists but also in the clinical context and from a public health perspective (1–7, 7a). Immunological memory is the basis for protective vaccines. Vaccinations against childhood diseases, such as poliomyelitis and smallpox, have been very successful, and smallpox has been eradicated by a worldwide campaign with the vaccinia virus (reviewed in 8–12). Nevertheless, efficient vaccines are still lacking against tuberculosis (TB), leprosy, and parasitic diseases, such as malaria, leishmaniasis, and schistosomiasis. Vaccines are also lacking against human immunodeficiency virus (HIV), dengue, respiratory syncytial virus (RSV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), rotaviruses, herpes simplex virus (HSV), and papillomavirus infections and against most tumors. In addition, some antiviral vaccines, such as those against measles 0732-0582/03/0407-0515$14.00

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and mumps, are far from offering complete protection since viral breakthroughs may occur (13, 14). These successes and failures or inadequacies demonstrate that our understanding of the nature of immunological memory is incomplete. This review considers the following questions: Have artificial vaccines been “foreseen” by nature? What is the physiological equivalent of our vaccines? What is missing in our knowledge not only about immunity but also about vaccines?


MECHANISMS OF IMMUNE PROTECTION Resistance to infections is based importantly on nonspecific mechanisms (interferons, complement, natural antibodies, natural killer cells, activated phagocytes) and many additional mechanisms (10, 15–17). These nonspecific resistance mechanisms are responsible for the major part (>95%) of host defense. For example, absence of interferon receptors increases susceptibility of mice to viral infections by several orders of magnitude (18). Specific immunity is phylogenetically a rather new fine-tuner of resistance, emerging as a result of coevolution between hosts and infectious agents. The two arms of the immune system, humoral and cellular, fulfill the following major tasks. The immunoglobulin receptor of B cells and secreted antibodies directly recognize complex folded proteins or carbohydrates. Protective antibodies inactivate and block the action of infectious agents or toxins by covering them and/or by facilitating their phagocytosis. Immunoglobulin M (IgM) and IgG protect against antigens in blood and the lymphatic system, IgA protects on mucosal membranes (19, 20), and IgE triggers mast cells and basophils in skin and mucosae. In contrast to B cells and antibodies, T cells recognize small peptides presented on the cell surface by major histocompatibility complex (MHC) antigens (21). Cytotoxic CD8+ T cells are specific for fragments of proteins synthesized by the cell itself and are presented by MHC class I (HLA-A, -B, -C) antigens; this pathway includes not only self-peptides but also viral, intracellular bacterial, and tumor antigens. Phagocytized antigens are processed in phagolysosomes and are presented by MHC class II antigens. Dendritic cells, which are either infected themselves or are able to take up infectious foreign antigen or decaying self-antigen, transport antigens to organized lymphatic tissues. They are therefore often of key importance in inducing T cell responses. Whereas antibodies act directly where they are released or transported to, T cells actively emigrate into peripheral solid tissues. T cells can act via direct contact or by specific release of immune mediators such as interferon or tumor necrosis factors (TNFs), or they can act nonspecifically via recruitment and activation of macrophages. Cytopathic viruses or bacteria that cause an acute lethal infection are in general most efficiently controlled by soluble diffusible factors including T cell–dependent cytokines [such as gamma interferon (IFNγ ) and TNF] and by specific neutralizing antibodies. Noncytopathic intracellular organisms usually cause no direct cell or tissue damage and therefore no disease, even though they tend to persist. In this case immune control is mediated by perforin-dependent, cytotoxic, and cytokine-releasing T cells that

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TABLE 1 General rules for induction and maintenance of effector cells and antibody responses T cells Ignore antigen not reaching lymph nodes, spleen, or Peyer’s patches in sufficient dose and for less than 3 days. Some T cells are induced against antigen reaching lymph nodes, Peyer’s patches, and/or spleen in moderate doses and dose kinetics for 3–15 days. All are induced and exhausted if antigen persists at sufficient levels everywhere. (This is negative selection, which occurs earliest in thymus, but also in secondary lymphatic organs, lymph nodes, and the spleen.) Maintenance of immediately effective cytotoxic or helper T cells depends on presence of antigen. B cells Induced only in secondary lymphatic organs (lymph nodes, spleen, Peyer’s patches, crypto patches). Induced by rigid, multimeric, highly concentrated antigenic determinants or multimeric antigens together with LPS-like polyclonal activators: IgM responses of T independent type 1. Induced by multimeric, mobile, or flexible antigens (on cell surfaces or linear flexible multimers) together with unlinked T help: IgM responses of T-independent type 2. Induced by limiting doses of mono- or oligomeric antigens to make IgM or IgG responses if conventionally linked T helper cell activity is provided: T help–dependent B cell responses. Switched to IgG-dependent upon carrier-specific conventionally linked T help. Maintenance of antibody titers in serum depends on antigen-driven B cell maturation to plasma cells.

cause inflammation and tissue damage (22, 23). Since the immune system cannot distinguish a priori between cytopathic and noncytopathic infections it cannot really “foresee” its beneficial and detrimental effects on the host; it merely responds to antigen. Therefore, protection by immunity represents an equilibrium between optimal resistance against the various cytopathic infections and avoidance of excessive immunologically mediated tissue damage. Clinical examples of unbalanced immunity against non- or weakly cytopathic infections causing disease by immunopathology are tuberculoid leprosy, fulminant aggressive hepatitis B virus (HBV), hepatitis C virus (HCV), or HIV infections leading to acquired immune deficiency syndrome (AIDS). T and B cell responses are initiated according to the following general rules defined by antigen structure, antigen localization, its dose, and how long antigen is available (5, 7a). (The rules are summarized in Table 1.) 1. Conventional immune responses of T and B cells can be induced only in organized secondary lymphoid organs (i.e., lymph nodes, Peyer’s patches, and the spleen). 2. T cells react against cell-associated antigens that are localized in secondary lymphoid organs in sufficient amounts and for a period of at least 3–5 days. Antigens that always stay outside of secondary lymphoid organs are

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immunologically ignored. At the other end of the spectrum, antigens that are always in primary or secondary lymphoid organs—such as serum proteins— induce and delete all potentially reactive T cells. This process is called negative selection (24–27). 3. B cells react against highly repetitive, rigidly ordered antigenic determinants with shortlived IgM responses independently of T help, particularly if combined with a polyclonal activator (28). These antigens are called Tindependent type I. Other multimeric but nonrigid antigens, including those on cell surfaces, will also induce B cell IgM responses (29) if presented together with indirect (or unlinked) T help (T-independent type II antigens). Usually, B cells react against monomeric or oligomeric antigens only if structurally linked specific T help is provided (30). It is important to note that all B cell responses become dependent upon linked T help if antigen doses are limited (29). Also, the switch to long-lived IgG and the maintenance of IgG responses are usually dependent upon linked carrier-specific T help. The highly repetitive paracrystalline identical determinants on most infectious agents distinguish them from the usually mono-oligomeric self-antigens accessible to B cells (28). A consequence of the tight T cell control of IgG responses is that B cells are in general not necessarily deleted by self-antigen. While they may not react against monomeric antigens, they are nevertheless potentially self-reactive. However, such autoreactive B cells are not readily induced to produce IgM or even switch to IgG responses because highly repetitive ordered self-antigens normally do not exist in the lymphatic system or in blood (31) and because self-antigens are usually not linked to polyclonal B cell activators (32). Specificity is a key issue in any discussion about immune protection, immunological memory, and vaccines. The specificity of immunity is most directly measured by protection or cross-protection in vivo, e.g., protection by immunity against poliovirus strain I (serotype A) is absent against a subsequent infection with poliovirus II (serotype B) (Table 2) (33). Since both cytotoxic and helper T cells against serotype-defined virus groups are shared between the various serotypes, the obvious lack of cross-protection between serotypes (e.g., poliovirus I, II, III) in human populations indicates that only preexistent neutralizing antibodies and not primed helper or cytotoxic T cells are responsible for protection (Table 2) (7, 34–36). Infectious agents that exhibit various serotypes are often highly cytopathic and cause acute diseases. Innumerable specificities of antibodies are usually induced by virus infection, but only neutralizing antibodies are protective (28, 37, 38); other antibodies, particularly those against internal viral antigens, are virtually irrelevant for protection. Those infections that tend to persist, including many viruses such as HBV, HCV, and HIV (23, 39, 40), facultative intracellular bacteria such as mycobacteria (9, 12, 41, 42), and intracellular parasites are usually controlled initially by T cells, but antibodies often play a controlling role also (e.g., 43, 44).

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TABLE 2 Protection against serotype-defined infections Virus

Serotypes

Specificity of T cells: CD4+, CD8+

Neutralizing antibodies

Polio

I, II, III

Largely shared Nonessential for protection

Highly specific Essential for protection

Immunization

Challenge

T cell response

Neutralizing antibody response

Serotype A

Serotype B

Secondary anti-A and secondary anti-B

Primary anti-B


Experimental evidence

Measuring Immunity How are immune responses best measured? This means measuring T cell and neutralizing antibody responses to assess essential mechanisms of protective immunity or so-called immunological memory and predict the efficiency of vaccines. Neutralizing antibody responses are measured in vitro by a virus plaque-reduction assay or by neutralization of the activity of bacterial toxins. These measurements correlate with the following observations: Neutralizing antibodies, usually of the IgG type, must possess an overall avidity of around 108 M−1 or more and must be available in serum at concentrations of around 10−8 M (≥1 µg/ml) to be protective in tightly controlled murine model infections (38, 45). The protective capacity of cytotoxic T cells correlates best with the direct measurable lytic activity of lymphocytes in a 4- to 5-hour in vitro assay tested against infected target cells or target cells pulsed with relevant T cell peptides at concentrations of around 10−9 to 10−10 M. The following tests done in vitro probably overestimate activities and relative precursor numbers of B and T cells: ELISA-binding antibodies assessing affinities or avidities of 105

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TCR:peptide-MHC Kd values at 37◦ C are typically 1.5–2.5 fold higher than at 25◦ C (see references cited in Table 1).

1 2

There is some variation between alleles but no difference between CD8αα and CD8αβ.

entropic changes are consistent with a decrease in conformational flexibility at the binding interface upon binding. RELATIONSHIP BETWEEN BINDING PROPERTIES AND FUNCTIONAL OUTCOME Several studies have examined the correlation between the solution-binding properties and functional consequences of TCR:peptide-MHC interactions. A broad correlation between affinity/half-life and functional effect [reviewed in (22, 23)] was observed, although there were some exceptions (24–28). This broad correlation supports models of TCR triggering in which the duration of binding, rather than a specific conformational change, determines the outcome of TCR:peptide-MHC interactions. What of the aforementioned exceptions to the correlation between affinity/halflife and functional outcome (24–28)? One possible explanation lies in the fact that TCR:peptide-MHC interactions, like all interactions between membrane-anchored molecules, are subject to mechanical stress (29, 30). Whereas mechanical stress will generally enhance dissociation, interactions can vary considerably in the degree of this enhancement (30). Thus, two TCR:peptide-MHC interactions that have identical half-lives in solution can have different half-lives when subjected to mechanical stress. Future studies that measure mechanical strength directly or measure solution-binding properties (such as the activation enthalpy of dissociation) that are thought to correlate better than half-life/affinity with mechanical strength are needed to address this issue (31).

