Annual Review of Immunology Volume 20 2002

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Annual Review of Immunology Volume 20, 2002

Annu. Rev. Immunol. 2002.20:1-28. Downloaded from arjournals.annualreviews.org by HINARI on 09/01/07. For personal use only.

CONTENTS FRONTISPIECE, Charles A. Janeway, Jr. A TRIP THROUGH MY LIFE WITH AN IMMUNOLOGICAL THEME, Charles A. Janeway, Jr.

xiv 1

THE B7 FAMILY OF LIGANDS AND ITS RECEPTORS: NEW PATHWAYS FOR COSTIMULATION AND INHIBITION OF IMMUNE RESPONSES, Beatriz M. Carreno and Mary Collins

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MAP KINASES IN THE IMMUNE RESPONSE, Chen Dong, Roger J. Davis, and Richard A. Flavell

PROSPECTS FOR VACCINE PROTECTION AGAINST HIV-1 INFECTION AND AIDS, Norman L. Letvin, Dan H. Barouch, and David C. Montefiori T CELL RESPONSE IN EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS (EAE): ROLE OF SELF AND CROSS-REACTIVE ANTIGENS IN SHAPING, TUNING, AND REGULATING THE AUTOPATHOGENIC T CELL REPERTOIRE, Vijay K. Kuchroo, Ana C. Anderson, Hanspeter Waldner, Markus Munder, Estelle Bettelli, and Lindsay B. Nicholson

55 73

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NEUROENDOCRINE REGULATION OF IMMUNITY, Jeanette I. Webster, Leonardo Tonelli, and Esther M. Sternberg

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MOLECULAR MECHANISM OF CLASS SWITCH RECOMBINATION: LINKAGE WITH SOMATIC HYPERMUTATION, Tasuku Honjo, Kazuo Kinoshita, and Masamichi Muramatsu

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INNATE IMMUNE RECOGNITION, Charles A. Janeway, Jr. and Ruslan Medzhitov

KIR: DIVERSE, RAPIDLY EVOLVING RECEPTORS OF INNATE AND ADAPTIVE IMMUNITY, Carlos Vilches and Peter Parham ORIGINS AND FUNCTIONS OF B-1 CELLS WITH NOTES ON THE ROLE OF CD5, Robert Berland and Henry H. Wortis E PROTEIN FUNCTION IN LYMPHOCYTE DEVELOPMENT, Melanie W. Quong, William J. Romanow, and Cornelis Murre

197 217 253 301

LYMPHOCYTE-MEDIATED CYTOTOXICITY, John H. Russell and Timothy J. Ley

SIGNAL TRANSDUCTION MEDIATED BY THE T CELL ANTIGEN x

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RECEPTOR: THE ROLE OF ADAPTER PROTEINS, Lawrence E. Samelson INTERACTION OF HEAT SHOCK PROTEINS WITH PEPTIDES AND ANTIGEN PRESENTING CELLS: CHAPERONING OF THE INNATE AND ADAPTIVE IMMUNE RESPONSES, Pramod Srivastava CHROMATIN STRUCTURE AND GENE REGULATION IN THE IMMUNE SYSTEM, Stephen T. Smale and Amanda G. Fisher PRODUCING NATURE’S GENE-CHIPS: THE GENERATION OF PEPTIDES FOR DISPLAY BY MHC CLASS I MOLECULES, Nilabh Shastri, Susan

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Schwab, and Thomas Serwold

395 427

463

THE IMMUNOLOGY OF MUCOSAL MODELS OF INFLAMMATION, Warren Strober, Ivan J. Fuss, and Richard S. Blumberg T CELL MEMORY, Jonathan Sprent and Charles D. Surh

GENETIC DISSECTION OF IMMUNITY TO MYCOBACTERIA: THE HUMAN MODEL, Jean-Laurent Casanova and Laurent Abel ANTIGEN PRESENTATION AND T CELL STIMULATION BY DENDRITIC CELLS, Pierre Guermonprez, Jenny Valladeau, Laurence Zitvogel, Clotilde Théry, and Sebastian Amigorena

495 551 581

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NEGATIVE REGULATION OF IMMUNORECEPTOR SIGNALING, André Veillette, Sylvain Latour, and Dominique Davidson

669

CPG MOTIFS IN BACTERIAL DNA AND THEIR IMMUNE EFFECTS, Arthur M. Krieg

PROTEIN KINASE Cθ

709 IN

T CELL ACTIVATION, Noah Isakov and Amnon

Altman

RANK-L AND RANK: T CELLS, BONE LOSS, AND MAMMALIAN EVOLUTION, Lars E. Theill, William J. Boyle, and Josef M. Penninger PHAGOCYTOSIS OF MICROBES: COMPLEXITY IN ACTION, David M. Underhill and Adrian Ozinsky

761 795 825

STRUCTURE AND FUNCTION OF NATURAL KILLER CELL RECEPTORS: MULTIPLE MOLECULAR SOLUTIONS TO SELF, NONSELF DISCRIMINATION, Kannan Natarajan, Nazzareno Dimasi, Jian Wang, Roy A. Mariuzza, and David H. Margulies

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INDEXES Subject Index Cumulative Index of Contributing Authors, Volumes 1–20 Cumulative Index of Chapter Titles, Volumes 1–20

ERRATA An online log of corrections to Annual Review of Immunology chapters (if any, 1997 to the present) may be found at http://immunol.annualreviews.org/

887 915 925


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Annu. Rev. Immunol. 2002. 20:1–28 DOI: 10.1146/annurev.immunol.20.080801.102422 c 2002 by Annual Reviews. All rights reserved Copyright °

A TRIP THROUGH MY LIFE WITH AN IMMUNOLOGICAL THEME


Charles A. Janeway, Jr. Section of Immunobiology, Yale University School of Medicine and Howard Hughes Medical Institute, 310 Cedar Street, New Haven, Connecticut 06520-8011; e-mail: [email protected]

KEYWDGRP> innate immunity, Toll-like reception, positive selection of T cells and B cells, suppressor T cells, adaptive immunity ■ Abstract In this essay, I make four points about the operation of the immune system. First, thanks to the innate immune system’s regulation of the main costimulatory molecules CD80 and CD86, the immune system rarely mistakes a pathogen for a self-antigen. Second, the adaptive immune system consisting of T lymphocytes and B lymphocytes can mistake self for non-self because adaptive immunity is selected in single somatic cells. Third, the adaptive immune system of T lymphocytes and B lymphocytes is always referential to self, as it is selected on self-ligands; it persists in the periphery on self-ligands; and at least for T cells, it is dependent on self-ligands to be able to mount a response. Fourth, it is becoming clear that regulatory or suppressor T cells are our main defense against autoimmunity, as my first boss, Richard Gershon, had predicted. These cells recognize antigen as do all T cells, but they secrete the immunoregulatory cytokines IL-10 and TGFβ.

INTRODUCTION My early life was one of health and wealth, although at the time I did not appreciate it. I grew up in Weston, MA, first on a highway on the south side of town (Massachusetts Route 30) and later on the north side of town, in a house that my parents had built for them by an architect friend after a careful search for sites within the same town. Throughout my early years, I was surrounded by love, kindness, and friends, especially after we moved to our new house on Concord Road in Weston. This neighborhood was very cohesive, held together by an unusual alliance between my father and the family next door, the Cummings family. They had bought the estate of the late Eddie Collins, a Hall of Fame second baseman for the Boston Red Sox, and over the ensuing years, they sold parcels to various people who became our neighbors, all about the same age and all with children. I made several very close friends, some of whom I still keep up with today, especially Kim Cummings. I had one other close friend named Rowley Elliston from my time on the south side of town who was then and continues to be my best friend. A medical experiment that I remember from my childhood, and still show 0732-0582/02/0407-0001$14.00

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A medical experiment that I remember from my childhood, and still show to medical students now, happened when I contracted measles, which my father diagnosed by seeing Kopplick’s spots in my pharynx. At the time he was working on agammaglobulinemia and treating it by injection of intramuscular gamma globulin. He lined up all the kids in the neighborhood, injected them with gamma globulin, and then systematically exposed each of them to me. Each one developed protection against the measles virus but not measles itself. Imagine getting that past an IRB today! This made a strong impression on me, but I did not understand it at the time or I should have become an infectious disease specialist rather then an immunologist. But the impression stayed with me throughout my life and is still with me today. The lesson that I draw from this, and later learned in medical school, was that humoral immunity exists to protect the body from external threats to its integrity. This also illustrates how tightly knit a community we were on Concord Road. How privileged we were to be able to go out alone at night and play with one another. It is hard to imagine parents letting their kids run around in the dark nowadays! Of course, as the town had a reputation for good schools, I went to school at the Weston Public Schools, first to elementary school, then to Junior High School, which in those days was housed in the same building as the Public High School, and then I went on to Weston High School. Thus, it was a surprise when my mother wanted me to go to a private school because I was so happy and doing so well in public school. I still do not know why she wanted me out of the house. Perhaps it was the graduation of my friend Kim from Weston High School, or perhaps it was the departure of my best friend Rowley to the Cambridge School of Weston, but she insisted that I go interview at two private schools. One of these was the Phillips Exeter Academy, in Exeter, New Hampshire, and the other was Milton Academy, in Milton, Massachusetts. I remember interviewing at Exeter and telling my interviewer, who asked me why I wanted to attend the academy, that I was not at all sure that I did. Perhaps this unexpected answer may have convinced them that I was at least honest enough to admit to them that I was unsure. I don’t know if this is true, but in any case they accepted me. Thus began two years of what felt like prison to me, and to many of my classmates, as I learned recently at my 40th reunion at Exeter. This came as somewhat of a revelation to me, but I had seen it earlier, at my 20th reunion. At this reunion, all the guys who looked just as they had during their years at Exeter turned out to have been happy there, and all the guys who looked totally transformed had been miserable. It was the uniformity of unhappiness that came out at the 40th reunion that struck me and all of my classmates. There was not one of the happy camper types there, or else they were well hidden. Suffice it to say that I hated being at Exeter. This was before they admitted girls, which was part of the problem with the school. In any case, I managed to get a good education there and got advanced placement in three subjects, so I could go through Harvard College in three years and go on to my calling, which was in medicine, like my father before me, and his father and grandfather before him.

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I was very lucky with my professors at Harvard College. In my freshman (or sophomore) year, I had two Nobel Prize winners, one of whom had already won the Prize, and one who was to win it later when I was living in Sweden. I then again got to marvel at his insights into the nature of chemical bonds. This was Eugene Lipscomb, and he taught those of us who were willing to listen to him all we needed to know about the nature of chemical bonds, using boron as his model molecule. It was for his work on boron that he won the 1976 Nobel Prize in chemistry, and he came to Sweden to give his lecture. I was then living in the city of Uppsala, Sweden, which is about an hour’s train trip north of Stockholm. It was wonderful to see him give the same flare to his Nobel Lecture that infused his lectures when I was a freshman chemistry student. After learning all about chemical bonds from Lipscomb, I found it relatively easy to breeze through the chemistry curriculum at Harvard, except for the labs, which still required some application. The second professor from whom I learned a great deal was George Wald, who taught freshman Biology, or Nat Sci 5 as it was called. He was particularly brilliant at explaining the central dogma of biology, which had just been formulated a few years before. This was that the genes were arranged on chromosomes and encoded information in DNA. The DNA code had to be transcribed into RNA, which was then translated into protein on ribosomes. This was really new at the time, and I was carried away with the excitement of these concepts. It is probably to George Wald that I owe my career in research, although there were many other central influences along the way: my lab instructor in Nat Sci 5, Hannah Gould, John Humphrey, Robin Coombs, Bill Paul, Dick Gershon, Susumu Tonegawa, and my many colleagues at Yale, to which I went in 1977. I want to give special credit to my wife, Kim Bottomly, to whom I would like to dedicate this reminiscence.

MY TRAINING AT MEDICAL SCHOOL AND IN IMMUNOLOGY When I finished Exeter and Harvard College, I knew that my destiny was to go to medical school, so I applied to several schools that had good reputations: Harvard, Johns Hopkins, and Yale. I never heard back from Yale, for some unknown reason, but I heard from Hopkins and from Harvard, which both accepted me. As I had a girl friend, who was later to become my wife, going to Wellesley College, that made the decision to go to Harvard Medical School easy. I worked hard there for the first two years, but I had trouble with the Biochemistry course, because I always solved problems by going back to first principles, whereas the instructors wanted me to use formulas that had been developed to make calculations easier for clinicians. But in any case, I basically learned my stuff in the first two years of medical school, and I passed with flying colors. I don’t actually know this, as we did not receive grades, but I could tell that I was doing well. The only thing that was wrong with this was

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that I forgot how to think; I felt like a sausage casing that had been stuffed full every day. This image stayed in my mind for the rest of my life and became useful later when I started to teach medical students at Yale. I think it made me a better teacher than I would have been if I had not been exposed to professors who talked fast and convincingly about all manner of subjects. The only courses I remember enjoying, besides Immunology, were the courses in anatomy and neurobiology. Anatomy was my first encounter with the human body and with teams of students dissecting it. I used Gray’s Anatomy to study for this course. The reason I liked Gray’s Anatomy was that it was well written, which may have influenced my later decision to write my own book on immunology, now in its fifth edition. The course in neurobiology was taught by a number of young, enthusiastic professors under the guidance of Steven Kuffler. This course challenged one to think, and think we did. We would come up with experiments to do and new ideas, rather than the dreary memorization that characterized most courses. Another problem was that we had basically no free time, the days went on forever, and the nights were spent studying what we had heard during the day. In the middle of my second year at Harvard Medical School, I got a message from my lab instructor in Nat. Sci. 5, Hannah Gould, who asked me if I would like to work with her on globin synthesis, which she was studying in London. This piqued my interest because I had visited London in 1956 with my family and had fallen in love with the city and England in general. I visited it again with my friend Rowley Elliston in 1960. So I went to the Dean of Student Affairs, whose name was Joseph Gardella, and I asked him if he thought this was a good idea. He told me that I could go anywhere I wanted. That sent me back to the drawing board, and because I had been interested in immunology, I began seeking positions in immunology in London. My lab instructor in the immunology part of the course in microbiology was Hugh McDevitt, and he had had us read a series of papers on the origin of antibody diversity that had me really excited about this subject. So I mentioned that I would like to work in London in the same laboratory that Hugh had just returned from. I asked Hugh to arrange a position there for me, and after some hunting, he found that Brigitta (Ite) Askonas had planned to have a woman postdoctoral fellow who had, at the last minute, dropped out. I went to work with Ite, but when I arrived, it turned out that John Humphrey had a lab for me, so in the end I worked for John. It was a great two years as it turned out, and I wrote several papers with John and with Michael Sela, who had provided the synthetic polypeptides that I worked on. These were called L-TGA (for the left rotating amino acids tyrosine, glutamic acid, and alanine) and D-TGA (for the rightward rotating enantiomers of the same amino acids). This was before the invention of the single letter code, which would have listed them as L-YEA, a neater acronym than L-TGA. I compared the response to L-YEA with the response to D-YEA, which gave very low but not insignificant responses. These findings took me two years to compile and confirm, but in the end I wrote up four papers, all of which were accepted for publication.

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Sally, my first wife and the mother of my daughter Katherine Anne, and I did other things while we were there. We traveled to Europe several times, to Wales and to Scotland, where by chance we met Chris Elson and his wife, talked about immunology and highland hikes, and ended up getting a ride back to Edinburgh. Overall, I had a good time. The only thing we could measure in those days was antibody production, and we measured it in great detail. It turned out that antibodies to L-YEA did not crossreact with D-YEA, and antibodies to D-YEA did not bind to L-YEA. That meant that they were specific. But how did they come by their specificity? Was a gene for each specificity found in the genome of the mice, or was there somatic mutation of a few or a single gene? John Humphrey himself was addressing the same question, in his case trying to rule out the “template hypothesis,” which proposed that the antigen was taken up by the lymphocytes and used as a template for the formation of specific antibodies. It was a wonderful environment for me to work in. After two years in John Humphrey’s lab, you can imagine my shock upon returning to medical school, where I discovered that what was called evidence in clinical medicine did not measure up to scientific standards. It was mainly guesswork and listening to one’s patients. I must say I was excited to see real live patients, and even some on whom I was allowed to perform simple procedures such as lumbar taps to measure the pressure in cerebrospinal fluid. I even performed a herniorraphy on a patient in the West Roxbury, VA, hospital where I did part of my surgical rotation. I had a really neat surgical instructor in my “Introduction to the Clinic” course at the beginning of my third year of medical school, so I thought for a while that I wanted to be like him. But after a while, I thought that going into surgery was going to condemn me to a life of routine procedures, so I looked elsewhere. I remember patients from those days quite clearly, especially one diabetic woman who went into a diabetic coma. She was on insulin treatment, and while Ralph Steinman (my former classmate and now fellow member of the National Academy of Sciences) bent over the woman, I asked for 50 ml of 50% glucose in water. The result of injecting this was very dramatic; the woman revived almost immediately, as Judah Folkmann had taught us she would, and Ralph was startled by the effect. But the main thing I remember was the endless hours of operating while functioning on adrenaline instead of glucose, due to the imperfect state of medical practice in those days, as well as in the present (though it has improved in many areas). The patients loved us for what we told them, not for what we could do for them. I began to feel as if my soul was rotting, and that was very distressing to me. I decided that I needed another dose of research, where no one would take my word for anything, and the results were always solid. Therefore, I began to ask around about research opportunities, especially when I realized that two of the elective courses I wanted to take in the second semester of my fourth year of medical school were not on offer that year. I had devised a trick to mark red blood cells, and I used it to show that blood group–specific antibodies could cross-link

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A RBC but not B RBC when a mixture of A and B RBCs were agglutinated with anti-A antibody. Using this as a bargaining chip, I ended up in Robin Coombs’s laboratory on Tennis Court Road in Cambridge, England, on my fourth trip to this fabled isle. During my visit with Robin Coombs, several very significant events occurred. The first was that I made a friendship, which I still treasure, with a wonderful Scott of Pictish origin named Ian McConnell. He took me under his wing, and he hosted Sally and I to several nice dinners, prepared by his wife, Anna McConnell. Ian was irreverent in a way that would not be tolerated in an American lab, and this taught me a lesson that I have long remembered: Don’t be stuck on yourself. The second event was the arrival one day, in a swirl of fur and knee-length boots, of Phillippa, or Pippa, Marrack, one of the best known English/American immunologists. Her grandfather was J. R. R. Marrack, a noted immunologist who devised the technique known as equilibrium dialysis to measure the affinity of antibodies. At the first meeting, I was overwhelmed by her attractiveness, but I did not let my feelings out. However, over the years, we have had frequent encounters, most of them extremely positive. She has become the model of success that most female and male scientists aspire to. I consider her to be one of my close friends in immunology. While in Coombs’s lab, I worked on B cell receptors, using a rosetting technique that Robin had worked out with Phillip Gell. Coombs had made antiimmunoglobulin antibodies that were first used to show that the hemolytic disease of the newborn was due to antibodies to Rh blood group antigens. The basic idea was to add anti-immunoglobulin antibody to rabbit lymphocytes and antibodycoated sheep erythrocytes, and then mix them in a tube and look for the number of rosetted cells. We also took advantage of rabbit allotypes to confirm the specificity of our observations. This was the first demonstration that a subset of peripheral lymphocytes had immunoglobulin on their surface; we also used rabbit thymocytes to show that these cells did not form rosettes, which we interpreted to mean that there was no immunoglobulin on their surface. Similar studies using fluorescent antibodies were published shortly after we presented our results at the annual meeting of the British Society of Immunology. In comparing the submission dates of these two papers, it is clear that ours was submitted before the meeting and theirs after, but theirs was published first, while ours, although not revised, was published months later. I learned an important lesson from this: It is not he who submits first that gets priority, but he who gets published first.

INTERNSHIP AT THE PETER BENT BRIGHAM HOSPITAL When my days in England were over (I did not attend my own graduation), I came home and moved into our old apartment. The next day, I began my internship at the Peter Bent Brigham Hospital (PBBH, now Brigham and Women’s). I had already planned a postdoctoral fellowship in Baruj Bennacerraf’s laboratory at the NIH as one of many “yellow berets.” This term was coined to describe people who avoided

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military service by going into the Public Health Service; I still have my honorable discharge certificate from the Public Health Service tucked away somewhere in my belongings. The year at the PBBH was at the same time one of the best and worst experiences of my life. What was good about it was serving my patients; what was bad was that I was forced to neglect my wife Sally to do so. In the end, we had a child and got divorced shortly thereafter, so I felt lonely for many years. However, there was a benefit to being single: I felt that I was free to do whatever I pleased. I had spent so much of my life living according to one or another person’s rules (the worst was at Exeter), and I needed time and freedom to rediscover myself and my own life. I know this will sound selfish to many who read this, but that was the way that I felt.

POSTDOCTORAL STUDIES UNDER WILLIAM E. PAUL AND HANS WIGZELL So for the next period of my life, I lived on my own and stayed up until all hours of the night. I was working hard, doing experiments that I believed in, getting results that I could interpret, and spending every other weekend with my young daughter Katherine Anne, whom I loved and still love very much. She was one year old when Sally and I split up. I was also living in an old log cabin on a dirt road outside of the town of Harmony, MD, that Sally and I had bought and fixed up. Eventually, when my postdoctoral years were over, I sold the property for about five times what it had cost. My daughter, Katie, could not understand why I would sell the house she knew as home. But I needed the money, which I later used to buy a home in New Haven, CT. During my postdoctoral fellowship at the Laboratory of Immunology, National Institute of Allergy and Immune Diseases, National Institutes of Health, I had the opportunity to work with several people who shaped my career, but the most important was Bill Paul, now head of the NIH Laboratory of Immunology, whom we called “the WEP” behind his back. This nickname was an affectionate abbreviation based on his initials. I first planned to work under Benacerraf, but he left the NIH the same day that I arrived from Harvard, leaving Bill in charge. Bill suggested that I work with Rose Lieberman, which I did for about a year, but it seemed that I was plowing old turf, and I wanted novelty. So Bill suggested that I try conjugating the hapten DNP to the heat-killed Mycobacterium tuberculosis known as H37Ra. I tried immunizing guinea pigs with this antigen, and I was surprised that I got what looked like hapten-specific T cell proliferation as a result of this immunization. I worked on this project for some time, and we published three or four papers on it over the five years that I spent happily in his lab. I also worked on a model of what was purported to be hapten-specific helper T cells, but it turned out to be an effect of antibody, as it could be passively transferred with antibody molecules purified over affinity columns.

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I was feeling restless about my progress in science, and I remember having serious conversations with several senior scientists about what I should do with my life. Should I go back to medicine, which I loved because my patients seemed to appreciate my spending time with them? But in medicine I felt I was taking the easier pathway, the easy way out, whereas if I went on with research, I would always face skeptical colleagues who did not believe my interpretations of my data nor I of theirs. This felt much healthier to me, and so I decided to go into full-time research; I could always get further training in internal medicine if my plans did not work out. After five plus years at the National Institutes of Health, I was looking at offers from Harvard, again with Baruj Bennacerraf, Johns Hopkins with Roberto Poljak, Washington University in St. Louis with Herman Eisen, or at Yale with Dick Gershon. Of all of these offers, I chose Yale because the opportunities seemed more wide open; there were only a few immunologists at Yale, and I felt more comfortable with them than with any other group. I presented a seminar on my work with hapten-specific T cells, which we had already demonstrated to recognize both the hapten and various peptides, later confirmed by a German scientist (1). I was offered a job by Vincent Marchesi, Chairman of Pathology, at a starting salary of $27,500. Although all the other programs made better offers to me, I decided to go with my gut feelings, and I am glad that I did. But before I could come to Yale, Vince told me that he had to have a lab renovated for me, and for two other faculty who would join me; they were not selected at the time of my hiring. Before I began my career at Yale Medical School, I wanted further training, so I decided I would go to work with Hans Wigzell, then a Professor of Immunology in Uppsala, Sweden. I was asked by my former wife if I could take Katie with me, so Katie and I flew to England, and then on to Sweden in the fall of 1975. When we arrived, I was met at the airport by Hans Binz, who I had earlier met at a scientific meeting in the former country of East Germany. It was the first scientific meeting to be held in East Germany, and I remember crossing the border into East Germany, where the border police were most interested in finding newspapers, which they confiscated as western propaganda. I also remember having to translate for the East German Minister of Health on a drunken evening in the Rasthskellar of the hotel we were staying in, which was in a former hunting lodge of the Kaiser. This was a very interesting experience for me, as I had not spoken a word of German since 1963, when I graduated from college. My suspicion was that the health minister knew English but was too proud to let on, and that was how I was roped into this conversation. I then flew back to England, collected Katie from Liz Simpson, who had kindly taken us in, and flew to Sweden. My initial impression of Sweden was not good. First, I thought I was going blind, as the evenings got shorter and shorter, so that I went to and from the lab in the dark. Second, everyone looked the same, and they all answered my question of why were they working with the same expression: “Ja, m˚an musta har pengar.” I translated this to mean: “Yes, one has to have money.” I later had a Swedish postdoctoral fellow who confirmed my translation. If you

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asked an American the same question, you would get a completely different type of answer, full of commitment to a cause or a career that was meaningful to them. I don’t yet know which I prefer, the Swedish or the American answer. At least the Swedes were honest enough to tell you that they were working for the pay. I also saw an article in the International Herald-Tribune that said that Sweden had the highest per-capita income in Europe, with the lowest standard deviation between the rich and the poor, and I believe that was true at the time. I loved working for Hans Wigzell. I even played squash with him (he always beat me), because I could get him to slow down enough after the match so that we could have a conversation. Another trick I learned was to drive him to the airport in my old SAAB which, although it had a hole in its floor, ran beautifully, far more smoothly than Hans’s own car. These were times when I could talk to him about science and about the experiments that I wanted to do. Although I published very little from this time, I learned how to isolate T cells and to grow them in vitro, so that later I could clone them, which seemed a wonderful thing to do once it became possible. Hans was always asking if I thought I would like it in “Jale,” which was, I am sure, a deliberate mispronunciation of “Yale.” I told him that I was sure that I would. Later, when he had become Rektor of the Karolinska Institut, he actually put his American friends in an old jail that had been converted to a hotel. I was finally able to tell him: “Ja, jag a¨ lska varend i F˚angelska” (Yes, I love being in jail).

MY CAREER AT YALE MEDICAL SCHOOL When I first came to Yale University School of Medicine in 1977, I was attracted by two things. One was the presence of Richard Gershon, whose early death at 57 in 1983 was one of the saddest moments of my life. The second reason was the opportunity that was given to me to build up an immunology program complete with a training grant, an immunology course, and the opportunity to interact with students at all levels. What has kept me here all these years was funding from the Howard Hughes Medical Institute. This came about as a result of a dispute between my Chairman, Vincent Marchesi, and the Chairman of Internal Medicine, Dr. Sam Thier. This dispute, which should have taught me a great deal about how the medical school operated, but did not, was over who should get the “big Hughes” and who should get the “little Hughes.” The terms were for the number of dollars attached to each: the “little Hughes” paid about $50,000 per year, while the “big Hughes” was supposed to pay all one’s expenses. Eventually, my chairman lost this argument, so the “little Hughes” came to him to support an investigator in one of the three disciplines which the Howard Hughes Medical Institute was then sponsoring: genetics, metabolism, and immunology. As I was the only junior faculty member in his department that fit this description, I became an HHMI investigator. Later,

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HHMI did not approve of the Department of Internal Medicine’s candidate, so the “big Hughes” went to Richard Gershon, giving us both HHMI positions. Because we had both HHMI positions at Yale, we became a force in the politics that surround money in academia. I like to think we used this influence well, or at least for the benefit of the school and for immunology research at Yale. Later, the Hughes Tool Company was sold to General Motors and HHMI got a stricter board of trustees, but as I was already an established investigator, I was kept at that rank during the time that each new regime came in at HHMI. Also recruited were Dr. Donal Murphy and Dr. Robert Rosenstein, both directly out of postdoctoral positions. We also recruited Al Bothwell, the first true molecular biologist in the Division of Immunology in the Department of Pathology. Don was my best friend at Yale, and we shared an office, which held a secretary and small offices for each of us. Because his father had been posted to New Haven during World War II, he had actually been born in New Haven, which was quite novel for a faculty member at Yale. It was a very upsetting moment for me when I could not persuade the tenure allotment committee, which was controlled by Dean Rosenberg, to offer Don a slot so that he could try for tenure. This happened because I was never given the powers of a chairman but was always referred to by Dean Rosenberg as “the senior immunologist at Yale.” The next significant event in my life was the recruitment of my wife, Dr. Kim Bottomly, Ph.D., to the faculty at Yale. She became my inspiration in all things, my career, scientific research, and teaching. I would often ask her about how to teach a particular topic, and she always had clever thoughts. I should also mention another side of her. She was, and is, a wonderful mother to my three daughters: Katie, who was with me when I met Kim, and Hannah and Megan, who were born in New Haven after Kim’s arrival at Yale. This brought much joy and much fighting to our house on Livingston Street, where we still live. Once Kim settled into our new house, we began making plans to have a family. It was a hectic time in our lives. I was beginning to be known, and Kim was too, for her discovery of what we called T helper cells (TH) because they could induce antibody responses, and T inflammatory cells (TI) because they caused inflammation at sites of antigen injection. These were later to become famous as Th2 cells and Th1 cells, the nomenclature proposed by Tim Mossman on the basis of the cytokines these cells could secrete. I still prefer Kim’s nomenclature because it fits the functions of these cells more closely, but the world has overtaken it. Then, just as we were settling in to New Haven, Dick Gershon was diagnosed with a small cell lung cancer, throwing all our plans up in the air. Dick was a heavy smoker, and he told his wife that this type of cancer was not caused by smoking, which was nonsense. But she believed him, and so she sued Yale and the HHMI for an outrageous sum of money, further deepening tensions between Yale, HHMI, and myself. Eventually, the suit was thrown out by a judge, Dick’s wife and young daughter left town, and we began talking among ourselves about whether we would remain independent or whether we would be

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swallowed up by the Pathology Department, of which we were then a division. When I was asked by Leon Rosenberg M.D., then the Dean of the Medical School, to meet him in his office, I thought he would offer me Dick’s job. Imagine my surprise when I found Vincent Marchesi, the Chairman of Pathology, sitting in his office with him! I thought this was going to be a chat with the Dean, but it turned into a diatribe by him against me and my behavior, and a set of demands that would have ended Immunology as an independent subject at Yale Medical School, and within the University as well. I was stunned. After this, Vin asked me to have a cup of coffee with him in the medical school cafeteria, where he laid it on even thicker than Lee Rosenberg had. I was further stunned. I felt as if I had just been put through a car wash without a car to protect me from all the blistering that I had undergone. I left Dean Rosenberg’s office saying that I would have to talk to my colleagues in the Division of Immunology. I did so, and they reacted with horror at the idea of our group becoming part of Pathology with no independence at all. When I went back to Lee Rosenberg, I told him that there was no enthusiasm for his idea about the immunology program, which at that time consisted of several investigators, as well as joint appointees Nancy Ruddle, Ph.D., Phillip Askenase, M.D., and Adrian Hayday, Ph.D. We all felt that we could not work with the Pathology Department any longer, and we wanted our own department. Leon’s reaction was that either he had chosen the wrong person, or that he had had the wrong idea. It is a tribute to his intellect, which is formidable, that he came to the latter conclusion. He was driven to this by an HHMI site visit shortly thereafter which consisted of people that he had to respect: Most of them were already in the National Academy of Sciences. This site visit told him he was off the mark in melding immunology into pathology, and to give him his due, he listened. I know because he called me the next day at my home (it must have been a Saturday), and he screamed at me for the better part of an hour, accusing me of setting him up with my friends. Nothing could be farther from the truth. I had carefully hidden my difficulties with Dean Rosenburg from my friends on the site visit team. They were stunned by his complaints that I was being resistant to his wishes. In any case, he calmed down and invited me to have lunch with him a few weeks later, at which he proposed the formation of a Section of Molecular Immunobiology, which I said was fine. He also said that he had appointed a close friend to head the search for a new chair whom he said was the best immunologist he knew. This turned out to be wide of the mark, but the basic idea seemed to me a lot better than the alternative, as long as the Immunobiology was an equal partner with the Molecular. We shook hands over that and ended our lunch. It later proved necessary to have lunch with him again, this time with all of my colleagues backing me up, to complain that his friend was not running a satisfactory search for a chair of our new section. He was, in fact, looking in the wrong places, and even scheduling seminars in rooms in which classes were taking place. In fact, we were able to point this out to Dean Rosenberg right after lunch, when a candidate for the job

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was found waiting outside a full lecture hall! Eventually, we got our way when Leon hired Richard Flavell as Chair of a new Section of Immunobiology.