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CORECEPTOR INTERACTIONS Given their functional similarity, the CD4 and CD8 molecules have surprisingly different structures (Figure 2). Whereas CD4 is a monomeric polypeptide with four IgSF domains in its ectodomain, CD8 is a disulphide-linked dimer in which each chain has a single IgSF domain supported on a stalk. In most T cells CD8 appears to exist exclusively as a heterodimer of an α and β chain (CD8αβ), but some T cells, notably intraepithelial lymphocytes, express the CD8αα homodimer (32). This pattern of expression, and the finding that CD8αα is substantially less effective than CD8αβ as a coreceptor (32), strongly suggests that CD8αβ is the primary coreceptor for conventional MHC class I–restricted T cells. What then is the function of CD8αα? Recently it was shown that CD8αα binds with a higher affinity than CD8αβ to a nonclassical MHC class I molecule, thymus leukemia antigen, and that this interaction modifies recognition of conventional peptideMHC class I by these T cells (33).

Structural Studies Both CD4 and CD8 bind to nonpolymorphic regions at the base of MHC molecules. Crystal structures have now been solved of human (34) and mouse (35) CD8αα in complex with MHC class I, as well as, at low resolution, a mouse CD4 fragment in complex with human MHC class II (36). When these structures are superimposed on structures of TCR:peptide-MHC complexes it is evident that the angle at which CD4 and CD8 engage peptide-MHC precludes direct association with a TCR that binds the same peptide MHC complexes (Figure 2). How can this be reconciled with evidence that CD4 and CD8 physically associate with the TCR:CD3 complex (37, 38)? One possibility is that the coreceptors and the TCR are linked indirectly through CD3 chains and associated molecules. This is supported by the observation that CD4 and CD8 associate with TCRs through ZAP-70 and Lck (39, 40). According to this model, nascent TCR triggering leads to recruitment of coreceptors via ZAP-70/lck, and the coreceptor thereby stabilizes further the TCR:peptideMHC interaction (41). A second possibility, proposed more recently (42), is that the coreceptor and the TCR that it associates with bind different peptide-MHC complexes. This was proposed as part of a new “pseudodimerization” model of TCR triggering, which was itself proposed to explain the observation that a single agonist peptide-MHC complex could lead to TCR triggering (42). It was suggested that, when a TCR with an associated coreceptor binds to an agonist-peptide-MHC, the coreceptor binds a distinct self-peptide-MHC complex. A TCR pseudodimer is formed when a second TCR binds to this self-peptide-MHC complex. Whereas it is believed that CD8αβ is likely to bind to MHC class I in much the same way as CD8αα, there is disagreement as to which CD8α chain the CD8β chain will “replace.” Based on the analysis of electrostatic surface potential, Gao et al. proposed that CD8 would bind in the position of the CD8α chain adjacent to the peptide-binding platform (34). A subsequent mutagenesis experiment provides

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some support for this (43). In contrast, Reinherz and colleagues have proposed that, because the CD8β stalk region is several amino acid residues longer than the CD8α stalk, CD8β is more likely to bind in the more “distal” position (35). The presence of O-linked carbohydrates suggests that the CD8 stalk region forms an extended structure, a possibility supported by low-resolution structural studies (44, 45). It was observed several years ago that glycosylation of the CD8β stalk changes during thymocyte maturation and upon activation of mature CD8 T cells (46). It was also shown several years ago that the binding properties of CD8 appear to be regulated by, for example, TCR engagement (47). Recent findings (48, 49) suggest that these phenomena are linked because changes in CD8β glycosylation correlate with changes in the binding of peptide-MHC tetramers, and manipulations decreasing sialylation of T-cell surface molecules increase the binding of peptide-MHC tetramers. These data have been interpreted to suggest that sialylation induces structural changes in the CD8β stalk that decrease the affinity of CD8αβ for MHC class I (48). However, much earlier data indicate that the structural effects of O-glycans depend only on steric interactions between the peptide-linked GalNAc and the adjacent amino acids of the polypeptide (50–52). The structural properties of the stalk region are therefore not expected to be affected by chain-branching or by changes in terminal sialylation. In light of this, and because binding was assessed using MHC multimers binding to cells, it is possible that changes in the valency of binding, rather than the CD8αβ:MHC affinity per se, were responsible for the observed changes in peptide-MHC tetramer binding.

Binding Properties A number of groups have studied the binding properties of the CD8αα:MHC class I and CD8αβ:MHC class I interactions using soluble, monomeric forms of these molecules (33, 53–56). The consensus is that CD8αα and CD8αβ bind with a similar affinity, that this affinity is very low (Kd = 50–200 µM at 25◦ C and >200 µM at 37◦ C), and that the binding of CD8 to peptide-MHC does not affect binding of soluble TCR to the same peptide-MHC complex. Analysis of the CD4:MHC class II interactions has proved to be more difficult. Whereas an early study measured a high affinity (57), attempts to reproduce this failed or could only measure very weak binding (58). Recently, Xiong et al. (59) reported binding of mouse MHC class II to human CD4 on the BIAcore with a Kd of ∼200 µM at 25C, but the exceptionally low binding response relative to background response (∼1%) and the low level of apparent CD4 activity (1000 times more efficient when DCs were given a protein as part of a dying cell relative to preprocessed peptide (84). In all of these examples, a foreign antigen is presented, but the entire cell is processed, and therefore the DCs should be loaded with MHC-self- and MHC-nonself-peptide complexes. An additional literature shows that DCs also capture soluble proteins in the steady state, in the absence of overt infection or adjuvants (88–91). In these early experiments, which used several different routes of antigen injection, DCs were isolated from the animals and added to activated antigen-specific T cells in culture; the T cells then proliferated without further addition of antigen. More recent experiments directly visualized in vivo DC uptake of soluble proteins administered into the airway (92) and of self-components from intestinal and gastric epithelial cells (93, 94). Therefore DCs are continually capturing and presenting self- and harmless environmental proteins. The endocytic and processing activities of DCs create a conundrum with respect to their function in innate and adaptive resistance to infection. If maturing DCs simultaneously capture a mixture of microbial antigens, self tissues, and harmless environmental proteins, how is the response limited to the microbe? To resolve this situation, it has been proposed that DCs are not immunologically quiescent

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in the steady state but use their antigen-handling capacities to play a major role in peripheral tolerance (95, 96).

LOW DOSES OF SOLUBLE ANTIGENS INDUCE PERIPHERAL TOLERANCE WHEN TARGETED TO DENDRITIC CELLS IN THE STEADY STATE


The MMR and DEC-205, Two Multilectin Endocytic Receptors DCs express several adsorptive endocytosis receptors, which could be used to target antigens for processing and presentation in vivo. For example, DCs express the MMR (macrophage mannose receptor) (CD206) and DEC-205 (CD205), a pair of homologous, large type-I membrane proteins. The MMR (97, 98) and DEC-205 (99, 100) have similar domain structures with an external cysteine-rich domain followed by a fibronectin II domain and several contiguous C-type lectin domains, 10 in the case of DEC-205 and 8 in the case of the MMR. The cytosolic domain of each receptor has a tyrosine-based coated pit localization sequence. These receptors localize to coated pits and are taken up into coated vesicles and endosomes (100, 101). The ligands for the MMR include mannosyl and fucosyl residues for the C-type lectin domains and select sulfated sugars for the terminal cysteine-rich domain (102). Endogenous self-ligands for the MMR include lysosomal hydrolases and certain collagen-like peptides in serum (103). Natural ligands for DEC-205 are not yet known. Nevertheless, antibodies to DEC-205 can be used as surrogate antigens and for antigen targeting to DCs (23, 24, 100, 104). Although both MMR and DEC-205 can be expressed by DCs, their distribution in vivo is distinct. Whereas the MMR is prominent on human monocyte–derived DCs in culture (105), this receptor has yet to be detected on DCs in the T cell areas of lymphoid organs in either mice (106) or humans (107). Instead, the MMR is found on the endothelium lining lymphatic sinuses and in macrophages of splenic red pulp and lymph node. Therefore the MMR may not provide a way to selectively target ligands to DCs in the steady state. In contrast, DEC-205 is expressed abundantly on T cell area DCs, as first shown by the development of the NLDC-145 monoclonal antibody (108), and it does provide a means to target antigens to DCs in vivo (see below). In terms of antigen-presenting function, the only study to simultaneously compare the function of the MMR and DEC-205 involved cultured mouse bone marrow DCs, and it yielded surprising results. Rabbit antibodies to DEC-205 were presented 30–100 times more efficiently than antibodies to MMR, even though both antibodies bound comparably to the cell surface and entered the endocytic system (104). The MMR recycled quickly through cells via early endosomes, as is the case for many adsorptive endocytosis receptors. In contrast, DEC-205 localized both to early endosomes and MHC class II+ late endosomes and lysosomes. An EDE sequence within the cytosolic domain of DEC-205 enabled this receptor to

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target MHC II compartments. This was shown in L cells transfected with a fusion receptor formed by the external region of the CD16 Fcγ receptor and the cytosolic tail of DEC-205. The targeting to MHC II compartments led to a marked increase in the efficiency of antigen presentation on MHC class II. In addition to improved MHC class II presentation, ligands for DEC-205 are processed via the exogenous pathway to MHC class I in a transporters for antigenic peptides (TAP)-dependent manner (24). Although the cell biology of the exogenous pathway has not been worked out, it has been proposed that a transporter in the DC endocytic system allows macromolecules to enter the cytoplasm. According to this model, such antigens would be processed by proteasomes in the cytoplasm and transported into the endoplasmic reticulum via TAPs (109). Finally, DEC-205 is an excellent antigen delivery vehicle because monoclonal antibodies to this receptor efficiently target DCs in vivo. When the purified antiDEC-205 IgG is injected subcutaneously, most CD11c+ DCs in the draining lymph node take up the antibody (23). Uptake is not detected in lymphocytes or macrophages, either in cell suspension or in tissue sections. In conclusion, DEC-205 is a valuable antigen-targeting receptor on DCs because antigens delivered to this receptor are processed for presentation on both MHC class I and II and because targeting is specific and efficient.

Other Receptors for Endocytosis on Dendritic Cells DCs express several other molecules capable of mediating adsorptive uptake. Many of these, in contrast to the MMR and DEC-205, are type II transmembrane proteins with a single external C-type lectin domain. Each of these lectins mediates uptake of its corresponding monoclonal antibody and, in some cases, presentation to mouse Ig-specific T cells. However, these monolectins have been studied primarily in human cell cultures and there is no information concerning antigen presentation in vivo, including the exogenous pathway to MHC class I. This is of some interest because the lectins are expressed by subsets of DCs. For example, Langerin or CD207 is expressed in Langerhans cells (110), the asialoglycoprotein receptor type 1 (111) and DC-SIGN or CD209 (112) in monocyte derived DCs, and the BDCA-2 molecule in plasmacytoid DCs (113). Additional endocytic receptors are shared with other cells. Nevertheless, these receptors are distinctive because uptake into DCs leads to presentation by the exogenous pathway to MHC class I. Some examples include the Fcγ R for immune complexes (114, 114a,b,c) and the αVβ5 and αVβ3 integrins for dying cells (79). In summary, there are many potential ways to enhance the efficiency of antigen presentation through receptor-mediated uptake, but for most of these, there is little in vivo validation at this time.