MY TEACHING CAREER AT YALE During the period from 1977, when I arrived at Yale University School of Medicine, until around 1980, we taught immunology as a subdiscipline of pathology, with a team of teachers coming from several departments, including Bob Cone from Surgery, Nancy Ruddle from Epidemiology and Public Health, Phil Askenase and John Dwyer from Internal Medicine, and Dick, Don Murphy, Bob Rosenstein, and myself from the Department of Pathology. The teaching of the subject was very disjointed and lacked focus, so I volunteered to give all the lectures in a short course in the first year of Medical School. This worked very well, and soon the students were making a real effort to get to my classroom and especially to the tutorials that I designed to go with the lectures. Although I taught undergraduates, graduate students, and medical students, my favorite students were always the medical students. At first, this was not true, and I was harshly criticized by them in their course critiques, which all Yale medical students have to write. I eventually taught Immunology to Yale medical students for about 15 years. This was a very happy period in my life, as I had total control over the course material, the tutorials that went with them, and the composition of the exam. It was always a short answer exam, with two questions on a page, and six pages of questions, which I would then have my secretary divide up and give one page to each of my five tutorial leaders and one page to me. In this way, I could guarantee that there was no bias in the grading of the exam. I used to have a curve set up that told me which student was learning and which one was not. In the early years, I learned that those who attended the tutorials would always pass the exam, while those that did not would often fail the exam. Later, under Dean Rosenberg, the exams were made mandatory in order to force the students to attend lectures. I thought this was a bad move, and I spoke up at the faculty meeting where this was decided. I think I offended many of my colleagues who had to teach longer courses covering more material than I did, but I think I was right in stating that if students were not attending lectures, one had only one’s self to blame. Make your lectures interesting and lively and the students come in droves. That was my experience, and I eventually won a Bohmfalk Teaching Award for my efforts. This took time and effort on my part and that of my colleagues who taught the tutorials, but in the end, at least for me, it was worth the effort. The prize for the most imaginative use of the tutorials went to my wife, Kim, who nearly won her own Bohmfalk Award for her outstanding tutorial teaching. The students used to crowd into her room, so that over time I had to assign the biggest room in our teaching building to Kim. It was a period of great happiness for me. After a relatively short time, I was a very successful teacher. It was the receipt, in 1983, of the best text that I have yet read, The Molecular Biology of the Cell

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(MBOC), that got me thinking about writing an immunology textbook. All the new information that was pouring out of my colleagues’ labs made me even more enthusiastic about writing my own book. By 1987, I sent a copy of my course notes to Gavin Borden at Garland Press, the publishers of MBOC. It was widely heralded as the best text ever published in cell biology. The reasons this book was so successful were two: First, a unique feature of the book was that it was written by a bunch of guys who loved their science and were unafraid of criticism. They sent all of their drafted chapters to world-renowned experts for a read-through, then modified them according to the comments they received. Second, this group was mother-henned by Miranda Robertson, the biology editor of Nature, at that time a leading journal in many areas of biology, including immunology. These two facts, along with Gavin’s good natured hosting of the authors in his house in a corner of London just off Abbey Road, made MBOC the most popular of cell biology texts. I picked up the essence of this book and adapted it to my immunology notes; shortly thereafter, I sent them to Gavin and thus began a long collaboration. Basically, our goal was to put Ivan Roitt out of the immunology textbook business, as up until that time, he had written the best book about the subject I loved so much. It turned out that Gavin knew Vitek Tracz, the originator of the Ivan Roitt publishing empire, and he set us up to work with Vitek. Another key player was soon on hand in the person of Miranda Robertson. So we had the whole MBOC team assembled to write a new textbook of immunology, except that we needed a person doing immunology in England; Miranda miraculously and brilliantly chose Paul Travers. Thus began a long collaboration between Paul, Miranda, Vitek, and myself, working out of Vitek’s London office. At first, I was happy to stay in the same house that the MBOC authors used, but it meant trooping across town on the London Underground, which was not ideal, and it also meant a series of long absences from home. These were mainly due to long wrangles with Miranda, usually over trivial results like complement lysis of red blood cells, which she thought was important and we did not. In any case, when the book was finally published in 1994, it was almost immediately adopted by many course directors, enabling me to relax about earning my keep in other ways. I was just trying to put Immunology in terms that I could understand, and it turned out that other people saw things much the way I did. Maybe I am just simpleminded, but, as it seemed to make sense to many brilliant students that I taught, it is likely to be more than that.

HOW MY THINKING ABOUT THE IMMUNE SYSTEM HAS CHANGED DURING MY YEARS AT YALE The main message I would like to convey is how I have grown intellectually during my years at Yale. As I said before, I was happy injecting antigens in complete Freund’s adjuvant or in alum with Bordetella pertussis, which gave me antibody production and later T cell proliferative responses. But I began to

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think that there must be more to immunology than just these responses, and I wondered where these influences were coming from. Not all features of adaptive immunity could be accounted for by the adaptive immune system consisting of T lymphocytes and B lymphocytes; there must be something else contributing to the signals that were necessary for immune induction. Wherever one looked, there were signs of this: T-independent antibody formation, T-independent-macrophage activation, and complement activation, especially by the so-called alternative pathway of complement activation. This pathway was very active against microbes, but it was quiescent with the cells of the host. I thought this might be due to innate immunity, about which I knew very little, and later that idea turned out to be right. Most people at that time were studying adaptive immunity. Even people who were studying invertebrates, which we now know cannot mount an adaptive immune response because they lack the genes required to make immunoglobulins and T cell receptors, were trying to prove that they made vertebrate-like adaptive immune responses. This was later shown to be wrong, most importantly by my colleague David Schatz, Ph.D., who along with Marjory Ottinger, Ph.D., defined the RAG genes. He later published what I think of as one of the best papers I have read, in which he described the acquisition of the two closely linked RAG genes and the sites upon which they act, the recombination signal sequences (RSS) (2). These turned out to come from a retroposon that invaded the germ line of some lowly vertebrate, as only vertebrates have adaptive immune systems. I return to this later when I introduce adaptive immunity in mice and humans.

INNATE IMMUNE RECOGNITION OF PATHOGENS AS A FIRST STEP IN ADAPTIVE IMMUNITY We performed several experiments that I like to think are crucial to the development of the idea that adaptive immunity was dependent upon a functioning innate immune system. This led eventually to the cloning of the first pattern recognition receptor, hToll or, more appropriately, hTLR4, for human toll-like receptor-4. The first of these experiments was performed by Yang Liu, who now has an endowed chair as Professor of Pathology at Ohio State University, who demonstrated that the ligand for the T cell receptor (TCR), which in our case was anti-CD3, had to be presented by the same cell as the so-called costimulatory molecules (3). This study had to be published in the Proceedings of the National Academy of Sciences, as it showed only a “quantitative” difference, according to a review we received from Nature. This was true, but the quantitative difference was of the order of 100–1000-fold, so overcoming it either by increasing the level of the costimulatory molecules or of the TCR ligand could not be expected to happen in vivo. The reason for this is that the TCR ligand is made up of a complex of a selfMHC molecule with an antigenic peptide, and there are limited numbers of MHC molecules on a cell. It also requires at least 100–200 specific ligands to activate

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a T cell. As the difference we found was in the neighborhood of 100–200-fold greater specific ligand when the ligand and the costimulator were presented on two different cells, it would be impossible to achieve this level. The finding that ligand and costimulator had to be presented by the same cell was subsequently confirmed by several other investigators using genuine T cell receptor (TCR) ligands, namely peptide:MHC complexes (4). What was not known at that time was what controlled the expression of the costimulatory molecules on the surface of an antigen-presenting cell. We thought it must be an infectious agent or a product of an infectious agent, because Yang and I had earlier shown that a whole variety of microbial substances could stimulate the antigen-presenting cells to express what we believed to be costimulatory activity (5). But I was not sure, so I pushed on with the theory that some innate immune receptor had to be responsible for the regulation of the costimulatory molecules expressed by adjuvant-primed antigenpresenting cells. This led to a very frustrating part of my life, in which a lot of work on adaptive immunity was done, but relatively little on innate immunity. Then, in a wonderful stroke of luck, I began receiving e-mails from a person by the name of Ruslan Medzhitov, who came from Tashkent in Uzbekistan and had spent an entire month’s stipend to obtain a copy of my introductory essay in the 1989 Cold Spring Harbor Symposium (6). I was interested in his ideas, but not overwhelmed with enthusiasm for hiring him. But then, in another stroke of blind luck, Ruslan earned a visiting fellowship to the University of California in San Diego, where he was to analyze protein sequences with Dr. Russell Doolittle. He went there and a short time later, I received a call from Dick Dutton, who was at that time the head of the biology department at UCSD and the president of the American Association of Immunologists. He had just met Ruslan, and he told me two things that convinced me to accept him in my laboratory. The first was that Ruslan still wanted to come to my lab. The second was that he was “a genius.” I immediately e-mailed Ruslan and told him that he could come to my lab. When Ruslan first arrived, he spent some time on various projects related to adaptive immunity. However, it was when he began turning his attentions to innate immunity that I knew that I had found the right colleague to test my ideas. He was what I had been waiting for all these years since I wrote the article in the Cold Spring Harbor Symposium: a man who was fixated on finding out what controlled the expression of costimulatory molecules on the surface of antigen-presenting cells. And even better than that, he was convinced that appropriate cytokines would be released as well. Ruslan had a degree in theoretical biochemistry, for which he had worked very hard, but he had no practical laboratory experience. I told him that he would need to develop such skills to be successful. I passed him one day in the lab, and I asked him how he was making out with the practical side of laboratory work. He said: “Fine, now I can tell a bottle of culture medium from a centrifuge.” I knew then that I had a winner, and I was right. Although I had trained many excellent graduate students and postdoctoral fellows, Ruslan was the first genuine superstar to work in my laboratory, and I have enjoyed interacting with him ever

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since. He was hired as an assistant professor in the Section of Immunobiology in 1999, and he was subsequently made a Howard Hughes Medical Institute Assistant Investigator. I had never had such a postdoctoral fellow, and I don’t expect to have another. Ruslan and I published several papers over the years that he was in my lab, and afterward when he had his own lab, but the first was the most influential (7). In it, he described the cloning of a human homologue of the Drosophila Toll protein, which clearly induced B7.1 and B7.2 (or CD80 and CD86 as they are now called). This protein was later renamed hTLR4 for human toll-like receptor-4. It turns out there are 10 TLRs in the mouse and human genomes. The genes that encode each of these are more closely related to one another in the mouse and humans than they are to other TLRs within a species, so that one can move from mouse to human and back with ease. We knew from Jules Hoffman, with whom I have collaborated for several years, that defects in the Toll system at any level would render the fruit fly Drosophila melanogaster susceptible to overwhelming fungal infection and subsequent death. This led to a dramatic cover of the journal Cell, in which a fruit fly that was clearly dead had fungal hyphi coming out of its cuticle, due to a defect in the Toll signaling pathway (8). It was seeing this picture that led us to focus on Toll cloning, although Ruslan had already started to clone it. I urged him on to do this, and he outdid my exhortations. He published one of the most important papers in biology, as shown by its inclusion in Ben Lewin’s compendium of “Great Discoveries in Biology.” What this paper did was to answer a crucial question: How does the body know when to respond to a foreign antigen, and how does it know how to avoid responses directed at self-antigens? The mechanism that Ruslan discovered is a simple one: It does so by recognizing molecular patterns associated with the outer coats of bacteria. In the case of TLR4, the pathogen associated molecular pattern, or PAMP, is LPS. Other pathogens are recognized by other TLRs, such as TLR2 (9), which recognizes lipoteichoic acid found commonly in the coat of gram positive bacteria, TLR5, which recognizes flagellin (10), or the unmethylated CpG DNA found in bacteria but not in mammals by means of TLR9 (11).

THE ANALYSIS OF ADAPTIVE IMMUNITY IN RECENT TIMES Once I realized that the problem of self:non-self discrimination had been solved in principle by Ruslan’s magnificent discovery, I felt liberated to go back to my first love, which was the study of adaptive immunity. I knew from my childhood experiences with my father, the original discoverer of X-linked agammaglobulinemia (12), that the adaptive immune system was essential to living a healthy life. The discovery, in my lifetime, of several vaccines that could protect one from infection and could thus improve health, all depended on inducing an adaptive immune

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response. Thus, I was left with the idea that both the innate immune system and the adaptive immune system were essential partners in living a long and healthy life (apart from the occasional cold). Of the two systems, I would rate the innate immune system the more important, as it is as old as the emergence of multicellular organisms from the “primordial slime,” and as defects in innate immunity are as rare as an undercooked steak. The requirement for the adaptive immune system of T cells and B cells was reinforced recently by the emergence of the acquired immune deficiency syndrome (AIDS), which attacks the adaptive immune system. It may also attack the innate immune system, as the initial infection occurs in dendritic cells or in macrophages (13). The rising toll of this deadly disease has given the lie to the easy assumption that we had beaten infectious diseases with the introduction of antibiotics, especially of the wonder drug penicillin. The discovery of antibiotics near the end of World War II led to the abolition of the Department of Microbiology at Yale and at several other highly regarded medical schools. But later, the Section of Molecular Pathogenesis was refounded in order to deal with epidemics like that of AIDS, antibiotic-resistant tuberculosis, and other infectious diseases. Unless we can devise a vaccine against HIV, the causative agent of AIDS, we will all become infected and eventually die. I used to show a cover of Newsweek which posed the question: “AIDS: The Public Health Threat of the Century?” And I would go on to say that I thought the public health threat of the century was living in the White House (Ronald Reagan was then the president). No longer do I find that joke to be funny; with well over 50,000,000 cases of HIV-1 infection worldwide, it is a true challenge that we must face or become extinct, like the Dodo bird. What AIDS does is to attack the adaptive immune system. Eventually, when the CD4 T cell count, normally around 5000 cells per milliliter, falls to 500 cells per milliliter or fewer, one dies of an infection with nonpathogenic bacteria, yeast, fungi such as Candida albicans, or from lymphomas that seem to be recognized and controlled by the adaptive immune system. We have learned a lot about the agent that causes this infection, but no one seems to have any idea how to make a vaccine to combat it. And time is running out. Thus, one needs both an innate immune system to watch out for pathogenic bacteria and viruses, and an adaptive immune system to make an effective response to those pathogens that can overcome the innate immune system. The other reason that we need an adaptive immune system is to protect us from re-infection with the same organism; such a system can make antibodies and T cells that can jump-start the adaptive immune response when the organism re-encounters the same or a closely related pathogen. This is a job that the innate immune system cannot do, as it has been selected over evolutionary time rather than in individual cells, as is true of the adaptive immune system. This means that the receptors for innate immunity are found in all multicellular organisms, whereas adaptive immunity is in evolutionary terms a new invention that is found uniquely in vertebrates.

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So I began to look more carefully at the adaptive immune system, and I eventually came to the conclusion that I had it all wrong. The adaptive immune system of lymphocytes depends on self-antigens for its development in the thymus, for its longevity in the peripheral lymphoid tissues, and even for its activation. I have only recently come to this realization, so convinced was I that Burnet (14), who first proposed the clonal selection hypothesis, was correct. This was a revolutionary thought for me, and it is still working its way through my thick skull. But I am convinced that it is true, and we have much evidence that supports this idea. I would like to start with the T lymphocytes with which I am more familiar, but I will end up with B lymphocytes and with regulatory T cells, which are the subject that drew me to come to Yale in the first place. Regulatory T cells are just a neologism for suppressor T cells, which Richard Gershon originally, and I now think correctly, predicted were a unique lineage of T lymphocytes that were needed to protect the host from autoimmune attack. So I will call them suppressor T cells in honor of Richard Gershon, my first real boss (15).

THE POSITIVE SELECTION OF T LYMPHOCYTES ON SELF-PEPTIDE:SELF-MHC COMPLEXES T cells are generated in the thymus by a series of rearrangements of their receptor gene segments. Once the complete T cell receptor is put on the T cell surface, the T cell has to undergo two processes before it can emerge into the peripheral lymphoid tissues. These are conventionally called positive (16) and negative (17) selection. It is still not clear to me that we know the sequence of these reactions, but it is easier to consider positive selection as coming first, and negative selection as happening at various times during T cell development. Positive selection raises the level of the TCR significantly, thereby making the developing thymocyte more sensitive to stimulation by self-peptides. The really key point is that the only peptides available to the developing T cells are self-peptides, and so from the start T cells are positively (and negatively) selected on self-peptide:self-MHC complexes. This seems to be an inescapable conclusion to me, so I state it boldly and without exception. I may be wrong, but everything I think I have learned over the years points in this direction. If this is so, how do any T cells ever escape from the thymus? The T cells that escape from the thymus have highly variable receptors, and some of them only recognize the self-peptide:self-MHC complexes weakly, enough to drive positive selection but not enough to activate the thymocyte, which leads to apoptosis or activation-induced cell death. The fortunate T cells that have such receptors then emigrate from the thymus and fill up the periphery. At least 85% of the T cells fail to recognize any ligand at all, probably due to a combination of T cell receptor (TCR) variability and MHC polymorphism, and thus these developing T cells fail positive selection and die within the thymic cortex. A few thymocytes have such avid receptors that the thymocytes are activated upon encounter with the

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self-peptide:self-MHC complex leading to activation-induced cell death. So the yield of thymocytes is between 1% and 5% of the initial numbers; for every thymocyte that is exported to the periphery, 20–100 thymocytes die. The upshot of these two processes is the selection of a TCR repertoire that is both self-MHC restricted and self-tolerant, as I have taught my students for years. So why do I think I see things differently now than I did when I arrived at Yale in 1977, 25 years ago? I now think that the recognition of self-ligands is important not only for selection of such a repertoire, but also for sustaining this repertoire in the periphery, and for signaling for activation when a pathogen or other antigen appears in the periphery. The reason I say a pathogen should be obvious to the informed reader, but I will reiterate that the presence of a pathogen or its products is essential to arm the antigen-presenting cells with the needed costimulatory molecules, as well as to stimulate the uptake, processing, and presentation of antigens. So that is how I now view the activation of T cells.

THE INTERACTION WITH SELF-PEPTIDE:SELF-MHC LIGANDS DRIVES PERIPHERAL T CELL SURVIVAL Peripheral T cells survive by interacting with a ligand that is almost certainly similar or identical to that which drove them to be positively selected in the thymus. The evidence for this is very strong, although some doubt it and claim that the homeostatic cell division observed in the periphery depends on the availability of so-called “space.” This impression is created by the use of either RAG−/− mice or irradiated recipients as hosts for the T cells. In our earlier experiments, we also used irradiated recipients to show that CD4 T cells could cycle to fill the space created by irradiation. However, we have now also performed identical experiments that show cycling of T cells in mice that are normal and not irradiated. Although the cycling was somewhat reduced, it still occurred. Thus, we believe that the T cells are recognizing self-peptide:self-MHC complexes on the surface of host cells (18; and C. Viret and C. A. Janeway, Jr., J. Immunol., in press). The cells involved in peripheral T cell survival are almost certainly the same cells that present foreign antigen to the T cell, that is dendritic cells (DCs). This was shown earlier by Brocker et al. (19), who expressed MHC class II molecules on MHC class II −/− dendritic cells under the control of a DC-specific promoter. These DCs are not activated by pathogens and therefore lack the costimulatory molecules or the foreign antigens necessary to trigger an adaptive immune response. Thus, these DCs can be considered as necessary for T cell survival but not for T cell activation by the self-peptide:self-MHC complexes they bear on their surfaces. I envision this being true for all T cells, as illustrated by the studies in Benedetta Rocha’s laboratory (20) for CD8 T cells, on which our original studies were modeled. We went on to examine the role of the peptide component and the specificity for MHC in our studies, also in mice that had been exposed to ionizing radiation (21).

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We found that using allogeneic MHC molecules would not support survival, nor would deletion of the α chain of the peptide exchange factor H-2M mice support survival of MHC class II–restricted CD4 T cells. Thus, I am reasonably certain that it is self-peptide:self-MHC complexes that provide the low-grade signaling that we see in our TCR transgenic CD4 T cells. Other interpretations of CD8 cell survival have been made by other authors (21), but as we have seen similar results in unmanipulated recipients of CD4 T cells, I feel certain that these T cells, at least, survive by constantly making and breaking contact with DCs bearing the essential self-peptide:self-MHC ligands (18). Thus, CD4 T cells, which I have spent much of my life studying, are not only positively selected on self-peptide:self-MHC ligands, they persist on self-peptide:self-MHC ligands. The real open question now is whether the recognition of self-peptide:self-MHC ligands also contributes to T cell activation? We examine that question in the next segment of this essay.

THE ADAPTIVE IMMUNE RESPONSE TO FOREIGN ANTIGEN ALSO SHOWS SIGNS OF REQUIRING CONTACT WITH SELF-PEPTIDE:SELF-MHC LIGANDS When na¨ıve CD4 T cells are stimulated with agonist ligands, which consist of a specific foreign peptide bound to a particular self-MHC molecule, they conventionally give a full response. This includes, but is not limited to, proliferation, cytokine secretion, and differentiation into either Th1 CD4 T cells or Th2 CD4 T cells. These cells secrete different sets of cytokines: Th1 cells secrete interferon γ and TNFβ, whereas Th2 cells secrete IL-4, IL-5, and IL-13. These cells also share at least two cytokines in common, GM-CSF and IL-3. Each cytokine is recognized by a different receptor, and these patterns are used to type CD4 T cells into these two lineages. This is in response to an agonist ligand, which, during intrathymic development, can delete cognate T cells totally. So what happens if you present an agonist peptide in a pure form by attaching it to the β chain of the I-Ab molecule and putting the transgene into a mouse that lacks endogenous Aβb? This question was initially tested with wild-type TCRs. In order to guarantee that the peptide, which my laboratory initially identified as a dominant binding peptide of the Eα molecule, residues 52-68, is the only peptide available to the cells, these mice had to be on a background that lacked the MHC class II invariant chain. Under these conditions, one could demonstrate that all of the MHC class II molecules were modified by a single peptide, Eα52-68. This was demonstrated by showing that the staining of the cells in the spleen was as intense with the Y-Ae antibody that we had produced to this peptide bound to I-Ab, but these same cells also bound an equivalent number of molecules of another antibody, which in our case was called Y-3JP, specific for related epitope that bound to all I-Ab molecules. However, really convincing evidence for this conclusion involved the blocking of Y-Ae antibody by the Y-3JP antibody and vice-versa, which proves that such mice bear exclusively the I-Eα chain peptide bound to I-Ab.

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The results of several studies by Kappler, Marrack, and Ignatowicz tell one several things. First, one derives a repertoire of T cell receptors that is basically diverse, although it is tolerant to the peptide:MHC complex carried artificially by this mouse (21). Second, there is a clear impact on the T cell receptor repertoire, in that many if not all of the T cells assayed as T cell hybrids have alloreactive potential. The commonest alloreactivity was to the I-Ab molecule itself, presumably by binding a variety of self-peptides and accounting for the majority of the T cell hybrids produced from these mice. The next commonest was reactivity to I-Ad, which has a very similar peptide binding groove that also binds a fair number of the same Eα peptides, although this could only be determined by mass-spectrometry analysis (22). These studies suggest that these mice inhibit negative selection to a host of self-peptide:self-MHC ligands, leading to reactivity to such ligands that normally select against such TCRs in the thymus and in the periphery. As we had studied a TCR that was strongly stimulated by the Eα52-68:I-Ab complex, we wondered whether it would be stimulated by this agonist peptide. Before I speak of the role of self-peptide:self-MHC complexes in T cell recognition, I need to discuss an experiment by Alam, Travers, and Gascoigne that appeared in Immunity in 1999 (23). In this study, the authors showed that agonist and antagonist peptide bound nearly identically to the several TCRs that they tested at room temperature (25◦ C), such that it seemed that differences in TCR binding could not explain the differences observed in functional assays, including thymic organ culture and peripheral T cell stimulation. However, when they ran their assays at physiological temperature (37◦ C), they got quite different results. They observed what they interpreted as TCR dimerization when they used an agonist peptide. These dimeric complexes were very stable, with a t1/2 of about 600 s, compared to a t1/2 of 20–30 s observed at 25◦ C. However, antagonist peptides, which can drive positive selection but also may inhibit responses to agonist peptides, had a t1/2 of 20–30 s whether they were assayed at room temperature or at the physiological temperature of 37◦ C. Thus, this assay clearly discriminated agonist from antagonist peptides. According to Alam et al. (23), their data fit best to a model of dimerization of the TCRs. But what would the second TCR recognize on a cell surface, where stimulation can occur with as few as 100–200 foreign peptides per cell? What seems most likely to me is that the second TCR would bind to a self-peptide:self-MHC complex. Such interactions have been known for many years in the response to self-APCs by autologous T cells in the so-called autologous MLR. This required the use of dendritic cells as stimulators for optimal responsiveness. The use of this assay has gone out of fashion, but it was studied by Glimcher, Shevach, and Paul in the mid 1970s, and I think it should be re-examined. I am virtually certain that the self-peptides that stimulate self-T cells in this assay will be the same self-peptides of which I speak; that is, self-peptides presented by self-MHC class II molecules to CD4 T cells that positively select in the thymus and contribute to “homeostatic” cycling in the periphery. However, what I would now like to address is the role of similar peptides in T cell activation. To examine this, we decided to cross our

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TCR specific for the Eα52-68:I-Ab complex, called 1H3.1, with the mice kindly provided to us by Pippa Marrack and John Kappler. The initial crosses, not surprisingly, showed marked deletion of 1H3.1 T cells, as there were plentiful self-peptide:self-MHC class II complexes available on the cell surface. It was only when we got to mice that lacked genes for invariant chain and the Aβ b chain of I-Ab, and had the covalently linked Eα52-68:Aβ b and the 1H3.1 transgene, that we got a big surprise. Instead of intrathymic deletion, we observed intrathymic positive selection, At first, I was mystified by this result, but on a trip to Italy in the summer of 1999, I told Antonio Lanzavecchia about it, and suddenly all the pieces fell into place. The reason we saw positive selection could be that all of the TCRs in the “immunological synapse” between the two cells were binding to the same self-peptide (or foreign peptide) and undergoing the same conformational change, so that they could not form dimers. I was very excited by this result, which, after two years of sitting on it, we finally decided to submit to the Proceedings of the National Academies of Science. Christophe Viret was afraid that any number of artifacts could have contributed to the absence of responses and he tested for many of these, but I finally got him to give the paper to Pippa Marrack, and she has communicated it to the Proceedings (C. Viret, C. Janeway, 24). This paper, together with my inaugural article in the Proceedings, which makes several of the same points, should make Christophe Viret a high-profile immunologist when he returns to France. His manuscript is much more conservative than my own, but I feel I am old and experienced enough to get egg on my face if I am wrong.

A TRIP TO PHILADELPHIA One time, about five years ago, I went to Philadelphia to give a seminar. One member of the audience was an old friend of mine from our days at the NIH; his name is Mike Cancro. I actually thought that he had given up research for administration, but I was wrong; he had simply taken a deanship but was still pretty interested in and actively looking at B cell development. He really liked my seminar, and more than that, it had given him an idea that we were ideally placed to investigate. He wondered what would happen if we performed the same analysis on B cells as we had previously used to probe T cell development. I asked what we should look for, and he told me to compare κ light chain sequences in peripheral B lymphocytes that were either immature or mature in mice transgenic for a single heavy chain transgene. He pointed us in the right direction, telling me to get mice from Mark Shlomchik, but neither Mark nor I had a free set of hands. Therefore, I asked a very bright M.D., Ph.D. student named Matt Levine to take a look at one mouse to see if it was feasible. So Matt tried the first mouse he could get his hands on, one that bore a Vh186.2 heavy chain transgene and was homozygous for a complete knock-out of its JH region, so that no other heavy

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chain could be formed. He sorted the cells based on the so-called E (immature) and F (mature) populations, extracted genomic DNA, and assayed light chain usage in the E and F populations. This required a great deal of sequencing, but it revealed one κ light chain that was five-fold higher in the mature population F as compared to the immature population E. He was encouraged by this result, so he did the same experiment using a different Ig-heavy chain transgene. This mouse yielded two different κ light chains that were expressed at about the same five-fold increase in population F as in population E, and they were different κ light chains than the one used by the first mouse. Matt eventually assayed seven mice, three for each heavy chain transgene, and one normal mouse. Each mouse showed the same difference in light chain usage, and so we wanted to publish our results in Immunity. However, we were blocked by a reviewer who wanted us to sequence both heavy and light chains on single cells. Finally, in frustration, we turned once again to Pippa Marrack. It turned out that her husband, John Kappler, had a slot for a communicated manuscript in PNAS, and so a paper appeared in the Proceedings of the National Academies of Science in 2000 entitled “Positive Selection in B Lymphocytes” (25). We included Mike Cancro as a couthor, as he basically told us what we needed to know, which was where to look and how. Having discovered positive selection in B cells, we then turned to the issue of what ligand(s) were doing the positive selecting? We wanted to test three hypotheses: The first was that they were influenced by the normal flora. To test this, we needed to get our mice made germ free. Thanks to an old friend named Ed Balish, whom I had met when I was looking at a job in Wisconsin, this turned out to be possible. We sent him several breeding pairs; he bred them and then delivered the pups by caesarian section. We assayed the κ light chain DNA, now by the more rapid technique of colony hybridization, and it turned out that germ-free mice showed the same positive selection that we had earlier seen in conventional mice. We next looked at whether prolonged expression of TdT, the enzyme that is used to synthesize nontemplated or N-nucleotides in the junctions between the V-D and D-J junctions, but only rarely to add nucleotides at Vκ -Jκ junctions, would affect positive selection. This led to the same results as the germ-free mice, but it took much longer to sort out because the Vκ - Jκ junctions were messed up by the enzyme. We next examined mice with the same heavy chain VH region as the mice used in our original experiment, but we engineered them in the cytoplasmic domain by removing the domain that permits secretion of the heavy chain. This gave us a big surprise. Mice that have only surface Ig but lack secreted Ig did not show positive selection, whereas our earlier results in the presence of secreted IgM showed clear signs of receptor-specific positive selection. Therefore, the ability to secrete Ig is essential for positive selection of the same Ig (unpublished data). This finding is consistent with Niels Jerne’s idiotypic network hypothesis (26), but it falls somewhat short of proving it. To prove that the idiotypic network is responsible for Matt Levine’s results, we need to try three experiments, all of

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which are now underway. The first, and easiest, of these is to provide pooled immunoglobulin to mice that cannot secrete their heavy chain transgene. If this works, we then need to confirm it with serum from mice with the same heavy chain that can be secreted. The best, and most difficult, experiment is to breed an H-2 marker into the secretion competent or the secretion incompetent (but not both), make a mixed bone marrow chimera, and then harvest cells from the spleen when the mouse is old enough to show the effects of secreted Ig on the nonsecreting background. These experiments are all underway in the hands of a very enthusiastic group of young scientists, who are performing all these analyses at the same time. One problem is that all of our heavy chain transgenes have to be bred to JH −/− mice, but we are fortunate to be collaborating with Mark Shlomchik, our colleague at Yale, who has several B10 background mice that lack JH regions. These mice are ideal for moving a heavy chain transgene around from one strain to another, as the JH−/− mice are absolutely essential for this experiment to prevent endogenous heavy chain rearrangement. I am particularly happy to be doing this test of Niels Jerne’s elegant hypothesis. Many years ago, he said that I and my predecessor in this series, Herman Eisen, had both done experiments that confirmed the existence of the idiotypic network. We put our heads together, as neither one of us agreed with him. We then designed an experiment to test Niels Jerne’s hypothesis, but again, it involved tolerizing mice to idiotypes used as immunogens. As in all such experiments, we observed the synthesis of anti-idiotype antibody to purified idiotype, but that got us no closer to the mechanism by which this occurred (27). Years later, when Alexander (Sasha) Rudensky came to the lab, he wanted to make T cells that were specific for what he called an “idiopeptide,” but that would only tell us that such T cells existed and not what they do in ongoing T cell responses. I think that Matt Levine has discovered the true role of idiotypic networks, or at least a part of that role, in choosing the correct H:L pairs in the primary B cell receptor repertoire. That makes sense to me; to discover what mechanism is at work in such situations is a new project requiring new tools, but at least we have a hint that we can probe the network scientifically, and that is all I ask.

THE DEMONSTRATION BY JUAN LAFAILLE AND JEFF BLUESTONE THAT SUPPRESSOR CELLS EXIST The existence of suppressor T cells was initially postulated by the late Richard Gershon on the basis of two fundamental ideas: The first was that anything in biology that can go up also has to come down; he called this the second law of thymodynamics. The second was that unopposed stimulation would lead to autoimmune disease. These ideas have been validated in recent years, especially in experiments by Juan Lafaille and independently by Jeff Bluestone. Although these investigators prefer to call these cells regulatory T cells (Treg), this is really

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a neologism for suppressor T cells, so I will stick with the original nomenclature throughout this analysis. Gershon was vindicated in a series of papers published over the last seven or eight years by Juan Lafaille, who was at that time working in Susumu Tonegawa’s lab at MIT. Using genes isolated from a clone of T cells that Jody Baron had prepared in my lab, called clone 19, which could produce a disease called experimental allergic encephalomyelitis (EAE), Juan prepared mice that were transgenic for the TCR of this clone. These mice could be induced to show EAE by stimulating the cells with the N-acetylated peptide 1-11 of myelin basic protein. To be sure that he had no endogenous genes for other TCRs, he crossed them to RAG−/−, I-Au mice, so this also required a great deal of breeding. Initially, they were breeding TCR transgenics with TCR transgenics, which explains a puzzling result in the first paper they published; the homozygous TCR transgenic mice got significant levels of spontaneous disease, much higher than in our own colony, where we bred only hemizygous mice. I really thought that they had infection in their colony, whereas our colony was specific pathogen free. It turned out later that they also did not observe disease in transgene hemizygous mice (28). Once they had mice that were TCR transgene positive, I-Au positive, and RAG-1−/−, they got a surprising result. The brain was flooded with MBP-specific T cells, and the mice succumbed to overwhelming EAE, even without immunization and in the absence of true signs of T cell activation. They published this result, we repeated it, and again saw overwhelming EAE. This result has been repeated many times, and always people see the same result. The real question was: What was missing in these mice that was present in the mice that could rearrange their T and B cell receptor genes? I heard Juan talk about this on many occasions, and he, and Susumu independently, later reported that what was missing was another kind of CD4 T cell that could suppress or protect the brain from attack (29, 30). One of the clear-cut messages of Juan Lafaille’s experiment was that the clone 19 TCR could not differentiate into a cell that suppresses the impetus of a clone that is autoreactive. Therefore, thanks to Juan’s effort to make an MBP TCR transgenic mouse that was bred onto a RAG-1 −/− mouse, we can infer the existence of the long-anticipated suppressor T cells. So you were right all along, Richard, and I personally apologize for years of my own skepticism. Having said that, it is not surprising that some T cells can escape from the thymus with autoreactive TCRs, but these are held in check by the existence of suppressor T cells. The question facing us now is whether these cells can be actively induced to make autoimmune disease even more unlikely. We believe that the answer is yes, as do several other laboratories who are working on the same question. So it appears that my thinking has come full circle, and that suppressor T cells do exist. We envision them as recognizing some self-peptide:self-MHC ligand as an agonist peptide, but secreting immunoregulatory cytokines such as TGFβ and IL-10. On the other hand, T cells will be activated to become autoaggressive in the case where a pathogen is involved, by putting CD80 and CD86 on the

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antigen-presenting cell’s surface or by activating a dendritic cell that produces a cytokine profile such that Th1 cells are produced. So, in conclusion, all our T lymphocytes and all of our B lymphocytes are referential to self-ligands, yet they are not activated by such ligands unless they are presented on an activated antigen presenting cell. Even then, a T suppressor cell may hold them in check with the same or similar peptide and organ specificity. In this way, I envision that suppressor T cells generally are dominant over autoaggressive T cells and thereby confine the number of autoimmune diseases to certain organs that are protected by other means, such as the blood-brain barrier in the case of EAE or in multiple sclerosis for which EAE serves as a model.

SUMMARY In conclusion, I want young scientists to continue up these same paths, and others, in studying how the immune system operates. I can imagine wonderful discoveries in the future, none of which is as important as discovering how HIV-1 produces AIDS and the devising of an effective vaccine against this deadly pathogen. In this essay, I have attempted to chart out the meanders of my own career, as an exemplar of how to conduct research, and also, where the results were wrong, how not to conduct research. I have tried to make four main points. First, that the immune system exists to protect the body from infection, and it does so in the first instance by using an ancient system of host defense. Second, that the adaptive immune system is referential to self-ligands, that is, T and B lymphocytes are selected on self-ligands, sustained in the periphery by self-ligands, and use recognition of self-ligands in mounting responses to foreign antigens. Third, never assume you know all things, as I did about autoimmune disease; always push that last mile, as Juan Lafaille did. And fourth, be inspired by the knowledge that exists at the time you enter research, but be irreverent toward this knowledge like my friend Ian McConnell, for this is the road to true understanding. ACKNOWLEDGMENTS First and foremost, I would like to acknowledge my great debt to my wife, Kim Bottomly, who, while working on Th1 and Th2 generation and function, took the time to care for my daughter Katie and our daughters Hannah and Megan. She also helped me in preparing my course notes, took care of me when I was sick, and generally gave everything that one scientist can give another scientist. Second, I would like to thank Jennifer Boucher-Reid for typing the same manuscript over and over again, and not complaining about it. Third, I would like to thank my whole laboratory, past, present, and I hope future for making my 25+ years at Yale so exciting and informative. Lastly, I would like to thank all the students I have taught at Yale Medical School.

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Visit the Annual Reviews home page at www.annualreviews.org


LITERATURE CITED 1. Padovan E, Mauri-Hellweg D, Pichler WJ, Weltzien HU. 1996. T cell recognition of penicillin G: structural features determining antigenic specificity. Eur. J. Immunol. 26:42–48 2. Agrawal A, Eastman QM, Schatz DG. 1998. Transposition mediated by RAG1 and RAG2 and its implications for the evolution of the immune system. Nature 394:744–51 3. Liu Y, Janeway CA Jr. 1992. Cells that present both specific ligand and costimulatory activity are the most efficient inducers of clonal expansion of normal CD4 T cells. Proc. Natl. Acad. Sci. USA 89:3845–49 4. Galvin F, Freeman GJ, Razi-Wolf Z, Hall W Jr, Benacerraf B, Nadler L, Reiser H. 1992. Murine B7 antigen provides a sufficient costimulatory signal for antigenspecific and MHC-restricted T cell activation. J. Immunol. 149:3802–8 5. Liu Y, Janeway CA Jr. 1991. Microbial induction of co-stimulatory activity for CD4 T-cell growth. Int. Immunol. 3:323–32 6. Janeway CA Jr. 1989. Approaching the asymptote? Evolution and revolution in immunology. Cold Spring Harb. Symp. Quant. Biol. 54:1–13 7. Medzhitov R, Preston-Hurlburt P, Janeway CA Jr. 1997. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388:394– 97 8. Lemaitre B, Nicolas E, Michaut L, Reichhart JM, Hoffmann JA. 1996. The dorsoventral regulatory gene cassette spatzle/ Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 86: 973–83 9. Takeuchi O, Hoshino K, Kawai T, Sanjo H, Takada H, Ogawa T, Takeda K, Akira S. 1999. Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-

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ory of the immune system. Ann. Immunol. (Paris) 125C:373–89 Sakato N, Janeway CA Jr, Eisen HN. 1977. Immune responses of BALB/c mice to the idiotype of T15 and of other myeloma proteins of BALB/c origin: implications for an immune network and antibody multispecificity. Cold Spring Harb. Symp. Quant. Biol. 41:719–24 Lafaille JJ, Nagashima K, Katsuki M, Tonegawa S. 1994. High incidence of spontaneous autoimmune encephalomyelitis in immunodeficient anti-myelin basic protein T cell receptor transgenic mice. Cell 78:399–408 Olivares-Villagomez D, Wang Y, Lafaille JJ. 1998. Regulatory CD4(+) T cells expressing endogenous T cell receptor chains protect myelin basic protein-specific transgenic mice from spontaneous autoimmune encephalomyelitis. J. Exp. Med. 188:1883– 94 Van de Keere F, Tonegawa S. 1998. CD4(+) T cells prevent spontaneous experimental autoimmune encephalomyelitis in anti-myelin basic protein T cell receptor transgenic mice. J. Exp. Med. 188:1875– 82

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THE B7 FAMILY OF LIGANDS AND ITS RECEPTORS:


New Pathways for Costimulation and Inhibition of Immune Responses Beatriz M. Carreno and Mary Collins Genetics Institute/Wyeth Research, 87 Cambridge Park Drive, Cambridge, Massachusetts 02140; e-mail: [email protected]; [email protected]

Key Words ICOS, PD-1, PD-L, T cell activation, tolerance ■ Abstract T cell activation is dependent upon signals delivered through the antigen-specific T cell receptor and accessory receptors on the T cell. A primary costimulatory signal is delivered through the CD28 receptor after engagement of its ligands, B7-1 (CD80) or B7-2 (CD86). Engagement of CTLA-4 (CD152) by the same B7-1 or B7-2 ligands results in attenuation of T cells responses. Recently, molecular homologs of CD28 and CTLA-4 receptors and their B7-like ligands have been identified. ICOS is a CD28-like costimulatory receptor with a unique B7-like ligand. PD-1 is an inhibitory receptor, with two B7-like ligands. Additional members of B7 and CD28 gene families have been proposed. Integration of signals through this family of costimulatory and inhibitory receptors and their ligands is critical for activation of immune responses and tolerance. Understanding these pathways will allow development of new strategies for therapeutic intervention in immune-mediated diseases.

THE B7/CD28/CTLA-4 PATHWAY: THE PARADIGM FOR THE FAMILY The CD28/CTLA-4/B7-1/B7-2 family provides a paradigm with which to define new related immune pathways. From this pathway, we find that multiple B7 ligands bind to both activating (CD28) and inhibitory (CTLA-4) receptors (1–3). These receptors do not function independently, but they modify responses delivered by engagement of the antigen-specific TCR on T cells. To date, members of the receptor family are type I transmembrane proteins with a single IgV extracellular domain, and the ligands are type I transmembrane proteins with both IgV and IgC extracellular domains. Interactions between the receptor-ligand pairs are mediated predominantly by residues in the IgV domains. Expression of both receptors and ligands is tightly regulated, allowing discrimination between signals that result in activation or inhibition of an immune response. The CD28 receptor is constitutively expressed on T cells. Engagement of CD28 on naive T cells by either B7-1 or B7-2 ligands on antigen-presenting cells 0732-0582/02/0407-0029$14.00

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provides a potent costimulatory signal to T cells activated through their T cell receptor (1–3). This results in induction of IL-2 transcription, expression of CD25, and entry into the cell cycle. CD28 engagement also confers critical survival signals to T cells through the Bcl-XL pathway (4). CD28 costimulation is necessary for the initiation of most T cell responses, and blockade of CD28 signaling results in ineffective T cell activation. This has therapeutic implications, in that blockade of CD28 costimulation can be profoundly immunosuppressive, preventing induction of pathogenic T cell responses in autoimmune disease models and allowing for prolonged acceptance of allografts in models of organ transplantation (1, 2). CTLA-4 (CD152) shares about 30% identity with CD28 at the amino acid level (Table 1). CTLA-4 expression is not detected on na¨ıve T cells but is transcriptionally induced after T cell activation (3). Cell surface expression of CTLA-4 is very tightly regulated, with most of the CTLA-4 protein residing within cytoplasmic vesicles (5). The critical role of CTLA-4 as a negative regulator of T cell activation is dramatically illustrated in CTLA-4-deficient mice, which die within 3 to 4 weeks of birth from massive lymphocytic infiltration and tissue destruction in critical organs (6–8). Both CD28 and CTLA-4 share binding to B7-1 (CD80) and B7-2 (CD86) ligands. B7-1 and B7-2 are capable of forming homodimers, allowing for interactions with homodimers of either CTLA-4 or CD28. The interaction of CD28 with its ligands is weaker than the interaction with CTLA-4. Human B7-1 binds to human CTLA-4 and CD28 with Kd values of 0.42 and 4 µM, respectively for the monomeric interactions (9). The B7-2:CTLA-4 interaction is of an affinity similar to B7-1:CD28, and the CD28:B7-2 interaction is of even lower affinity (10). Mice

TABLE 1 B7 family receptors: Amino acid identities (%) were calculated using the Wisconsin Package (GCG) Version 10 Gap program, and the Blosum 62 scoring matrix. For sequence comparisons indicated with an asterisk, the Structgappep scoring matrix was used, as the sequences were too distantly related to align with Blosum 62. Comparisons between all mouse (m) and human (h) proteins are shown Protein identities (%) among B7 family receptors Protein

mCD28

hCD28

69

mCD28 hCTLA-4 mCTLA-4 hICOS mICOS hPD-1

hCTLA-4

mCTLA-4

hICOS

mICOS

hPD-1

mPD-1

33

34

29

29

15*

18*

30

32

24

25

12*

20*

74

18

18

20

18*

21

18

22

18*

69

13*

16*

12*

14* 60

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deficient in B7-1 and B7-2 have significant abnormalities in both humoral and cellular immune responses, again illustrating the fundamental role of this pathway (11). The recent crystal structures of CTLA-4-B7 complexes are characterized by homodimers of CTLA-4 that contain B7-binding sites located distally to the CTLA-4 dimer interface (12, 13). The crystal structures suggest that the CTLA-4 homodimer can bind to noncovalent homodimers of B7-1 or B7-2 to form a lattice of CTLA-4-B7 interactions (12, 13). Formation of such a lattice could function to form a stable signaling complex at the T cell–APC interface. CD28 also forms homodimers, with a conserved cysteine located just proximal to the transmembrane domain linking the monomers in CD28 and CTLA-4. By homology, CD28 may also form lattice structures with B7-1 and B7-2, which could serve to potentiate the costimulatory signals delivered through CD28. With the determination of the human genome sequence, and with significant accumulation of mouse genomic sequences, algorithms for gene homology can be used to identify genes encoding proteins with structural homology to B7 and CD28/CTLA-4. The Ig superfamily represents a large number of proteins, including BCR and TCR, and thus it does not provide sufficient criteria to identify a protein as a new costimulatory or inhibitory receptor or ligand. Instead, proteins with the highest homology to known gene family members are identified and tested for functional relationships. In addition, gene mapping can suggest evolutionary relationships. Recently, several proteins have been identified as new members of the B7 and CD28/CTLA-4 families (Figure 1). The B7 family ligands have been selected based on identities of about 20% to 30% in the extracellular domains, and they are characterized by an amino terminal signal peptide, one Ig-V and one Ig-C domain, a transmembrane domain, and a cytoplasmic tail. Inclusion in the B7 family of ligands is based upon degree of homology, as well as on evidence of costimulatory or inhibitory function in immune assays. New transmembrane proteins related to the CD28 receptor family have been identified based upon homology, IgV extracellular domain structure, functional activity, and binding to a B7-like ligand.

THE ICOS PATHWAY: IDENTIFICATION OF ICOS AND ITS LIGAND ICOS (AILIM) is a costimulatory receptor homologous to CD28 and CTLA-4 (14, 15) (Table 1). Human ICOS is a 55–60 kDa, disulfide-linked, glycosylated homodimer when isolated from activated human T cells. The protein has two putative N-glycosylation sites, and the unglycosylated monomer has a molecular weight of about 20 kDa (16). Mouse ICOS is a 47–57 kDa, disulfide-linked, N-glycosylated homodimer (17). Rat ICOS is highly homologous (85% identity) to the mouse protein (18). In rat, a differentially spliced form of ICOS containing a longer cytoplasmic tail has been identified, which suggests that additional forms may exist in human and mouse (18).

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Figure 1 Summary of B7 family ligands and their receptors: The names of receptors and ligands are indicated, as well as a brief summary of predominant expression patterns for each. The conserved structure of a single IgV extracellular domain for receptors and IgV and IgC extracellular domains for ligands is depicted at the top. Function arrows summarize whether the pathway is thought predominantly to costimulate or inhibit the response of the receptor-bearing cell.

ICOS lacks the canonical MYPPPY motif, which is present in the extracellular domains of CD28 and CTLA-4, but contains a related FDPPPF sequence in the analogous position in the protein. The crystal structures of the CTLA-4/B7-1 and CTLA-4/B7-2 complexes implicate the MYPPPY site as the major contact site in CTLA-4 with B7-1 and B7-2 (12, 13). Although the structure of CD28 has not yet been solved, amino acid homologies, mutation data, and modeling support the concept that this motif will also be a major B7-binding site for CD28 (19). The related FDPPPF site in ICOS is not sufficiently conserved to allow for detectable binding of ICOS to B7-1 or B7-2 (16, 20, 21). However, structural homology raises the possibility that this motif might be important in the binding of ICOS to its ligand, which is a member of the B7 family (see below). The ICOS gene is closely linked to the genes for CD28 and CTLA-4 on human chromosome 2q33 and mouse chromosome 1 (17, 22). In humans, the three genes form a tightly linked cluster, with a gene order of CD28-CTLA-4-ICOS within a 300-kb region (22a), suggesting that these genes originated by gene duplication. This tight clustering suggests that expression of these genes may be coordinately

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regulated, with alterations in chromatin structure affecting the entire locus, as observed for tightly linked cytokine genes (23). Moreover, disease susceptibility loci mapped to this region must include ICOS in addition to CD28 and CTLA-4 as candidate genes. ICOS is an inducible costimulatory receptor expressed on activated, but not resting, T cells (14); ICOS is identical to the H4 T cell activation antigen (24). Expression of ICOS on T cells is dependent upon both TCR and CD28 signals, in that activation of T cells in the absence of CD28 engagement results in diminished levels of ICOS on T cells (25). This suggests that signals delivered by ICOS will typically occur distally to those delivered through CD28. However, ICOS expression is not absolutely dependent upon CD28 signals because activated human CD8+ T cells that do not express CD28 can express ICOS (14), and some T cell responses in CD28-deficient mice can be modulated with ICOS.Fc (26). Interestingly, although blockade of the CD28-B7 interaction opposes induction of ICOS expression, blockade of the CD40-CD40L pathway has no effect on ICOS induction on activated human T cells (16). The costimulatory functions of both CD28 and ICOS raise the possibility that they may share overlapping signaling pathways. The cytoplasmic tail of CD28 contains a YMNM motif, which is a PI-3 kinase binding site, and which can bind Grb2 and a Grb2-related protein GADS/GRID (27). Mutations in this site result in a failure to effectively recruit PI3K and a failure to induce Bcl-XL expression in response to CD28 ligation (27, 28). In addition, a consensus SH3-kinase binding site, PYAP, distal to the YMNM site is critical for costimulation of proliferation and IL-2 production (28, 29). The cytoplasmic tail of ICOS contains a YMFM motif, which binds the p85 subunit of PI3K, although binding of Grb-2 to ICOS was not detected (22). The cytoplasmic tail of ICOS lacks the PXXP site implicated in IL-2 production by CD28 engagement, which may account, in part, for the distinct functions of CD28 and ICOS. ICOS is expressed on T cells in lymphoid organs, such as spleen, lymph node, and Peyer’s patches in human and mouse (14, 16, 17, 21). ICOS+ T cells are found in germinal centers and surrounding T cell zones, and ICOS expression in these areas is enhanced after immune priming (21). CXCR5+ T cells, a subset of CD4+ T cells that are found in B cell follicles and germinal centers, are highly enriched in ICOS expression, as compared with T cells from peripheral blood (30, 31). This activated T cell subset is likely to be involved in enhancement of antibody responses. ICOS is expressed in the medulla and the cortico-medullary junction of the thymus (17). However, mice deficient for ICOS have a normal thymus and normal numbers of peripheral CD4+ and CD8+ T cells, suggesting that ICOS does not play a critical role in T cell development (32–34). In human, ICOS expression was detected in fetal and newborn thymuses, with expression primarily in the medulla (16). These data suggest that ICOS could contribute to thymic development, but analysis of ICOS-deficient mice indicates that ICOS is not obligatory. Similarly, mice deficient in CD28 have normal thymuses and normal numbers of peripheral

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TABLE 2 B7 family ligands: Amino acid identities (%) were calculated using the Wisconsin Package (GCG) Version 10 Gap program, and the Blosum 62 scoring matrix. Comparisons between all mouse (m) and human (h) proteins are shown. At the time of this review, the sequence for mouse B7-H3 was not available Protein identities (%) among B7 family ligands Protein

mB7.1

hB7.2

mB7.2

hICOS-L

mICOS-L

hB7H3

hPD-L1

mPD-L1

hPD-L2

mPD-L2

hB7.1

45

26

24

26

26

27

25

25

23

24

30

27

24

27

26

26

25

24

25

51

23

27

26

21

26

22

20

25

25

28

24

24

20

21

48

33

25

27

27

28

31

25

25

22

29

31

28

27

26

70

41

39

43

38

mB7.1 hB7.2


mB7.2 hICOS-L mICOS-L hB7H3 hPD-L1 mPD-L1 hPD-L2

70

T and B cells, indicating that these costimulatory pathways are not obligatory for normal T cell development (35). The ligand for ICOS (B7h, GL50, B7RP-1, LICOS, B7-H2, KIAA0653), which is called ICOS-L for clarity and nonpartisanship in this review, was identified as a B7-like molecule capable of binding to ICOS and delivering a costimulatory signal to T cells (17, 20, 21, 36–38). Comparisons indicate that mouse and human ICOS-L share about 48% amino acid identity, and about 25% identity with other members of the B7 family (Table 2). Human ICOS-L maps to chromosome 21q22.3 and has been annotated on the chromosome 21 DNA sequence at position 31156109 (39, 40). The location of the human gene suggests that mouse ICOS-L should map in the syntenic region of mouse chromosome 10. Thus, in contrast to the CD28/CTLA-4/ICOS gene cluster, ICOS-L is in a distinct location from the B7-1 and B7-2 genes, which are on human chromosome 3q13.3-21 and mouse chromosome 16, respectively (41). Interestingly, the transcripts identified by the different groups as ICOS-L are not identical but result from differential splicing patterns (42). Two predicted forms of the human protein have been identified from immune sources, encoding proteins with differences in their cytoplasmic tails. It will be interesting to determine whether there are functional implications for this differential splicing. Differential splice variants have also been observed for the B7-1 and B7-2 genes (43–45). Measurement of the binding affinity of ICOS to ICOS-L indicates that the affinity of this interaction is very comparable to that of CD28 and B7-1 (21, 38). Davis and colleagues (38) measured a Kd of 4 µM for the interaction of monomers of ICOS-L with immobilized ICOS.Fc at 37◦ C, similar to their estimates of the affinities for B7-1 and CD28 (9, 38). Although they detected weak binding of tetramers of ICOS-L to CD28 and CTLA-4 at 25◦ C, there was no binding of monomers at 37◦ C; other groups have failed to detect this association using dimeric

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reagents. This suggests that ICOS-L is not a physiological ligand for CD28 or CTLA-4. ICOS-L is expressed constituitively on unstimulated B cells, splenic and peritoneal macrophages, and peripheral blood–derived dendritic cells by binding of ICOS.Fc to murine cells (20, 21, 36). It has also been detected on a small subset of CD3+ T cells, like other B7-family members (20). In humans, ICOS.Fc bound to peripheral B cells from some donors, and to monocytes and monocytederived dendritic cells (46). Interestingly, INFγ , but not LPS or TNFα or antiCD40L, augmented the expression of ICOS-L on CD14+ monocytes. ICOS-L expression has been detected by analysis of mRNA in many nonlymphoid tissues, such as kidney, liver, heart, and brain (20, 36). TNFα further induces expression of ICOS-L on B cells and monocytes, and most interestingly, it induces the expression of ICOS-L on fibroblasts by mRNA analysis. LPS treatment of mice, which induces TNF-α production, induced ICOS-L mRNA expression in testes, kidney, and peritoneum (36). ICOS-L expression was downmodulated in spleen cells from mice treated with LPS. These data suggest that ICOS-L may be induced by inflammatory signals in peripheral sites, although this must be confirmed at the protein level. Examination of ICOS-L protein in rejecting heart transplants showed that ICOS-L protein was expressed by interstitial dendritic cells in normal myocardium, and additionally on large inflammatory macrophages in rejecting hearts (47).

MODULATION OF IMMUNE RESPONSES BY THE ICOS PATHWAY Engagement of ICOS on T cells that have been stimulated through the TCR results in augmented proliferative responses and cytokine production (14). In comparisons of costimulation mediated through CD28 and ICOS, production of IL-2 is most effectively induced by CD28 (21, 48, 49), although modest enhancement of IL-2 production by ICOS engagement has been reported (37, 50). Costimulation of human CD4+ T cells by ICOS does not produce sustained proliferative responses due to limiting IL-2 production (50). Costimulation through ICOS is particularly effective in enhancing IL-10 production; in direct comparisons, ICOS is more potent than CD28 in inducing the production of IL-10 (14, 16, 25, 37). Both pathways augment the production of other effector cytokines such as IFNγ , IL-4, IL-5, and TNFα (25, 49, 50). Thus, CD28 costimulation appears to have a nonredundant role in the initial costimulation of IL-2 and is critical for initiation of immune responses. In contrast, the subsequent expression of ICOS and engagement by ICOS-L is more important for augmentation of IL-10 and enhancement of effector functions. ICOS engagement can augment induction of both Th1 and Th2 cytokines, but under some circumstances it may more effectively costimulate Th2 responses. ICOS is expressed similarly on both Th1 and Th2 lines after primary stimulation but remains high only on Th2 lines after repeated activation steps (22, 25). This

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suggests that blockade of the ICOS pathway could affect both primary Th1 and Th2 responses, but that highly polarized Th2 responses might be more affected by ICOS blockade. Kopf et al. (26) show that both the Th1 cytokine IFNγ and Th2 cytokines IL-4 and IL-5 are reduced by administration of ICOS.Fc at the time of infection with N. brasiliensis, indicating that the ICOS pathway can enhance production of both Th1 and Th2 cytokines in vivo. In addition, Coyle et al. (22) show that production of both Th1 and Th2 cytokines can be suppressed by addition of ICOS.Fc at the time of reactivation in vitro. However, for highly polarized lines, only the production of Th2 cytokines is reduced by ICOS blockade (22). In addition, adoptive transfer of polarized Th2 lines in a lung inflammatory model results in a dependence upon ICOS engagement in vivo for optimal production of Th2 cytokines and resulting eosinophilia. The highly polarized Th1 line, which induces a neutrophilic infiltrate, is not ICOS dependent (22). Thus, it appears that Th2 cytokine production will generally be more dependent upon ICOS costimulation, whereas the dependence upon ICOS for Th1 cytokines will be determined by the precise conditions under which that Th1 response is elicited. Recent studies in EAE suggest that ICOS costimulation may play a larger role in the effector phase of a Th1 response, in that disease is ameliorated by blockade of ICOS only during the effector phase (51). CD28 costimulation augments production of both Th1 and Th2 cytokines as well, but Th2 responses are also more dependent upon CD28 costimulation (1). Thus, it may be that Th2 responses simply require a higher threshold of costimulatory signals, and that both CD28 and ICOS contribute to the induction of Th2 responses. Interestingly, CTLA-4 engagement can oppose T cell activation with costimulatory signals delivered by either CD28 or ICOS (50), and thus the outcome of the immune response will also depend upon whether CTLA-4 is engaged concomitantly with ICOS by APC that express both B7 ligands and ICOS-L. ICOS engagement can also influence CD8+ T cell responses. Expression of ICOS-L in an immunogenic, MHC class I+ tumor resulted in enhanced tumor rejection in mice (52). In these studies, ICOS-L costimulation of CD8+ T cells was found to enhance IL-2 and IFN-γ production preferentially in recall responses compared with naive responses. No enhancement of CD8+ lytic effector function was observed (52), which is consistent with studies showing that inhibition of the ICOS pathway had no effect on CTL responses after LCMV or VSV infection in mice (26). Thus, generation of lytic effector functions in CD8+ T cells does not appear to be ICOS-dependent. The ICOS pathway appears to play a large role in antibody responses and germinal center formation. ICOS is expressed by germinal center T cells, and its ligand is expressed by splenic B cells (21). Transgenic mice expressing a secreted form of ICOS-L.Fc protein are characterized by lymphoid hyperplasia in the spleen, lymph nodes, and Peyer’s patches, and have high serum levels of IgG (21). Evaluation of ICOS-deficient mice by three independent groups supports a critical role for ICOS in humoral immunity (32–34). ICOS-deficient mice have a consistent decrease in serum IgG1 levels (32, 34), and immunization of mice with TNP-KLH in the absence of adjuvant or with alum or IFA reveals a deficit in IgG1 and IgG2a antibody

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production (32). This deficit could be overcome by the use of CFA as an adjuvant (32). However, in another study, immunization with KLH in CFA also resulted in decreased isotype switching (34), suggesting that the precise immunization conditions and antigen used may affect the outcome. Immunization of mice with NP-OVA in alum (34) or with aerosolized antigen in the lung (33) also revealed a deficit in IgE production in ICOS-deficient mice. Thus, under several conditions of immunization, deficits in isotype switching have been observed. This deficit is not rescued by secondary immunization and appears to be due to a lack of T cell help, as isotype switching to T cell–independent antigens is intact (34). Activation of CD40 was able to rescue the defect in isotype switching in ICOS-deficient mice (32). Engagement of ICOS can enhance anti-CD3-mediated induction of CD40L on T cells, indicating that both CD28 and ICOS can contribute to activation of the CD40L-CD40 pathway. ICOS-L is highly expressed in the B cell–rich areas of the spleen (21), consistent with the proposal that activation of the CD40 pathway may be the critical event mediated by the ICOS pathway in the development of a humoral response (32). However, expression of CD40L is clearly not absolutely dependent upon ICOS expression, as ConA-activated T cells from ICOS-deficient mice can express normal levels of CD40L (53). Consistent defects in germinal center formation are also observed in ICOSdeficient mice (32–34). Mice form fewer and smaller germinal centers in response to both primary and secondary immunization (32–34). Mice deficient in CD28 (35) or in both B7-1 and B7-2 (11) are also defective in isotype switching and germinal center formation. Similarly, mice deficient in CD40L have severe deficits in isotype switching and germinal center formation (54, 55). It will be interesting to determine precisely how these three pathways intersect in directing the humoral immune response.

THERAPEUTIC IMPLICATIONS: THE ICOS PATHWAY The apparent increased dependence of Th2 responses on the ICOS pathway and the clear role for ICOS in isotype switching raise the possibility that targeting this pathway may be useful in generation of therapeutics for diseases with antibodymediated and Th2-mediated pathologies. Interestingly, antagonism of ICOS appears to be more effective late in an immune response. Blockade of ICOS at the time of antigen priming for lung inflammation had little effect upon subsequent airway challenge in normal mice (49). In addition, ICOS-deficient mice are still susceptible to induction of inflammatory lung disease induced by airway challenge with OVA in primed mice (33). The absence of ICOS in this model results in lower production of IL-4 and IL-13, but no change in the lung histology (33). In contrast, antagonism of ICOS 21 days after priming significantly reduced lung inflammation after airway antigen challenge (49). Similarly, ICOS-blockade decreased lung inflammation and airway hyperreactivity after adoptive transfer of highly polarized Th2 cells to naive mice (22). Blockade of the B7/CD28 pathway

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also prevents airway hyperreactivity after adoptive transfer of either Th1 or Th2 cells lines and, further, does not show the Th2-bias of the ICOS blockade. Antagonism of CD28 is more effective at the time of antigen priming and less effective at later times in these models of lung inflammation (49). This suggests that the major contributions of CD28 and ICOS in costimulation of Th2 responses occur at different times during generation of this response. Induction of EAE, a Th1-mediated autoimmune disease, is not dependent upon ICOS, in that antagonism of ICOS at the time of antigen priming does not prevent disease (51). Surprisingly, ICOS-deficient mice are more susceptible to EAE (33), and blockade of ICOS at the time of priming for EAE results in more severe disease (51). The enhanced disease could be due to the absence of a protective Th2 response, as a deficiency in IL-13 production was noted (33). Other explanations are possible at this time, including defects in IL-10 production or defects in regulatory cells, both of which would be expected to result in enhanced disease (56, 57). CD4+ CD25+ regulatory cells are dependent upon CD28 for maintenance in the periphery (58) but have not yet been examined in ICOS-deficient mice. ICOS costimulation in EAE does appear to be critical at the time that encephalitogenic T cells begin to migrate into the CNS (51). Antagonism of ICOS at this time reduced disease severity. Mice deficient in CD28 or in both B7-1 and B7-2 are resistant to EAE (59). Interestingly, adoptive transfer of primed encephalitogenic T cells into B7-double deficient mice also results in reduced disease, implicating CD28 engagement in both priming and effector stages of the disease (59). The ICOS pathway plays a role in graft rejection, in that blockade of the ICOS pathway results in prolongation of heart allograft survival in mouse models (47). Combination of anti-ICOS antibody and anti-CD40L antibody in this heart transplant model also reduced vasculopathy in the cardiac grafts compared with antiCD40L alone, suggesting that the ICOS pathway is contributing to chronic allograft rejection (47). Similarly, combinations of agents that block the B7/CD28 pathway and anti-CD40L promote long-term cardiac allograft survival in mice and prevent development of vascular lesions associated with chronic rejection (60). These data suggest that both ICOS and CD28 are contributing to inflammatory stimuli underlying the chronic rejection pathology. IL-4 production has been linked to development of transplant arteriosclerosis in mice deficient for CD40 (61). It is possible that both CD28 and ICOS contribute to chronic rejection pathology by costimulation of Th2 responses that could accompany the Th1 response mediating graft rejection. In addition, these pathways may function in promoting alloantibody responses, which could contribute to inflammatory responses in vessels.

THE PD-1/PD-L PATHWAY PD-1 (program death-1) is a 50–55 kDa type I transmembrane receptor that was identified in a T cell line undergoing activation-induced cell death (62). PD-1 is a member of the Ig superfamily that contains a single Ig V–like domain in

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its extracellular region (Table 1); it lacks the MYPPPY motif, a sequence critical for CTLA-4 and CD28 binding to B7.1 and B7.2 (63). The PD-1 cytoplasmic domain contains two tyrosines, with the most membrane-proximal tyrosine (VAYEEL in mouse PD-1) located within an ITIM (immuno-receptor tyrosinebased inhibitory motif) (62). The presence of an ITIM on PD-1 strongly suggested that this molecule could function to attenuate antigen receptor signaling by recruitment of cytoplasmic phosphatases (64). Human and murine PD-1 proteins share 60% amino acid identity with conservation of four potential N-glycosylation sites, and residues that define the Ig-V domain (65, 66). The ITIM in the cytoplasmic region and the ITIM-like motif surrounding the carboxy-terminal tyrosine (TEYATI in human and mouse) are also conserved between human and murine orthologues. There is 62% amino acid identity in the PD-1 cytoplasmic region between human and murine proteins. This contrasts with the 100% conservation observed between human and murine CTLA-4 cytoplasmic regions. The genome location of human PD-1 has been mapped to chromosome 2q37.3 (65); CTLA-4, CD28, and ICOS mapped on the same chromosome at 2q33 (17, 22, 22a). In normal murine tissue, PD-1 mRNA expression is confined to the thymus (67). Approximately 1% of thymocytes are PD-1 positive with expression restricted to a subset within the double negative (DN) population (67). In vivo administration of anti-CD3 mAb results in marked apoptosis of CD4+CD8+ (DP) cells in the thymus; however, such treatment leads to induction of PD-1 protein expression on the surviving CD4−CD8− (DN) and single positive (SP) thymocytes. Significantly anti-CD3 mAb, but not dexamethasome, treatment induces PD-1 expression. These observations strongly suggest that T cell activation, and not induction of apoptotic death per se, results in expression of PD-1. The potential role of PD-1 in thymic selection has been studied in detail (68). An increased percentage of PD-1+ DN thymocytes has been reported in neutral and positively selecting backgrounds. Interestingly, PD-1 deficiency in positive selecting backgrounds resulted in an increase in DP cells and a decrease in SP thymocytes (68). These findings suggested a role for PD-1 in thymic positive selection, in which PD-1 engagement could increase the threshold of pre-TCR/CD3 complex signals required for transition from the DN to DP stage. In PD-1-deficient mice, a lower pre-TCR/CD3 complex threshold would allow for a higher number of cells to transition from DN to DP. In addition, PD-1 could also affect the efficiency of positive selection by modulating the threshold of TCR αβ signals. In contrast, PD-1 deficiency has a negligible effect on negative selection of TCR transgenic T cells (69). These studies suggest that TCR thresholds of activation can be modulated upon engagement of PD-1. Moreover, these studies suggest that PD-1 plays no significant role in central tolerance. Analysis of murine spleen and lymph node populations indicated that within each of these populations a small percentage of PD-1 positive cells could be detected (63). Under resting conditions, neither T nor B cells expressed PD-1. However, activation of T or B cells through the antigen receptor or with PMA and

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ionomycin resulted in cell surface expression of the PD-1 receptor (63). PD-1+ T and B cells are large in size and co-express CD25 and CD69, correlating expression of PD-1 with cell activation. PD-1 protein expression can be detected as early as 24 h after TCR activation. Both CD4+ and CD8+ T cell populations expressed cell surface PD-1 upon activation, and the pattern of PD-1 expression on human T cells is similar to that observed for murine T cells (B. Carreno, unpublished observations). PD-1 is also expressed in activated macrophages (70). The ligands for PD-1 are the B7 family members PD-L1 (B7-H1) and PD-L2 (B7-DC) (71–74). Interaction of PD-1 with either PD-L1 or PD-L2 results in inhibition of T and B cell responses (70–72). An alternatively spliced PD-L2 variant lacking the Ig V–like domain has been described, but this variant does not bind to PD-1 (72). PD-L1 and PD-L2 share 40% amino acid identity and thus are more homologous to each other than to other ligands of the B7 family (Table 2). Human and murine orthologues of PD-L1 or PD-L2 share 70% amino acid identity. Interestingly, murine PD-L2 has only 5 amino acids in its cytoplasmic tail, whereas the human PD-L2 cytoplasmic region is 28 amino acids in length. Both human PD-L1 and PD-L2 genes map to chromosome 9p24.2, and these genes are separated by only 42 kb (72). This genomic proximity is reminiscent of that observed for B7-1 and B7-2, which are tightly linked on human chromosome 3q13.3-21 (41). Murine PD-L2 maps to a region located between 19C2 and 19C3 (74). A variety of normal tissues have been examined for expression of PD-L1 and PD-L2 transcripts (71–73). The pattern of expression of these molecules is significantly broader than that reported for other B7 family ligands. The overall distribution of PD-L1 and PD-L2 transcripts is similar in human and murine tissues, with high levels of expression in placenta, low expression levels in spleen, lymph nodes, and thymus, and the absence of expression in brain. Transcripts for both PD-L1 and PD-L2 are detected in human heart; in murine hearts, transcripts for PD-L1 are abundantly expressed whereas PD-L2 transcripts are absent (71, 72). PD-L2 but not PD-L1 transcripts are detected in human pancreas, lung, and liver (72). Identification of the cellular populations that express PD-L1 and PD-L2 in these tissues awaits further investigation. Expression of PD-L1 and PD-L2 in both lymphoid and nonlymphoid tissues suggests that the PD-1/PD-L pathway may modulate immune responses in secondary lymphoid organs as well as in peripheral sites. Expression of PD-L1 and PD-L2 on antigen presenting cell (APC) populations has also been examined in detail (71–73). Resting B cells, monocytes, and dendritic cells do not express either PD-L1 or PD-L2. Transcripts for these ligands can be detected upon activation of these populations by antigen receptor, LPS, or IFN-γ . In human B cell populations, LPS or BCR activation results in induction of PD-L1 and PD-L2 (71–73). In human monocytes, IFN-γ , but not TNF-α, treatment results in expression of both ligands; PD-L1 expression precedes that of PD-L2. Interestingly, IFN-γ treatment also upregulates B7-1 transcripts and ICOS-L protein expression on human monocyte populations (46, 71). On dendritic cells, LPS plus IFN-γ treatment induces PD-L1 and PD-L2 mRNA expression (71–73). B7-1 and B7-2 transcripts are also upregulated in these cells by LPS plus IFN-γ . Tseng

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et al. (74) reported that PD-L2 (B7-DC) transcripts are exclusively expressed on spleen and bone marrow–derived murine dendritic cells. These authors postulate that some of the unique functions of dendritic cells could be attributed to PD-L2 expression. Further studies will be necessary to sort out these discrepancies in expression patterns. Interestingly, mitogen or TCR activation of murine and human T cells results in cell surface expression of PD-L1 in addition to PD-1 (73) (L. Carter, B. Carreno, unpublished observations). Similarly, BCR-activated B cells express PD-L1 and PD-L2 in addition to PD-1 (63, 71, 72). Thus, B and T cell function can be modulated by engagement of cell-surface PD-1. Additionally, upon activation, both T and B cells can engage PD-1 on other cells through expression of PD-1 ligands. This suggests that at points of T:B contact, modulation of antigen receptor signals can occur bidirectionally through PD-1. The consequences of such an interaction are unknown but might serve to limit TCR and BCR receptor signaling after activation. IFN-γ can also modulate PD-L1 expression in nonlymphoid cells. Endothelial cells constituitively express cell-surface PD-L1, and in vitro treatment with IFN-γ , but not LPS or TNF-α, results in its rapid upregulation (M. Eppihimer, J. Leonard, personal communication). Furthermore, IL-12 challenge of IFN-γ +/+ but not IFN-γ −/− mice results in enhanced expression of PD-L1 in blood vessels of various tissues (M. Eppihimer, J. Leonard, personal communication). Thus in vivo, IFN-γ upregulation of PD-L1 expression on endothelial cells may play a significant role in attenuation of lymphocyte function at peripheral sites. Consistent with these findings, several studies have suggested an immunosuppressive role for IFN-γ . IFN-γ receptor–deficient mice develop accelerated collagen-induced arthritis (75), and IFN-γ blockade enhances EAE (76). In addition, IFN-γ treatment has been reported to confer resistance to EAE (77). Thus, the beneficial effect of IFN-γ reported in some autoimmune settings could be partly attributed to induction of PD-L1 ligand expression at sites of inflammation and subsequent downregulation of immune responses by PD-1 engagement. Finally, PD-L1 and PD-L2 transcripts have been detected in various tumor cell lines (72). Additionally, cell-surface expression of PD-L1 has been reported in human breast cancer cell lines (72). These observations have led to the suggestion that tumors may escape immunosurveillance by attenuation of T cell responses upon PD-1 engagement. This hypothesis has implications for the development of new strategies for tumor immunotherapy, as one would predict that blockade of PD-1/PD-L interactions could enhance tumor-specific T cell responses.