Delivery of Peptides Engineered into the Anti-DEC-205 Antibody To test the idea that antibodies to DEC-205 efficiently target antigens to DCs in vivo, the heavy chain of the antibody was engineered to include a sequence

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for a hen egg lysozyme (HEL) peptide presented on I-Ak molecules (23). The constant regions of the rat heavy chain also were replaced with mouse C regions carrying mutations to block binding to Fcγ receptors. Submicrogram amounts of the engineered antibody were then injected into mice that were adoptively transferred with HEL-specific TCR transgenic T cells. In spite of the low doses of antigen injected (2n DNA, whether from young normal mice (109), transplanted hosts (110), or aged mice, (75) are less efficient as transplantable entities. Thus, serial transplantation introduces an artifact(s) unrelated to lifespan, so direct inferences regarding HSC lifespan from these studies must take such caveats into account.

Apoptosis Programmed cell death (apoptosis) also regulates the size of the HSC pool (111). Ectopic expression of the antiapoptotic protein BCL2 in transgenic mice leads to an increase in the steady-state frequency of HSCs and progenitor cells in the bone marrow and an increase in competitive repopulation potential (112). In addition, BCL2-expressing HSCs show enhanced survival in vitro and may be maintained in serum-free media containing only SlF, IL-3, or Tpo (113). Importantly, BCL2 transgenic HSCs are not prevented from differentiating under such conditions, and each of the single cytokines capable of evoking HSC proliferation in vitro differentially biases the outcome of such divisions. The precise physiologic regulators of apoptosis in HSCs have remained elusive. RT-PCR analysis showed that murine HSCs do express BCL2 family members; however, BCLxL, rather than BCL2 itself, appears to be the primary antiapoptotic protein expressed by HSC (112). CD95 (Fas), which can trigger apoptosis of cells after ligand binding, is not expressed by murine HSCs (114), and bone marrow hematopoiesis does not appear to be affected by Fas deficiency (115). However, Fas expression may be

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inducible on HSCs or hematopoietic progenitors following exposure to certain cytokines including IFN-γ or TNF-α (115–117) and in these cases appears to reduce hematopoietic repopulating potential, although rigorous studies of purified HSC populations have not been reported.


Hematopoietic Stem Cell Migration: Physiological Circulation and Enforced Mobilization As discussed above, HSC migration is an intrinsic aspect of the development of the hematopoietic system and is critically required for the success of bone marrow and peripheral blood progenitor (PBPC) transplantation in the treatment of multiple hematopoietic and nonhematopoietic diseases. While the capacity of HSCs to migrate from blood to bone marrow (homing) and from bone marrow to blood (mobilization) has been conserved through evolution, the biological role of this phenomenon in HSC function remains unknown. Surprisingly, migration of HSCs to and through the circulation appears to occur physiologically in normal animals. Using genetically marked parabiotic mice, which are surgically joined such that they develop a shared circulatory system, we recently demonstrated that HSCs rapidly migrate through the blood and play a functional role in the reengraftment of unconditioned bone marrow (118). Thus, in the steady state HSCs redistribute via the bloodstream among distinct anatomical locations and therefore are likely to be found in all tissues of the body. Importantly, their presence in tissues may confound interpretation of studies designed to detect hematopoietic activity from nonhematopoietic tissues, as unless purified populations are used, hematopoietic activity derived from itinerant HSCs may be attributed to developmental plasticity of tissue-specific progenitor cells (119, 120). The constant flux of HSCs in the circulation further suggests an explanation for the unexpected success of bone marrow transplantation. This clinically important process likely exploits an already existing mechanism of HSC migration that in unmanipulated animals allows the constitutive recirculation of HSCs through bone marrow, blood, and other tissues. Likewise, the induced mobilization of HSCs, stimulated by treatment with cytotoxic agents and/or cytokines, may occur via an amplification of normal HSC migration, either increasing HSC exit from the bone marrow or inhibiting HSC reentry into the tissues from the blood. Yet the question remains: What is the physiological relevance of constitutive circulation of HSCs in adults? One possibility is that the capacity for migration, which is vital to the seeding of HSCs to appropriate hematopoietic organs in the developing fetus, is retained, somewhat by default, into adulthood. Alternatively, constant flux of adult HSCs may provide an immediate source of rapidly recruitable progenitor cells for initiating extramedullary hematopoiesis in case of catastrophic blood loss. Migration could also be a fundamental step in HSC development that is required to determine HSC cell fate decisions (i.e., differentiation), via the relocation of daughter HSCs to distinct marrow niches. Finally, as HSCs have, in some instances, been shown to contribute to the regeneration of chronically injured

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nonhematopoietic tissues, circulating HSCs may represent a source of pluripotent cells in normal animals, which can be recruited for repair of damaged tissue under appropriate circumstances (see below). Given the clinical significance of HSC migration in transplantation and in mobilization regimens, much research has focused on identifying the molecular mediators of this process. As in paradigms described for the migration of mature inflammatory cells involving the sequential action of tethering receptors, activating chemoattractants and strongly adhesive proteins (121, 122), HSC migration appears to invoke the function of particular cell adhesion and chemokine receptors. HSC homing to bone marrow likely begins by the initial tethering of cells to endothelium, which may involve the function of vascular selectins (E- and/or P-selectin) and/or the integrin very-late-adhesion molecule-4 (VLA-4, α4β1) (123–125). Bone marrow endothelial cells constitutively express the VLA-4 ligand, vascular cell adhesion molecule-1 (VCAM-1), and E-selectin (126, 127), and irradiation enhances adhesion molecule expression by bone marrow endothelium (126). Murine and human HSCs express the chemokine receptor CXCR4 (128, 129), and in in vitro chemotaxis assays, murine HSCs show selective responsiveness to the CXCR4 ligand SDF-1α (129). Consistent with a role for SDF-1α/CXCR4 interactions in HSC homing to bone marrow, engraftment of NOD/SCID mice by human HSCs is blocked by inhibitory antibodies to CXCR4 (130). SDF-1 is constitutively expressed by bone marrow endothelium (131, 132), and its expression in both the bone marrow and spleen increases following irradiation (133). Although mice deficient in either CXCR4 or SDF-1α exhibit perinatal lethality (134–136), CXCR4deficient HSCs appear to be capable of seeding the fetal liver, and CXCR4−/− fetal liver HSCs can successfully engraft the bone marrow of irradiated recipients and undergo multilineage differentiation (59, 60). Interestingly, CXCR4-deficient hematopoietic progenitors are present at an increased frequency in the circulation of chimeric mice, perhaps indicating that these cells are not properly retained in the bone marrow in the absence of CXCR4/SDF-1α interactions (60). In addition to its role as a chemoattractant, SDF-1α may also directly affect the proliferation and differentiation of primitive hematopoietic cells (137, 138) and may enhance the activity of adhesion receptors, particularly integrins, on both HSCs and progenitor cells (131, 139). In particular, SDF-1α induces the function of VLA-4 and LFA-1 on HSC-enriched human cord-blood cells (139) and enhances their NOD/SCID repopulating ability following in vitro culture (140). SDF-1mediated integrin activation may play an important role in converting rolling adhesion to firm arrest, thereby allowing extravasation of circulating HSCs into the bone marrow extravascular space (131). During normal development, colonization of fetal liver, bone marrow, and spleen by HSCs requires the expression and function of β1 integrin, likely to direct the homing of yolk sac– or AGM-derived HSCs to these tissues (141, 142). In the adult transplantation setting, function-blocking antibodies against VLA-4, VLA-5 (α5β1), or their shared subunit β1 integrin prevent engraftment of NOD/SCID mice by human HSC-enriched CD34+ cord

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blood, and inhibition of VLA-4 blocks bone marrow homing and engraftment by murine HSCs (139, 143, 144). However, the targeted deletion of either α4 or α5 integrin fails to block the localization of stem cells and early hematopoietic progenitor cells to the bone marrow (145), although fetal liver chimeras generated with α4−/− fetal liver cells show cell-autonomous defects in lympho- and myelopoiesis (146). Thus, combinatorial or compensatory functions of αβ integrin heterodimers are likely involved in stem and progenitor cell homing from blood to bone marrow. HSC and progenitor cell mobilization can be stimulated by systemic treatment with certain cytotoxic drugs (including cyclophosphamide (Cy), hydroxyurea (HU), and 5-fluorouracil (5-FU)) and/or cytokines (including G-CSF, GM-CSF, IL-11, IL-3, IL-8, SlF, Flt3L, and others), which substantially increase the frequency of HSCs and progenitor cells in the bloodstream (147). Induced HSC mobilization is often associated with increased HSC proliferation (148–150), and in some cases, HSC division may be required for mobilization (149). Interestingly, the same molecules that play a role in stem and progenitor cell homing to bone marrow have often been implicated in the pharmacological mobilization of these cells from the bone marrow. For example, following cyclophosphamide/G-CSF treatment of mice, expression of both VLA-2 (α2β1) and VLA-4 is significantly reduced on mobilized peripheral blood (MPB) HSCs (144, 151). In addition in vivo administration of blocking antibodies against the integrin VLA-4 induces the mobilization of colony-forming cells (CFC), CFU-S, and long-term repopulating activity in both mice and primates (152–154). Conversely, blocking antibodies to the integrin LFA-1 appear to prevent progenitor cell mobilization induced by IL-8 (155). In addition, induced overexpression of SDF-1α in the circulation mobilizes stem and progenitor cells (156), and antibodies against CXCR4 or SDF-1α can block mobilization induced by G-CSF administration (132). Regulated proteolysis has recently emerged as an important mediator of induced HSC egress from the bone marrow and of hematopoietic recovery following cytoreductive treatment. In vivo treatment of mice with various HSC-mobilizing agents including G-CSF, SlF, and cyclophosphamide correlates with an increase in neutrophil-associated proteolytic activity within the bone marrow (132, 157, 158). Neutrophil function had previously been implicated in induced HSC mobilization, as neutropenic mice are unable to mobilize hematopoietic progenitors in response to multiple agents (159, 160). Neutrophil-expressed proteases degrade bone marrow–expressed VCAM-1 and SDF-1α in vitro, and reduced expression of these proteins in the bone marrow correlates well with increased progenitor frequency in the circulation (132, 157, 158). Furthermore, in vivo treatment of mice with an inhibitor of neutrophil elastase significantly inhibits G-CSF-induced progenitor cell mobilization and ameliorates the loss of SDF-1α in the bone marrow (132). Similarly, proteolytic release of soluble SlF from membrane-bound SlF by the matrix metalloproteinase MMP-9 is induced following 5-FU, SDF-1α, VEGF, or G-CSF administration and appears to be required for efficient mobilization of hematopoietic progenitor cells and for hematopoietic recovery following 5-FU-induced cytoreduction (150). These data indicate that, in addition to direct

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effects on HSCs and progenitors, mobilizing stimuli also cause profound changes in the bone marrow microenvironment that ultimately influence HSC motility and function.