ATTENUATION OF IMMUNE RESPONSES BY THE PD-1/PD-L PATHWAY Identification of the PD-1 ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC), and assessment of their interaction with PD-1 confirmed the negative regulatory function of PD-1 in immune responses (71, 72). Neither PD-L1 nor PD-L2 bound to CD28,

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CTLA-4, or ICOS. Reciprocally, soluble forms of B7-1 and B7-2 did not bind PD-1. Experiments using T cells from PD-1-deficient mice formally demonstrated the functional consequences of PD-1/PD-L1 interactions (71). Proliferation of wild-type, but not PD-1-deficient, T cells was inhibited in the presence of PD-L1. Similarly, proliferation of human T cells was decreased in the presence of PD-L1. Cross-linking of PD-1 by PD-L2 upon TCR activation also resulted in decreased proliferation (72). Furthermore, PD-1 cross-linking by either ligand resulted in decreased IFN-γ , IL-10, IL-4, and IL-2 secretion (71, 72). These results indicate that, upon TCR activation, cross-linking of PD-1 by PD-L1 or PD-L2 leads to diminished immune responses. Thus, PD-1 cross-linking by either PD-L1 or PD-L2 results in similar outcomes, suggesting that these two ligands may have overlapping functions in vivo. However, additional information regarding their affinities for PD-1, as well as the generation of PD-L1- and PD-L2-deficient mice will be necessary to discern their potential roles in vivo. Consistent with an inhibitory function, studies on PD-1+ B cell lymphomas have shown that PD-1/BCR co-engagement results in inhibition of Ca2+ influx as well as hypophosphorylation of BCR downstream signaling molecules syk, phosphatidyl inositol-3, phospholipase C, and vav (70). SHP-1 and SHP-2, Src-homology-2 (SH-2) domain containing phosphatases, have been implicated in inhibitory signals mediated by NK receptors, with recruitment of both phosphatases upon tyrosine phosphorylation of ITIM motifs (64). A role for SHP-2 in CTLA-4 inhibitory signals has also been reported (78). Co-ligation of the BCR and PD-1 resulted in increased phosphorylation of SHP-2 and recruitment of SHP-2 to the PD-1 receptor (70). Interestingly, the carboxy-terminal tyrosine (TEYATI), and not the tyrosine within the canonical ITIM, has been implicated as necessary for SHP-2 binding (70). Similarly, TCR/PD-1 co-ligation resulted in increased phosphorylation of SHP-2 (72). Thus, these studies point to SHP-2 as a likely candidate involved in transducing inhibitory signals initiated by PD-1. PD-1 appears to function as an attenuator of T cell responses, and the process by which this regulation occurs is distinct from activation-induced cell death (72). PD-1 engagement by either PD-L1 or PD-L2 results in cell cycle arrest. Activation of cells in the presence of either ligand leads to an accumulation of cells at the G0/G1 phase of the cell cycle. IL-2 production is drastically inhibited upon PD-1 engagement, while exogenous IL-2 can rescue PD-1-mediated cell cycle arrest (L. Carter, B. Carreno, unpublished observations). These observations suggest that PD-1 may affect T cell activation and proliferation by regulating IL-2 transcription. These data parallel those reported for CTLA-4, which most likely inhibits T cell cycle entry by regulating IL-2 transcription and mRNA stability (79–81). The interplay between PD-1 and CD28 on T cell activation has also been examined. Optimal, but not suboptimal, CD28 costimulation can rescue PD-1-mediated inhibition (71). At low antigen concentrations, PD-1 signals can antagonize costimulation mediated by CD28. At high antigen levels, CD28 costimulation overrides the PD-1 inhibitory effect. Interestingly, PD-1 cell surface expression is highest

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at low antigen concentration. Thus, a correlation can be established between antigen concentrations required for highest PD-1 expression and conditions in which downregulation of T cell responses are more readily observed (72). Interestingly, two separate groups have concluded that PD-L1 (B7-H1) and PD-L2 (BC-DC) can function to costimulate T cell responses (73, 74). Both groups have reported that activation of T cells in the presence of suboptimal TCR signals, and either PD-L1 or PD-L2 results in increased proliferation. Additionally, anti-CD3 plus PD-L1.Fc activation results in increased secretion of IL-10, IFN-γ and GM-CSF but not IL-2 or IL-4 (73). Activation of T cells with anti-CD3 and PD-L2.Fc increases IFN-γ but not IL-4 or IL-10 (74). The discrepancy between these results and those of Freeman and colleagues (71, 72) raises the possibility that there may be additional receptors for PD-L1 and PD-L2. If, indeed, a second receptor with costimulatory function exists for PD-L ligands, this pathway would have symmetry with that of CD28/CTLA-4/B7. As speculated for CD28/CTLA-4, temporal regulation of receptor expression and affinities of ligands would then determine whether the costimulatory or inhibitory signals prevail.

PD-1 DEFICIENCY LEADS TO AUTOIMMUNE DISORDERS AND BREAKDOWN OF PERIPHERAL TOLERANCE Consistent with its negative regulatory function, PD-1 deficiency in vivo results in the development of autoimmune disorders (69, 82, 83). C57BL/6- PD-1−/− mice consistently displayed splenomegaly, increased numbers of B lymphocytes and myeloid cells, and increased serum IgG2b, IgG3, and IgA (82). Antibody responses to T-independent, but not T-dependent, antigens were greatly enhanced in PD-1−/− mice relative to control littermates. Furthermore, PD-1−/− B cells displayed enhanced proliferation in response to BCR cross-linking. These mice spontaneously developed a lupus-like disease with age (69). At 6 months, PD-1-deficient mice displayed elevated serum IgG3 levels and increased IgG3 and C3 deposition in the glomeruli. At 14 months, approximately 50% of these mice had lupus-like glomerulonephritis and histological evidence of arthritis as well as granulomatous inflammation. Introduction of the lpr mutation (B6-lpr/lpr-PD-1−/− ) accelerated the onset and severity of disease. Thus, PD-1 deficiency in the C57BL/6 background resulted in the development of a late onset, chronic, progressive, lupuslike glomerulonephritis and arthritis, and the severity of disease was exacerbated by the absence of FAS-mediated apoptosis. Interestingly, introduction of the PD-1 deficiency into the Balb/c background resulted in a distinct autoimmune phenotype, with cardiomegaly, diffuse IgG1 deposition in cardiomyocytes, and high circulating levels of heart-tissue reactive IgG1 (83). This disease developed rapidly, and Balb/c-PD-1−/− mice died as early as 5 weeks of age. By 30 weeks, two thirds of mice had succumbed to disease. In contrast, no disease was observed in Balb/c-PD-1−/− -RAG−/− mice, indicating that T and B cells are required for disease development. The distinct severity and phenotype of disease observed in

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these two strains indicated that other genetic modifier genes in addition to the PD-1 deficiency contribute to the pathologies observed. However, in both genetic backgrounds, PD-1 deficiency appeared to alter the balance between activating and inhibitory signals, resulting in a loss of peripheral tolerance. Further support for a role for PD-1 in the maintenance and/or induction of peripheral tolerance has come from studies examining responses to self-antigens (69). PD-1 deficient 2C TCR transgenic mice bred to the autoreactive background (H-2b/d) exhibited growth retardation, splenomegaly, and lethal graft-vs-host disease. Massive infiltration of inflammatory cells in liver, heart, and lung was observed. Additionally, an increase in the total number of cells, most notably activated CD8+ T cells, in the spleens was also reported. As PD-1 has a minimal, if any, role in negative selection in the thymus (69), these results point to PD-1 as negative regulator of self-reactivity in the periphery. Altogether, the outcome is a breakdown of peripheral tolerance to selected tissue antigens. In vitro, PD-1 and CTLA-4 functions are quite similar. Engagement of either PD-1 or CTLA-4 results in inhibition of T cell proliferation, cytokine production, and cell cycle progression (3, 71). In vivo, deficiency in these molecules results in development of lymphoproliferative disorders, albeit with different degrees of severity. CTLA-4-deficient mice display very aggressive lymphoproliferative disorders and die at 21–28 days of age (6, 7, 84). Lymphocytic infiltration is observed in multiple organs. The disorder is characterized by a high frequency of T cell blasts (CD25, CD69, CD44hi, CD45ROlow), with signs of T cell activation detected as early as 5–6 days after birth. In contrast, a less aggressive disorder is observed in PD-1-deficient mice (69, 83). Of the phenotypes reported, the most aggressive is the cardiomyopathy in Balb/c-PD-1−/− mice (83). As deficiencies in either of these receptors result in breakdown of peripheral tolerance, both receptors must have critical and nonredundant functions in the maintenance of tolerance. These pathways may control T cell responses at two distinct points, with the first at the time of T cell activation in lymphoid tissues, and the second upon reactivation in peripheral sites. CTLA-4 would have a predominant role in regulating the threshold for T cell activation in lymphoid sites, where B7-1 and B7-2 are primarily expressed. Because both PD-1 and PD-1 ligands have broader expression patterns, PD-1 could be important in regulating thresholds of activation for T and B cells in both lymphoid and peripheral sites during inflammation. By controlling the magnitude of T cell responses at initiation and again at reactivation, these pathways could function as independent checkpoints to safeguard against self-reactivity.

B7-H3: A NEW B7-LIKE LIGAND Human B7-H3 was recently identified as a new costimulatory member of the B7 family (85). B7-H3 shares from 26% to 33% amino acid identity with other members of the B7 family (Table 2). Northern analysis indicated that this gene is broadly expressed, with mRNA detected in most organs, as well as in immune tissues

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including spleen, lymph node, thymus, bone marrow, and fetal liver. Tumor cell lines derived from nonlymphoid tissues were also positive for B7-H3 mRNA, but Molt-4, a lymphoblastic leukemia, and Raji, a Burkitt’s lymphoma line, were not. B7-H3 expression was not detected in unstimulated PBL but could be induced in lymphoid cells by activation. B7-H3 protein was detected on GM-CSF-stimulated monocytes and IFN-γ activated dendritic cells, as well as on CD3+ T cells activated with PMA and ionomycin. B7-H3-transfected 293 cells did not bind CTLA-4.Fc, ICOS.Fc, or PD-1.Fc, which suggests that this B7 member binds a distinct receptor (85). Binding studies with the B7-H3.Fc protein further suggest that this receptor is present on activated T cells. Activation of human T cells with plate-bound anti-CD3 plus increasing concentrations of B7-H3.Fc resulted in a dose-dependent enhancement of proliferation relative to cells activated with anti-CD3 plus control Ig. B7-H3 enhancement of proliferative responses was not as profound as that observed with B7-1.Fc. B7-H3.Fc enhances proliferative responses of both CD4+ and CD8+ T cells. In addition, B7-H3.Fc stimulation increased secretion of IFNγ in 50% of cell donors tested. Interestingly, expression of B7-H3 in a melanoma line resulted in increased lytic activity of melanoma-specific T cells. The receptor for this newest member of the B7 family remains to be identified, and further elucidation of B7-H3 function in immune responses is needed.

NEW MEMBERS OF THE B7 AND CD28 FAMILY? The recent expansion of the B7 family and its receptors raises the question of whether there are additional members of this family with immune function. Certainly, the receptor for B7-H3 remains to be identified. Conflicting results indicating that PD-L1 and PD-L2 can both costimulate and inhibit immune responses raise the possibility of additional receptors for these ligands. In addition, experiments demonstrating B7-dependent responses in mice deficient for CD28 and CTLA-4 suggest that there are additional receptors for the B7 molecules (86, 87). Finally, experiments evaluating ICOS-L often use the ICOS.Fc reagent to define this ligand. Antibody reagents for ICOS-L are needed to confirm that these interactions do not include contributions of additional ligands for ICOS. In addition to the B7-like ligands and their receptors described above, molecules with homologies to these families have been described. In each of these cases, data are too limited to currently include these molecules as bona fide members of the B7-family and their receptors. A key question is whether these related proteins have a role in immune responses. Members of the butyrophilin gene family share homology with the B7-family of immunoregulatory receptors (88, 89). Butyrophilin is a 66-kDa type I transmembrane protein that forms a major component of milk fat globule membrane (90, 91). It has no reported role in the immune system. Six additional family members were identifed by homology and genomic location and form two subfamilies (92, 93). The seven members of the butyrophilin gene family are located in a cluster

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on human chromosome 6p22.1, telomeric to the class I MHC (89, 93). Like the B7 proteins, butyrophilins are type I transmembrane proteins with IgV and IgC extracellular domains. However, they also have unique cytoplasmic domains, including a series of heptad repeats and a B30.2 domain, which may mediate protein interactions (89, 93). Butyrophilin genes are expressed at low levels in most tissues (93). Interestingly, the predominant transcripts of the BTN3 subfamily do not express the B30.2 domain due to alternate splicing (93), making the BTN3 subfamily proteins more similar to the B7 gene family. Linsley et al. (88) also noted that the myelin protein MOG has homology with B7-1, B7-2, and butyrophilin, although MOG contains only an IgV extracellular domain. MOG is an autoantigen in EAE, and immunization of susceptible rats with the IgV domain of butyrophilin also results in an inflammatory response in the CNS due to cross-reactivity of T cells to MOG-derived epitopes (94). MOG maps to 6p21 in humans, centromeric to the butyrophilin genes and telomeric from the class I and II gene clusters (95, 96). Although MOG can act as an autoantigen in animal models of multiple sclerosis, as yet there are no data defining an interacting protein for MOG, nor any data implicating MOG as a costimulatory protein in immune function. Beyond these defined family members, other potential homologous sequences have been identified in database searches (97). These include SIRPα and β, transmembrane proteins expressed in the immune system; HHLA2, an endogenous retroviral sequence; MCAM, a melanoma adhesion protein; and VEJAM, vascular endothelial junction–associated molecule, as well as a few novel sequences with similar levels of homology. Additionally, Linsley et al. (88) noted the homology of the chicken B-G gene to the B7 family. Homology searches to the CD28 receptor family can also be carried out, and there is sufficient homology between CD28, ICOS, and CTLA-4 to detect these as related sequences. However, PD-1 is less homologous to this family, suggesting that the receptors for the B7 family of ligands may be less conserved. Indeed, as each of these receptors contains only a single extracellular IgV domain, many proteins of the Ig-superfamily can be detected with weak homology, including TCRα and Igκ proteins. This suggests that functional tests of members of the Ig superfamily will be critical in identifying new members of costimulatory and inhibitory receptors and ligands related to CD28 and B7.

PERSPECTIVES The sequencing of the human genome has led to an explosion of gene identification, along with information about gene clusters. The new challenge is to ascribe function to new genes with sequence relationships to known genes. For the family of genes related to the CD28 and B7 molecules, this has led to an appreciation that these receptor-ligand interactions will include both costimulatory and inhibitory receptors, and that multiple receptor-ligand interactions are possible. In addition, regulation of the immune response can be affected by signals delivered through

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multiple costimulatory or inhibitory receptors, each of which is simultaneously expressed by the interacting cells. Integration of these various signals with signals through antigen-specific receptors then determines the outcome of that cellular interaction. Regulation of immune responses in both the priming events in immune sites and in activation or attenuation in peripheral sites can occur through these pathways. An understanding of which interactions are critical at various steps in immune responses will allow intervention in immune-mediated diseases through precise manipulation of these pathways. ACKNOWLEDGMENTS We would like to thank Dr. Vincent Ling, Dr. Laura Carter, and Dr. Arlene Sharpe for critical reading of the manuscript and for many helpful discussions. We would also like to thank our colleagues at Genetics Institute/Wyeth Research and our academic collaborators for many enjoyable and thought-provoking discussions in the field of costimulation and immune responses. Visit the Annual Reviews home page at www.annualreviews.org

LITERATURE CITED 1. Salomon B, Bluestone JA. 2001. Complexities of CD28/B7: CTLA-4 costimulatory pathways in autoimmunity and transplantation. Annu. Rev. Immunol. 19: 225–52 2. Greenfield EA, Nguyen KA, Kuchroo VK. 1998. CD28/B7 costimulation: a review. Crit. Rev. Immunol. 18:389–418 3. Chambers CA, Kuhns MS, Egen JG, Allison JP. 2001. CTLA-4-mediated inhibition in regulation of T cell responses: mechanisms and manipulation in tumor immunotherapy. Annu. Rev. Immunol. 19: 565–94 4. Boise LH, Minn AJ, Noel PJ, June CH, Accavitti MA, Lindsten T, Thompson CB. 1995. CD28 costimulation can promote T cell survival by enhancing the expression of Bcl-XL. Immunity 3:87– 98 5. Linsley PS, Bradshaw J, Greene J, Peach R, Bennett KL, Mittler RS. 1996. Intracellular trafficking of CTLA-4 and focal localization towards sites of TCR engagement. Immunity 4:535–43

6. Tivol EA, Borriello F, Schweitzer AN, Lynch WP, Bluestone JA, Sharpe AH. 1995. Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity 3:541–47 7. Waterhouse P, Penninger JM, Timms E, Wakeham A, Shahinian A, Lee KP, Thompson CB, Griesser H, Mak TW. 1995. Lymphoproliferative disorders with early lethality in mice deficient in CTLA4. Science 270:985–88 8. Chambers CA, Sullivan TJ, Allison JP. 1997. Lymphoproliferation in CTLA-4deficient mice is mediated by costimulation-dependent activation of CD4+ T cells. Immunity 7:885–95 9. van der Merwe PA, Bodian DL, Daenke S, Linsley P, Davis SJ. 1997. CD80 (B7-1) binds both CD28 and CTLA-4 with a low affinity and very fast kinetics. J. Exp. Med. 185:393–403 10. Ikemizu S, Gilbert RJ, Fennelly JA, Collins AV, Harlos K, Jones EY, Stuart

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MAP KINASES IN THE IMMUNE RESPONSE Chen Dong,1 Roger J. Davis,2 and Richard A. Flavell3


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Department of Immunology, University of Washington School of Medicine, Seattle, Washington 98195-7650; e-mail: [email protected] 2 Howard Hughes Medical Institute and Program in Molecular Medicine, Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, Massachuetts 01605; e-mail: [email protected] 3 Howard Hughes Medical Institute and Section of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520; e-mail: [email protected]

Key Words JNK, p38, ERK, innate immunity, helper T Cells ■ Abstract MAP kinases are among the most ancient signal transduction pathways and are widely used throughout evolution in many physiological processes. In mammalian species, MAP kinases are involved in all aspects of immune responses, from the initiation phase of innate immunity, to activation of adaptive immunity, and to cell death when immune function is complete. In this review, we summarize recent progress in understanding the function and regulation of MAP kinase pathways in these phases of immune responses.

INTRODUCTION Immune responses involve a number of cell types that function as initiators, regulators, and effectors. These cells interact with and cross-regulate each other, and the target cells respond using signal transduction pathways to mediate gene expression and immune function. The MAP kinase cascade is one of the most ancient and evolutionarily conserved signaling pathways, which is also important for many processes in immune responses. There are three major groups of MAP kinases in mammalian cells—the extracellular signal-regulated protein kinases (ERK) (1), the p38 MAP kinases (2), and the c-Jun NH2-terminal kinases (JNK) (3, 4) (Figure 1). These MAP kinases are activated by dual phosphorylation at the tripeptide motif Thr-Xaa-Tyr. The sequence of this tripeptide motif is different in each group of MAP kinases: ERK (Thr-Glu-Tyr); p38 (Thr-Gly-Tyr); and JNK (Thr-Pro-Tyr). The dual phosphorylation of Thr and Tyr is mediated by a conserved protein kinase cascade. The ERK MAP kinases are activated by the MAP kinase kinases (MKK) MKK1 and MKK2; the p38 MAP kinases are activated by MKK3, MKK4, and MKK6; and the JNK pathway is activated by MKK4 and MKK7. These MAP kinase kinases are activated, in turn, by several different MAP kinase kinase kinases (MKKK). 0732-0582/02/0407-0055$14.00

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Figure 1 Mammalian MAP kinase pathways.

Different upstream signals can lead to the activation of these MKKK. However, prominent roles for small G proteins have been identified. Thus, the ERK pathway can be activated by Ras via the Raf group of MKKK. In contrast, the p38 and JNK MAP kinases are activated by Rho family GTPases, including Rac and Cdc42. Candidate MKKK that are activated by Rho proteins include members of the MEKK and mixed-lineage protein kinase (MLK) groups. Signal transduction along the MAP kinase pathways can be facilitated by scaffold proteins. The JIP group of proteins has been recently identified as scaffolds for the JNK pathway, which connects MLK to MKK7 and to JNK [recently reviewed by Davis (3)]. MAP kinases play important functions in lymphocyte development and have been well reviewed recently (5–7). In this review, we focus on recent progress on the function and regulation of MAP kinases in different components or phases of immune responses, with particular focus on the studies achieved through mouse genetic approaches. Due to space limitations, we apologize that we are not able to cover all aspects of MAP kinase research here.

MAP KINASES IN INNATE IMMUNE RESPONSES Immune responses to foreign organisms can be traced back to invertebrates. In the fruit fly, Drosophila, a group of 8 proteins known as the Toll family is central to innate defense, which involves the recognition of bacteria and fungi, and the induction of antimicrobial peptides to kill an invader (8). It is well established that the

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NF-κB pathway is an important downstream target of Toll receptors in Drosophila innate immune responses. MAP kinases such as JNK and p38 are activated by pathogens in insect cells (9, 10). Fly p38 appears to attenuate antimicrobial peptide gene expression following exposure to lipopolysaccharide (LPS) (9). The role of JNK in Drosophila innate immune response has not been defined genetically due to the embryonic lethality of the flies deficient in components of this pathway. In mammalian systems, 10 Toll-like receptors (TLR) exist. Recent years have seen rapid progress in defining the role of these TLRs in recognition of “pattern” molecules on microbial organisms and, as a consequence, induction of inflammatory responses (11). For instance, TLR4 is critical for LPS recognition (12–14); TLR2 for lipoteichoic acids (LTAs) and peptidoglycan (PGN) (15, 16); TLR5 for bacterial flagellin (17); and TLR9 for CpG-containing DNA (18). TLR cytoplasmic domains resemble that of the interleukin 1 (IL-1) receptor, and hence they are called Toll-IL-1-Receptor (TIR) domains. After ligand-mediated dimerization, TLR recruits an adaptor protein MyD88 (19–24). MyD88 then assembles a signalsome containing IRAK, TRAF6, and ECSIT (24–26). Together, they mediate activation of NF-κB and MAP kinases such as p38 and JNK, which leads to the production of inflammatory cytokines such as tumor necrosis factor (TNF) α, IL-1, and IL-12. In support of the roles of these signaling intermediates in mediating MAP kinase activation, genetic ablation of MyD88 resulted in defective MAP kinase activation in response to endotoxin (19), and IRAK−/− and TRAF6−/− cells exhibited deficiency of p38 and JNK activation in response to IL-1 (27, 28).

MKK3-p38 Mediates IL-12 Production in Innate Immune Responses To examine the functional roles of p38 MAP kinase in immune responses, Lu et al. analyzed mice deficient in the p38 kinase MKK3 (29). Mkk3 disruption caused an approximately 40%–70% reduction of total p38 activity in macrophages treated with LPS. The residual activity could be contributed by MKK6 or perhaps MKK4. Mkk3−/− macrophages exhibited a selective defect in LPS-induced IL-12 production at both protein and RNA levels. The same results were achieved by using p38-specific inhibitors SB 203580, SB 202190, and SB 202474. Production of other cytokines such as TNFα, IL-6, IL-1α, and IL-1α were comparable between wild-type and knockout cells, and addition of p38 inhibitors appeared to have an additive inhibitory effect only on IL-1α and IL-1β expression by knockout cells, but not on TNF or IL-6 expression. The MKK3-p38 pathway seems therefore to play a specific role in the activation of IL-12 production in macrophages; p38 activation by other kinases may compensate during the IL-1 regulation. The essential function of MKK3 in IL-12 regulation was reported in the same study to exist in dendritic cells (DC). CD40-CD40L interaction, a receptor-ligand pair upstream of p38 and JNK, is the main activator of IL-12 production in these cells. MKK3 deficiency resulted in a severe reduction of IL-12 secretion by bone marrow–derived DC activated by CD40L.

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Regulation of TNFα Production by MAP Kinases p38 MAP kinase activates many protein kinases such as MAP-kinase-activated protein kinases (MAPKAP) MK2 and MK3. Kotlyarov et al. generated and analyzed mice deficient for MAPKAP MK2 (30). These mice showed increased stress resistance and survived LPS shock. Interestingly, production of TNFα protein in the serum of LPS-treated animals in vivo, and by LPS-treated splenocytes in vitro, was greatly compromised, whereas the mRNA levels were not affected. Therefore, these investigators proposed that MAPKAP MK2 regulates TNFα biosynthesis at the posttranscriptional level. p38 and MAPKAP MK2 may exert their regulation of TNFα translation through AU-rich elements (ARE) in the 30 untranslated region of TNFα transcripts. Targeted deletion of these elements in the mouse genome resulted in abnormal TNFα gene expression and development of two types of autoimmunity, i.e., inflammatory arthritis and inflammatory bowel diseases (31). While p38 inhibitors blocked TNFα production in normal mice, ARE-deleted mice did not respond to the drug, indicating a requirement for the ARE region in the p38-mediated activation of TNF translation. In addition, IL-10, an anti-inflammatory cytokine, was recently found to repress TNFα production by the inhibition of p38-MAPKAP MK2-mediated ARE activity (32). Evidence for ERK regulation of TNFα induction was also recently reported. Tpl2/Cot is a proto-oncogene that serves as a MAP kinase kinase kinase (MKKK) (33, 34). Mice deficient for this kinase were found deficient in TNFα production when exposed to LPS (35). The macrophages from these mice exhibited selective ERK activation deficits, and the ERK inhibitor PD98059 had a similar effect. Deletion of the ARE motif in TNFα mRNA minimized the effect of Tpl2 deficiency, suggesting that ERK may target the ARE region as well. However, ERK may work at a different phase than p38/MAPKAP MK2. TNFα mRNA transport from the nucleus to the cytoplasm was inhibited by ERK inhibitor or Tpl2 inactivation.

Cellular Responses to Inflammatory Cytokines: The Roles of p38 and JNK p38 and JNK MAP kinases are preferentially activated by inflammatory cytokines such as TNF and IL-1, and they have critical functions in cellular responses to these cytokines. p38 MKK3-mediated p38 activity was reported by Wysk et al. to be important also for TNF-induced cytokine production (36). p38 activation was selectively inhibited in Mkk3−/− mouse embryonic fibroblasts (MEF) in response to TNFα, but not to IL-1, UV, or sorbitol. As a result of this deficiency, Mkk3−/− MEF failed to upregulate IL-1α, IL-1β, IL-6, and TNFα mRNA or to downregulate IL-1 receptor antagonist (RA) in response to TNF. This study clearly indicates the essential function of MKK3-p38 in the cellular response to TNF, but not to

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IL-1. However, p38, especially p38α, does play an important role in IL-1-mediated inflammatory response. Allen et al. examined p38α −/− MEF and found that IL-1induced IL-6 production was greatly compromised (37). It is still unclear which MKK may mediate p38 activation in IL-1 signal transduction. Given the absence of deficiency in Mkk3−/− MEF, then MKK6, or MKK4 are candidates. It is also possible that these three MKKs function redundantly. To support a role for MKK4 in p38 activation, Ganiatsas et al. reported that Mkk4−/− MEF exhibited profound reduction of p38 activation in response to IL-1 but not to anisomycin, although JNK activation is affected in both cases (38). JNK activity can be strongly induced in multiple cell types by liposaccharides (LPS) or inflammatory cytokines such as TNF and IL-1 (3, 4). It is interesting that Drosophila cells activate the JNK pathway in response to LPS (10), suggesting that the JNK pathway is evolutionarily conserved in innate immune responses. However, the functional roles of JNK have not been elucidated in these systems. In fibroblasts, JNK can also be activated by inflammatory cytokines and doublestranded viral RNA (39). Using JNK2−/− fibroblasts, Chu et al. demonstrated that JNK is required for production of multiple cytokines including type I interferon and IL-6 (39). It is intriguing that JNK1 did not compensate in the above experiments. Recently, Han et al. studied the role of JNK in rheumatoid arthritis fibroblast-like synoviocytes (40). By use of a novel JNK-specific inhibitor and synoviocytes from JNK1 or JNK2 knockout mice, they reported that JNK is required for IL-1-mediated collagenase-3 expression and joint inflammation. JNK is activated by TRAF2 in the TNF signaling pathway and by TRAF6 following IL-1 activation (27, 41). Further downstream, one JNK kinase—MKK7, but not the other—MKK4, appears to play an essential function in activating JNK in TNF- or IL-1-treated MEF, although both are involved in JNK activation in response to physical stress (42). Tournier et al. examined JNK activation in MEF deficient in either MKK4 or MKK7, or both; they found that MKK7 is solely required for JNK activation in these cells in response to inflammatory cytokines. It is interesting that MKK4 and MKK7 preferentially phosphorylate different target amino acids of the tripeptide motif in JNK, whereas MKK4 phosphorylates tyrosine, MKK7 phosphorylates threonine. JNK activation by MKK7 appears to be an essential part of inflammatory responses. JNK

MAP KINASES IN HELPER T CELL ACTIVATION AND DIFFERENTIATION CD4+ helper T (Th) cells play a central regulatory role in immune responses. Like CD8 cytotoxic T cells, Th cells develop in the thymus, with specificity to recognize specific MHC-peptide complexes on antigen-presenting cells (APC). Following the receipt of signals from these innate immune cells through the T cell receptor and CD28 costimulator, Th cells are triggered to produce interleukin 2 (IL-2) and

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Figure 2 The role of MAP kinase pathways in Th cell differentiation and cytokine production.

enter the cell cycle. Following or during several days of rapid cell division, these T cells differentiate into effector cells (Figure 2). During this process of differentiation, a new pattern of cytokine expression is established that provides the gene products responsible for the specific effector functions of these cells, and therefore for their ability to protect the host from a variety of pathogens. Two classes of effector CD4 T cells have been defined on the basis of the cytokines that they secrete and the immunomodulatory effects conferred by these cytokines (43–45). Effector Th1 cells produce proinflammatory cytokines such as interferon-γ (IFN-γ ) and lymphotoxin-α (Lt-α). These cytokines organize inflammatory centers and enhance cellular immune response; moreover, intracellular pathogens such as Mycobacterium, Salmonella, and other intravesicular agents are killed by IFN-γ through the activation of antimicrobial defenses. Th1 cytokine production is also characteristic of many organ-specific autoimmune diseases, including rheumatoid arthritis, insulin-dependent diabetes mellitus, experimental autoimmune encephalomyelitis (EAE), etc. Effector Th2 cells, in contrast, produce different cytokines (IL-4, IL-5, IL-9, IL-10, IL-13, and so on) that together instruct B cells to proliferate and differentiate into antibody-secreting plasma cells, as well as potentiate the function of several cell types in the antiparasite responses. As such, Th2 cells play an important role in the provision of protection against certain extracellular pathogens such as bacteria and a variety of parasites, and they are also involved in asthmatic reactions. Proper differentiation of na¨ıve Th cells into Th1 or Th2 cells is critical for a T-dependent immune response.