Hematopoietic Stem Cell Plasticity The idea that the developmental potential of HSCs may not be limited to hematopoietic outcomes has emerged from several published reports indicating that cells derived from bone marrow are capable of giving rise to multiple “unexpected” cell types. These include neural cells (161–163), skeletal muscle (164–166), cardiac muscle (166–169), and hepatic cells (170–173), as well as epithelia of the gut, skin, lung, and kidney (174). Some investigators have suggested that “transdifferentiation” of bone marrow HSCs underlies these events; however, direct evidence supporting such claims is, for the most part, lacking. Most studies have assayed unpurified or partially purified cell populations, and almost none have performed analysis on single cells, a requirement for rigorous proof of multipotency. Bone marrow harbors both hematopoietic and mesenchymal stem cells, which give rise to multiple differentiated cell fates, and it is conceivable that additional tissue-specific stem cells also reside there. Furthermore, recent evidence suggests that apparently multipotent progenitor cells, which may contribute to nearly every tissue in the body, can be isolated from adult bone marrow (175), providing further impetus for future studies to employ only highly purified, well-characterized cell populations. To try to clarify the true potential of HSCs, we have analyzed the progeny of single, rigorously purified and transplanted HSCs (16) and found little evidence to support the idea that these cells contribute significantly to the production of nonhematopoietic cells, at least in the steady state (i.e., in the absence of any acute or chronic tissue injury aside from the initial irradiation required for HSC engraftment). Our data argue against the hypothesis that bone marrow HSCs possess a robust, intrinsic capacity for the production of nonhematopoietic cell outcomes; however, we cannot rule out the potential of HSCs to be recruited into atypical functions in the face of selective pressure induced by tissue injury. Strong selective pressure may facilitate HSC-derived nonhematopoietic cell outcomes, whether as a result of transdifferentiation or cell fusion with endogenous progenitors, by rescuing host cells with donor-derived gene products. Spontaneous fusion in vitro of embryonic stem cells with bone marrow– or brain-derived cells, with subsequent acquisition of “stem cell” function in the hybrid cells, has recently been demonstrated (176, 177), but the possibility that such a mechanism may underlie transdifferentiation in vivo has yet to be addressed.

Gene Expression Profiling at the Population and Single Cell Levels The hallmark of stem cells is their ability to balance self-renewal and differentiation. Whether the differentiation of HSCs through lineage-restricted progenitors

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to mature effector cells occurs as the result of exogenous or intrinsic signals remains unclear, but in either case the molecular mechanisms of this process are reflected in the gene expression profiles of the stem and progenitor cells along the differentiation hierarchy. At each decision point, genes associated with the adopted pathway are upregulated, while the genes necessary for the lineage(s) not chosen are silenced. Insight into these mechanisms has been provided by studies examining the relative expression levels of known lineage-associated genes. Early studies used multipotential hematopoietic cell lines to model HSC behavior. Factor-dependent cell Patterson mix cells (178) have been widely used for this purpose, as they are karyotypically FDCP normal, nonleukemogenic cells that respond to cytokines with appropriate lineage readout, i.e., granulocytes and monocytes, in response to G-CSF and GM-CSF; and erythrocytes in response to erythropoietin (EPO) (179). Further studies revealed that the EPO receptor (EPOR) and GATA-1 erythroid transcription factor were expressed at low levels (180, 181) and that hypersensitive sites in the EPOR promoter and β-globin control locus were present, indicating that the chromatin structure of these genes is in an active, open configuration in these cells prior to differentiation (182). Single-cell RT-PCR analysis of individual FDCP mix cells and human CD34+ Lin− progenitors revealed coexpression of erythroid and granulocyte/macrophagespecific genes (183). These results suggested that stem and progenitor cells are “primed” for multilineage differentiation by expressing low levels of lineageaffiliated genes. It has also been suggested that priming may likewise account for the expression of multiple lineage markers on some leukemic cells (184). More precise analysis has been done with freshly isolated HSCs and lineagecommitted progenitors, showing coexpression of lineage-affiliated genes in a single developing cell (185). Sixteen percent of single HSCs coexpressed erythroidspecific (β-globin and EPOR) and granulocyte/macrophage-specific [myeloperoxidase (MPO), granulocyte colony-stimulating factor receptor (G-CSFR)] genes, whereas 39% of single CMPs, the next lineal descendant of HSCs committed to the myeloid lineage (22), show a promiscuous gene expression. Importantly, the downstream progenitors of CMPs, GMP, and MEP only expressed lineageappropriate transcripts, MPO/G-CSFR, and β-globin /EPOR, respectively. Similarly, 23% of single CLPs coexpressed both B cell–specific (λ5 and/or Pax5) and T cell–restricted (CD3 or GATA-3) genes, whereas proB and proT cells only expressed T or B lineage-affiliated genes, respectively (22). Taken together these results reveal that low-level, promiscuous expression of lineage-specific genes precedes commitment to a particular lineage and may be requisite of multipotent progenitors. However, it is interesting to note that promiscuous lymphoid and myeloid gene expression has not been reported except in Pax5−/− proB cells as discussed below (186). This may indicate that myeloid differentiation is the default developmental pathway and that the gene expression programs required for lymphoid development must be actively induced. The reports described above used RT-PCR on few or, most informatively, on prospectively isolated single cells to examine the expression of known

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lineage-specific genes. However, precise knowledge of the complete gene expression programs of the stem and progenitor populations is necessary to elucidate fully the molecular mechanisms associated with development and lineage decisions. Recent advances in microarray and genomic approaches have already begun to facilitate these studies and will likely provide global gene expression data. Lemischka and colleagues have used non-PCR-based subtracted libraries of fetal liver HSCs to identify thousands of genes selectively expressed in AA4.1+ fetal liver HSCs (187). Many of the transcripts identified correspond to expressed sequence tags (ESTs) that had not been previously characterized. Additionally, known genes not previously detected in HSCs were identified. For example, CD27, previously described in T cells, was identified as selectively expressed in both fetal liver and bone marrow HSCs and has subsequently been shown to be preferentially expressed on short-term repopulating HSCs (188). These studies as well as an additional microarray analysis of enriched short-term and LT-HSCs (189) revealed an overlapping set of genes, including studies of CD34, CD27, and evi-1 that were preferentially expressed in HSCs in each of the studies. The comparison of gene expression of HSCs to the downstream lymphoid and myeloid progenitors (CLP, CMP, GMP, and MEP) reveals clusters of genes preferentially expressed in each compartment along the developmental hierarchy (A.V. Terskikh, T. Miyamoto, I.L. Weissman, unpublished data). These results nicely document the shifts in the gene expression programs that correlate with the different potentials associated with each of the progenitor populations. Terskikh et al. also showed an overlap in the genes expressed in hematopoietic and neural progenitors, supporting the notion that some genes responsible for stem cell properties, such as those enabling selfrenewal, would be shared among stem cells from different somatic tissues (190). The coordination of the silencing of some genes with the activation of others is the mechanism by which cells choose a differentiation pathway to the exclusion of others. The strength and weakness of these genomic approaches are the large number of genes identified by these methods. What remains to be established are the roles these gene candidates play in shifting the intricate balance that exists between self-renewal, pluripotency, and differentiation and discerning those genes that make the cell competent to receive/respond to differentiating signals and those genes that are deterministic for differentiation to a given lineage. Many of these studies confirm the currently known model of the hierarchy of blood development by validating the expression of expected genes. In addition to having implications for the roles of the promiscuously expressed genes in the differentiation/commitment process, these studies raise questions as to the mechanisms of the priming itself in terms of the chromatin status of the loci. Additional information gained from stem- and progenitor-specific libraries and microarray analyses are likely to be more powerful in identifying the genes, novel or otherwise, that comprise the self-renewal and differentiation programs. These approaches will not readily reveal the role of genes that are not differentially transcribed as part of the developmental program but that are modified posttranslationally as part of the orchestrated changes.

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Lineage Commitment and Plasticity In general, the process of development from pluripotent progenitors to mature cells with specific functions involves the progressive loss of developmental potential to other lineages. Over the past several decades, researchers have exploited a variety of technological advances in biomedical research to elucidate the precise developmental steps in blood cell formation. From this data, a hierarchy is emerging in which each successive developmental stage loses the potential to become a specific cell type or class of cells (191). This stepwise developmental process has been considered linear in the sense that once a cell has made a developmental choice it cannot revert. We are still a long way from a complete understanding of the molecular basis for lineage commitment in hematopoiesis, but testable models for this complex process have emerged and are currently under investigation. In this section we detail a few specific experimental systems and significant results that provide insight into the mechanisms that control lineage commitment, thereby shedding light on the issue of lineage infidelity. The earliest known lymphoid-restricted cell in adult mouse bone marrow is the common lymphocyte progenitor (CLP) (21), and the earliest known myeloidrestricted cell is the common myeloid progenitor (CMP) (22). Importantly, these cell populations possess an extremely high level of lineage fidelity in in vitro and in vivo developmental assays. The only “unexpected” developmental outcome observed to date from either of these progenitor populations is the infrequent generation of low numbers of B cells from CMPs in vivo. It is not known whether this result is due to lineage infidelity of CMPs or impurities (i.e., B cell progenitors) in the sorted CMP population. Regardless, the prospective isolation and developmental characterization of these progenitors support the linear model of hematopoiesis in which a cell that loses the potential to develop into a specific lineage never regains that potential. As it now stands, the earliest lineage-potential decision that a developing HSC/multipotent progenitor population must make is whether to become a lymphoid or myeloid cell type, and once it does, that decision is permanent. Developmental hierarchies have been elucidated to a remarkable extent for both the T cell and B cell lineages with the number of developmental stages growing still. Unfortunately, this level of understanding has not yet been achieved in regard to NK cell development. To begin to explore the signals that promote NK cell development, CLPs genetically engineered to express the human IL-2 receptor β chain (hIL-2Rβ) were studied (192). This receptor was chosen because it had previously been demonstrated that IL-15 was indispensable for NK cell development, the receptor for which is composed of three chains: the IL-15Rα chain, the common γ chain (γ c), and the IL-2Rβ chain (193). The hypothesis was that CLPs stimulated through an ectopically expressed hIL-2Rβ chain (which couples with an endogenous γ c) would preferentially attain a NK cell fate because this signal would mimic an IL-15R signal. Surprisingly, when hIL-2Rβ-expressing CLPs were stimulated with hIL-2 in vitro they developed into B cells, NK cells,

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granulocytes, and macrophages. As mentioned above, myeloid cell development (i.e., granulocytes and macrophages) had never been observed in CLPs in previous experiments. What signal was IL-2 providing that led to this dramatic cell fate conversion? Further investigation determined that hIL-2Rβ signaling activated the expression of the granulocyte/macrophage colony–stimulating factor receptor (GM-CSFR), which could drive CLP development toward myeloid cell fates when activated. In follow-up experiments, ectopic expression and activation of the GM-CSFR could also induce lineage conversion in CLP, bypassing the need for hIL-2Rβ. Importantly, this IL-2R-induced lineage conversion effect could be recapitulated in proT cells from the thymus but not in proB cells (192). What does this data suggest about the process of lineage commitment, at least to the lymphoid lineage? When considering this, it is important to note that in RT-PCR assays the GM-CSFR was detected in HSCs, the only known precursor to CLP (185). CLPs, however, never express the GM-CSFR under normal developmental conditions, whereas it is frequently expressed in CMPs (9, 192). Therefore the simplest model is one in which the first step in commitment to a specific lineage is downregulation of cell-surface receptors that drive development to alternate lineages. At the CLP/CMP lineage checkpoint, HSCs commit to the lymphoid lineage by shutting down expression of the GM-CSFR (and likely other genes), thereby preventing myeloid cell outcomes. Expression and stimulation through the GM-CSFR disturbs this program and can redirect CLPs to the myeloid lineage. Therefore, CLPs have not irreversibly committed to the lymphoid lineage but rather have taken only the first step in this process. The question then becomes, at what point does a cell become fully committed to a specific lineage? One clue comes from the data mentioned above in which proB cells cannot be diverted from their lymphoid cell fate in response to hIL-2Rβ signaling, whereas proT cells can (192, 194). Therefore, proB cells must have developed beyond the next developmental hurdle and become stabilized in their lineage choice. What is this stabilizing factor(s)? Fortunately, an ideal lineage-stabilizing factor candidate has been studied extensively: the transcription factor Pax-5. In a series of elegant studies, Pax-5 was not only shown to play a central role in the early development of the B cell lineage, but it also turned out to be required for stabilizing the commitment process itself (195). In Pax-5−/− mice B cell development is arrested at the transition between the pro-BI and pro-BII stage (prior to V to DJ rearrangement at the IgH gene locus). Unlike their wild-type counterparts, Pax-5−/− pro-BI cells also have the ability to grow extensively in vitro and develop into a number of distinct hematopoietic lineages including T cells, NK cells, and macrophages in various in vitro and in vivo assays (186). The incredible developmental plasticity of Pax-5−/− pro-B cells can be attributed to the role of Pax-5 in silencing and activating specific genes during development. Relevant to this discussion, Pax-5 downregulates genes associated with the myeloid lineage, including c-fms and PD-1, and activates genes that promote B cell development including CD19 and Igα (186). Therefore, without Pax-5 to orchestrate lineage-appropriate gene expression, pro-B cells from