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Leishmania major infection in mice represents one of best-studied animal models for Th differentiation and function. Most common inbred strains can mount a Th1 response and resolve the lesions; Balb/c mice, however, develop an infection that never heals and a Th2 response that renders them susceptible to the infection. On the other hand, immunodeviation of Th1 responses to the Th2 direction has been proposed as a means to alleviate the symptoms in autoimmune diseases.


ERK Regulation of T Cell Activation and Differentiation The ERK pathway was first identified downstream of oncogenic Ras and is often involved in the regulation of cell growth and differentiation. There are two isoforms of ERK, ERK1 and 2, which are sometimes referred as p44/p42 MAP kinases. They can be activated by MEK1 and MEK2 upstream kinases. ERK activation is an important event of T cell activation. TcR engagement leads to the recruitment of multimolecular components to the cell surface, including adaptor proteins SLP76 and Grab2, which in turn activate the sos-ras-MEK-ERK pathway (46). Deficient ras and ERK activation was reported to exist in clones that are anergized, i.e., stimulated without CD28 costimulation (47, 48). However, there is also evidence that ERK inhibition alone had no influence on anergy induction (49); thus, it is unclear whether ERK deficiency is merely the result of anergy, or together with other pathways it contributes to anergization. Although there is a strong consensus on the role of ERK pathway in thymocyte selection—ERK1-deficient mice exhibited defective thymocyte maturation (50)—its function in peripheral Th cell differentiation was not studied until recently. Using dominant H-RAS transgenic mice in which ERK activation by TcR was severely compromised, Yamashita and colleagues showed that this pathway is required for Th2 differentiation (51). Similar results were found using wild-type cells treated with inhibitors against MEKs. They went further to show that the ERK pathway functions to enhance IL-4-induced STAT6 and IL-4R phosphorylation, which suggests a mechanism of cross-regulation among different signaling pathways.

p38 in Th1 Differentiation The p38 MAP kinase pathway was first reported by Rincón et al. to be selectively activated in mouse Th1 effector cells (52). Recent studies also suggest the role of pro-inflammatory cytokines IL-12 and IL-18 in p38 activation in T cells (53, 54). Growing evidence supports the role of the p38 pathway in Th1 differentiation and cytokine production. Imidazole inhibitors of the p38 kinases block IFN-γ production by Th1 cells in a dose-dependent manner but have no effect on IL-4 production by Th2 cells (52). Furthermore, transgenic mice in which a dominant negative p38α transgene was directed by the lck distal promoter showed reduced IFN-γ cytokine secretion and mRNA production (52). T cells from mice deficient in the p38 upstream kinase MKK3 have a defect in IFN-γ production, even when provided antigen-presenting cells from a wild-type B6 mouse (29). The effect

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of the p38 pathway on IFNγ is probably transcriptional since IFN-γ promoter reporters are also inhibited by dominant negative p38-α in Jurkat cells, indicating that p38 regulation is necessary for IFNγ expression. On the other hand, transgene-encoded constitutively active MKK6, one of the upstream MAP kinase kinases that activates p38 kinase, led to the activation of p38 MAP kinase and the consequent activation of IFN-γ transcription to higher levels than seen in control transgene negative cells (52). Candidate downstream targets of p38 regulation are likely to include transcription factors of the ATF family. Studies of the IFN-γ promoter, the prototype Th1 specific cytokine, led to the identification of c-jun/ATF2 sites and a series of other ATF binding sites within two functionally active elements called the proximal and distal IFN-γ elements (55, 56). Transgenic mice in which these two elements were linked to luciferase reporter constructs (57, 58) showed that the proximal element that carries a c-jun/ATF2 site, factors which are p38 targets, exhibited Th1 specificity, whereas the distal element did not. However, ATF2-mutant cells did not exhibit an IFNγ defect (59); therefore, it is not clear at this stage whether other ATF2 isoforms or ATF factors may play compensatory roles.

JNK Regulation of Th Differentiation Three JNK-encoding genes have been identified in mammals: JNK1, 2, and 3 (3). JNK3 is selectively expressed in neuronal tissues, while JNK1 and 2 are ubiquitously expressed (3). Surprisingly, however, when examined by Northern blot analysis, expression of JNK1 and JNK2 in peripheral lymphoid tissues was only weakly detectable (60). The same results were found for the JNK kinases, MKK4 and MKK7 (42). Consistent with these results, JNK activity in na¨ıve T cells and in T cells activated for a short time (2% are generally numbered in series (Table 1). The inhibitory KIR have long cytoplasmic tails containing pairs of immune tyrosine-based inhibitory motifs (ITIMs), whereas the KIR with short cytoplasmic tails are activating receptors that associate with the DAP12 signaling molecule via a positively charged lysine residue in their transmembrane domain (37). The prototypical KIR from which all others can be derived is a long-tailed KIR with three extracellular Ig-like domains, represented in humans by KIR3DL1 and KIR3DL2. These KIR are encoded by separate genes that span 14–16 kb and are organized in nine exons, which roughly correspond to different functional regions of the protein (38) (Figure 1). Exons 1 and 2 code for the signal peptide plus the first two amino acids of the mature polypeptide. Exons 3, 4, and 5 each encode an Ig-like domain, named D0, D1, and D2, respectively. A stem, encoded by exon 6, connects the D2 domain to the transmembrane region encoded by exon 7. The cytoplasmic tail is encoded by exons 8 and 9. The majority of human KIR have two extracellular Ig-like domains. These are of two types: type 1 KIR2D having domains homologous to D1 and D2 of KIR3DL, and type 2 KIR2D having domains homologous to D0 and D2 of KIR3D. Genes

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TABLE 1 Basic structural and functional features of KIR

KIR

Extracellular region (Ig-like domains)

Charged amino acid in tm region

Cytoplasmic tail (a.a.); ITIMs

Known ligands

mRNA

3DL1

D0-D1-D2

—

84; 2

HLA-BBw4

+

3DL2

D0-D1-D2

—

95; 2

HLA-A3, others?

+

3DS1

D0-D1-D2

Lys

22,27; 0

?

+ Lys80

+

2DL1

D1-D2

—

84; 2

HLA-C

2DL2,3

D1-D2

—

84,76; 2

HLA-CAsn80

+

Lys80

2DS1

D1-D2

Lys

39; 0

HLA-C

+

2DS2

D1-D2

Lys

39; 0

HLA-CAsn80

+

2DS3,5

D1-D2

Lys

39; 0

?

+

2DS4

D1-D2

Lys

39; 0

HLA-C?

+

2DL4

D0-D2

Arg

115; 1 or 11; 0

HLA-G

+

2DL5

D0-D2

—

115; 2

?

+(−)a

KIRC1

D0-D1-D2 (no stem)

—

67; 1

?

−(+)b

a

Some KIR2DL5 variants are not transcribed.

b

Although generally nontranscribed, a cDNA sequence for KIRC1 has been recently deposited in the GenBank (47a).

encoding type 1 KIR2D are similar in organization to those encoding KIR3D and contain a region homologous to exon 3 encoding the D0 domain of KIR3D (78.7– 79.8% sequence identity with 3DL1 and 79.8–81.2% with 3DL2) (39, 40). This region, called “pseudoexon 3,” is spliced out of the RNA transcript even when it maintains the correct reading frame and has correct splicing sites, as is the case in half of the genes encoding type 1 KIR2D (39). The one feature that distinguishes exons 3 from the pseudoexons 3 is that they are longer by one codon. If the three nucleotides missing from pseudoexons are part of an exonic splicing enhancer necessary for inclusion of exon 3 in mature mRNA (39), then their absence could

Figure 1 Organization of the KIR3DL1 gene (38). Exons (boxes) and introns (lines) are represented approximately to scale.

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explain the lack of expression of type 1 KIR2D pseudoexons. In humans, type 2 KIR2D are represented by 2DL4 and 2DL5. In comparison to genes encoding KIR3D, the 2DL4 and 2DL5 genes have a deletion >2 kb that includes exon 4 encoding the D1 domain (41, 42). KIR cytoplasmic tails can have different lengths, despite that exons encoding them have similar lengths and sequences. This is because single-nucleotide substitutions or short indels produce stop codons at diverse positions within the exonic sequence (43). The varied nature of the polymorphisms leading to short cytoplasmic tails is one of several striking examples of convergent evolution encountered in studying NK-cell receptors for MHC class I. It argues strongly in favor of there being important functions for activating KIR. No other major variations in the reading frames of functional KIR genes have been described. All other polymorphic structural features, with known or suspected importance for MHC class I specificity or signal transduction, are due to simple nucleotide replacements. Among these are ■

■

■

Charged residues in the transmembrane region: lysine in activating KIR and arginine at a different position in KIR2DL4. This and the following feature suggest that 2DL4 may be an activating receptor despite its long cytoplasmic tail (44). Indeed, ligation of KIR2DL4 has been shown to activate production of interferon-γ , but not cytotoxicity (44a). Inactivation of the carboxy-terminal ITIM in 2DL4 due to substitution of cysteine for tyrosine. Residues conferring specificity for HLA-C allotypes to 2DL1–3 and activating KIR2D.

In addition to the ∼13 expressed genes, three other KIR genes or gene fragments (usually not transcribed) have been defined, and for these a uniform nomenclature has yet to be adopted. The structure of KIRC1 (45), also known as KIR44 (46) or 3DL3 (47), is similar to that of KIR3D genes, except for absence of a stem-encoding exon and presence of a stop codon before the second ITIM. It is expressed at a very low level (47a). The nucleotide sequence of KIRC1 is phylogenetically about equidistant from KIR3D and the two types of KIR2D (sequence identity to other KIR in exons 1–9: 88.2% to 3DL1, 89.7% to 2DL5.1, 86.3 to 2DL4, 91.1% to 2DL3). Further, phylogenetic analysis does not consistently assign KIRC1 to any of the aforementioned KIR groups (38, 46, 48) (Figure 2); rather, it suggests that this gene defines a different KIR lineage. KIR48 (46), also called KIRX (47) or 2DS6 (38), is a gene fragment lacking exons 6–9 (also lacking exon 2 in many haplotypes). The KIR48 sequence is close to those of type 1 KIR2D, but its third exon lacks the three-nucleotide deletion characteristic of pseudoexons in the latter genes. KIR48 could thus retain features of the ancestral KIR3D gene from which type 1 KIR2D genes were derived (39). Finally, KIR15 is an inactivated type 1 KIR2D gene (46) and could correspond to the gene fragment known as KIRZ (47) or KIRY (49). Other genes and cDNA sequences corresponding to KIR15 indicate

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Figure 2 Phylogenetic relationships among representative KIR genes of four primate species. A distance tree was constructed by a neighbor-joining method (77) using the Pileup and PAUP search applications of the Wisconsin package (Genetics Computer Group), and this tree is represented using TreeView (by Roderic D.M. Page). Confidence of groupings was estimated by 1000 bootstrap replicates (78), and its percentage is shown besides each branch. The following sequences were analyzed: Homo sapiens 2DL3 (L76662), 2DL4 (AF034773), 2DL5 (AF20903), 3DL1 (L41269), KIRC1 cDNA (AF352324), KIRX (exons 2–5 from AC011501); Pan troglodytes 2DL4 (AF258804), 2DL5 (AF258805), 2DL6 (AF258806), 3DL1/2 (AF258798), 3DL4 (AF258800); Pan paniscus 2DL4 (AF266736), 3DLa (AF266732), 3DL4 (AF266731); Macaca mulatta 2DL4 (AF334644), 2DL5 (AF334646), 3DL1 (AF334616), 3DL6 (AF334621). For the sake of sequence-length homogeneity, sequences 50 and 30 of the regions homologous to the motifs AGGGCCGGTC (exon 2) and TCTAGGGAGA (exon 9) of Hs-3DL1, respectively, were trimmed in all entries. KIRC1 was excluded from KIR-lineage 1 due to its different exon organization and variable phylogenetic clustering with different other KIR genes (38, 46, 48). Similarly, KIR3D of the rhesus monkey have not been included in lineage 2 due to the low confidence of its grouping with genes of this lineage.

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that some alleles of this locus are not fragments but complete genes (38). What all the sequences share is a frame-shift in exon 4.

VARIABLE ORGANIZATION OF THE KIR-GENE COMPLEX


KIR-Haplotype Diversity In humans, KIR are encoded by a compact family of genes that occupies ∼150 kb of the leukocyte receptor cluster (LRC) on chromosome 19q13.4 and contains no other types of gene (38, 47, 49, 50). A characteristic feature of KIR haplotypes is variability in the quantity and quality of the genes they contain (47, 51). Of the nine and ten genes and pseudogenes present in two sequenced KIR haplotypes, only two expressed genes (2DL4 and 3DL2), one unexpressed gene (KIRC1), and one gene fragment (KIRX) are held in common (47). Genomic typing in populations and in families is consistent with 2DL4, 3DL2, KIRC1, and KIRX being the common components of KIR haplotypes (46, 51–54). Inferred from the diverse KIR genotypes obtained in such studies are many additional KIR haplotypes from the two sequenced, which differ in the presence, absence, duplication, and hybridization of particular genes. The variable number of genes in KIR haplotypes is likely the result of extensive gene duplications and nonreciprocal crossing-over events that are facilitated by the sequence similarity of KIR introns and intergenic regions and the proximity of the genes (38, 47, 50). Of the genes common to KIR haplotypes, KIRC1 and 3DL2 define the ends of the KIR-gene region and KIRX-2DL4, the middle. These genes have been called the framework genes, which then define two intervals containing genes that vary between haplotypes (47, 49). Two distinct forms of haplotype can be distinguished on the basis of several features (Table 2). The A haplotype has fewer genes, with 2DS4 being the only gene encoding a short-tailed KIR (51). B haplotypes have more genes than A haplotypes, and these include 2DL5 as well as various combinations of the genes encoding short-tailed KIR (46, 51, 55). The presence of the 2DL5 gene in B haplotypes is associated with a large (∼24 kb) HindIII fragment on Southern blots that is not produced from A haplotypes. The A haplotypes are characterized by having the 2DL3-KIRZ-2DL1 linkage group in interval one, whereas B haplotypes

TABLE 2 Summary of dimorphisms of typical KIR haplotypes, arranged from centromer to telomer according to Martin et al. (38) and Wilson et al. (40) KIR-cluster interval

A haplotype

“Complete” B haplotype

1

2DL3-KIRZ-2DL1 —

2DL2 2DL5.2

2

3DL1 — 2DS4

3DS1 2DL5.1 Combinations of 2DS1–3, 2DS5

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have 2DL2 and, often, 2DL5.2 (42, 47, 51). In interval two, A haplotypes have 3DL1 and 2DS4, whereas B haplotypes are typified by 3DS1, 2DL5.1, and various combinations of 2DS1–3 and 2DS5 (42, 47, 51, 55). In terms of gene content, the A haplotypes are less variable than B haplotypes, although the frequencies of A and B haplotypes in populations are relatively even (51). Because of the propensity for recombination within the KIR genes, it is likely that certain haplotypes will not place comfortably in either A or B category because they represent hybrids. Also possible is that the gene order and placement in some haplotypes may significantly differ from those published so far (38, 40). From comparison of KIR haplotypes, various consequences of unequal recombination have been appreciated. First is the duplication of genes, as illustrated by the presence of haplotypes having one or two copies of 2DL5 differing by eight or fewer nucleotide substitutions (42, 56; M. Gómez-Lozano, C. Vilches, unpublished). Second is the recombination of one copy of a duplicated gene with other KIR genes to evolve pairs of KIR with similar ligand-binding specificity, but distinct pathways of signal transduction. Examples of such pairs are 2DL1/2DS1 and 2DL2/2DS2. When a similar recombination involves a single-copy gene, it can create alleles of the locus that differ in their potential for signal transduction. That appears to be the case for 3DL1 and 3DS1, which generally segregate as alleles (47, 51, 53, 54). The mutual exclusivity of the genes 2DL3-KIRZ-2DL1 (A haplotypes) and 2DL2 (B haplotypes) can also be attributed to an operational allelism caused by asymmetric recombination. 2DL2 is a hybrid produced by recombining the centromeric part of 2DL3 with the telomeric part of 2DL1, an event that would have deleted the KIRZ pseudogene from 2DL2-containing haplotypes (47). In this sense, 2DL2 can be considered an allele of the whole 2DL3-KIRZ-2DL1-gene region. The plasticity and rapid evolution of the KIR-gene family can have the effect of blurring distinctions between alleles and loci, as illustrated by the examples cited here. However, these complications and the questions they raise can all be successfully resolved by performing selected analysis and comparison at the level of the KIR haplotype.

Allelic Polymorphism Allelic polymorphism, as well as haplotypic difference in gene number and content, is also a significant component of human KIR diversity (43, 54, 57–60). Most KIR genes, like their Ly49 analogues (28), show allelic polymorphism, but the extent varies from one KIR gene to another. Most individuals are heterozygous at one or more KIR genes, and consequently the frequency with which unrelated humans have the same KIR type is very low (54, 57–59), perhaps approaching that of HLA. Allelic diversity for KIR genes is much higher than for CD94 and the NKG2 family of genes, in terms of both the number of alleles and the differences between them (61). Allelic diversity is generated by point mutation and homologous recombination (54, 57–60), but the relative contribution of these two mechanisms varies between the KIR genes (54). Allelic polymorphism has been described for all the

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KIR genes encoding inhibitory HLA class I specific receptors, and it appears most extensive for 3DL1 and 3DL2 (54, 62, 63). The number of differences between KIR alleles is less than for polymorphic HLA class I loci. This property and the relatively small number of allelic sequences determined have limited the application of statistical methods in assessment of a role for natural selection in generating diversity. Although amino acid residues distinguishing KIR allotypes are distributed throughout the primary structure, their frequent localization in loops of the Ig-like domains is consistent with selection of polymorphisms that contribute to direct interaction with ligands or other molecules (54, 64, 65). However, the functional consequences of KIR polymorphism, for example, in modulating the strength or specificity of binding to MHC class I ligands have yet to be determined.

PHYLOGENETIC COMPARISON OF KIR The gene and allele diversity in human KIR haplotypes suggest that the KIRgene family has evolved rapidly in comparison to most human genes (25). Also pointing to this conclusion is the distribution of Alu repetitive elements in KIR haplotypes, which reveal a recent expansion of the gene family (38, 47). This expansion could have occurred subsequently to the divergence of primates from other mammalian orders. Comparison of KIR in several primate species (common chimpanzee, pygmy chimpanzee, rhesus monkey) has clearly demonstrated dramatic change in the KIR-gene family over time periods as short as a few million years (48, 61, 66). The most extensive comparison is that of humans and common chimpanzee (61), and it is used here to describe principles emerging from all such comparisons. Three KIR lineages are conserved in humans and chimpanzees, showing that the lineages predate divergence of the two species from a common ancestor (Figure 2) (61). The three lineages comprise: 2DL4 and 2DL5 receptors with D0-D2 domains (lineage 1); KIR3D specific for MHC-A and B allotypes (lineage 2); KIR recognizing MHC-C allotypes (lineage 3). In addition, the divergent KIRC1 is also found in the two species (61). Lineage 1 is the most conserved phylogenetically; 2DL4 and 2DL5 constitute two of the three orthologous genes in these species. Conservation of 2DL4 correlates with trans-species conservation of HLA-G (67), its ligand in humans (68, 69). In addition, the putative signaling motifs of 2DL4 provide an example of convergent evolution. In humans and both chimpanzee species, but not macaques (66), the cytoplasmic tail of 2DL4 has a single ITIM, the second one having lost its critical tyrosine through mutation. This property, combined with the presence of an arginine in the transmembrane region, could prevent SHP-1 binding and render 2DL4 an activating receptor (44). Surprisingly, different ITIMs are mutated in human (N-terminal) and chimpanzee (C-terminal) 2DL4 (48, 61, 70, 71). Furthermore, a common human 2DL4 variant has a short cytoplasmic tail with no ITIMs due to a frameshift (72). Thus, the three mutations were independently selected: either to evolve a new, and possibly

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similar, function for human and chimpanzee 2DL4, or to eliminate a function that was no longer useful. The second lineage shared by humans and common chimpanzees comprises KIR3D specific for MHC-A and B. Humans have two KIR of this type, 3DL1 and 3DL2, whereas chimpanzees have a single receptor that shares sequence motifs with both (thus named 3DL1/2) and recognizes MHC-A and B allotypes of the two species (61). KIR recognizing MHC-C in humans (2DL1–3) and common chimpanzees (2DL6, 3DL4) belong to the third lineage, which is apparently specific to hominoids (Figure 2), as is the MHC-C gene (73, 74). This heterogeneous KIR lineage includes both KIR3D and type 1 KIR2D, the latter having evolved from a KIR3D ancestor by functional inactivation of the exon encoding the D0 domain (39, 40). The final stage of this evolutionary trend is seen in the human species, where all KIR recognizing HLA-C and their paralogs have nonfunctional pseudoexons 3 (39); the only remnant of the KIR3D ancestor is the KIR48/KIRX gene fragment (Figure 2). In the common chimpanzee, this lineage has two KIR with pseudoexons 3 (including 2DS4, the single other ortholog of a human KIR gene) and several genes encoding KIR3D (61). Loss of the D0 domain could relate to the lower expression of HLA-C than of HLA-A or -B (75, 76), and the loss could give more sensitive detection of cells in which HLA-C has undergone selective downregulation (39). Thus, both HLA-C and its specific receptors could have co-evolved to achieve a specialized function in NK-cell-mediated defense. In summary, comparison of KIR recognizing MHC-A, -B, and -C molecules between humans and common chimpanzees reveals conservation of functions, which contrasts with extensive species-specific evolution of the receptors exerting these functions. The mechanisms responsible for this divergence, which include gene duplication, recombination, point mutation, and inactivation of functional regions, have operated rapidly, perhaps at rates comparable to or even greater than associated with MHC class I genes (61). Extending the comparison of KIR genes to other primates (Figure 2) reveals that only 2DL4 has orthologs in all species studied; the phylogenetic relationship of rhesus 2DL5 to its homonyms with D0-D2 domains of other primates is less well supported. Thus, rhesus 2DL5 may represent a paralog of hominoid 2DL5. Of note, MHC-G is a pseudogene in the rhesus monkey (79, 80), but the Mamu-AG gene of this species, related genetically to its MHC-A, has structural features and expression patterns similar to HLA-G (81), making it of interest in determining whether Mamu-AG is a ligand for 2DL4. Genes encoding KIR with 3 Ig-domains, though diversified in several lineages (Figure 2), are also found in all species. Thus, from the known specificities of these two KIR families in humans and common chimpanzees, separate recognition of classical and nonclassical MHC molecules seems fundamental in the KIR system. Bonobos exemplify this best, since some animals have minimal haplotypes containing just 2DL4 and a KIR3D gene besides KIRC1 (48). It is also a constant that KIR3D and type 1 KIR2D are more diverse than KIR with D0-D2 domains, as are their respective ligands, which suggests a

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cause-and-effect relationship between the polymorphisms of KIR and MHC, or the existence of a common force driving the evolution of both. KIR with similar extracellular domains but opposite signaling motifs coexist in all primate species studied (48, 61, 66), further supporting the hypothesis that activating KIR have important, but as yet unknown, roles in NK-cell function. The genes encoding activating KIR have expanded and diversified to greatest extent in humans (20, 21, 82, 83). Despite the diversity of KIR haplotypes, all humans have at least one gene for an activating KIR (51), with some donors bearing up to six receptors of this kind. In contrast, some common and pygmy chimpanzees lack short-tailed KIR without obvious impairment of their health and fecundity (48, 61). This could mean that activating KIR exert a redundant or accessory function in these species but, also, that they serve for defense against pathogens not encountered in captivity, or that additional activating KIR await discovery in these animals.

STRUCTURAL BASIS OF KIR-HLA RECOGNITION The structural heterogeneity of KIR and their specificity for polymorphic determinants of HLA class I molecules were established from study of NK cell cytotoxicity. In these analyses the susceptibility of allogeneic targets, and, later on, HLA class I– deficient cell lines transfected with single class I alleles, were tested against NKcell clones and KIR-transfected cells (25, 84–87). More recently, chimeras containing the extracellular portion of KIR and the activating cytoplasmic tails of the FcεRI γ -chain (88) or CD3ζ (89) have been used to overcome the technical difficulty of studying inhibitory signals. Further refinement in analysis of KIR-HLA interaction was achieved by synthesizing soluble forms of the receptor or its ligand. First, fusion proteins comprising the extracellular portion of KIR and the Fc of human IgG1 were used in flow cytometry to study cells expressing different HLA class I molecules (87, 90, 91). Direct interaction between recombinant KIR and HLA molecules was subsequently demonstrated by native gel electrophoresis, which revealed the basic 1:1 stoichiometry of KIR-HLA complexes (92). This technique was also used to study the specificity of KIR-HLA binding and the influence on it of other molecules (e.g., bivalent cations, antigenic peptides) (92–95). More recently, surface plasmon resonance (SPR) methodology has permitted quantitative assessment of KIRHLA interactions (96–99). The kinetics and thermodynamics of their binding have been measured by SPR, as well as the specificity and peptide-dependence of the interaction. The affinity of KIR for HLA-C is in the range shown by TCR for specific peptide-MHC complexes, but the kinetics of KIR-HLA-C binding and detachment are much faster. The fast on and off rates may facilitate progressive NK cell surveillance of MHC class I expression on a succession of potential target cells. Most recently, X-ray diffraction studies of crystallized 2DL2-Cw3 and 2DL1Cw4 complexes (65, 98), preceded by others on crystals of 2DL1–3 alone (64, 100,

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101), have given three-dimensional pictures of HLA-C being recognized by inhibitory KIR. In several aspects of their three-dimensional structure KIR2DL1-3 resemble hemopoietic receptors. Most importantly, an acute angle between the D1 and D2 domains creates an elbow that constitutes the interface with HLA-C. This angle ranges from 55◦ to 84◦ , depending on the KIR and the method used to interpret raw data (64, 65, 98, 100, 101). Two loops of D1, three of D2, and the loop connecting the two Ig-like domains contribute to the interaction. All six loops bear negatively charged glutamate or aspartate residues that face a positively charged surface on HLA-C (Figure 3). The part of the HLA-C surface that becomes buried by interaction with KIR2DL1–2 includes the C-terminal end of the alpha-1 domain helix, the N-terminal end of the alpha-2 domain helix, and the C-terminal residues of the peptide. The shape complementarity of KIR-HLA-C surfaces, as assessed by the median shape correlation statistics value, is similar or greater than those of antigen-antibody or MHC-TCR complexes (65).

Recognition of HLA-C by KIR2DL1–3 MOLECULAR BASIS Much of the research to understand KIR-HLA interaction has concentrated on inhibitory KIR that recognize HLA-C. The specific recognition of HLA-C alleles with either lysine or asparagine in position 80 by 2DL1 and 2DL2-3 (84–86, 102), respectively, has been basically confirmed by studies with recombinant receptors and ligands (87, 90–92, 96, 103, 104). Nonetheless the latter studies have repeatedly shown weak to moderate cross-reactivity of 2DL1–3 with HLA-C alleles bearing the “wrong” epitope, an effect that depends partially on bound peptides (93, 97, 104). A few of these cross-reactions have been confirmed by cytotoxicity studies (104), but their relevance in vivo is unclear. The opposite phenomenon, failures in detecting expected interactions (such as that of Cw8 or Cw1 with 2DL2 and 2DL3), are less consistent and, in part, seem the result of a lower sensitivity of flow cytometry with KIR-Fc constructs in comparison with cytotoxicity assays (90, 104).

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− → Figure 3 (a) Amino acid sequence alignment of the regions participating in contacts in the 2DL2:Cw∗ 0304 and 2DL1:Cw∗ 0401 complexes (65, 98). The basis for the locus specificity of KIR2D is illustrated by the amino acid sequences of a typical Bw6 allele (B∗ 0702), the recombinant HLA-C/HLA-B allele B∗ 4601 and A∗ 0201. Contact amino acids in HLA-C and KIR2D are shaded. (b) Conserved and variable contacts in the KIR:HLA-C interface. Residues of Cw∗ 0304:GAVDPLLAL and Cw∗ 0401:QYDDAVYKL that participate in contacts with 2DL1 or 2DL2 are paired with their contacting amino acid(s). KIR residues are labeled according to the type of interaction they establish: salt bridges (bold), hydrogen bonds (underlined) or hydrophobic contacts (italics); other polar interactions (65) are not shown; amino acids implicated in more than one type of interaction are labeled with a combination of styles; for simplification, all HLA-C amino acids are in plain style. Residues differing between 2DL1 and 2DL2 are marked with asterisks.


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Site-directed mutagenesis experiments and crystallographic analysis have clarified how KIR2D recognize HLA-C allotypes. Allospecificity is mostly determined by dimorphisms of KIR-residue 44 (2DL1Met44/2DL2–3Lys44) and amino acid 80 of HLA-C (Lys/Asn) (103–105), which establish direct interactions with each other (Figure 3) (65, 98). All other KIR-contact amino acids of Cw3 and Cw4 are completely conserved among HLA-C allotypes. However, the binding of 2DL2 to Cw3 and 2DL1 to Cw4 are based on largely different interactions (Figure 3a), which result in Lys80 of Cw4 being accommodated by a cavity of 2DL1 that is not seen in 2DL2 (65, 98). The nature of KIR-HLA-C contacts explains the profound effects of mutations affecting KIR2DL1-3 residues 44, 45, 68, 70, 105, 106, 135 and 183 (89, 91, 98, 103, 104). HLA-C residue 77, for which the Ser/Asn dimorphism is in strong linkage disequilibrium with that of residue 80, does not contribute directly to KIR recognition, as expected from the results of mutagenesis experiments (105). In contrast, the suggested influence of two additional HLA-C dimorphisms (Ala/Thr73 and Ala/Asp90) in the strength of binding to 2DL1–3 (106) cannot be explained by the crystallographic data. Furthermore, Cw∗ 1503, one of few natural alleles encoding the Ala73-Ala90 combination suggested to abolish KIR interaction (106), is recognized by both 2DL1-Fc and 2DL2-Fc constructs (104). Further research should establish definitively whether polymorphic motifs other than Asn/Lys80 can modulate recognition of HLA-C alleles by KIR2D. KIR recognizing HLA-CAsn80 do not cross-react with Bw6-positive HLA-B allotypes, although they have the same amino acid sequence at positions 77–80 as the HLA-C ligands (104). The locus-specificity of 2DL2 and -3 is determined, at least partly, by amino acid Val76 of HLA-C (Glu in HLA-B) because these KIR recognize B∗ 4601, an exceptional HLA-B allotype carrying Val76 (104, 107) (Figure 3a). Since Val76 and neighboring amino acids establish several hydrophobic contacts, the negative charge of HLA-B Glu76 would tend to disrupt these and thus change or destabilize the structure. Arginine 69 of Cw3 can also contribute to locus-specific recognition because it establishes hydrophilic interactions with Glu21 of 2DL2; HLA-B allotypes have nonpolar amino acids (Ala or Thr) at position 69 (Figure 3b). Lack of KIR2D cross-reactivity with HLA-A is explained by the fact that all allotypes of this locus bear three or more nonconservative changes in the contact residues: positions 69, 76, 79, 80, 145, and 149–151 (Figure 3a) (108). THE ROLE OF PEPTIDE The observation that certain antigenic peptides favor or prevent KIR recognition of HLA class I (86) raised the possibility that NK cells might discriminate between cells presenting self and foreign peptides. More detailed analysis showed that KIR recognition is indeed influenced by the peptide bound to HLA, but that there is no truly peptide-specific recognition, since KIR can bind to a same HLA molecule carrying rather different peptides (109–111). According to experiments with synthetic peptides, residue Ä–1 (P8 in nonamers) is most critical for recognition of HLA-B and HLA-C (98, 109, 110, 112–114). In the 2DL2:Cw3 complex, steric hindrance limits the peptide repertoire to amino acids with small side chains at position Ä–1 (98). In the 2DL1:Cw4 interface, P8

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forms no contact with the KIR; the electronegative surface of 2DL1 seems to repel peptides with acidic residues in that position (65). Of note, the influence of peptides on KIR interaction varies for different HLA-C molecules (65, 93, 98), which may contribute to the variable recognition of allotypes sharing the same nominal specificity according to the residue at position 80 (104). The concept that emerges from the cited experiments is that peptides can be either permissive or prohibitive to KIR recognition of HLA. Despite the lack of peptide specificity in the KIR:HLA interaction, it is still relevant to ask whether peptides can influence NK cell recognition of infected cells. A priori it might seem unlikely that peptides provided by an intracellular pathogen are predominantly of a type that prevent recognition of HLA molecules. However, transfection of a cell line with a plasmid vector skewed the profile of peptides bound by HLA-B to an extent that prevented engagement of KIR3DL1, thus making the cells susceptible to lysis by NK cells expressing this KIR (114). One should perhaps wonder whether the basal set of peptides presented by healthy cells contains a proportion of permissive peptides tuned just above the threshold that permits inhibition of NK cells; even subtle changes in that pool might then trigger NK cell activation. This could be particularly relevant for HLA-C molecules, which are expressed at one tenth to one third the level of HLA-A and B (75, 76, 115) and are therefore closer to the threshold necessary for NK-cell inhibition. FROM HLA-C RECOGNITION TO NK CELL INHIBITION. THE ROLE OF BIVALENT CATIONS AND KIR AGGREGATION The mechanism by which HLA-C binding triggers phos-

phorylation of KIR ITIMs has yet to be identified. The angle between the D1 and D2 domains of 2DL1–2 seems to vary slightly upon HLA-C binding [1◦ –11◦ depending on the report (64, 65, 98, 100)]. Unknown is whether this variation induces a conformational change in the cytoplasmic tail that facilitates tyrosine phosphorylation. There is more experimental evidence for HLA-C inducing a KIR-aggregation process mediated by bivalent metallic cations: ■

■

■

■

Clustering of KIR on cell surfaces follows recognition of a protective HLAC molecule on a target cell, a process that is blocked by addition of zincchelating agents (116). Inhibitory function of 2DL1 is impaired by mutation at a Zn-binding motif of its N-terminal end or by addition of Zn-chelating agents, but its binding to HLA-C is unaffected by either manipulation (88). Zn2+ and other bivalent cations, including Cu2+ and Co2+, induce homo- or hetero-aggregation of recombinant KIR2D (95, 117). Soluble dimers of KIR2DL1, either engineered or Co2+-induced, bind more strongly to HLA-C than the monomer, which could be due either to higher affinity or avidity caused by proximity of twin binding sites (94, 95).