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Pax-5−/− mice are not stabilized (or committed) in their lineage choice. Another important result regarding Pax-5 is that it can block hIL-2Rβ-induced myeloid differentiation when overexpressed in CLP but not in proT cells (A.G. King, M. Kondo & I.L. Weissman, unpublished observations). This suggests that there also exists a T lineage stabilization factor(s) yet to be identified (although there are candidates). It is not known what signal(s) is responsible for controlling subsequent lineage fate decisions downstream of CLP (i.e., between B, T, and NK cells), but research into this question is ongoing. There has been some speculation as to whether the lineage infidelity occasionally observed in acute myeloid leukemia cells occurs during the normal course of hematopoiesis in vivo. Evidence for lineage conversions has come from experiments using genetically modified cells or transformed cell lines [reviewed in (196)], and thus has not answered the question of whether this process is common and significant in vivo. A more recent approach has utilized technology that allows one to permanently mark cells as soon as they express a specific developmental marker and then determine whether that cell later transdifferentiates into an alternative lineage. This technology relies on the lineage-specific expression of a recombinase (Cre or FLP) that when expressed removes an inhibitory element in a target gene that is then permanently turned on in that cell; GFP and β-galactosidase are common reporters that are used. If such a marked cell decides to switch lineages, it can easily be detected. These studies are no doubt informative, but it is important to keep in mind that multilineage gene expression (or priming) is common in progenitor cells, as mentioned above. If a progenitor has the capacity to develop into both lymphoid and myeloid lineages, it may express myeloid-specific genes prior to committing to the lymphoid lineage. For example, HSCs express the GM-CSFR prior to deciding whether to proceed down the lymphoid or myeloid lineage (185). If the GM-CSFR promoter was used to express the recombinase, it would be expressed in and mark as myeloid-committed an HSC that ultimately became a lymphoid cell. Therefore, one would have the false impression that a committed myeloid cell transdifferentiated to the lymphoid lineage. Although this caveat may not apply to all of these in vivo cell-marking strategies, it is an issue that must be factored into the interpretation of such data.

HUMAN HEMATOPOIETIC STEM CELLS AND PROGENITORS Dilemmas of Experimental Systems Identification and characterization of human hematopoietic stem and progenitor cells have been impaired by the lack of optimal assay systems. As in the mouse, short-term in vitro assays are sufficient to demonstrate clonal myelo-erythroid, B-, NK-, and dendritic cell but not T cell, read out. However, competitive in vivo repopulating assays used in mice to demonstrate sustained self-renewing and multipotent differentiation capacity of HSCs, as well as T cell development from

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HSCs and committed progenitors, cannot be performed in humans for ethical reasons. Therefore, surrogate assays have been developed. In various forms of the long-term culture-initiating cell assay (LTC-IC), candidate cells are primarily cultured for 5–10 weeks on adherent, bone marrow–derived stromal cells that presumably resemble a bone marrow–like microenvironment (197, 198). In a second step cells are transferred into semisolid medium containing cytokines. Cells of the primary culture that retain their proliferative capacity, the LTC-IC, will generate myelo-erythroid or B cell colonies (199–201). This assay is very useful to define primitive or primarily quiescent progenitors and provides proof of self-renewal capacity or multilineage differentiation potential. To study human hematopoiesis in in vivo models, two essential prerequisites need to be met: The host should not eliminate the xenograft via an immune reaction and should provide a permissive microenvironment for engraftment and multilineage differentiation of donor cells. Spontaneously occurring immunodeficient mouse strains partially meet these criteria and have been modified to improve their model function. Early studies were done in SCID mice that display a T and B cell defect (202, 203) and beige/nude/xid (bnx) mice that display an NK, T, and B cell defect (204). However, both mice strains can mediate rejection of xenografts owing to macrophages and residual NK cells. Therefore, SCID mice were backcrossed to nonobese diabetic (NOD) mice that display partially deficient NK cell, antigen-presenting cell, and macrophage functions (205). To improve the microenvironment for human cells and consecutively, proliferation and multilineage read out, SCID mice were transplanted with human fetal bone, thymus, liver, or lymph nodes (SCID-hu model) (203, 206), and recipient mice were injected with recombinant human cytokines (204, 207) or were genetically modified to produce human cytokines (208). Owing to good engraftment capacity (10–20 times better than SCID), easy handling, ready availability, and economic aspects, the NOD-SCID mouse is currently used by most groups studying human hematopoiesis in in vivo models, and engrafting cells are termed SCID repopulating cells (SRCs) (92, 209– 211). However, SCID mice have a high radiation sensitivity, human hematopoiesis in the NOD-SCID model shows a bias towards B-cell development, T cell development is rare, and engraftment can only be monitored for about 6 months owing to the limited life-span of these mice. To address some of these deficiencies additional mouse models such as NOD-SCID β2 microglobulin knockout mice (212, 213), NOD-RAG1 knockout mice (214), NOD/SCID/γ c triple-mutant mice (214a), and RAG2/common cytokine receptor γ chain (γ c) double-mutant mice (215, 216) have been developed. The specific utilities of these mouse strains still need to be determined. As an alternative to the murine xenotransplantation models, Zanjani et al. established a large animal transplantation model in which human hematopoietic stem cells are transplanted intraperitoneally into unconditioned, early gestational sheep fetuses (217–219). In this model low numbers of selected progenitors can engraft, and myelo-erythroid as well as T and B lymphoid read out can be monitored over several years.

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Isolation of Candidate Human Hematopoietic Stem Cells Since the generation of monoclonal antibodies against the sialomucin CD34 almost two decades ago (220), the CD34 antigen has become the major positive marker for human hematopoietic stem and progenitor cells. Among nonhematopoietic tissues, CD34 is expressed on endothelial cells of small vessels and is a ligand for L-selectin (CD62L) (200, 221). The biological function of CD34 on hematopoietic cells is poorly understood: Expression of human CD34 in mouse hematopoietic cells suggests a role of CD34 in adhesion to the stromal microenvironment (222), and CD34 mutant mice show reduced colony-forming activity in bone marrow; however, mutant mice keep up normal peripheral blood counts and respond to hematopoietic stress as well as wild-type mice, showing that CD34 is not essential for hematopoiesis (223). Of hematopoietic cells in human fetal liver, cord blood, and bone marrow, 0.5–5% express CD34 (220, 224). CD34+ cells harbor virtually all in vitro clonogenic potential (220, 224, 225) (see also Table 1); however, the CD34+ population is heterogeneous. Only a small fraction (1–10%) of CD34+ cells that do not express mature lineage markers (Lin−, as CD3, CD4, CD8, CD19, CD20, CD56, CD11b, CD14, and CD15) and CD38 (226) contains single cells with in vitro bilineage, lymphoid (B/NK) and myeloid differentiation potential (227–230) (Table 1). The majority of CD34+ cells (90–99%) coexpress the CD38 antigen, and this subset contains most of the lineage-restricted progenitors (discussed below). CD34+CD38− cells and not CD34+CD38+ cells are highly enriched for LTC-IC (201, 231) and contain SCID-hu-repopulating (227) and NODSCID-repopulating cells (209, 210), with some of them able to read out even in secondary NOD-SCID transplants (211, 232). However, the Lin−CD34+CD38− cell fraction is still very heterogeneous with regard to surface marker expression and biological functions. Single Lin−CD34+Thy-1+ cells and not Lin−CD34+Thy-1− cells generate B/ myeloid progeny in culture and produce B/myeloid progeny in SCID-hu mice transplanted with 104 sorted cells (227). Also, Lin−CD34+Thy-1+ cells and only few if any Lin−CD34+Thy-1− cells generate T cells in SCID-hu thymi (227). Although not evaluated in this study, virtually all Lin−CD34+Thy-1+ cells reside in the CD38− fraction. The highest LTC-IC activity (63%), NOD-SCID (SRC 1/5) and fetal sheep repopulating ability, resides in a CD34+KDR (VEGFR2)+ fraction (0.1– 0.5% of CD34+ cells) in cord blood, bone marrow, and G-CSF-mobilized peripheral blood (211). CD34+KDR+ cells but not CD34+KDR− cells generated myeloid, T, B, and NK cells in mice and myeloid/T cells in primary and secondary fetal sheep transplants. These results show a >100 × enrichment of SRC in CD34+KDR+ cells compared with CD34+CD38− cells. Only ∼30% of CD34+KDR+ cells are CD38−, suggesting that depletion of CD38+ cells would further increase SRC purity. With this high purification of potential human HSCs, it will now be essential to do critical clonal in vivo experiments, as has been done for mouse HSC (18). An alternative approach to assess clonal read-out is to mark putative HSCs genetically with retroviral vectors that randomly and permanently integrate into

Surface marker

lin−CD34+, retrovirally marked

+

CD45RA Thy-1 HLA-DR

−

+

8c

Stroma cell culture

Stroma cell culture SCID-hu

NOD-SCID

Pre-culture, NOD

Clonal B/NK/DC, no myeloid

Clonal B/NK/DC, no myeloid T cells

Clonal B/myeloid SRC Long/short-term SRC

B/myeloid

T/myeloid, primary+ secondary host

Fetal sheep NOD-SCID

Frequency 63%

LTC-IC

(Continued )

(264)

(263)

(232)

(254)

(211)

(245) (244)

(209, 210)


+

CD34+CD38−CD7+IL-7Rα −

0.09a

0.2 Hu DNA detection

d

8

B/T/myeloid, SRC (1/10 ) T/myeloid, primary+ secondary host

B/myeloid, SRC (1/106)

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CB

Candidate lymphoid progenitors FBM/BM lin−CD34+CD38+CD10+ CD45RA+Thy-1−HLA-DR+

CB

lin CD34 CD38 CD133 -SCID

−

lin−CD34+CD38−CD133+

CB −

88c

lin−CD34+KDR+ B/NK/T/myeloid,

CB/BM/MPB NOD-SCID

NOD-SCID Fetal sheep

NOD-SCID

(228) (255) (229) (230)

(227)

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−

0.1-0.5b SRC(1/5)

lin CD34 CD38 lin−CD34−

−

CB BM

−

lin−CD34+CD38−

CB/BM

0.01a

T/B/myeloid Clonal B/myeloid Clonal B/myeloid Clonal B/myeloid Clonal B/myeloid Clonal B/ NK/myeloid

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−

CD34+CD38−HLA-DR+ CD34+CD38loCD10−CD19− CD34+CD38−CD33−CD10− lin−CD34+CD38−

SCID-hu Long-term culture Seq. colony formation Liquid culture Seq. Colony formation Seq. Colony formation

Assay system

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FBM CB CB BM

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TABLE 1 Surface marker expression on candidate human hematopoietic stem and progenitor cells


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CD34+CD45RA+ CD34+CD45RA−

lin−CD34+CD38+CD45RA+IL-3Rlo 0.35a lin−CD34+CD38+CD45RA−IL-3R− 0.13a

lin−CD34+CD38+CD45RA−IL-3Rlo 0.28a

80b 20b

CD34+CCR1+ CD34+CCR1−

Methylcellulose NOD-SCID Methylcellulose Methylcellulose NOD-SCID No B/NK progeny detected

Methylcellulose Methylcellulose


No lymphoid read-out tested.