These findings provide evidence for KIR2D aggregation in the cell membrane after recognition of HLA-C. Two types of KIR2D homoaggregates have been

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proposed as possible signaling mediators: binding of two molecules through their D2 domains, which would bring together their cytoplasmic tails; and higher order aggregates in which the N-terminal ends would be linked by a bivalent cation (95). The first model is appealing because of the structural similarity of KIR to hematopoietic receptors (100), which themselves form homo- or heterodimers. However, although crystallography has revealed KIR-KIR and KIR-HLA-C interactions different from the one believed to be physiological in ligand binding (64, 65, 98), none of them resembles the proposed aggregates nor seems capable of contributing to a higher affinity receptor or bringing together the cytoplasmic tails of several KIR. On the other hand, since KIR crystals were formed in the absence of zinc, or other bivalent cations, complexes of KIR bound through their N-terminal end were unlikely to have been seen. The Zn-binding capacity of KIR2D was inferred from the presence of an HEGVH motif in the N-terminal sequence (118). This sequence fits the zincbinding motif of neutral metallopeptidases, in which the two histidines coordinate with zinc and the glutamate is an essential part of the catalytic site (PROSITEDatabase of protein families and domains: http://www.expasy.ch/cgi-bin/nicedoc. pl?PDOC00129). In contrast, only His1 of KIR seems essential for Zn-binding, consistent with the cation bridging two KIR molecules through their N-terminal histidines (88, 95). Although Glu2 would in this model be unnecessary for metalinduced homodimerization (95), it is conserved in all inhibitory human and chimpanzee KIR with D1-D2 configuration, but not in KIR3D (43, 61). Further exploration is needed to see if Glu2 is necessary in other steps of KIR2D function. For example, mutation in Glu2 might reveal unsuspected interactions or even show that KIR2D can enzymatically degrade proteins involved in regulating NK-cell activity.

Specificity of Activating KIR2D The binding of HLA-C to KIR2DS1 and -2, although not quantified, is clearly weaker than to their inhibitory counterparts 2DL1–3 (91, 104, 119). Nonetheless, binding specificity of KIR2DS1 for Lys80 HLA-C and KIR2DS2 for Asn80 HLAC allotypes could be reproduced in flow cytometric assays using Fc constructs of 2DS1 (91) and a 2DS2Y45F mutant with enhanced affinity (104). All 2DL1 residues that contact Cw∗ 0401 are conserved in 2DS1 (Figure 4), but the nonconservative substitution of Lys for Thr70 seems to determine the lower affinity of the latter receptor (91). Similarly, only a Phe-to-Tyr change distinguishes the interacting loops of 2DL2 from those of 2DS2, but swapping this residue enhances the affinity of 2DS2 for HLA-CAsn80 allotypes (104, 119). Among the other human KIR2D with activating character, 2DS3 and 2DS5 resemble 2DL1 at residues implicated in HLA-C recognition, whereas 2DS4 is more similar to 2DL2 (Figure 4). The actual specificities of 2DS3 and 2DS5 have yet to be investigated, and as their extracellular domains bear one or more substitutions in the loops implicated in HLA-C recognition, reliable prediction

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Figure 4 Activating KIR2D bear nonconservative amino acid substitutions for HLA-C-contacting residues of inhibitory KIR2D (shaded ).

of their specificity is not possible. As for 2DS4, some reports show recognition of certain, but not all, HLA-C allotypes (120–122), while other studies failed to demonstrate interaction with any HLA-A, -B or -C allotypes (93, 104, 119, 123). The existence of more KIR2DS receptors than known ligands challenges current understanding of KIR:HLA interactions and requires further investigation on the specificity and function of these KIR.

The Recognition of HLA-B and HLA-A by KIR3D KIR3DL1 was described as a specific receptor for HLA-B allotypes expressing the serological Bw4 epitope (124–129). This epitope, encoded by amino acids 80–83 in the highly polymorphic alpha-helix of the alpha-1 domain (130), is polymorphic itself: Three sequences (IALR, TALR, TLLR) have been found in positions 80–83 of different HLA-B allotypes recognized by anti-Bw4 sera (Figure 5). (A fourth

Figure 5 Comparison of HLA-B allotypes expressing the Bw4 epitope with HLA-A and HLA-B allotypes not recognized by KIR3DL1. The alignment shows the alpha helices in the regions homologous to those recognized by KIR2D in HLA-C molecules (contact positions of the latter are shaded).

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Bw4 motif, TAAR, only found in the now deleted B∗ 4401 allotype proved to be the result of sequencing error (131). HLA-B alleles encoding the alternative serological Bw6 epitope and the majority of HLA-A molecules have NLRG and TLRG sequences, respectively, in positions 80–83. However, the HLA-A antigens A23 (9), 24 (9), 25 (10), and 32 (19) have a Bw4-associated motif (IALR) (Figure 5) and cross-react with anti-Bw4 antibodies (132). Specific NK-cell recognition of cells expressing the Bw4-positive HLA-B allotypes was demonstrated and shown to correlate with staining by the DX9 and Z27 antibodies, which turned out to recognize KIR3DL1 (124–129, 133). Early reports suggested that NK cells discriminate not only between Bw4 and Bw6 targets, but also among Bw4 alleles having either Thr80 or Ile80 (134, 135); also some NK cells recognize both HLA-A and HLA-B alleles with the IALR motif (134). Unfortunately, possible participation of KIR3DL1 or other MHC-receptors in NK-cell inhibition was not tested in these experiments, which have not been reproduced. KIR3DL1 discrimination of Bw4- and Bw6-allotypes was confirmed subsequently and shown to depend on amino acids Thr/Ile80, Leu82, and Arg83 by site-directed mutagenesis (57, 136, 137). In contrast with previous studies (134), HLA-A allotypes with the IALR motif did not inhibit NK cells expressing 3DL1 (136), suggesting that recognition of additional polymorphic residues outside the 80–83 region of HLA-B (Figure 5) confers locus-specificity to this KIR. As is the case for KIR2D:HLA-C complexes, the interaction of 3DL1 with HLA-B was susceptible to changes in the antigenic peptide repertoire (112–114). On the receptor side, study of deletion mutants containing only D0, D0 + D1, and D1 + D2 domains demonstrated requirement for all three Ig-domains of KIR3DL1 (133). In the context of the three-dimensional structure of KIR2D (64, 65, 98, 100, 101), the dependence of HLA-B recognition on the 3DL1 D0 is intriguing because the structure provides no clues regarding the role of this domain, even if it is assumed that the D1-D2 domains of KIR3D and KIR2D adopt similar spatial conformations. In comparison with the KIR2D:HLA-C interaction, knowledge of KIR3D:HLA-B binding has benefited less from the new methods for studying molecular interactions. Further study is needed to define which 3DL1 residues are essential for Bw4 recognition and how the polymorphisms of the many 3DL1 variants so far identified influence its function (54, 62, 63). The Ig-like domains of 3DS1, the short-tailed allotype of 3DL1, differ by only 6–12 amino acid changes from their 3DL1 counterparts. However, most of them are in loops of the Ig-like domains (54), including ones corresponding to the binding site of KIR2D (Figure 6), and they could therefore affect the binding properties of 3DS1. Indeed, no interaction of 3DS1 with Bw4 HLA-B alleles has been reported. Identification of KIR recognizing HLA-C and HLA-B prompted search for a specific HLA-A receptor. Independent investigations reported KIR3DL2 to be such a receptor. In one study, NK-cell clones expressing 3DL2 were inhibited by targets expressing HLA-A3 and A11, whereas targets expressing HLA-A1 or A2 were killed (129). In another study HLA-A3 was again shown to be a 3DL2 ligand, both through inhibition of cytotoxicity and using a KIR-Fc construct. In

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Figure 6 Amino acid sequence alignment of human KIR3DL1 (L41269), KIRDS1 (L76661), and KIR3DL2 (L41270), and common chimpanzee KIR3DL1/2 (AF258798) in the regions homologous to the ligand-interacting loops of 2DL1 and 2DL2. Numbering refers to human KIR3D.

contrast with the first study, HLA-A11 seemed not to be recognized by 3DL2, and neither were A2, A68(28), A24(9), A31(19), and A33(19) (138). An inhibitory effect of HLA-A3 compared to -A2 mirrored the results of earlier work performed before identification of killer-cell receptors for HLA (139, 140). Although some investigations failed to observe recognition of HLA-A3 by 3DL2 (57), further evidence that HLA-A allotypes are recognized by KIR3D came from study of the common chimpanzee receptor KIR3DL1/2. As mentioned above, this receptor shares sequence motifs with the extracellular domains of human 3DL1and 3DL2 (Figure 6) and recognizes MHC-A, as well as MHC-B molecules of both humans and apes (61). Inconsistency in demonstrating 3DL2:HLA-A interactions might reflect a lower affinity than those other KIR:HLA pairs. In any case, the actual ligand of KIR3DL2 is currently a matter of controversy that needs to be resolved.

The Recognition of HLA-G by KIR2DL4 Inhibition of NK cells by HLA-G presents an attractive hypothesis to explain maternal tolerance of the trophoblast, a tissue that lacks HLA-A and HLA-B: expressing only HLA-C and the nonclassical class I molecules HLA-G and HLAE (141–143). There is agreement that HLA-G can directly inhibit NK cells as a ligand for the ILT-2 receptor (23, 144, 145) and through recognition of its leader peptide in complex with HLA-E by CD94:NKG2A (17–19). By contrast, the role of KIR in recognizing HLA-G is confused, with virtually every inhibitory KIR having been at some time proposed to be an HLA-G receptor (120, 146, 147). On the basis of the evidence currently available the best candidate is KIR2DL4 (69, 71, 148). In flow cytometry HLA-G transfected cells exhibited moderately brighter staining with a 2DL4-Fc construct than did other HLA class I transfectants (71). A more specific binding of 2DL4-Fc to HLA-G was shown by Rajagopalan & Long but “nonspecific binding” to other HLA class I molecules was also consistently detected (69). These authors reasoned that the D0 domain of 2DL4 was responsible for the nonspecific binding, because other constructs containing D0 domains had similar nonspecific binding (69). Contrasting with these results, an HLA-G tetramer

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failed to bind to NK cells (144), which ubiquitously express 2DL4. Of several interpretations of this result, one possibility is that 2DL4 protein is not present on the cell surface of peripheral blood NK cells as reported in one study (148), but not in another (69). KIR2DL4 was proposed to mediate HLA-G-induced inhibition of decidual NK-cells, since protection by HLA-G was abrogated by an antiserum raised against 2DL4 (148). NK92 cells infected with vaccinia-2DL4 also appeared to be inhibited by HLA-G, but these experiments were later shown to lack a negative control, since mock infection produced occasionally the same effect (69). A further complication is the suggestion that 2DL4 activates, rather than inhibits, NK cells (44). Recognition of nonclassical, rather than classical MHC class I molecules is consistent with 2DL4’s divergent structure compared to KIR recognizing HLA-A, -B, and -C (70, 71). Unlike these KIR, 2DL4 is phylogenetically conserved in humans and Old World primates (Figure 3), fitting well with recognition of conserved, nonpolymorphic class I. However, what remains obscure is the physiological function of interaction between HLA-G and 2DL4. The recently reported KIR2DL5 gene is structurally related to 2DL4 (46) and similarly conserved in primates (Figure 3). These similarities raise the possibility that 2DL5 also recognizes a nonclassical MHC class I molecule. That the amino acid sequences of 2DL4 and 2DL5 are only 80% identical suggests they have different ligands (46).

THE EXPRESSION OF KIR GENES A diverse NK cell repertoire in which each cell expresses a subset of KIR is itself evidence for a model in which these receptors survey for abnormal and selective downregulation of HLA class I allotypes. With few exceptions, all KIR genes in the genome of every individual are transcribed in his/her polyclonal NK cell population (51). However, individual NK-cell clones express only some of the KIR genes, in apparently stochastic combinations that are regulated mainly at the transcriptional level (57) and stably maintained (140, 149, 150). Randomness in the activation of KIR genes is supported by their combinatorial frequencies (57), which fit a “product rule” (151). Similarly for alleles, individual NK cells from the same donor have been shown to express one, two, or no KIR3DL1 alleles, there being no allelic exclusion (54). Correlation of NK cell patterns of KIR expression with KIR and HLA genotype in siblings shows that the KIR repertoire is largely determined by the KIR genotype and that the modifying effect of HLA genotype is comparatively small (H. G. Shilling, L. A. Guethlein, N. W. Cheng, C. M. Gardiner, D. Tyan, and P. Parham, submitted for publication). This situation contrasts with the αβ T cell receptor repertoire, which is strongly biased as a result of thymic selection by autologous MHC type. The apparent weakness of the selection can in large part be attributed to the fact that CD94:NKG2A can also serve as an inhibitory receptor

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for self-MHC class I. The evidence suggests CD94:NKG2A expression is being regulated so that it fills the holes in the KIR repertoire, i.e., it is expressed on those cells that do not express an inhibitory KIR with specificity for autologous HLA class I. Fitting with this interpretation is the inverse correlation seen between the proportions of total NK cells expressing KIR or CD94 (57; H. G. Shilling, L. A. Guethlein, N. W. Cheng, C. M. Gardiner, D. Tyan, and P. Parham, submitted for publication). Common principles seem to apply to the expression of most KIR genes, regardless of whether they encode inhibitory or activating receptors. KIR2DL4 is exceptional in being expressed ubiquitously, whereas all other KIR are expressed in clonotypic manner (57, 68, 69); controversial is whether the 2DL4 protein is also expressed ubiquitously (69) or restricted to decidual NK cells (68). Dissociation between transcription and protein expression has occasionally been reported for other KIR genes (152). There are also exceptions to the rule that KIR genes are expressed by at least some NK cells in every donor who has the gene: 2DL5 is silent in some individuals (42) and KIRC1 is generally not transcribed (45, 46), although a cDNA sequence was recently reported (GenBank AF352324). Patterns of KIR gene expression correlate with sequence variation in their promoter regions (42, 47), and the lack of expression of some genes appears intrinsic, not a result of downregulation. KIR-gene expression in T lymphocytes and NK cells seems governed by similar rules. One key difference is that T-lymphocytes acquire KIR after having participated in an immune response; activation of KIR genes takes place after rearrangement of the Tcr genes, possibly during transition to becoming memory cells, a process to which KIR may contribute (152–156). Since KIR inhibit the effector functions of T-cells (157), an important issue is whether and how memory T-lymphocytes are activated by a renewed contact with antigen after they express KIR. Requirement for a strong stimulus to overcome KIR-mediated inhibition seems antagonistic to effective memory response. Whereas KIR expression appears to be as stable in T-lymphocytes as in NK cells (149, 150, 154), one report described progressive downregulation of T-cell KIR in the absence of stimulation through the TCR (34). The molecular mechanisms that regulate expression and cell distribution of KIR remain unknown. Comparison of the promoter sequences of transcribed and silent variants of the KIR2DL5 gene points to consensus binding sites for AML1 and Ets-1 as necessary for KIR transcription (42). In addition, TCF-1 has been proposed to participate in the stochastic activation of some Ly49 genes (158); the core sequence of the TCF-1 binding site [(C/A)A(C/A)AG] appears in the promoter of several ILT (159) and KIR genes (42), making possible the participation of TCF-1 in their regulation. Comparative promoter analysis of expressed and silent KIR genes and variants should identify which cis- and trans-acting elements are essential for stochastic and tissue-specific activation of KIR genes. Because of the convergence of Ly49 and KIR to analogous functions and mode of expression, despite considerable structural difference (151), parallel study of both systems is

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essential for appreciation of the mechanisms that have produced such surprising convergence.


KIR DIVERSITY: WHAT IS IT GOOD FOR? The variability of the KIR system in human populations raises the obvious question: What is it good for? The most extreme aspect of KIR variability is that particular genes can be absent from a person’s genome. In actual fact, only 3DL2, 2DL4, and KIRC1 are ubiquitously present in KIR haplotypes and are thus framework genes of the cluster (49). Of these genes, KIRC1 is silent in most humans (45, 46). For 2DL4, there is uncertainty regarding its signaling potential (44, 69, 72) and even on its membrane expression in nondecidual NK cells (68). Furthermore, an adult fertile woman appears to lack a 2DL4 gene (N. Gómez-Lozano, R. de Pablo, C. Vilches, unpublished), and embryos homozygous for HLA-G1 defects develop normally (160), although products derived from alternatively processed mRNA of these mutants have been suggested to interact with 2DL4 (161). The single conserved inhibitory KIR could thus be 3DL2, one for which most humans seem not to have a ligand. If the polymorphism of both the HLA system and the KIR-gene complex are considered together, inactivation of KIR:HLA pairs appears a common natural phenomenon (Table 3). However, although no particular KIR seems essential for survival until reproductive age, no deficiencies affecting the expression of all KIR genes have been reported. It is unlikely that having more KIR genes confers a

TABLE 3 Natural “knock-outs” of KIR genes and KIR-HLA pairs KIR:HLA pair

Inactivating event

2DL1:HLA-CLys80

HLA-CAsn80 homozygotes (common) Lack of 2DL1 gene (uncommon)

2DL2,3:HLA-CAsn80

HLA-CLys80 homozygotes (common) No known examples of negatives for both 2DL2 and 2DL3

3DL1:HLA-BBw4

Bw6 homozygotes (common) 3DS1 homozygotes (uncommon)

3DL2:HLA-A3,others?

Combinations of other HLA-A alleles (most humans) No known examples of negatives for 3DL2

2DL4:HLA-G

2DL4 variants without known signaling motifs (common) HLA-G mutations (rare) Lack of 2DL4 gene (rare)

2DL5:unknown ligand

Lack of the gene or lack of expression (common)

3DS1, 2DS1-5:HLA-C or unknown ligands

Lack of individual genes (common) Mutation of DAP12 (rare)

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strong evolutionary advantage in the long term, since haplotypes bearing fewer KIR genes are as frequent as those with a higher number (51). However, the presence of different numbers and qualities of KIR genes in the genome likely influences the NK-cell mediated immune response. For instance, four inhibitory KIR:HLA pairs would function in individuals having a KIR ‘A’ haplotype whose HLA phenotype included A3, a Bw4+ HLA-B allotype, and both Asn80 and Lys80 in their HLA-C allotypes. On the contrary, donors homozygous for the KIR ‘B’ haplotype shown in Table 2 could have no single functional inhibitory KIR:HLA pair if they were homozygous for HLAC-Lys80 and lacked HLA-A3. These people could still use the CD94:NKG2A system for sensing levels of HLA class I expression, but it is conceivable that their NK cells would be less able to defend against pathogens inducing selective downregulation of certain HLA molecules, or that the use of different receptors confers qualitative differences to their response against pathogens. It has been argued in the latter direction that the faster progression of HIV infection in B35 individuals may be related to the Bw6 condition of this antigen (31); the rationale for this proposal is that the downregulation of HLA-B by HIVNef would not be detected by NK cells of an individual who is homozygous for Bw6. More recently, Bw6 homozygosity has indeed been proposed to correlate with a prognosis for a person with HIV infection (162). In comparison to other species, an intriguing aspect of the human KIR cluster is the expansion of genes encoding activating KIR; some donors have up to six genes of this type (A/B genotypes). As discussed above, convergent evolution has produced multiple activating versions of KIR and other MHC class I receptors, which suggests they have an essential, but as yet unknown, role. Activating KIR have been suggested to participate in the lysis of HSV-infected cells (36) and in the pathogenesis of rheumatoid arthritis (163, 164). Paradoxically, no obvious impairment in number or phenotype of NK-cells or T-cells has been observed in patients homozygous for mutations affecting the DAP12 gene (165). In contrast, both NK-cell phenotype and function, and T-lymphocyte-mediated, antigen-specific responses, are altered in DAP12-targeted mice (166). Furthermore, the activating Ly49H receptor confers resistance to potentially lethal infection by murine CMV (167, 168), which constitutes the strongest evidence for the relevance of activating MHC class I– specific receptors in immunity. Activating KIR might participate in NK-cell function in two ways: first, by being truly specific for HLA; second, by recognizing HLA class I-cross-reactive molecules encoded, induced, or modified by pathogens. Some viral proteins indeed mimic MHC class I (169) or mask cellular MHC class I–homologues that activate NK cells (9). Similar proteins could be recognized by activating KIR and still be beneficial to the pathogen by favoring a balance that permits the host to survive and, hence, the virus to continue its life cycle. If, on the other hand, the more parsimonious possibility is assumed of activating KIR being specific for HLA class I, these receptors could complement the role of inhibitory KIR in surveillance against selective downregulation of MHC class I; altered cells that lose some

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fraction of their HLA class I molecules could activate NK cells co-expressing inhibitor and activating receptors with different HLA class I specificities even in the absence of any other marker of transformation. Since T-lymphocytes express inhibitory and activating KIR (120, 157, 163, 164, 170) and NK cells secrete immunomodulatory factors (2), antigen-specific immune responses could vary among humans bearing dissimilar KIR receptors. KIR might achieve this by raising or lowering the activation threshold of T-lymphocytes, promoting termination or continuation of immune responses, favoring or hindering presentation of antigens by particular HLA loci due to competition with TCR for overlapping surfaces of MHC, thereby skewing decision-making (tolerance/activation, effector/memory/apoptosis, Th1/Th2) by T-lymphocytes. In summary, KIR diversity has the potential to contribute variability in both innate and adaptive immunity. Availability of simple methods to study KIR genes makes it likely that the influence of their diversity on the immune response will be analyzed in the short term by means of epidemiological studies. These should illustrate whether KIR polymorphisms behave as factors of susceptibility or protection that influence the response to infections, malignancy, autoimmune and inflammatory diseases, and transplanted tissue. ACKNOWLEDGMENT The unpublished research by N. Gómez-Lozano and C. Vilches that is referred to here is supported by grant FIS 01/0381 from the Instituto de Salud Carlos III, Spain. Visit the Annual Reviews home page at www.annualreviews.org

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Zafer A, Belouchi A, Gros P, Vidal SM. 2001. Susceptibility to mouse cytomegalovirus is associated with deletion of an activating natural killer cell receptor of the C-type lectin superfamily. Nat. Genet. 28:42–45 169. Cosman D, Fanger N, Borges L. 1999. Human cytomegalovirus, MHC class I

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ORIGINS AND FUNCTIONS OF B-1 CELLS WITH NOTES ON THE ROLE OF CD5 Annu. Rev. Immunol. 2002.20:253-300. Downloaded from arjournals.annualreviews.org by HINARI on 09/01/07. For personal use only.

Robert Berland and Henry H. Wortis Department of Pathology, Tufts University School of Medicine and the Program in Immunology, Sackler School of Graduate Biomedical Sciences, Boston, Massachusetts 02111; e-mail: [email protected], [email protected]

Key Words autoimmunity, tolerance, repertoire, B cells, T independent type 2 (TI-2), antigen ■ Abstract Whether B-1a (CD5+) cells are a distinct lineage derived from committed fetal/neonatal precursors or arise from follicular B-2 cells in response to BCR ligation and other, unknown signals remains controversial. Recent evidence indicates that B-1a cells can derive from adult precursors expressing an appropriate specificity when the (self-) antigen is present. Antibody specificity determines whether a B cell expressing immunoglobulin transgenes has a B-2, B-1a or marginal zone (MZ) phenotype. MZ cells share many phenotypic characteristics of B-1 cells and, like them, appear to develop in response to T independent type 2 antigens. Because fetal-derived B cell progenitors fail to express terminal deoxynucleotidyl transferase (TdT) and for other reasons, they are likely to express a repertoire that allows selection into the B-1a population. As it is selected by self-antigen, the B-1 repertoire tends to be autoreactive. This potentially dangerous repertoire is also useful, as B-1 cells are essential for resistance to several pathogens and they play an important role in mucosal immunity. The CD5 molecule can function as a negative regulator of BCR signaling that may help prevent inappropriate activation of autoreactive B-1a cells.

INTRODUCTION Any one of three types of antigen—thymus independent type 1 (TI-1), thymus independent type 2 (TI-2), or thymus dependent (TD)—can initiate B cell proliferation, differentiation, and ultimately antibody secretion. B cells activated by antigens of different types enter distinct differentiation pathways. Thus, a B cell responding to a TD antigen plus CD40 ligand matures in a germinal center, is able to somatically mutate, and gives rise to memory cells as well as plasma cells. In contrast, a TI-1-responding cell produces IgM, perhaps IgG2b, no memory, and little or no somatic mutation. Thymus independent type 2 antigens induce multivalent cross-linking of the B cell receptor (BCR). This is not sufficient to drive responding B cells into antibody production because additional signals supplied by noncognate interaction 0732-0582/02/0407-0253$14.00

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with T cells, NK cells, and perhaps other cells are necessary (1). Ultimately, TI-2responding B cells mainly produce IgM and some IgG3 antibodies containing few or no somatic mutations. They do not usually generate long-term memory cells (no secondary response) (2, 3). We propose that the phenotype of TI-2 responding cells differs from that of B cells triggered by a TD or innate (TI-1) antigen. We further propose that depending on the circumstances of interaction of the B cell with TI-2 antigen, the responding cells will be found in the spleen as marginal zone (MZ) B cells, in the peritoneal or pleural cavity as B-1a or B-1b cells, or as anergic cells destined for elimination. [Although differing in detail, a model broadly similar to ours was developed independently by Martin & Kearney and published earlier in an elegant review (4).]

Definition of B-1 Cells B-1 cells can be distinguished from all other B cells by surface phenotype. In contrast to recirculating follicular (also B-2 or B-0) cells, they are CD45 (B220lo), IgMhi, CD23−, CD43+, and IgDlo. They are also larger and exhibit more side scatter than do B-2 cells. In the peritoneal cavity (PerC), but not the spleen, B-1 cells express C3 (CD11b, Mac-1). B-1 cells are absent from peripheral lymph nodes (LN) and variably make up about 5% of splenic B cells. However, they constitute a substantial fraction of B cells in the peritoneal and pleural cavities (5, 6). Originally, B-1 cells were identified by their expression of CD5. Subsequently, a population of peritoneal CD5− B cells was identified whose surface phenotype was in other respects identical to that of B-1 cells. By consensus, CD5+ B-1 cells are referred to as B-1a cells and CD5− B-1 cells as B-1b cells. Most of the work reviewed in this article was with B-1a cells or did not distinguish between B-1a and B-1b. Furthermore, it is concerned almost exclusively with B-1a cells from the PerC. B-1a cells from the spleen have not been much studied because of their low frequency. However, they differ from PerC B-1a cells in two important respects. PerC B-1a cells contain constitutively active STAT-3, an inducible transcription factor (6a), while splenic B-1a cells do not (6b). Splenic B-1a cells, unlike those isolated from the PerC, flux calcium normally upon BCR ligation (26). The work reviewed here almost exclusively concerns mice. In humans there exist two classes of CD5+ B cells, only one of which appears to share other phenotypic properties with murine B-1a cells (6c). Like murine B-1a cells, CD5+ human peripheral blood B cells have been reported to produce polyspecific autoreactive antibody (173, 174). However they have not been much studied and it is not clear to what extent they are equivalent to B-1a cells in the mouse. In other species the situation is even more uncertain. CD5+ B cells have not been reported in rat (180) except for a recent report of rats expressing a transgenic neuropeptide Y (6d). In rabbits (6e), sheep (6f), cattle (6g), and chickens (6h), all or most peripheral B cells express CD5.

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Properties of B-1 Cells


In addition to surface phenotype, B-1 cells have a number of distinguishing properties discussed in more detail later in the review. In vitro, they are long lived (7), refractory to activation through B cell receptor (BCR) ligation (8–10), and, in contrast to B-2 cells, induced to proliferate by treatment with phorbol esters (11). The repertoire of B-1 cells is decidedly skewed toward reactivity with common bacterial and self-antigens. Typical B-1 immunoglobulin genes have fewer N insertions than those of most B-2 cells (12), and they usually do not contain somatic mutations (13).

Functions of B-1 Cells The segregation of B cells with a particular repertoire into a population with the B-1 phenotype is presumably of significance to the animal. Some aspects of the B-1 phenotype may assure that weakly autoreactive B cells are not recruited into germinal centers where affinity maturation could result in high-affinity, pathogenic autoreactivity. Other aspects of the phenotype may have been selected, as they enable responses to certain TI-2 antigens or provide natural antibody. We return to these issues after an examination of B-1 development.

ORIGINS OF B-1 CELLS B-1 and B-2 cells were originally proposed to derive from different, committed, precursors and therefore to represent the end products of two distinct lineages. This view was based on cell transfer studies in which fetal liver reconstituted both the B-1 and B-2 compartments of irradiated mice, while adult bone marrow was generally limited to the generation of B-2 cells (14–16). Subsequently, the fetal omentum (17) and paraaortic splanchnopleura (18) were shown to contain precursors exclusively for B-1 cells. Thus, the B-1 lineage appeared to be predominantly of fetal and the B-2 lineage of adult origin. That a fetal-derived B cell population would persist for the life of the animal was explained by additional studies showing that B-1 cells were self-renewing. Transfer of B-1 cells into neonatal (19) or irradiated (20) mice resulted in long-term reconstitution of the B-1 compartment (discussed in 14). According to this lineage model, the progenitors of B cells either have or do not have the potential to become a B-1 cell. We (21) and others (22, 23) have proposed an alternative model to account for the properties of B-1 cells. In this induced-differentiation model, the B-1 phenotype was proposed to be a consequence of TI-2-like activation. This model was based on our observation that BCR cross-linking, in the absence of an innate antigen, T cell help, or CD40 ligation, resulted in the induction of CD5 on splenic B-2 cells (21, 24). It was subsequently shown that BCR cross-linking also induced in B-2 cells the ability to proliferate in response to phorbol ester, another feature of B-1 cells (25). The combination of BCR ligation and IL-6 treatment induced two additional

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features of B-1 cells, the downregulation of CD23 and of IgD (21). However, an in vitro treatment that induces the full B-1 phenotype has not been found, which suggests that additional unknown signals are involved. According to the induced differentiation model, encounters with naturally occurring TI-2 antigens account for the appearance of B-1 cells in vivo. Differences in the ability of precursors from adult and fetal sources to generate B-1 cells are due to differences in the repertoires and hence specificities of B cells derived from these sources. The fetal/neonatal repertoire is skewed toward the expression of immunoglobulins that bind frequently encountered TI-2 antigens. The adult repertoire rarely generates these specificities and therefore generates few B-1 cells. In contrast, the lineage model postulates that differences in the repertoires of B-1 and B-2 cells are a result of antigen-driven selection for survival or expansion. Interestingly, IgM expression per se is not required for B-1a development. Mudeficient mice that can still express IgD have a population of PerC CD5+ B cells that express high levels of surface IgD (25a). This is particularly striking given the fact that B-1 cells normally express only very low levels of IgD. The functional properties of these cells were not examined. We now review evidence, some of it very recent, that we believe is most consistent with the induced differentiation model of B-1 development.

B Cell Receptor Signaling is Essential for B-1 Development The study of gene targeted and transgenic mice has generated strong evidence that BCR signaling is critical for B-1 development. Mutations that disrupt BCR signaling result in substantial depletion of the B-1 subset while largely sparing B-2 cells (see Table 1). Conversely, mutations or transgenes that enhance BCR signaling result in an expanded B-1 compartment (Table 1). These results indicate that B-1 cells require a BCR-generated signal for development, survival, or expansion. If there is a B-1 lineage, the phenotype is not expressed unless there is ligation of the BCR by antigen.

B Cell Receptor Specificity Determines the Likelihood that a Given B Cell Acquires the B-1 Phenotype In mice transgenic for B-1-derived immunoglobulin genes, transgene expressing B cells are predominantly CD5+ (Table 2). In one recent study, such cells were shown to have the B-1 property of enhanced in vitro life span (26). Conversely, in mice carrying B-2-derived transgenes, transgene expressing B cells are almost exclusively of the B-2 phenotype (Table 2). The importance of BCR specificity in the generation of B-1 cells suggests that the requisite BCR signaling is ligand-dependent. Conclusive evidence that B-1 development is dependent on a ligand-driven process comes from recent work of Hayakawa, Hardy, and colleagues (27). They had previously cloned a B-1-derived hybridoma (SM6C10) expressing an anti-T cell antibody (ATA) specific for the T cell glycoprotein Thy-1 (28). When transgenic mice were made that expressed

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TABLE 1 Genetic alterations that affect B-1 cell numbers Alterations that decrease B-1 cell numbers Mutation of positive regulators of BCR signalingPoint mutation (xid) or deletion of btk Deletion of PKCβ Deletion of PLCγ Deletion of P85α of PI-3 Kinase Deletion of CD19 Deletion of BLNK (SLP-65) Deletion of CD21/35 Deletion of vav-1 Mutation of B cell transcription factors Deletion of Oct 2 Deletion of Aiolos Deletion of NFATc

(33, 251, 252) (253) (254, 255) (256, 257) (258, 259) (260–263) (264) (but see 265) (266, 267) (268) (269) (Berland & H. Wortis, unpublished observation)

Mutation of growth factors or growth factor receptors Deletion of IL-5 Deletion of IL-5R

(270) (271, 272)

Other Deletion of Cyclin D2

(273)

Alterations that increase B-1 cell numbers Mutation of negative regulators of BCR signaling Loss of function of SHP-1 (motheaten, motheatenv) Deletion of CD22 Deletion of Lyn Deletion of CD72

(274) (155, 156, but see 153, 154) (151) (275)

Overexpression of positive regulators of BCR signaling CD19 transgenic

(276)

Overexpression of transcription factors Fli-1 transgenic

(277)

Overexpression of growth factors IL-5 transgenic Osteopontin transgenic IL-9 transgenic

(278) (162) (279)

the SM6C10 heavy chain, they had high titers of ATA, all of which was produced by transgene-expressing B-1a cells (27). The majority of these cells expressed an endogenous light chain identical to that in the original SM6C10 hybridoma (27). Thus, as in other transgenic mice, expression of a B-1a immunoglobulin specificity correlated with acquisition of the B-1a phenotype. Strikingly, when SM6C10 µ transgenic mice were crossed onto a Thy-1−/− background, ATA producing B cells

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TABLE 2 Effect of Ig-transgene expression on B cell development

Transgene

Specificity

Source

Phenotype of transgene expressing B cells

3-83

H-2KkDk

Normal Adult Spleen

B-2

(26, 280)

HyHEL-10

Hen Egg Lysozyme

Normal Adult Spleen

B-2

(40, 281)

References

VH81X

Unknown

Normal Fetal Liver

MZ

(109)

M167

Phosphoryl choline

Normal Adult Spleen

MZ

(109)

VH12/Vκ4

Phosphatidyl choline

CD5+ Lymphoma

B-1a

(32)

VH11/Vκ9

Phosphatidyl choline

B-1 cell

B-1a

(26)

SM6C10

thy-1

B-1 cell

B-1a

(27)

4C8

Mouse Red Blood Cells

NZB Spleen

B-1

(119)

2-12H

sM snRNP

MRL/lpr mouse

B-1a

(37)

and transgene expressing B-1 cells were absent (27, 29, 30). Thus, engagement of the BCR by antigen, in this case Thy-1, is necessary for B-1a development. Ligand-mediated BCR signaling could be required to induce the B-1 phenotype or for the survival or expansion of cells already committed to the B-1 lineage. Evidence for the former comes from a series of studies in Steve Clarke’s laboratory. This group has been working with mice carrying B-1-derived rearranged genes encoding the heavy (VH12) and light (Vκ4) chains, which together produce an antibody specific for phosphatidyl choline (PtC). Antibody to PtC is frequently produced by B-1 but not B-2 cells (31). In VH12/Vκ 4 transgenic mice, B cells expressing both transgenes are almost exclusively B-1 (32). In order to determine if B-1 commitment preceded or followed transgene expression, Clarke & Arnold bred the X-linked immune deficiency (xid) mutation into these mice. Xid is a mutation in Bruton’s tyrosine kinase (Btk), a gene which encodes a kinase necessary for BCR signaling. Xid is known to prevent B-1, but not B-2, development (33, 34). As expected, in transgenic xid mice, there were no transgene-expressing B-1 cells (23). Significantly, transgene-expressing B-2 cells (B-0, in the nomenclature of Clarke and colleagues) were now found (23). The most straightforward interpretation of this result is that on a wild-type background, transgene expression in a B-2 (B-0) cell rapidly results in the acquisition of the B-1 phenotype. On an xid background, B-1 differentiation cannot occur because there is no effective BCR signaling, and this allows accumulation of transgene-expressing B-2 (B-0) cells. However, in xid mice most B cells are transitional or immature, not mature. The argument can then be made that this developmental arrest prevents lineage committed B cells from maturing into B-1 cells (35). In a subsequent study, Arnold et al. identified splenic B cells in VH12/Vκ 4 transgenic mice that had a phenotype intermediate between B-1 and B-2 (B-1int) (36).

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B-1 cells are CD5+, CD43+, IgMhi, FSChi, CD45(B220)lo, and CD23−, where low and high refer to staining intensity relative to B-2 cells. B-1int cells are CD5+, CD23int, CD45(B220)int, and FSCint. B-1int cells also have IgM and CD43 levels slightly lower than in B-1 cells. When these intermediate phenoptype cells were transferred into sublethally irradiated mice and analyzed five days later, they had acquired the B-1 phenotype, with the exception of the loss of CD23 expression. This differentiation to B-1 was blocked by cyclosporin A (CSA) treatment, which suggests that it requires BCR signaling (see below) (36). The discovery of B-1int cells that can progress to a fuller B-1 phenotype in a CSA sensitive manner is consistent with the notion that B-1 cells are derived from cells with a B-2 (or B-0) phenotype after BCR ligation. Presumably B-1int cells are detectable in VH12/Vκ4 transgenic mice, but not in nontransgenic mice, because of the much larger number of B cells undergoing differentiation to B-1 in the transgenic mice. Using a different immunoglobulin transgenic model, Clarke’s group has recently shown that splenic B cells with a B-2 phenotype can differentiate into PerC B-1 cells. These mice express an anti-Sm snRNP heavy chain derived from an MRL/lpr mouse. Transgenic, anti-Sm B cells are found in the spleens as transitional, rapidly turning over B-2 cells and in the PerC as B-1a cells. Splenic B-2 cells assume a B-1a phenotype after transfer to sublethally irradiated recipients (37). One important question is the nature of the signals that drive B-1 differentiation. The nature of the B-1 repertoire, enriched as it is for weak autoreactivity, suggests that B-1 cells are usually selected by weak interactions with self-antigens. This is further supported by the example of anti-Thy-1 transgenic mice discussed above. However, when mice expressing transgenes encoding an antibody (3-83) with specificity for H-2Kk were crossed with mice expressing ligands with very low affinity for the 3-83 immunoglobulin (KA ∼ 1 × 104 M−1), the progeny deleted or receptor edited the 3-83+ B cells (38). Thus, as suggested by Chumley et al. (26) either B-1 cells are selected by even weaker autoreactivities, or the signals driving B-1 cell development are of a qualitatively different nature than those leading to tolerance. One possibility is that cells targeted for tolerance by self-reactivity can be rescued by an additional signal or signals that drive them into the B-1 compartment. This is suggested by work from Tim Behren’s laboratory examining tolerance in anti-HEL/sHEL transgenic mice (39). In this system, mice express both an immunoglobulin specific for hen egg lysozyme (HEL) and a gene encoding a soluble form of HEL (sHEL). Anti-HELexpressing B cells become anergic as a result of encountering sHEL (40). Behrens and colleagues recently showed that these anergic cells express CD5, albeit at a level below that seen in typical peritoneal B-1 cells (39). Also, unlike B-1 cells, these anergic cells express low levels of surface IgM (39). Whatever the precise nature of the signals leading to the B-1 phenotype, B-1 development appears rather sensitive to the strength of the signal received from the BCR. This is suggested by two studies showing that in mice transgenic for a B-1 BCR specificity, reduction of BCR surface density, even by a factor of only two, significantly reduces the number of transgene expressing B-1 cells (41, 42).

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Adult Bone Marrow Can Give Rise to B-1 Cells Cell transfer studies clearly indicate that fetal precursors more readily give rise to B-1 cells than do adult precursors. However, examples exist in which B-1 cells were generated after adoptive transfer of adult bone marrow cells. The Herzenberg laboratory reported sporadic generation of PerC B-1 cells after transfer of adult bone marrow to SCID recipients. They attributed this to the possible persistence of fetal precursors in some preparations of adult bone marrow (43). We reported recovery of splenic B-1 cells after transfer of adult (>5 month old) bone marrow into neonatal SCID recipients (44). In this study, reconstituted B-1 cells were shown to have a high frequency of N-insertions, suggesting that they truly were derived from adult precursors (see below). Others, too, have reported that adult bone marrow contains some progenitors for B-1 cells (45–47). Finally, Whitmore et al. reported that transferred bone marrow (but not PerC cells) gave rise to cells that responded to the TI-2 antigen polyvinyl pyrrolidinone (PVP) after immunization, and that the anti-PVP antibody-producing cells had acquired the B-1 phenotype (48). These workers also demonstrated the (likely) presence of N-insertions in 10 of 16 hybridomas derived from these anti-PVP producing cells. Despite these results, the ability of adult precursors to give rise to B-1 cells remained in question (see for example 49). These doubts were in part based on studies showing that purified BM pro-B cells that had begun but not yet completed heavy chain rearrangement could give rise to B-2 but not B-1 cells after transfer into SCID recipients (50). Conversely, pro-B cells at the same developmental stage isolated from day-16 fetal liver gave rise predominantly to B-1 cells. Similar results were obtained when pro-B cells were allowed to differentiate in vitro in stromal cell cultures (50). However, a recent study indicates that the ability to generate B-1 cells is not uniquely a property of fetal precursors. In this study, mice were made transgenic for VH11/Vκ 9, a B-1-derived anti-PtC specificity. As in other studies (Table 2), expression of these B-1-derived transgenes resulted in B cells with the B-1 phenotype (26). Significantly, CD45(B220)−/CD19− precursors isolated from the bone marrow of adult VH11/Vκ 9 transgenic mice exclusively gave rise to B-1 cells when transferred into irradiated recipients or placed in culture with IL-7. These cells had the surface phenotype and enhanced ability to survive in culture typical of B-1 cells. In contrast, equivalent cells isolated from the bone marrow of mice transgenic for 3-83, a B-2 specificity, gave rise exclusively to B-2 cells, both in vivo and in vitro. This study would seem to demonstrate beyond any doubt that adult BM precursors, when expressing an appropriate specificity, differentiate into CD5+ long-lived cells, i.e., B-1 cells.

Fetal vs. Adult B Cell Development The fact remains that fetal precursors more readily give rise to B-1 cells than do adult precursors. Based on the studies discussed above, this most likely reflects differences in repertoire between fetal and adult-derived B cells. That such differences exist is well established. Pro- and pre-B cells in the fetal liver rearrange

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their heavy chains with the preferential use of J-proximal V genes (51). V gene usage in adults is approximately random, that is, the frequency of use of genes from a given VH family is proportional to the number of VH genes in the family (52). In adults, terminal deoxynucleotide transferase (TdT) makes nontemplated nucleotide insertions (N insertions) during VDJ rearrangements. This enzyme is not expressed in fetal pre-B cells (53, 54). As a result, fetal-derived B cells contain germ-line encoded heavy and light chains, whereas adult-derived B cells contain heavy and light chains that diverge from germ line. Recent evidence indicates that fetal vs. adult differences in V region usage are selected for during pre-B cell development (55). This was seen by examining productive and nonproductive rearrangements of heavy chain genes in single, sorted pre-B cells from adult BM. In nonproductively rearranged alleles there is, as in the expressed repertoire of neonatal B cells, preferential use of J-proximal V genes. This skewing is absent in productively rearranged alleles, indicating that there is either selection against use of J-proximal V regions or selection for use of other V regions. This selection is dependent on surrogate light chain (SLC) expression since in pre-B cells of λ5 knockout mice, which lack a functional SLC, both productive and nonproductive rearrangements exhibit preferential use of J-proximal V genes (55). The SLC, which consists of V-pre B and λ5, associates with µ heavy chains to form the pre-BCR. Successful assembly of this complex is necessary for light chain rearrangements to begin (56; but see below for a possible exception to this). Evidence to support the role of the SLC in randomizing V gene usage comes from examination of the J-most heavy chain gene, VH81X. This gene is common in the neonatal repertoire but rare in the adult (57, 58). In adults productive rearrangements of this gene were seen only in pre-B cells that expressed c-kit, the receptor for stem cell factor (55). C-kit is only found on early B cell progenitors, at a stage prior to SLC-dependent pre-B cell expansion (59). Of seven productively rearranged VH81X heavy chains cloned from adult pre-B cells, none was able to associate with the SLC when transfected into an SLC expressing cell line (55). What then is the basis for the successful generation of VH81X expressing B cells in the neonate? One possibility is that the germ line–encoded VH81X gene can associate with the SLC, but that this is prevented by introduction of N insertions. This notion is supported by the fact that an N-less, fetal-derived VH81X transgene could efficiently drive adult B cell development (60). In addition, introduction of this VH81X transgene into λ5 knockout mice did not relieve the block to B cell development caused by the absence of λ5 (60). Thus, this transgene cannot promote B cell development in a λ5-independent manner. Additional evidence that it is N insertion that prevents VH81X expression in adult B cells comes from transgenic mice in which expression of a TdT transgene is forced in fetal pre-B cells. In nontransgenic neonatal mice the ratio of productive to nonproductive VH81X rearrangements (P/NP) was 2.4, indicating strong selection for this gene. In TdT transgenic neonates, the P/NP dropped to 0.32, indicating that N insertion inhibits selection for VH81X heavy chains (61). Taken together,

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these studies suggest that in the course of evolution the VH81X transgene was selected to be maintained in its germ-line configuration. This may be generally true of members of the fetal/neonatal repertoire. For these genes, the absence of N insertions may be sufficient to explain their restriction to fetal/neonatal B cells. Recently, Wasserman et al. proposed another possible mechanism to ensure the generation of different repertoires in fetal/neonatal and adult B cells (62). They found that a B-1-derived VH11 heavy chain is unable to associate with the SLC, yet contributed to the fetal-derived mature B cell population. They went on to suggest that it is the inability of this heavy chain to form a pre-BCR that promotes expansion of fetal pre-B cells (62). In contrast, B-2-derived heavy chains were able to form a pre-BCR and inhibited the expansion of fetal pre-B cells (62). This contrasts with the situation in adult bone marrow where association with SLC has been shown to be necessary for B cell development. In other words, Wasserman et al. propose, pre-B cells expressing a heavy chain/SLC complex fail to expand in a fetal microenvironment, but the complex is required for expansion in the adult marrow. These conclusions are hard to reconcile with the demonstration that in fetal liver organ cultures, efficient B cell development is dependent on λ5 expression (63) or with the observation that a VH81X transgene failed to drive pre-B cell development in λ5 knockout mice (60). In addition, Ye et al. demonstrated that a B-1-derived VH12 heavy chain was able to associate with the SLC and form a pre-BCR (64). Despite the superior ability of fetal precursors to generate B-1 cells, the B-1 repertoire in adult mice is not the same as the fetal/neonatal B cell repertoire. For example, VH81X, commonly expressed on fetal/neonatal B cells, is largely absent from the adult. This may reflect the loss in the adult of a self-antigen necessary for the maintenance of VH81X-bearing B-1 cells because after transfer into adult SCID mice, PerC B-1 cells transgenic for VH81X failed to expand and survive (60). Furthermore, analysis of VDJ genes isolated from PerC B-1 and B-2 cells by single cell PCR found that about 60% of genes cloned from B-1 cells contained N-insertions (12). Although this contrasts with genes cloned from B-2 cells, where greater than 90% contain N insertions (12), it suggests that many B-1 cells may be derived from adult precursors. Preferential use of J-proximal V genes was not observed in B-1 cells (12), again a fact consistent with the possible adult origin of many B-1 cells, although this may also be explained by post Ig–rearrangement selection. Any contribution to the B-1 compartment by newly arising adult B cells is constrained by the existence of negative feedback regulation of B-1 development (65).

Other Factors Influencing B-1 Development While appropriate antigen receptor signaling is crucial for the generation of B-1 cells, other signals also play important roles in their development and localization. B-1 cells, unlike B-2 cells, constitutively express the IL-5 receptor (65a). IL-5 transgenic mice that overexpress IL-5 have increased numbers of B-1 cells (278). Conversely, mice containing targeted disruptions of the IL-5 (270) or IL5R (271)

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genes have decreased numbers of B-1 cells early in life. B-1 numbers recover by 6–8 weeks of age, indicating that another factor can substitute for IL-5 later after birth. IL-9 might be the relevant factor, as its overexpression causes an increase in the fraction of B-1 cells (279). A subfamily of TNF-related ligands and their receptors have been discovered to be important in B cell development and function. These ligands are BLys (also called TALL-1, BAFF, THANK, and zTNF4) and APRIL. They both bind with high affinity to the receptors TACI and BCMA (reviewed in 65b). Disruption or overexpression of these ligands and receptors results in complex B cell phenotypes that are discussed in Siegel and Lenardo (65c). BlyS, is essential for B-2 but not B-1 development (65d). Nonetheless, in one study, BlyS overexpression resulted in an expanded splenic B-1 population (159). Perhaps because they express higher levels of the CXCL13 receptor CXCR5 than do splenic B-2 cells, PerC B-1 cells are more sensitive to this chemokine (65e). In mice containing a targeted disruption of the CXCL13 gene there was defective homing of B-1 cells to the PerC (K. Ansel and J. Cyster, personal communication). Splenic B-1 cells were still present. The responses of PerC, but not splenic, lymphocytes (B-1, B-2, and T), to CXCL13 (as well as to another chemokine, CCL21) are reduced in aly/aly mice which contain a point mutation in the NIK tyrosine kinase gene (85). This point mutation results in defective processing of NF-κB2 (65f ).

FUNCTIONS OF B-1 CELLS Production of Natural Serum IgM B-1 cells adoptively transferred into irradiated mice (43, 66), unmanipulated neonatal mice (19), or B cell–depleted neonatal mice (65) produce quantities of IgM approximating the levels seen in unmanipulated animals. Based on such studies, B-1 cells are believed to be the primary source of natural IgM. This antibody, which is produced in the absence of exogenous antigenic stimulation (67, 68), is polyreactive, weakly autoreactive, and reactive with many common pathogen-associated carbohydrate antigens. Several recent reviews highlight the important roles of natural antibody in adaptive immune responses, protection from bacterial infection, and protection from autoimmunity as well as in a model of ischemia reperfusion injury (69, 70). Consistent with a major role for B-1 cells in natural IgM production, a number of natural IgM specificities have been identified in the B-1 repertoire. These include specificities for phosphorylcholine (PC) (71), phosphatidyl choline (PtC) (31, 72, 73), thymocytes (28), LPS (74), and influenza virus (75). In mice mutated so that they express membrane IgM (mIgM) but are unable to produce secretory IgM (sIgM), there is an increase in the number of B-1 cells, suggesting that the size of the B-1 compartment is regulated by serum IgM levels (76, 77). The ability to produce natural IgM is not a property unique to cells with the B-1 phenotype. Purified small resting lymph node B cells have been shown to

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differentiate into long-lived activated IgM-producing cells and to reconstitute normal serum IgM levels after transfer into SCID recipients (78). These cells remained CD5− over the course of the experiment. Interestingly, they acquired a phenotype similar (and perhaps identical) to that of MZ B cells (78). Once established, these IgM producing, B-2-derived cells were resistant to replacement by a second injection of purified B-2 cells. They also resisted replacement by newly emerging B cells when adult bone marrow was injected along with LN B cells. In these mice, a normal-size B cell compartment, derived from injected bone marrow, coexisted with an activated, IgM-secreting population largely derived from injected lymph node B cells (78). Replacement did slowly occur, possibly reflecting competition between established and newly emerging cells for access to factors needed for differentiation into IgM-secreting cells. These experiments suggest that it may be the repertoire of B-1 cells, rather than the B-1 phenotype per se, that selects them into the pool of natural IgMsecreting cells. As discussed earlier, this repertoire also drives cells to assume the B-1 phenotype. It would be interesting to see if injected B-1 cells would be more efficient than mature B-2 cells in displacing lymph node–derived IgM-producing cells in experiments similar to those discussed above. Several studies demonstrate the importance of demonstrably B-1-derived natural antibody. For instance, antibodies expressing the T15 idiotype, which are almost exclusively of B-1 origin (71), play an important role in protection from infection by Streptococcus pneumoniae (79, 80). Furthermore, it was recently shown that antibodies with the T15 idiotype bind oxidized low-density lipoprotein (LDL) (81). This binding may aid in clearance of LDL from the blood and thus play a role in prevention of atherosclerosis. Alternatively, these antibodies may bind to atherosclerotic plaques, initiate an inflammatory response, and contribute to the pathogenesis of vascular disease. Anti-PtC antibody is critical for protection from acute septic peritonitis after cecal ligation and puncture (CLP) (82). Mice unable to secrete IgM (sIgM−/−) because of a targeted mutation are highly susceptible to death after CLP. Transfer of normal mouse serum or purified B-1-derived monoclonal anti-PtC protected sIgM−/− mice from CLP-induced death (82). B-1 cells are responsible for almost all natural antibody reactive with LPS (74). This antibody is important in clearance of LPS as indicated by the increased susceptibility of antibody-deficient mice to death after LPS injection (83). Administration of normal mouse serum prior to LPS injection protects mice from death (83). Uninfected mice contain natural anti-influenza IgM, which is derived exclusively from B-1 cells (75). Upon influenza infection, titres of this antibody do not increase. Rather, an increase in B-2-derived immune IgM is observed (75). Reconstitution experiments demonstrate that resistance to influenza infection is dependent on both B-1- and B-2-derived IgM (84). In the absence of B-1-derived natural anti-influenza IgM there is a delayed T-dependent IgG2a response and increased mortality. Thus, natural IgM is necessary for a normal adaptive response to influenza (84). Similarly, IgG responses to model TD antigens are impaired in

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sIgM−/− mice (76, 77) but can be rescued by administration of normal serum IgM prior to immunization (77). The role of natural IgM in adaptive responses could be to facilitate complementmediated localization of antigen to germinal center follicular dendritic cells (FDCs), to lower the threshold of B cell activation by allowing simultaneous engagement of the BCR and the CD21/CD19 complement receptor, or both (see 69 for discussion).


Production of IgA in the Mucosal Immune System Several groups demonstrated that transfer of total peritoneal B cells (66, 85), or sorted B-1 cells (86), into lethally irradiated (66, 86), or RAG2−/− (85) mice resulted in the appearance of donor-derived IgA plasma cells in the gut lamina propria (LP) and mesenteric lymph nodes (MLN). Transfer of fetal omentum into SCID mice, which reconstituted the B-1 but not the B-2 compartment, also resulted in the appearance of IgA secreting plasma cells in the LP (17). In mixed radiation chimeras, where the relative contributions of PerC- and BM-derived donor cells could be followed, PerC cells were estimated to give rise to about 40% of the IgAproducing cells in the gut (66). In a second, independent study in which purified B-1 cells were transferred, a similar fraction of LP IgA producing cells appeared to be B-1 derived (86). The role of B-1 cells in gut IgA production is further supported by the fact that CD19-deficient and xid mice, both of which have a selective reduction in B-1 cell numbers, have decreased numbers of IgA-secreting plasma cells in the LP (but not in the spleen) (86). IgA production is dependent on exposure to exogenous stimuli because mice raised under germ-free conditions largely lack gut IgA (67, 86–88). This is in contrast to B-1-derived natural IgM, which is produced even in germ-free mice (67, 87, 88). B-1, but not B-2, gut IgA production is independent of T cell help because it is unaffected by mutations that eliminate T cells (nu/nu or TCRβ −/−/ δ −/−) (86). Whether T cell–independent IgA production by B-1 cells requires particular gut associated lymphoid structures was addressed by Macpherson et al., who examined TNFR-1−/− mice (86). TNFR-1−/− mice have lymph nodes (LNs), but their LNs lack FDCs, primary B cell follicles, and GCs (89). They either lack PPs or contain a reduced number with altered architecture (89, 90). Despite these defects, TNFR-1−/− mice have near normal numbers of LP IgA secreting cells (86), which suggests that neither B-1- nor B-2-derived IgA production requires organized follicular lymphoid structures. This raises the question of whether LNs or PPs are required at all for gut-associated IgA. However, mice lacking all LNs and PPs due to targeted disruption of the LTα gene (91, 92), or double knockout of LTα and TNF genes (93), exhibit severely reduced gut IgA production (92, 93). Thus, both B-1 and B-2 IgA production is compromised by the loss of LNs and PPs, despite the fact that B-1 IgA production is independent of T cells. LTβ −/− mice (94), or mice treated in utero with an LTβ-Ig fusion protein (95, 96), lack PPs and peripheral LNs, but they do contain MLNs. In a recent

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study, gut IgA production in LTβ-Ig treated mice (PP−/MLN+) was compared to that of TNF−/−LTα −/− mice (PP−/MLN−) (97). TNF−/−LTα −/− mice were again found to produce very little gut IgA. In contrast, LTβ-Ig-treated mice had both a nearly normal frequency of gut-associated IgA-producing cells and nearly normal gut IgA production. This suggests that MLNs provide something necessary for gut IgA production, including IgA production from B-1-derived cells. However, these data conflict with those of Koni et al. who found that LTβ −/− mice (also PP-/MLN+) produce very little fecal IgA (94). Perhaps LTβ has a function in gut IgA production independent of its role in MLN formation, and LTβ-Ig treatment does not interfere with this putative function Another strain of mouse with defects in lymphoid organ development is the naturally occurring alymphoplasia (aly) mouse. These mice carry a point mutation in the tyrosine kinase NIK (98) that results in the absence of all LNs and PPs as well immune deficiencies (99), including the absence of IgA-secreting plasma cells in the lamina propria and MLN (85). These mice also have increased numbers of PerC B-1 cells (85). PerC cells from aly/aly mice, in contrast to those from aly/+ mice, failed to generate IgA-producing cells in the lamina propria or MLN after transfer to Rag2−/− mice (85). Thus, defects in gut IgA production are at least partly a PerC cell-intrinsic effect. Recipients of aly/aly cells also had the elevated PerC B-1 cell levels characteristic of aly mice (85). This suggested the possibility that failure of B-1 cells to emigrate from the PerC might at least partly explain the lack of gut IgA-producing cells in aly mice. This idea is supported by the fact that aly/aly peritoneal B cells (both B-1 and B-2) have defective in vitro chemotactic responses to two chemokines; secondary lymphoid tissue cytokine (CCL21) and B lymphocyte chemoattractant (CXCL13) (85). Reduced chemotaxis was not seen in splenic B-2 cells from aly/aly mice. Consistent with an important role for CXCL13 in B-1 cell homing, it was recently reported that in wild-type mice PerC B-1 cells exhibit an enhanced migration response to CXCL13 in vitro and express higher levels of CXCR5, the receptor for CXCL13, compared to splenic B cells (65e). What is the significance of B-1-derived sIgA in the gut? Given the difference in repertoire of peritoneal B-1cells versus splenic B-2 cells, it is natural to ask if IgA derived from B-1 cells has a different range of specificities than that derived from B-2 cells. Support for this possibility comes from a study of mice containing allotype marked B-1 and B-2 cells (100). Examination of fecal bacteria from these mice using flow cytometry revealed that about 65% of bacteria were coated exclusively with B-1-derived IgA. Thirty per cent of bacteria were coated exclusively with B-2-derived IgA, and only about 5% were coated with IgA from both sources (100). It is unclear how this situation arises, or what its significance is. Bos et al. suggest that B-1-derived sIgA helps maintain commensal bacteria in the gut, whereas B-2-derived sIgA, because of recognition of different epitopes and/or binding with higher affinities, mediates elimination of potentially pathogenic bacteria (100).

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Role in TI-2 Responses Several lines of evidence suggest that B-1 cells are involved in TI-2 responses: (a) Their repertoire is skewed toward reactivity with TI-2 antigens; (b) they lack the somatic mutations that are the hallmark of affinity maturation in germinal centers; (c) xid mice, which lack B-1 cells, are unable to respond to TI-2 antigens; (d ) they produce IgM and IgG3, the major isotypes produced in response to TI-2 antigens; (e) TI-2, but neither TD nor TI-1, activation of B cells in vitro results in a partial induction of the B-1 phenotype. B-1 cells have in fact been shown to participate in TI-2 responses. Transferred B-1 cells respond in vivo to the TI-2 antigen α1-3 dextran (19). The best established role for B-1 cells in a TI-2 response is the production of T15 idiotype anti-PC antibody in response to infection by S. pneunoniae (71, 103, 110). This occurs even in TCRα −/−δ −/− mice, proving its T independence (103). The production of T15 antibody generated in response to S. pneumonia infection is in addition to the constitutive T15 antibody B-1 cells produce as a component of natural antibody. The T15 idiotype is generated by the association of unmutated VH1 and Vκ22 heavy and light chains. The fact that the murine germ line encodes such a useful specificity, that B cells expressing this specificity are selected and maintained in the apparent absence of external antigen, and that these B cells are readily recruited to respond to antigen in the absence of T cell help, has led to the proposal that B-1 cells are in essence “natural memory” cells (4). B-1 cells are not the only cells able to respond to TI-2 antigens. In fact, B-1 cells do not participate in the response to immunization with the TI-2 antigens TNP-Ficoll (72) and NP-Ficoll (19). Rather, splenic marginal zone (MZ) B cells appear to respond to these TI-2 antigens (104). Consistent with a role for MZ B cells in some TI-2 responses, Pyk-2−/− mice, which have normal numbers of PerC B-1 cells and MZ macrophages but lack MZ B cells, exhibit severely impaired responses to both TNP-Ficoll and dextran (107). MZ B cells are another phenotypically and functionally distinct subset of B cells (reviewed in 4). Entry into the MZ subset also appears to be a consequence of antigen receptor specificity. Thus, in M167 and VH81x µ transgenic mice, B cells expressing, respectively, the M167 and 35-1 idiotypes are greatly enriched in the MZ subset and largely missing from the recirculating follicular population. In contrast, in MD2 µ transgenic mice, B cells expressing an anti-HEL specific BCR are under-represented in the MZ subset (109). MZ B cells have been shown to be activated more rapidly by polyclonal stimuli than are follicular B cells, both in vitro (108) and in vivo (103). This suggests that this subset is comprised of cells selected based on their antigen-specificities and poised to provide a rapid first line of response to pathogens. Together with B-1 cells, they provide “natural memory” (4). What are the respective roles of B-1 and MZ B cells in TI-2 responses? A recent study by Martin et al. casts light on this question (103). They examined the response of M167 idiotype bearing B cells to infection by S. pneumoniae in M167 µ transgenic mice. M167 idiotype antibodies react with PC on S. pneumoniae. Using

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adoptive transfer of sorted cells, Martin et al. demonstrated that the M167 id response to S. pneumoniae was almost exclusively due to the rapid differentiation of MZ B cells into IgM plasmablasts in this transgenic system. This is perhaps not surprising since prior to immunization 83% of M167 id+ cells in these mice had a MZ phenotype (109). However, when equal numbers of MZ and FO M167 id+ cells were transferred, MZ cells were more efficient at generating M167 id+ plasmablasts than were FO cells (103). Despite the ability of M167 id+ MZ B cells to respond to immunization with S. pneumoniae, most antibody to these organisms is produced by B-1 derived plasmablasts secreting T15 id+ IgM. To better understand what determines the magnitude of the B-1 vs MZ B cell response, Martin et al. transferred increasing numbers of M167 id+ MZ cells along with excess non-transgenic splenocytes. When small numbers of transgenic id+ MZ cells were transferred, the predominant response to iv immunization with S. pneumoniae was the secretion of T15 antibody. With increasing numbers of transferred transgenic MZ cells, there was an increase in the M167 response and a concomitant decrease in the T15 response. This suggests that the relative number of cells in MZ and B-1 subsets able to respond to an antigen determines, at least in part, the relative contribution of each subset to immunization with that antigen. In the experiment just discussed, antigen was administered iv and the response of splenic cells examined. T15 id+ B-1 cells exist in the spleens of unimmunized mice and presumably are the source of the T15 producing plasmablasts observed after iv immunization. However, since B-1 cells are enriched in the PerC, Martin et al. went on to ask whether this anatomical segregation of B-1 cells had any functional consequences. They found that although iv administration of S. pneumoniae led to production of both M167 and T15 antibodies, low dose ip administration resulted in a T15 response only in the PerC. It failed to elicit any splenic response, presumably because antigen was cleared by the PerC response and never became blood borne. In contrast, high dose ip administration of S. pneumoniae gave rise both to a T15 PerC response and a T15 plus M167 response in the spleen. Thus B-1 cells, because of their enrichment in the PerC, are ideally suited to respond to antigens that enter the organism through the gut epithelium. In contrast, MZ B cells are situated so as to be able to efficiently encounter and respond to blood borne pathogens. To the extent that different antigens may be encountered on blood borne versus gastrointestinal pathogens, it would be useful to select different repertoires into the MZ versus B-1 subset. It is unclear to what extent this is the case since little is known about the MZ repertoire. It is however true that some particular heavy/light chain combinations, such as that responsible for generating the T15 idiotype, are found only on B-1 cells (71). Since MZ and B-1 cells are selected into their respective compartments based on BCR specificity, it would be interesting to know what signals are responsible for this selection and how they differ between the subsets. In the case of the response to S. pneumoniae discussed above, both T15 B-1 cells and M167 MZ B cells are responding, as far as is known, to the same antigen on the bacterium, PC. Since

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PC is also present on mouse cells, it is plausible, but by no means certain, that PCreactive B-1 and MZ B cells are initially selected into their respective compartments by interaction with this self-antigen. This raises the question of why expression of each idiotype results in selection into a different compartment. This could be due to different affinities of these antibodies for PC, with T15 antibodies having a higher affinity for PC than do M167 antibodies (105). Selection into the B-1 compartment might require higher strength BCR signaling than selection into the MZ (or FO) population. Alternatively, selection of a cell into the MZ or B-1 compartment may depend on other, unknown signals in addition to BCR ligation, and the required signals may be different for B-1 and MZ differentiation. Recently Cariappa et al. reported results that they interpreted as indicating that B cells assume the MZ phenotype as a consequence of receiving lower levels of BCR signaling than are required for entry into the recirculating FO B cell pool (106). This would appear to contradict the idea that MZ B cells, in contrast to FO B cells, are a (self-) antigen selected population. Their conclusions are based on the fact that mice lacking the transcription factor aiolos have B cells that are hyperresponsive to BCR signaling and essentially lack MZ B cells. In contrast, xid mice, which have defective BCR signaling, have normal numbers of MZ B cells. (The authors show that there is an increase in the number of putative MZ precursors in xid mice; however, the fraction of cells with the full MZ phenotype is essentially the same as in WT.) Other interpretations of their data are possible. Aiolos deficient mice, despite the hyperresponsive phenotype of their B cells, have decreased numbers of B-1 cells (269). Yet, as discussed above, there is compelling data showing that B-1 development is dependent on stronger, rather than weaker BCR signaling. The effect of aiolos deficiency on positive selection of B-1 and MZ B cells may be independent of its effect on sensitivity of BCR signaling in vitro. As for xid mice, Martin and Kearney have shown that on an xid background VH81x transgenic B cells expressing the 35-1 id fail to be selected into the MZ subset in contrast to such cells on a WT background (109). This suggests that although xid mice have a normal sized MZ compartment, the repertoire of cells in this compartment may be altered. In conjunction with the absence of B-1 cells in xid mice, this could explain the inability of xid mice to respond to TI-2 antigens. Also inconsistent with weaker BCR signaling being necessary for MZ B cell development is the fact that both MZ and B-1 cells are absent from CD19 knockout mice (109, 259, 276). Distinct from the question of whether or not B-1 cells respond to TI-2 antigens is the issue of whether or not the response to TI-2 antigens induces a B-2 cell to express the B-1 phenotype as suggested by in vitro experiments. In an attempt to address this issue, Houghton and colleagues reconstituted lethally irradiated mice with allotype marked B-1 and B-2 cells, immunized them with the TI-2 antigen polyvinylpyrollidine (PVP), and determined the allotype and phenotype of responding cells (48). Responding cells were predominantly bone marrow derived but with the surface phenotype (CD5+, CD23−, CD43+, IgDlo, IgMhi) of B-1 cells. Since bone marrow only inefficiently reconstitutes the B-1 compartment, the

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responding cells were most likely B-2 cells that had been driven to assume the B-1 phenotype by activation with PVP. This raises the issue of the extent to which the B-1 compartment is composed of cells that arose in the adult in response to a TI-2 antigen.