FL, fetal liver; FBM, fetal bone marrow; CB, cord blood; BM, bone marrow; PB, peripheral blood; MPB, mobilized peripheral blood; SRC, SCID repopulating cells.

∗

All myeloid colonies All myeloid progeny CFU-G/M/GM restricted BFU-E/CFU-Meg restricted Erythroid progeny

CFU-GM enriched∗ BFU-E enriched∗

CFU-GEMM/GM enriched∗ BFU-E enriched∗

All myeloid colonies∗ CFU-GM enriched∗ BFU-E enriched∗

(271)

(279)

(278)

(277)

(275, 276)

(274)

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Percentage of frequency of progenitors in aMNCs, bCD34+ cells, cCD34+CD38− cells, dlin−CD34−CD38− cells.

BM/CB

60–90b 10–40b

CD34+Flt3+ CD34+Flt3−

Methylcellulose Methylcellulose Methylcellulose

CFU-G/M/GM no E/Mix∗ CFU-M enriched no E/Mix∗ CFU-G enriched no E/Mix∗

CFU-GM enriched∗ CFU-GEMM/BFU-E enriched∗

(273)

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CB/BM

60–80b 11–17b 7–20b

CD34+IL-3Rlo CD34+IL-3R+ CD34+IL-3R−

Liq. cult./methylcell. Liq. cult./methylcell. Liq. cult./methylcell.


CFU-GM enriched∗ LTC-IC/BFU-E enriched∗

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Fetal BM/BM CD34+CD64−M-CSFRhi CD34+CD64+M-CSFRhi CD34+CD64−M-CSFRlo

CB/BM/PB

Methylcellulose, LTC-IC Methylcellulose, LTC-IC

Read-out

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Candidate myeloid progenitors BM CD34+CD45RO− CD34+CD45RO+

% frequency Assay system

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the host genome, allowing the follow-up of single-cell progeny (233, 234). A disadvantage of this method is that viral integration requires cycling of target cells, which could induce commitment and loss of HSC capacities. Using this approach, Guenecha et al. showed for the first time that single-marked Lin−CD34+ clones transplanted into NOD-SCID animals together with other Lin−CD34+ cells generated multilineage B and myeloid progeny (232). This study also suggests that short-term (10 weeks) are mostly CD34− but acquire CD34 expression upon “activation” through G-CSF mobilization or 5-FU treatment. Finally, transplanted CD34+ HSCs can revert to a CD34− phenotype that upon secondary transfer displays full HSC potential [(248–250); reviewed in (251)]. If the murine CD34+ expression on HSCs model CD34 expression on human HSCs, cord blood up to teenage HSCs and G-CSF as well as chemotherapymobilized HSCs should be mostly CD34+. Only untreated adult bone marrow HSCs would be mostly CD34−. However, Okuno et al. suggested that human and mouse HSCs differentially regulate the CD34 gene (252): Artificial expression of the entire human CD34 genomic locus in mice revealed that HSCs of 10-week-old mice were murine CD34− but expressed human CD34.

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If CD34+ and CD34− human HSCs exist, is there any other marker that would positively identify both? CD133, a recently described glycoprotein, might meet this criterion. CD133 is expressed in all CD34+CD38− and on some CD38+ progenitors (253). Also, 0.2% of Lin−CD34−CD38− cells express CD133, and these are the only cells within the Lin−CD34−CD38− fraction with SRC potential (254). Therefore, it was suggested that this CD34+ and CD34− “unifying” antigen might be a better target for HSC enrichment than CD34 (254). To clarify this issue, further preclinical studies are needed. All or most clinical trials that have tested CD34+ enrichment methods in an autologous or allogeneic transplantation setting used chemotherapy and/or cytokine-mobilized CD34+ cells and according to both mouse models should harbor most of the HSCs. Indeed, to date, no higher incidence of late graft failure has been reported.

Identification of Human Early Lineage Committed Progenitors The existence of clonal lymphoid- and myeloid-committed progenitors that harbor all lymphoid and myeloid potential, respectively, has long been proposed and was finally shown in the mouse model (21, 22). This also suggests that HSCs or multipotent progenitors do not commit directly to monospecific lymphoid or myeloid progenitor cells. Although good assays for myeloid development are available, combined T, NK, and B cell read-out from single or low numbers of cells remains a major unresolved challenge. Because early lymphoid and myeloid commitment is discussed in the mouse section of this review, we focus here on studies that addressed clonal read-out of proposed progenitors in human. All described bipotent lympho-myeloid progenitors reside in the CD34+CD38− fraction (see above) (227–230, 255). To segregate those from the earliest lymphoid or myeloid progenitors, additional candidate antigens need to be identified. Based on combined phenotypic and functional analysis of fetal liver and fetal and adult bone marrow, terminal deoxynucleotidyl transferase (TdT), CD7, CD10, and IL-7Rα are such candidate antigens (256–262). So far, the closest definition of a potential human CLP population showed that fetal and adult bone marrow Lin−CD34+CD38+CD10+ cells generate no myeloid progeny and contain clonal progenitors of B, NK, and dendritic cells (263). Also, these cells as a population give rise to T cells in the SCID-hu thymus assay (263) (see Table 1). In another similar rigorous study it was shown that cord blood CD34+CD38−CD7+ cells contain ∼40% clonal B, NK, and dendritic cell precursors (264). However, T cell read-out was not evaluated. Because the described CD7+ progenitors reside in the CD38− fraction and ∼30% of them are CD10−, it was suggested, but not formally proven, that these cells are developmentally upstream of the CD34+CD38+CD10+ cells (264). In mice genetic disruptions of IL-7 or the IL-7Rα chain demonstrate nonredundant, essential functions of this cytokine/cytokine receptor pair for T and B cell development (265), IL-7Rα expression distinguishes the earliest lymphoid committed cells (21), and in vivo IL-7 administration might improve T, B, and NK

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cell reconstitution after murine allogeneic bone marrow transplantation (266). In humans, however, IL-7 signaling is not essential for normal B cell development because in vitro B cell development is possible without IL-7 (267), and IL-7R disruption in vivo causes T cell but not consistently B cell deficiencies [268, 269; reviewed in (270)]. It would be of interest to know whether human early lymphoid progenitors express IL-7Rα and depend on IL-7 signaling. Lin−CD34+IL7Rα +CD19−Pax-5+ bone marrow cells are reported to contain cells with high Blymphoid developmental capacity, but clonal and multilymphoid potential was not tested (260). In contrast, CD34+CD38−CD7+ lymphoid progenitors are reported to be cell surface IL-7Rα −, IL-7Rα transcripts were only occasionally detected, and Pax-5 was not detectable by PCR (264). Although no data about IL-7Rα and Pax-5 expression was reported in the original publication of the lymphoidcommitted Lin−CD34+CD38+CD10+ cells, we now know that this population contains cells that express IL-7Rα and Pax-5 as assessed by RT-PCR (271). Furthermore, we can subdivide the Lin−CD34+CD38+CD10+ cells into a CD10+IL7Rα − and a CD10loIL-7Rα + fraction, with the latter being highly enriched in clonal B-cell progenitors (M.G. Manz & I.L. Weissman, unpublished observations). It shall be important to determine whether CD10loIL-7Rα + cells, but possibly not CD10+IL-7Rα − cells, contain both T and B cell progenitors and whether thymus seeding cells share some of these phenotypes [for review of human early thymocyte development see (272)]. As in the case of lymphoid progenitors, multiple studies report on surface marker–associated enrichment of myeloid-colony forming cells (273–279) (see Table 1). Collectively this data shows that CD34+ cells that are either CD45RO−, CD45RA+, CD64+, IL-3Rα +, flt3+, or CCR1+ are enriched for CFU-GM-forming cells, whereas CD34+ cells that are either CD45RO+, IL-3Rα −, flt3−, or CCR1− are enriched for erythroid-colony forming cells (see Table 1). When reported, cloning efficacy ranged between 10–35% (274) and 26–50% (276). No data on alternative lymphoid potential are available from these studies. In an attempt to identify human early myeloid commitment as precisely as in murine bone marrow (22), we identified three cell populations that are likely counterparts of the murine CMPs, GMPs, and MEPs (271). These cells are CD34+ CD38+, they are negative for multiple mature lineage markers including early lymphoid markers such as CD7, CD10, and IL-7Rα, and they are further distinguished by the markers CD45RA, an isoform of CD45 that can negatively regulate at least some classes of cytokine receptor signaling (280), and IL-3Rα, a cytokine receptor that when activated supports proliferation and differentiation of primitive progenitors (96, 281, 282). CD45RA−IL-3Rα lo (CMPs), CD45RA+IL-3Rα lo (GMPs), and CD45RA−IL-3Rα − (MEPs), display cloning efficacies of 84%, 75%, and 87%, respectively, and show no in vitro B or NK cell read-out. Importantly, CD45RA−IL3Rα lo cells give rise to GMPs and MEPs and at least one third generate both GM and MegE colonies on a single-cell level. By focusing on a limited number of hematopoiesis-associated genes, we found expression profiles generally consistent with the distinct in vitro read-out capacities of the different progenitors. This

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data, documenting for the first time myeloid progenitor purification and placement in a developmental hierarchy, is largely in line with previous reports on enrichment of myeloid progenitor fractions (Table 1).


Clinical Implications of Hematopoietic Stem Cells The current common clinical application of hematopoietic cell transplantation (HCT) is for patients with malignancies, bone marrow failure states, and immunodeficiencies. Two types of transplantations are performed—autologous and allogeneic. Patients who receive autologous grafts have an underlying malignancy that is either at high risk of relapse or has already failed following standard chemotherapy. Thus, the therapeutic principle behind an autologous transplantation is that significantly increased doses of radiation and/or chemotherapy can be delivered to the patients to achieve maximal tumor kill, with the dose-limiting toxicity being death of the hematopoietic organ, and the patients are rescued by the HCT. Dose escalation studies have been performed to determine the maximum tolerated doses that result in ablation of malignancies and host hematopoiesis without conferring untoward toxicities to the other organ systems (283). Standard clinical practice is to use mobilized peripheral blood (MPB), which is collected from patients following cytoreductive cycles of chemotherapy to minimize the tumor burden. Mobilization schemes generally involve the administration of cyclophosphamide in conjunction with G-CSF or with G-CSF alone. The MPB product is collected by apheresis and stored frozen. Most transplant centers do not manipulate the autologous product; thus the potential exists for the MPB product to be contaminated with tumor cells. Whether or not these tumor cells contribute to relapse in patients that fail autologous transplantation remains controversial. Often individuals who fail autologous transplantation relapse in the sites of their original disease, suggesting that the lack of efficacy for those individuals was owing to insufficient ablation of the tumor by the preparative regimen rather than reinfusion of tumor cells. With the advent of devices that allow selection of hematopoietic stem and progenitor cells using the CD34+ marker, it is becoming more prevalent to perform CD34+ cell selection attempts to obtain grafts that are reduced in tumor cell burden. A more stringent method for purifying human HSCs that can result in significantly greater purging of tumor cells was described above and utilizes the same approach taken to isolate HSCs from mouse bone marrow. Human HSCs are enriched by magnetic bead selection for CD34+ cells and are isolated by fluorescence-activated cell sorting for CD34+ Thy-1+ cells (227). Three clinical trials have been performed using HSCs from MPB that were isolated by CD34+Thy-1+ selection: The first cohorts were patients with widely metastatic (stage IV) breast cancer (241), the second, patients with multiple myeloma (240), and the third, patients with subsets of non-Hodgkin’s lymphoma (284). The goals of these studies were to determine if adequate numbers of CD34+Thy-1+ HSCs could be collected and purified from patients with these different malignancies to yield rapid and sustained hematopoietic cell engraftment, to assess the tumor contamination in the MPB product, and to test for

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treatment-related toxicities. The amount of CD34+Thy-1+ cells collected was based upon mouse studies (285) that indicated >2–4 × 105 HSCs (Thy-1loLin−/lo Sca-1+c-Kit+) per kg recipient body cells would be sufficient for rapid engraftment. In all three studies it was possible to collect and purify adequate numbers of CD34+Thy-1+ cells. The median time to obtain absolute neutrophil counts >500/mm3 was between 10 and 12 days, and the best results were seen if doses >8 × 105 CD34+Thy-1+ cells/kg were infused. Significant reduction in tumor contamination was observed in MPB products that showed evidence of tumor cells prior to the HSC isolation. In the breast cancer studies 37% of MPB samples had contaminating cytokeratin+ cells, whereas none of the purified products had evidence of contaminating breast cancer cells as determined by an assay sensitive to one in one million cells (241). In the lymphoma studies tumor contamination was reduced between 3 and 6 logs (284), and in the myeloma studies the reduction was between 2 and 4 logs (240). The clinical outcome was most impressive in the stage IV breast cancer patients, who with a median follow-up time of 1.4 years still had a survival rate of ∼60% (241). A recent reanalysis of data from these stage IV breast cancer patients from one institution (Stanford University) demonstrated that with a median follow-up time of >4 years the results continue to look promising. These data are particularly impressive given a prior stage IV breast cancer study (286) that demonstrated a median event free survival time of 9.6 months for patients treated with high-dose chemotherapy plus autologous MPB rescue as compared with 9.0 months for patients who received conventional dose chemotherapy. In this study >90% of the patients died by 2 years posttransplantation. Taken together, these three clinical studies demonstrated that it is possible to obtain sufficient numbers of CD34+Thy1+ cells with significant reduction in tumor burden and achieve rapid and sustained hematopoietic cell engraftment.

Allogeneic Hematopoietic Stem Cell Transplants The therapeutic concept for allogeneic HCT differs from that of autologous HCT because hematopoietic cells obtained from an appropriate HLA-matched donor not only can rescue patients who undergo myeloablative radiation combined or not with chemotherapy, but the allogeneic graft also can confer an effect that has been termed graft-versus-tumor (287–289). Unmanipulated allogeneic bone marrow contains ∼107 CD3+ cells per kilogram of recipient weight, whereas MPB contains one log greater (108) T cells per kilogram. The significance of the T cell content contained within a graft is multifacted. One of the major complications of an allogeneic transplantation using an unmodified hematopoietic graft is graftversus-host disease (GVHD) (290–292). GVHD is caused by mature T cells that recognize host antigens as foreign and mount an immune attack against the host organs. For this reason all allogeneic transplantations are placed on systemic posttransplant immunosuppressive therapy. Attempts to reduce the morbidity and mortality of GVHD by depletion of T cells from the hematopoietic grafts resulted in reduced GVHD, but other untoward complications were noted. In clinical trials

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T cell depletion of bone marrow resulted in significantly increased incidences of graft failure (293, 294). Furthermore, it has been observed that immune reconstitution as well as loss or reduction of graft-versus-tumor activity is increased in recipients of T cell–depleted grafts. Thus, the current clinical standard is to transplant either allogeneic bone marrow or MPB into recipients with the knowledge that the mortality owing to GVHD and related complications is ∼15–20% in HLA-matched sibling transplants and even greater in other more genetically disparate transplants. Studies were performed in mice to resolve the problem of resistance to engraftment of purified allogeneic HSCs (295). These studies showed that there are at least three ways to achieve successful allogeneic HSC engraftment: (a) Escalation of the numbers of HSCs transplanted allows engraftment across most genetic disparities. Doses in excess of 30–60-fold the amount required to only rescue lethally irradiated mice across congenic barriers are often needed to rescue mice transplanted across major and minor histocompatibility complex barriers, although the doses of fully allogeneic HSCs required for rapid (100-fold by coculture with a clonal yolk sac endothelial cell line. Proc. Natl. Acad. Sci. USA 93: 14782–87 Muller AM, Dzierzak EA. 1993. ES cells have only a limited lymphopoietic potential after adoptive transfer into mouse recipients. Development 118:1343–51 Palis J, Yoder MC. 2001. Yolk-sac hematopoiesis: the first blood cells of mouse and man. Exp. Hematol. 29:927–36 Yoder MC, Hiatt K. 1997. Engraftment of embryonic hematopoietic cells in conditioned newborn recipients. Blood 89: 2176–83 Yoder MC, Hiatt K, Mukherjee P. 1997. In vivo repopulating hematopoietic stem cells are present in the murine yolk sac at day 9.0 postcoitus. Proc. Natl. Acad. Sci. USA 94:6776–80 Yoder MC, Hiatt K, Dutt P, Mukherjee P, Bodine DM, Orlic D. 1997. Characterization of definitive lymphohematopoietic stem cells in the day 9 murine yolk sac. Immunity 7:335–44 Wolf N, Bertoncello I, Jiang D, Priestley G. 1995. Developmental hematopoiesis from prenatal to young-adult life in the mouse model. Exp. Hematol. 23:142–46 Harrison D, Astle C. 1997. Shortand long-term multilineage repopulating hematopoietic stem cells in late fetal and newborn mice: models for human umbilical cord blood. Blood 89:2176–83 Ema H, Nakauchi H. 2000. Expansion of hematopoietic stem cells in the developing liver of a mouse embryo. Blood 95:2284–88 Matsuoka S, Tsuji K, Hisakawa H, Xu M, Ebihara Y, et al. 2001. Generation of definitive hematopoietic stem cells from murine early yolk sac and paraaortic splanchnopleures by aortagonad-mesonephros region-derived stromal cells. Blood 98:6–12 Kyba M, Perlingeiro RC, Daley GQ. 2002. HoxB4 confers definitive lymp-

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hoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors. Cell 109:29–37 Sauvageau G, Thorsteinsdottir U, Eaves CJ, Lawrence HJ, Largman C, et al. 1995. Overexpression of HOXB4 in hematopoietic cells causes the selective expansion of more primitive populations in vitro and in vivo. Genes Dev. 9:1753– 65 Antonchuk J, Sauvageau G, Humphries RK. 2002. HOXB4-induced expansion of adult hematopoietic stem cells ex vivo. Cell 109:39–45 Antonchuk J, Sauvageau G, Humphries RK. 2001. HOXB4 overexpression mediates very rapid stem cell regeneration and competitive hematopoietic repopulation. Exp. Hematol. 29:1125–34 Thorsteinsdottir U, Sauvageau G, Humphries RK. 1999. Enhanced in vivo regenerative potential of HOXB4transduced hematopoietic stem cells with regulation of their pool size. Blood 94:2605–12 Helgason CD, Sauvageau G, Lawrence HJ, Largman C, Humphries RK. 1996. Overexpression of HOXB4 enhances the hematopoietic potential of embryonic stem cells differentiated in vitro. Blood 87:2740–49 Kawabata K, Ujikawa M, Egawa T, Kawamoto H, Tachibana K, et al. 1999. A cell-autonomous requirement for CXCR4 in long-term lymphoid and myeloid reconstitution. Proc. Natl. Acad. Sci. USA 96:5663–67 Ma Q, Jones D, Springer TA. 1999. The chemokine receptor CXCR4 is required for the retention of B lineage and granulocytic precursors within the bone marrow microenvironment. Immunity 10:463–71 Wang LC, Swat W, Fujiwara Y, Davidson L, Visvader J, et al. 1998. The TEL/ETV6 gene is required specifically for hematopoiesis in the bone marrow. Genes Dev. 12:2392–402

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Annu. Rev. Immunol. 2003. 21:807–39 doi: 10.1146/annurev.immunol.21.120601.141135 c 2003 by Annual Reviews. All rights reserved Copyright °

DOES THE IMMUNE SYSTEM SEE TUMORS AS FOREIGN OR SELF? Annu. Rev. Immunol. 2003.21:807-839. Downloaded from arjournals.annualreviews.org by HINARI on 09/01/07. For personal use only.

Drew Pardoll Sidney Kimmel Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231; email: [email protected]

Key Words immune surveillance, tumor tolerance, NKG2D, immune escape, NKG2D ■ Abstract Given the vast number of genetic and epigenetic changes associated with carcinogenesis, it is clear that tumors express many neoantigens. A central question in cancer immunology is whether recognition of tumor antigens by the immune system leads to activation (i.e., surveillance) or tolerance. Paradoxically, while strong evidence exists that specific immune surveillance systems operate at early stages of tumorigenesis, established tumors primarily induce immune tolerance. A unifying hypothesis posits that the fundamental processes of cancer progression, namely tissue invasion and metastasis, are inherently proinflammatory and thus activating for innate and adaptive antitumor immunity. To elude immune surveillance, tumors must develop mechanisms that block the elaboration and sensing of proinflammatory danger signals, thereby shifting the balance from activation to tolerance induction. Elucidation of these mechanisms provides new strategies for cancer immunotherapy.

INTRODUCTION Successful immunotherapy of cancer will ultimately require understanding the natural relationship between the immune system and tumors as they transform, invade, and metastasize. As is discussed below, there is evidence that immune responses against tumor antigens bear both similarities and differences to immune responses against “self” tissue antigens. That immunity to tumors would resemble immunity to normal tissues is not surprising because tumors are of course transformations of normal cells in which growth control has become dysregulated. However, dissection of the molecular events of tumorigenesis together with the pathophysiology of cancer progression teaches us that there are significant features that distinguish cancer cells from their normal counterparts. Some of these differences significantly impact the nature of their interaction with the immune system. It is these distinctive features that may well represent the immunologic Achilles’ heel of cancer, capable of being exploited therapeutically. Specifically, tumors differ fundamentally from their normal cell counterparts in antigenic composition and biologic behavior (Table 1). The molecular hallmark 0732-0582/03/0407-0807$14.00

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TABLE 1 Differences between normal and cancer cells that can affect interaction with the immune system Normal cell

Cancer cell

Implications of cancer immunity

Stable genome

Multiple genetic alterations

Multiple neoantigens arising

Stable transcriptome

Major epigenetic instability

Altered levels of antigen density

No tissue invasion

Invasion and metastasis a hallmark

Potential induction of inflammation activating innate and adaptive immunity

Stable pattern of cytokine and growth factor expression

Abnormal expression of growth factors such as cytokines and VEGF and TGF-b

Potential local inhibitory effects on innate and adaptive immunity

of carcinogenesis is genetic instability (1). Genetic instability in cancers is a consequence of deletion and/or mutational inactivation of genome guardians such as p53 (2). Indeed, many of the genetically defined familial cancer syndromes, such as hereditary nonpoliposis colon cancer and familial breast cancer, are due to mutations in genes that mediate responses to DNA damage (3–8). The genetic instability of cancer cells means that new antigens are constantly being generated in tumors as they develop and progress. This does not occur in normal, nontransformed tissues, which maintain a stable antigenic profile. In addition to the thousands of mutational events that occur during tumorigenesis, hundreds of genes that are either inactive in the normal tissue of origin or expressed at relatively low levels are activated dramatically in cancers. Although these epigenetic changes do not formally create tumor-specific neoantigens, they raise the concentration of encoded proteins many orders of magnitude, thereby dramatically affecting antigenicity. Whereas uncontrolled growth is certainly a common biological feature of all tumors, the major pathophysiologic characteristics of malignant cancer responsible for morbidity and mortality are the ability of transformed malignant cells to invade across natural tissue barriers and to metastasize. Both of these characteristics, which are never seen in normal tissues or benign tumors, are associated with dramatic disruption of tissue architecture. One of the important consequences of tissue disruption, even when caused by noninfectious mechanisms, is the elaboration of proinflammatory signals. These signals, generally in the form of cytokines and chemokines, are central initiators of both innate and adaptive immune responses. Thus, unlike normal tissues, cancers are constantly confronted with inflammatory responses as they invade tissues and metastasize through the body. How they handle and modulate these responses dictates the interplay with the host immune system. This review summarizes the evidence for both immune surveillance and immune tolerance of cancer. These apparently disparate views can in fact be reconciled into a unified hypothesis in which tumors must develop specific mechanisms to locally

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inhibit the activation of innate and adaptive immunity to progress successfully through invasive and metastatic stages. Identification of these mechanisms will define a whole new category of targets for cancer immunotherapy.


THE REBIRTH OF TUMOR IMMUNE SURVEILLANCE Ever since its initial proposal in 1959 by Thomas (9) and then Burnet (10), the immune surveillance hypothesis has come under relentless attack. The fundamental tenet of the immune surveillance hypothesis is that tumors arise with similar frequency to infection with pathogens and that the immune system constantly recognizes and eliminates these tumors based on their expression of tumor-associated antigens (TAAs). Originally, the existence of TAAs was surmised based on the finding that tumors induced in animal models were frequently rejected when transplanted to syngeneic hosts, whereas transplants of normal tissue between syngeneic hosts were accepted (11–14). Modern molecular oncology teaches us that TAAs represent the consequences of the genetic and epigenetic alterations in cancer cells. In fact, both spontaneously arising and chemically induced tumors display diverse properties, with some being rejected effectively (termed regressor tumors) and others growing progressively (termed progressor tumors) after transplantation to syngeneic hosts (15). A corollary to the original immune surveillance hypothesis is that progressor tumors in animals, as well as clinically progressive spontaneous cancers in all species, are not eliminated because they develop active mechanisms of either immune escape or resistance (Figure 1). A fundamental prediction of the immune surveillance hypothesis is that immunodeficient individuals would display a dramatic increase in tumor incidence. The two major challenges to this hypothesis thus involved observational analyses of tumor incidences in immunodeficient mice and patients with heritable immunodeficiencies. Initially some investigators reported no increased incidence of tumors in nude mice (16–20), which have atrophic thymi and therefore significantly reduced numbers of T cells and T cell–dependent immune responses. A caveat to the interpretation of these experiments is that nude mice still produce diminished numbers of T cells and therefore are capable of some degree of T cell–dependent immunity. In addition, they frequently display a compensatory increase in innate immunity including natural killer (NK) cell function. In the 1970s and 1980s, epidemiologic studies of patients with heritable immunodeficiencies revealed a more complex pattern of cancer risk (21). Uncommon cancers such as lymphoblastic lymphomas and Kaposi’s sarcoma were observed at significantly increased frequency; however, the common epithelial cancers seen in adulthood (such as colon cancer, lung cancer, prostate cancer, etc.) were not increased in this population. As more was learned about the microbial—particularly viral—etiology of some malignancies, it became clear that the cancers most commonly found in immunodeficient individuals were virus-associated. Virtually all

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Figure 1 Implications of immune surveillance and resistance mechanisms for tumor survival. If the immune system can successfully survey the body for tumors based on their acquisition of neoantigens consequent to genetic alterations, tumors will be eliminated prior to becoming clinically apparent. According to this notion, tumor survival requires that a tumor actively acquires resistance mechanisms that either cloak it from or inactivate effector arms of the immune surveillance system. Alternatively, a tumor can survive by inducing tolerance to its neoantigens at an early stage of development.

lymphomas are Epstein-Barr virus in origin, resulting from a failure of T cells to control Epstein-Barr virus–transformed B cells. Kaposi’s sarcoma is consequent to human herpesvirus 8 infection (22–26). Other virus-associated cancers such as cervical cancer (from human papillomavirus) were also increased in frequency (27, 28). Interestingly, the major non-virus-associated cancer observed at increased frequency in immunodeficient individuals is stomach cancer. It is now appreciated that stomach cancer is commonly a consequence of ulcer disease related to infection with the Helicobactor pylori bacterium (29). From these studies the notion emerged that immune surveillance indeed protects individuals against certain pathogen (mostly virus)-associated cancers by either preventing infection or checking chronic infection by viruses and other pathogens that can eventually lead to cancer. However, the failure to observe an altered incidence in non-virus (pathogen)-associated cancers was taken as a strong argument against the classic immune surveillance hypothesis. An important caveat to the interpretation of cancer incidence data in heritable immunodeficiency patients is that individuals with more severe immunodeficiency tend not to live past their thirties or forties. Thus, a more subtle effect of immunodeficiency on the incidence of non-pathogen-associated cancers later in life would not be observable. Indeed, a number of recent studies reevaluating tumor immune surveillance in genetically manipulated mice has revealed clear-cut evidence that various components of the immune system can indeed modify both carcinogen-induced and spontaneous carcinogenesis. In a series of studies Schriber, Old, and colleagues reexamined cancer incidence in mice rendered immunodeficient via genetic knockout of either the RAG-1 gene [necessary for immunoglobulin and T cell receptor (TCR) gene rearrangement], the γ -interferon receptor (γ -IFN-R) or STAT-1 (primary signal transducer for the γ -IFN-R) genes (30, 31). RAG−/− mice have no

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T or B cells, and γ -IFN-R−/− and STAT-1−/− mice have diminished innate and adaptive immunity. Initially these researchers monitored tumor incidence in either RAG-deficient or γ -IFN-R mice either treated with carcinogens or crossed onto a cancer-prone p53−/− background. In both cases the incidence of observable cancers was slightly but significantly increased relative to nonimmunodeficient counterparts when observed over an extended period (≥1 year). Furthermore γ IFN-insensitive p53-knockout mice also developed a broader spectrum of tumors than mice lacking p53 alone. Transplantation of metholcholanthrine-induced tumors from γ -IFN-R-knockout mice suggested that direct γ -IFN sensitivity by the tumor played a significant role in the defect in immune surveillance. These results have prompted an analysis of γ -IFN sensitivity of human tumors. Although loss of γ -IFN sensitivity has been documented in a number of cases (owing to mutation or loss of expression of various components of the γ -IFN-R signaling pathway), the overall incidence in human cancer appears to be quite low (65% of the nonIgM+ leukemic cells exhibit >5% difference from the closest germline sequence. However, the presence and level of mutation is not consistent among B-CLL cases; rather they follow a hierarchy based on the VH family (VH3 > VH4 > VH1) and the specific VH genes within these families (VH 3-07 >4-34 >1-69) that are expressed by the leukemic cells (8). Furthermore, these mutations demonstrate selection for replacement (R) mutations in the complimentarity determining region (CDR) and selection against R mutations in the framework region (FR) (8). These findings suggest prior antigen selection via an intact BCR (40–42) of the B cells that eventually became leukemic. Based on these data, B-CLL cases are now considered heterogeneous at the cellular level and classifiable into two subgroups based on the presence or absence of significant numbers of V gene mutations. These differences may indicate that the B-CLL precursors received contrasting stimulations by distinct types of antigen prior to leukemic transformation (8) and/or that the precursors were transformed into leukemic cells at distinct maturational stages (8, 11, 43).

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The V Gene B-CLL Exhibits Different and Relatively Unique CDR3 Characteristics The high degree of sequence variability in the CDR3 of both the H and L chains (HCDR3 and LCDR3, respectively) leads to major differences in CDR3 amino acid composition, length, and charge among individual B cell clones (44, 45). In B-CLL cases as a whole, the use of D genes is comparable to that of other antibodies in GenBank (8, 46). These D segments are expressed in the hydrophilic reading frame (8) as in the normal adult B cell repertoire (46, 47). However, there is evidence for distinct subgroups of B-CLL cases characterized by the use of certain specific D segments and their association with specific VH and/or JH segments. For example, the D3-3 segment is found most often among B-CLL cells with BCRs comprised of VH 1-69 and JH6 genes (8, 13), although this finding has not been universal (11). Similarly, the usage of JH gene families in B-CLL cases as a whole does not differ significantly from the normal B cell repertoire (8), although JH use does differ among B-CLL cells that express different VH family genes (8, 13). The vast majority of cases that express the VH 3-07 gene (∼90%) use a JH4 gene segment, whereas VH1 > VH3) (8). These differences are most obvious when comparing the most frequently used genes in the three major families. The average HCDR3 length of 3-07-expressing cases is very short (8), whereas the length of 1-69-expressing B-CLL cells is much longer (8, 13). Indeed both lengths are significantly different from those of normal B cells. Interestingly, the HCDR3 lengths of the 4-34+ leukemic cells segregate into two groups: unmutated 4-34-expressers with long HCDR3s that usually contain a JH6 or JH5 segment and mutated 4-34-expressers with shorter lengths that usually contain a JH4 segment (8). The HCDR3 of IgM+ VH1-expressing B-CLL cases frequently contain relatively long stretches of tyrosine (Y) at their 30 ends, coded for in part by the JH6 segment (8, 13). Furthermore, the charge of the HCDR3, as defined by an estimated pI, indicates another distinction based on VH gene use (8). VH1-expressing B-CLLs have the lowest estimated pI, whereas leukemic cells that express a VH3 gene have a much higher estimated pI. The VH4-expressing B-CLL cells display intermediate values. Thus, the length, amino acid composition, and charge of HCDR3 in B-CLL cells is variable, and each tends to vary according to the VH gene expressed in the B-CLL cell (8). These findings suggest that the observed differences in BCR structure among the B-CLL clones are the result of selection by distinct antigenic epitopes, which may or may not have driven the respective clones down distinct differentiation pathways.

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Composite Structural Similarities of These BCRs When the structural characteristics of the rearranged VHDJH and VLJL of B-CLL cases are considered in toto, the leukemic cells can be divided into two subgroups that differ in specific VH gene use, the presence of significant numbers of V gene mutations, and HCDR3 features (Figure 1). The prototypic rearranged VHDJH of the unmutated group (

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