B-1 Cells and Autoimmunity That B-1 cells often produce autoreactive antibodies, although nonpathogenic and of a low affinity, has led to interest in these cells as possible sources of the highaffinity, pathogenic autoantibodies seen in polysystem autoimmune disease. Indeed, there are examples, in both human and mouse, of an association between autoimmune disease and B-1 cells. In humans, elevated numbers of B-1 cells have been reported in patients with Sjorgen’s syndrome (111) and rheumatoid arthritis (112). In mice, increased numbers of B-1 cells have been observed in a number of naturally occurring and genetically manipulated strains that develop autoimmune manifestations (see below). How might B-1 cells contribute to autoimmune disease? In several respects Per-C B-1 cells have a phenotype similar to that of anergic B cells. Like anergic B cells they do not flux Ca2+ or proliferate in response to BCR ligation (10). Furthermore, anergic B cells have recently been shown to express low levels of CD5, which helps to maintain anergy (39). In addition, both anergic B cells (113) and B-1 cells (114) have elevated nuclear levels of the transcription factor NFATc. It is therefore possible that the induction of the B-1 phenotype serves to tolerize B cells expressing certain specificities while still keeping them available for certain responses. Autoimmune disease could occur as a result of failure to induce the B-1 phenotype. Alternatively, susceptibility to autoimmune disease might result from diminished negative regulation of B-1 cells. As a result B-1 cells producing low-affinity autoantibodies would receive T cell help, enter germinal centers, class switch, undergo somatic mutation, and as a result of affinity maturation, produce highaffinity IgG autoantibodies. B-1 cells are able to respond to T cell help and switch class (115). Somatic mutations are not seen in peritoneal B-1a cells (13), but they are found in some human CD5+ cells (116). Of course, it is also possible that once B-1 cells are recruited into germinal centers they lose the B-1 phenotype, which makes it difficult to establish the phenotype of the antecedent cell. In addition to the possibility of producing pathogenic autoantibody, B-1 cells may contribute to autoimmune disease by presenting self-antigen to autoreactive T cells or by virtue of their ability to secrete IL-10. This is discussed more fully below. We now turn to an examination of several mouse models of autoimmunity in which B-1 cells play a role. AUTOIMMUNE HEMOLYTIC ANEMIA (AHA) IN NZB MICE NZB mice spontaneously develop autoimmune hemolytic anemia as a result of the production of anti–red blood cell (RBC) antibodies (117). Even before overt disease, these mice exhibit immune system aberrations, including an increase in the number of B-1 cells (118).

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Evidence that B-1 cells may play a causative role in development of disease comes from a series of studies by Honjo and colleagues using mice transgenic for the heavy and light chains of 4C8, an NZB-derived, pathogenic anti-RBC immunoglobulin (119). Expression of 4C8 on a C57/BL6 (nonautoimmune) background resulting in deletion or anergy of transgene-expressing B cells in the spleen. Normal numbers of transgene-expressing B-1 cells were found in the PerC (119). About 50% of 4C8-transgenic mice develop autoimmune hemolytic anemia when housed under conventional conditions. Mice housed under germ-free or SPF conditions remained healthy, suggesting that exposure to pathogens (or perhaps certain commensal organisms) is necessary for disease (120). In additional studies, oral administration of LPS induced peritoneal as well as lamina propria B cells to secrete IgM and resulted in anemia in previously SPF-maintained, nonanemic 4C8 transgenic mice (121). Exogenous IL-10 or IL-5, but not IL-4, also activated peritoneal B cells leading to disease (122). Intra-peritoneal (ip) injection of mouse RBC’s induced apoptosis of the transgene-expressing peritoneal B-1 cells. Thus, the survival of these autoreactive cells is dependent on their sequestration from antigen (123). Strikingly, injection of RBCs into the peritoneum resulted in recovery from anemia, indicating that B-1 cells or their descendants might be the source of the secreted 4C8 autoantibody (123). This possibility was directly tested by enumerating 4C8-secreting cells in the bone marrow, spleen, and peritoneum of transgenic mice with severe, moderate, or no anemia. 4C8-secreting cells were found only in the peritonea, and their frequency correlated with the severity of anemia (123). 4C8-secreting cells were reported (data not shown) to have the surface phenotype of B-1 cells (123). Another indication that B-1 cells are involved in the development of disease is the fact that F1 males produced by crosses between xid mice and mice transgenic for 4C8 failed to produce 4C8 antibody or to develop disease (121). Xid, or X-linked immunodeficiency, is a spontaneous mutation of the X-linked Bruton’s tyrosine kinase (Btk) gene, which prevents B-1 development (33). Interpretation of this result is complicated by the fact that the xid mutation also affects, although less dramatically, B-2 development and function (34). These results suggest that activation of autoreactive B-1 cells by pathogens or bystander effects could lead to autoimmunity. Interestingly, in 4C8 transgenic mice B-1 development is dependent on T cells because when the 4C8 transgenes are crossed onto a Rag−/− background very few peritoneal B cells develop (124). Transfer of fetal thymus or administration of IL-5, IL-10, or LPS rescued B-1 development and resulted in anemia (124). T cells are not required for B-1 development in nontransgenic mice. These studies provide compelling evidence that autoreactive B-1 cells, sequestered from antigen in the peritoneum, can be activated by pathogens or cytokines to secrete autoantibodies and thereby cause disease in a nonautoimmune strain of mouse. However, in nontransgenic mice, B-1 cells, although the predominant source of nonpathogenic autoantibodies, do not produce pathogenic autoantibodies. In fact, in young 4C8 transgenic mice there are few peritoneal B cells, suggesting that transgene-expressing B-1 cells (or their progenitors) are

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efficiently deleted in these mice (123). Presumably, a small number of cells that escape deletion end up sequestered from antigen in the peritoneum where they expand. The question, then, is what role, if any, do B-1 cells play in pathogenesis of autoimmunity in nontransgenic, NZB mice. Support for the idea that B-1 cells do play a role is provided by experiments in which peritoneal B-1 cells are selectively eliminated by repeated intraperitoneal injection of H2O (125). This treatment prevents development of AHA (125). It selectively targets B-1 cells because peritoneal B-2 cells eliminated by hypotonic shock are replenished from bone marrow precursors, whereas B-1 cells are not. In NZB mice, B-1 cells could, as in 4C8 transgenic mice, actually produce pathogenic autoantibody. Alternatively, they may play an accessory role, for example by presenting antigen to T cells (126) or secreting IL-10 (127). MURINE LUPUS ERYTHEMATOSUS (SLE) IN NZB/NZW F1 (NZB/W) MICE F1 hybrids of NZB and NZW mice spontaneously develop an autoimmune syndrome similar to human systemic lupus erythematosus (SLE) (117, 128). The murine disease (like that in human) is more common in females and is characterized by the production of somatically mutated IgG autoantibodies with high-affinity for nuclear antigens such as dsDNA (129) and nucleosomes (130). The presence of these antibodies results in deposition of immune complexes in the kidneys, and the development of glomerular nephritis, proteinurea, and death due to kidney failure (128). Like NZB mice, (NZB X NZW)F1 mice have an enlarged B-1 compartment (118). Breeding the xid gene into (NZB X NZW)F1 mice prevents B-1 development and development of disease (131). Similarly, selective depletion of peritoneal B-1 cells by ip injection of H2O reduces the severity of disease (125). These results suggest a role for B-1 cells in the development of SLE in NZB/W mice. In MRL/lpr, another murine model of SLE, there is no increase in B-1 cells and adoptive transfer experiments indicate that B-2 cells, not B-1 cells, are necessary for disease pathogenesis (132). (NZB X NZW)F1 mice exhibit both T and B cell abnormalities. Yet, transfer into SCID mice of cultured pre-B cells derived from (NZB X NZW)F1 fetal liver was sufficient to generate many manifestations of SLE including renal disease (133). Since T cells do not develop from these selected donor cells, it appears that B-lineage intrinsic defects play a primary role in the pathogenesis of lupuslike disease in (NZB X NZW)F1 mice. It is not known specifically what role, if any, B-1 cells play in this process. One possibility is that they (or their derivatives) produce pathogenic autoantibody. Before the development of disease, both (NZB X NZW)F1 mice and SLE patients have large numbers of B cells spontaneously producing nonpathogenic, low-affinity, IgM anti-DNA antibody (134). Disease progression is believed to entail an antigen-driven switch in these cells to the production of pathogenic, high-affinity, IgG anti-DNA antibody (135–137). There is one report that most of the spontaneous anti-DNA IgM was produced by B-1 cells in (NZB X NZW)F1 mice (138). However, another study found that in

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NZB.H-2bm12 mice, which are similar to (NZB X NZW)F1, there was no difference in the frequency of splenic B-1 versus B-2 cells secreting IgM anti-DNA IgM (139). Consistent with this, yet another group showed that sorted (NZB X NZW)F1 splenic B-2 and peritoneal B-1 cells were equally competent to produce IgM or IgG anti-DNA when transferred into SCID mice along with (NZB X NZW)F1 T cells (140). Interestingly, transferred B cells from both sources produced equivalent levels of IgG anti-DNA and were able to cause disease. Three of three mice reconstituted with splenic B-2 cells and two of three mice reconstituted with peritoneal B-1 cells developed proteinurea (140). Leaving aside questions of the purity of the transferred cells, which was not rigorously controlled for, this suggests that (NZB X NZW)F1 B-1 cells are capable, although not uniquely so, of producing pathogenic autoantibody. Therefore, one aspect of (NZB X NZW)F1 disease susceptibility may be a breakdown in the mechanisms preventing B-1 cell hypermutation and affinity maturation. In NZM2410 mice, an autoimmune strain derived from (NZB X NZW)F1 mice, B-1 cells express high levels of the costimulatory molecules B7-1, B7-2, and CD24 in comparison to B-2 cells or to B-1 cells from C57/BL6 mice (126). B-1 cells from these mice, compared to B-2 cells, exhibited enhanced antigen-presenting ability to T cells in vitro (126). This might enable them to better elicit the cognate T cell help presumably necessary for somatic mutation. Unfortunately, the antigenpresenting capabilities of NZM2410 B-1 cells were not compared to those of B-1 cells from a nonautoimmune strain. In addition to being a source of pathogenic autoantibodies, B-1 cells could contribute in other ways to autoimmunity. Their enhanced antigen-presenting ability might better activate T cells to provide help to B-2 cells. Enhanced T cell activation could also contribute to disease by a mechanism independent of antibody production. Chan et al. reported that in the MRL/lpr model of SLE, B cells expressing mIgM, but unable to secrete Ig, can activate T cells resulting in renal cellular infiltrates and nephritis (141). In addition, in (NZB X NZW)F1 (but not C57/BL6) mice, a significant fraction of splenic CD1hi B cells have a B-1a phenotype (142). CD1 is able to present sugar and/or lipid antigens to some subsets of T cells (143, 144). Splenic T cells transgenic for a CD1-reactive T cell receptor induced a lupuslike disease when transferred into irradiated nude mice along with nude BM (145) Although the CD1hi B-1a cells present in (NZB X NZW)F1 spleen appear not to secrete pathogenic autoantibody (N. Baumgarth, personal communication), they may contribute to autoimmunity by activating CD1-restricted splenic T cells in (NZB X NZW)F1 mice. Finally, B-1 cells could contribute to SLE by their secretion of IL-10 (127). This interleukin is important in SLE pathogenesis. Continuous injection of anti-IL-10 antibody delayed the development of autoimmune disease in (NZB X NZW)F1 mice while continuous administration of IL-10 accelerated it (146). AUTOIMMUNITY IN GENE TARGETED AND TRANSGENIC MICE A number of gene knockout and transgenic mice have increased B-1 cell numbers and manifestations of autoimmune disease. Targeted disruption of the src family tyrosine kinase lyn

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results in B cells that are hyperesponsive to BCR ligation because of impaired CD22 (147–149) and Fcγ RII (149–151) mediated downregulation of BCR signaling. Lyn knockout mice have large numbers of B-1 cells in their lymph nodes (151) and exhibit increasing levels of IgG autoantibodies and glomerular nephritis with age (149, 151, 152). The targeted disruption of the CD22 gene leads to enhanced BCR signaling (153–156) and, in one of four independently generated lines, to an increase in number of peritoneal B-1 cells (156). In another of the four, the number of peritoneal B-1 cells was not increased to a statistically significant extent, but the fraction of peritoneal cells with a B-1 phenotype was significantly increased (155). In the line found to have increased B-1 cell numbers, mice were followed for up to 20 months and found to develop high titers of IgG antibody specific for dsDNA, cardiolipin, and myeloperoxidase (157). These antibodies were of high affinity and somatically mutated (157). The mice did not develop other manifestations of autoimmune disease. Mice transgenic for the tumor necrosis factor (TNF)-ligand family member Blys (also termed TNF4, BAFF, TALL-1, and THANK), which is a costimulator of B cells, exhibit immune system dysregulation including increased serum IgM and IgG as well as the appearance in some older animals of anti-DNA antibodies, rheumatoid factor, and high levels of circulating immune complexes. Some animals exhibited Ig deposition in the kidneys and others some frank glomerular nephritis (158, 159). Transgenic lines were developed by two groups. One placed Blys under the control of a liver-specific promoter (158), while the other used a B cell specific promoter (159). In the latter line, but not the former, there was an increase in the number of splenic B-1 cells (159). The fact that autoimmune manifestations occur in Blys transgenic mice whether or not there was expansion of the B-1 compartment may indicate that B-1 cells are not responsible for the autoimmunity. It is possible, however, that there is B-1 dysregulation that does not consistently lead to an increase in B-1 number but is important in pathogenesis. Mice in which a TNFα transgene was introduced under the control of the lungspecific surfactant protein C promoter (SP-C/TNF) developed pulmonary fibrosis (160). Subsequently it was determined that lung interstitial mononuclear cells contain an elevated fraction of CD5+/IgM+ cells (161). This may be downstream of an increase in osteopontin (OPN). SP-C/TNF transgenic mice exhibit elevated levels of OPN mRNA in their lungs (160, 161); in a different study OPN transgenic mice were shown to have an increased fraction of PerC B-1a cells (162).

CD5+ ON B CELLS The transmembrane glycoprotein CD5 (historically, Lyt-1 or Ly-1 in mice) was first identified on T cells (163) by staining with a polyclonal antibody. Creation of a monoclonal antibody permitted the identification of CD5 on Thy-1− cells in

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the B cell areas of murine lymphoid tissue (164) and subsequently on a minor population of spleen cells with high levels of mIgM that was particularly evident in NZB mice (165–167). Fetal and neonatal tissues of normal mice were found to be rich in CD5+ B cells (168). At the same time Hayakawa and coworkers showed that CD5+ B cells had unique functions (72). Virtually parallel observations were made concerning a human lymphocyte surface antigen. A monoclonal antibody identified an antigen (Leu 1) on thymusdependent T cells and B cell–type chronic lymphatic leukemia cells (169). Others confirmed finding this 67-kDa protein on mature T cells (170) and normal adult B cells (171). As in mice, fetal and neonatal tissues were enriched for these cells (168, 172), and they had unique functions (173, 174). CD5 has now been identified and at least partially characterized in humans, mice, rabbits, rats, cattle, sheep, pigs, and chickens. The chicken CD5 (69 kDa with a 57-kDa protein core) is expressed on all T and B cells, although there are fewer molecules on the latter (175). Porcine CD5, of which only a small, highly (96%) homologous region has been sequenced, is expressed on T cells and a minor fraction of B cells (176). In cattle (177) and sheep (178) there appear to be minor populations of B cells that express surface CD5. All rabbit B cells are CD5+ (179). In the rat, CD5 is detected on T cells but is not observed on any B cells (180). In mice and humans the genes encoding CD5 and the related CD6 (181) are about 45 kb apart in a conserved syntenic group (182) that maps to mouse chromosome 19 (183) and human chromosome 11 (184, 185).

Expression As thymocytes mature, in particular as the amount of surface CD3 increases, the amount of CD5 also grows (186, 187). An in vitro experiment suggested that this could be a response to extracellular ligands: Murine CD4+CD8+ lymphoma cells responded to anti-CD3, concanavalin A, or phorbol esters plus ionomycin with a marked increase in surface CD5 (188). Double positive thymocytes also responded to TCR engagement by increasing their CD5 levels (189). Compelling in vivo evidence from several systems also shows that CD5 expression in thymocytes is driven by TCR binding of ligand (190). In a system employing mice transgenic for a TCR with anti–HY specificity, expression of the antigenic ligand in the context of the appropriate MHC class I caused thymocyte deletion. Dutz and coworkers went on to show that when β2-microglobulin was not present, H-Y expression did not delete the majority of CD4+ CD8+ thymocytes but did allow the development of thymocytes with an unusually low level of CD5 (191). These conclusions were confirmed (192–194) and further extended in an important paper by Azzam et al. (195) that showed that low level expression of CD5 on double negative cells did not require receptor gene expression as it was seen in Rag −/− mice. However, even this level of CD5 is likely to be CD3 receptor ligation–dependent, as CD3 is expressed on DN thymocytes of Rag −/− mice (196). Treatment in vivo with various amounts of anti-CD3 resulted in proportional

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increases in CD5 expression as well as progression to the double positive stage. Lack of lck reduced but did not prevent CD5 expression. In their system, too, MHC-deficient mice expressed low levels of CD5. In toto their results show that the level of expression of the TCR, the avidity of ligation, and the strength of signal transduction (number of ITAMs in the CD3/TCR complex) collectively determined the amount of CD5 expressed. This supports a model in which the specificity of the TCR and the availability of appropriate antigen determine the avidity of TCR/ligand binding and thus the level of expressed CD5. In contrast, ligation of the TCR on mature peripheral CD4 or CD8 T cells does not seem to induce higher levels of CD5 expression (195). As CD5 decreases the sensitivity of the TCR (see below) its expression would alter the selected T cell repertoire. CD5 expression would block negative selection of some T cells with receptors that bind self-antigen with high avidity. At the same time CD5 would cause some low avidity cells to die from neglect. Conversely, loss of CD5 expression would allow deletion of some T cells that ordinarily bind selfantigen with insufficient avidity to induce tolerance. Loss of CD5 would also allow some low avidity self-reacting cells to be selected. These postulates were validated in experiments with TCR transgenic mice that co-expressed self antigens (196a). Similar evidence exists regarding B cell expression of CD5. The transformed murine pre-B cell line 70Z/3 expresses a low level of CD5. Expression is increased by treatment of the cells with LPS and decreased by IL-4 (197). CD5 expression can be induced by treatment of human peripheral B cells with phorbol esters (198, 199), and this can be blocked by simultaneous addition of IL-4 (200). In our hands murine CD5− B cells were not converted to CD5+ by this treatment (M. Teutsch, H. Wortis, unpublished). Ligation of the BCR of murine splenic B-2 cells by anti-IgM induces CD5 expression. In contrast, no induction occurs consequent to activation with LPS, T helper cells or CD40 ligand. In fact, CD40 ligand or IL-4 diminishes the induction of CD5 by anti-IgM (21, 201). Induction is at least in part at the level of transcription as it is blocked by actinomycin D and correlates with the accumulation of CD5 mRNA (24). In addition, CD5 50 flanking sequences allow anti-IgM treatment to induce expression of a luciferase reporter gene in transient transfection experiments (202). If anti-IgM mimics the ligation of the BCR by classic TI-2 antigens, then TI-2 antigens themselves should induce CD5 expression. This was tested by Whitmore, Haughton and Arnold who, after immunizing mice with the TI-2 antigen polyvinyl pyrrolidinone (PVP), used flow cytometry to sort CD5+ cells and CD5-cells (48). Similar regulation appears to occur in humans as incubation of CD5-tonsillar B cells with Staphlococcus aureus Cowan strain 1–induced CD5 expression (203). As in mice, IL-4 decreased CD5 expression on cells maintained in culture (204). PROMOTER Weichert et al. cloned a 1700-bp fragment 50 of the murine CD5 gene (205). By CAT assays they found that sequences between −125 and −27 were sufficient and necessary for transcriptional activity in transformed B and T cell

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lines. In contrast, the same CD5 flanking sequences could not drive expression in (nonlymphoid) NIH-3T3 cells. The region between −125 and −27 has no TATA box but does have potential Sp1, initiator and other recognizable elements. To date no elements in this proximal region have been identified by mutational analysis as important for B cell expression. Tung, Kunnavatana, Herzenberg & Herzenberg find that approximately 200 bp 50 of the translation start site there is a less than 50-bp region that is important for optimal expression of CD5 in transformed B and T lymphocytes. An ets binding site within this region appears to be essential for expression (205a).

Enhancer In order to identify regulatory sequences responsible for the induction of CD5 by BCR cross-linking, reporter constructs containing CD5 50 flanking sequences were introduced into murine splenic B cells. In all cases, reporter activity of unstimulated transfected cells was similar to background activity obtained by transfection with a promoterless reporter construct. A construct containing about 2200 bp 50 of the start site was induced about 10-fold by BCR ligation. Induction was abolished by deletion to −1965. Further analysis revealed that a 122-bp element comprised of sequences from −1919 to −2040 was sufficient to confer inducibility on the CD5 promoter proximal region (−6 to −277 relative to the ATG) even when placed in the reverse orientation and downstream of the reporter. Four complexes formed on this element after incubation with extracts from both induced and noninduced cells. Four additional complexes formed only in extracts from anti-IgM-treated B cells. Among these inducible complexes were two that were shown to contain NFAT. Point mutation of two putative NFAT binding sites severely compromised the activity of the enhancer in the transfection assay (202). This is consistent with the earlier observation that CD5 induction on splenic B cells was calcium dependent and cyclosporin A sensitive (201). A virtually identical element is now known to be located about 9.5-kb upstream of the CD5 gene in humans (cf. 114). Peritoneal CD5+ B cells have considerably more NFATc and in particular more nuclear NFATc than do their companion splenic B-2 cells. This raises the possibility that the constitutive expression of CD5 on B-1 cells may be NFAT-dependent. Preliminary data indicate that in NFATc knockout mice B-1a cell development and CD5 expression is compromised (R. Berland, H. Wortis, unpublished).

The CD5 Molecule EXTRACELLULAR DOMAINS CD5 is a type 1 transmembrane glycoprotein with a relative molecular mass of 67 kDa (206, 207). Comparison of the murine and human genes reveals 43% distal and 63% proximal identity in the extracellular region, and 90% identity for the intracellular carboxylterminal portion of the molecule (206). The extensive conservation of CD5, particularly of the cytoplasmic regions, suggests that it has important functions.

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Partial characterization of the extracellular region of CD5 reveals it to be a rod-like structure with N-linked and sialylated O-linked glycans (208). It contains three scavenger receptor cysteine-rich (SRCR) domains (209, 210). CD5 and CD6 (see below) comprise a closely related group of cell surface molecules with three SRCR domains of about 110 amino acids (181). There is no known structural homology between the cytoplasmic regions of CD5, CD6, or any of the scavenger receptors. A third protein with a similar SRCR structure, Spα, is secreted by hematopoietic cells (181, 211). Spα was also independently cloned as AIM, an apoptosis inhibitor that is produced by macrophages (212, 213) and binds to unknown ligands on lymphocytes and macrophages. A natural soluble form of CD5 was recently identified in human serum (214). Soluble CD5, which may be generated by proteolytic cleavage, contains the extracellular SRCR domains of membrane-bound CD5. Factor I of the complement system is another secreted protein that contains an SRCR (215). Amino acid sequence comparison of SRCR domains allows them to be divided into two types, those with three (type A) or four (type B) disulfide bridges (216). Type B–containing proteins include CD5 and CD6 as well as Spα, CD163, and others variously expressed by lymphocytes, macrophages, and gut-associated tissues (181). Type A SRCR domains are found in Class A macrophage scavenger receptors and other proteins (cf 217). Physiological ligands of the type B SRCRs have not been established, with the notable exceptions of CD163, which binds haptoglobin-hemoglobin complexes (217a), and CD6, which binds (218) with ALCAM (activated leukocyte adhesion molecule) via its membrane-proximal SRCR domain (219, 220). Interestingly, CD5 and CD6 appear to differ at residues critical for binding to ALCAM (220a). Several cell surface molecules have been reported to be ligands of CD5. The first identified ligand was CD72 (221, 222). This was shown by a demonstration that CD5 purified from lysates of human or murine T cell lines bound to target B cells. This binding was specifically blocked by antibodies to CD72. However, Bikah and colleagues reported that a soluble CD5-Ig fusion protein bound to B cells and this binding was not blocked by either of two anti-CD72 antibodies. Nor did this fusion protein bind to L cells expressing transfected CD72 (223). Therefore, either CD5/CD72 is not a receptor/ligand pair or the fusion protein differed in ligand specificity from CD5 purified from membranes. Bikah reported that small resting B cells proliferated in response to the CD5 fusion protein, suggesting this receptor-ligand binding might have physiological significance. They named the putative ligand CD5L and reported that it was found only on B cells. It was just detectable on small resting cells but readily found on ex vivo peritoneal B cells and splenic B cells stimulated in vitro with LPS or anti-IgM. This may be the same ligand as described by Biancone et al., but there are differences in the characteristics of the partially purified proteins (10, 224). Evidence also suggests that a broadly expressed surface protein (225), as well as a region of the immunoglobulin VH framework (226), are CD5 ligands.

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To date, none of these has been shown to be a functional physiological ligand. As noted above, there is some evidence that CD5L can activate B cells. Also, a soluble recombinant form of CD5 acts in vitro as a modest costimulator of BCR-mediated proliferation, perhaps by binding to CD72 (227). Administration of a recombinant form of CD5 to mice with an antibody-induced membranous glomerulonephritis partially inhibited the disease (224), although the mechanism by which this effect is mediated has not been established. CYTOPLASMIC DOMAINS The five characterized mammalian (human, mouse, sheep, bovine, rat) CD5 molecules typically have cytoplasmic regions containing 96 amino acids. Of these, over 91 are identical or conserved. Each CD5 contains four tyrosines, four threonines, and eleven serines. This basic conservation is also preserved in the rabbit (C. Raman, K. L. Knight, unpublished). ASSOCIATION OF CD5 WITH TCR AND BCR Coimmunoprecipitation of Brij 96 lysates of human T cells revealed association of CD5 and CD3 with lck and fyn. Cross-linking of CD5 by antibody induced phosphorylation on the tyrosines of numerous proteins including CD5 itself. Treatment with anti-CD3 also caused phosphorylation of CD5 (228). Osman and colleagues (229) calculated that 10%– 20% of CD5 was CD3 associated. Fluorescence resonance energy transfer (FRET) demonstrated CD3 and CD5 to be within 10 nm of one another on human peripheral T cells (230). Based on co-immunoprecipitation of Brij lysates of normal human thymocytes, another group concluded that CD5, Zap-70, and CD3 were associated (231). CD phosphorylation correlated with the induced association with CD5. Human CD5 and IgM were co-immunoprecipitated from digitonin lysates of CD5+ T cells (232). This group also showed that CD5 is phosphorylated on tyrosine in response to CD5 ligation. In keeping with the idea that the BCR and CD5 are physically associated, CD5 and mIgM co-capped in response to cross-linking (233). CONSEQUENCES OF LIGATION: CANDIDATE EFFECTORS Within two minutes of ligation of human peripheral T cell CD3, CD5 is phosphorylated on tyrosines, threonines, and serines (234). In fact, as Pani and co-workers reported (235), the CD5 molecules in thymocytes and mature T cells have a basal level of phosphorylation on tyrosine that is increased by TCR cross-linking. They also found that a known negative regulator of activation, SH2-containing hematopoietic cell phosphatase (SHP-1), was associated with CD5 in both resting and TCR-activated cells. Perez-Villar (236) found that in the transformed human T cell line Jurkat, CD5 was basally phosphorylated on tyrosine and SHP-1 and CD5 could be coimmunoprecipitated (both ways) as could in vitro phosphatase activity. The association of CD5 and SHP-1 was not mediated by CD3 as a CD3− variant also showed this association. They failed to find CD5-associated SHP-2. TCR crosslinking increased both CD5 phosphorylation on tyrosine and the amount of

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associated SHP-1. To explore the functional significance of these associations, stable transfectants expressing a fusion protein in which extracellular CD6 was combined with the transmembrane and cytoplasmic domains of CD5 were created. Cross-linking of the CD6/CD5 chimera with the TCR partially reduced calcium transients and phosphorylation on tyrosine of CD3, ZAP-70, Syk, and PLCγ 1 but did not affect tyrosine phosphorylation of lck. (However, it should be noted that this Jurkat line expressed endogenous, functional CD5.) Single Y > F mutations in the CD5 intracellular domain revealed that deletion of the juxta-membrane tyrosine was sufficient to ablate SHP-1 association, in vitro phosphatase activity, and the observed alterations in calcium transients and protein phosphorylation. Deletion mutations of the CD5 cytoplasmic region showed that a truncated protein with only the single membrane-proximal tyrosine retained all the observed negative regulatory activities and the ability to associate with SHP-1. The Perez-Villar study raises an interesting question about the mechanism by which CD5 might associate with the phosphatase SHP-1 and effect its activation. Current evidence indicates that optimal activation of SHP-1 requires engagement of both of its SH2s with P-TYRs (237). Yet, here mutations of CD5 show that phosphorylation of the juxta-membrane tyrosine is both necessary and sufficient for CD5 to mediate activation of SHP-1. It is known that loss of SHP-1, as in motheaten mice, results in T cells with increased sensitivity to activation by TCR cross-linking (235). Therefore, it is possible that SHP-1 is necessary for the negative regulatory activity of CD5. If so, either submaximal activation of SHP-1 via CD5 association is sufficient for negative regulation of T cells or association with CD5 may maximally activate SHP-1 despite the presence of only a single ITIM. Alternatively, the involvement of a third protein with a phosphorylated tyrosine may be involved. Conceivably, SHP-1 could bridge between CD5 and a phosphorylated tyrosine on a second protein or an adapter SH2 could bind with CD5 via a single SH2 and present two phosphorylated tyrosines to SHP-1. In B cells, CD5 negatively regulates signals through the BCR, as indicated by studies with CD5−/− mice. B-1 cells from these mice, in contrast to those from wild type, can flux Ca2+, activate NF-κB, and proliferate in response to BCR cross-linking. In the same study, B-1 cells from CD5+ mice were activated after BCR cross-linking if cells were first treated with biotinylated anti-CD5 and streptavidin. This was interpreted to be a consequence of sequestration of CD5 from other cell surface proteins, a likely candidate being the BCR complex itself (10). To examine the basis of the negative regulation of BCR signaling by CD5, a novel fusion protein containing the extracellular and transmembrane domains of Fcγ RIIB and the cytoplasmic region of CD5 without the juxta-membrane tyrosine was introduced into an FcR− variant of the transformed CD5-B cell line A20 (238). Co-cross-linking the BCR and this recombinant protein significantly decreased calcium transients, ERK2 activation, and the late but not the early phosphorylation of PLCγ 1. In contrast with other studies, anti-CD5 failed to co-immunoprecipitate SHIP, SHP-1, or SHP-2. Removal of the 16 aa segment Y429 through L444 ablated the negative regulation of the BCR by the CD5 recombinant protein. Taken

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together, these results suggest that CD5 may have an inhibitory element in addition to the juxta-membrane ITIM. Other experiments suggest that in B1a cells negative regulation by SHP-1 can be relieved by ligation and sequestration of CD5 (239). Using the two-hybrid system, two potential ligands of the CD5 cytoplasmic region were identified: CAM kinase IIδ and Tctex-1 (240). Tctex-1 is a component of the dynein motor complex. The association of CAM kinase IIδ and CD5 was further explored in vitro using fusion proteins. A 33 amino acid fragment containing residues of the juxta-membrane portion of the cytoplasmic region of CD5 bound to CAM kinase IIδ. Interestingly, this segment partly overlaps with a sequence that has homology with a CAM kinase IIδ association domain. CAM kinase II was previously shown to be activated through the BCR (241). Co-immunoprecipitation experiments failed to reveal any association of CD5 and CAM kinase IIδ in B cell lines in which both proteins are expressed. Nevertheless, it is attractive to think that CD5 might regulate the ability of CAM kinase II to phosphorylate calcineurin, thereby inhibiting the calciumdependent activation of proteins such as NFAT. Other yeast two-hybrid experiments suggested that casein kinase II (CK2) interacts with the cytoplasmic region of CD5. Co-immunoprecipitation of proteins from human B and T cell lines as well as mouse splenocytes revealed constitutive association of these two proteins. CD5 was phosphorylated in vitro by CK2. In B or T cell lines, cross-linking of CD5, but neither the B nor TCR, leads to the activation of CK2. A functional role for CK2 was not demonstrated (242, 243), and the significance of this protein-protein interaction remains to be established. It is intriguing that the cytoplasmic portion of CD163, a macrophage transmembrane protein with type B SRCRs, also associates with CK2 (243a).

FUNCTIONS OF CD5 Role of CD5 in B Cells In response to anti-TCR+ PMA or to concanavalin A, single positive thymocytes but not peripheral T cells from CD5 knockout mice proliferated more than wildtype cells. Yet, they proliferated equally well in response to PMA + ionomycin. Therefore, CD5 acts downstream of the TCR and, presumably, upstream of PLC. Thymocytes from CD5 knockout mice also show heightened Ca2+ transients and phosphorylation on the tyrosines of PLCγ 1 in response to TCR ligation. While candidate immediate targets were not identified, it is interesting to note that there appeared to be an alteration in the ratio of the phosphorylated isoforms of the ζ chain of CD3 and that there was markedly elevated tyrosine phosphorylation of Vav in the knockouts (245). Following TCR ligation there was phosphorylation on tyrosines of both the p21 and p23 isoforms of CD3. Previously it was observed that agonists induce similar levels of phosphorylation of CD3 p21 and p23, whereas anergizing agonists induce more phosphorylation of p21 than p23 (cf. 244). Interestingly, following treatment

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with anti-TCR in the absence of CD5, phosphorylation of p23 was greater than that of p21. This suggests a model in which CD5 acts to alter a TCR ligation (agonist) signal toward anergy rather than activation. In the same study, absence of CD5 altered positive or negative selection of thymocytes in mice expressing any of three transgenic TCRs. That selection was variously affected suggests that CD5 acts to modify the strength of signal mediated through the TCR. Given that CD5 acts as a negative regulator, the loss of CD5 would alter the response to some ligands such that they would now be above the threshold of negative selection. Responses to other, weaker, antigen/TCR combinations would now be increased to the point that ligating cells would be positively selected (245, 246). In another system, in the presence of antibody to the TCR, antibody to CD5 provided a costimulatory stimulus that induced apoptosis of CD4+ CD8-medullary thymocytes (247). The ability of CD5 to act as a negative modifier of CD4 development by thymocyte selection was shown in a second transgenic TCR system. Here, the ability of CD5 to act as a negative regulator required the cytoplasmic portion of the molecule (248).

B Cell Function in CD5 Knockout Mice Studies of CD5 knockout mice showed that CD5 also acts as a negative regulator of B-1a cell activation (10). Purified B-2 and B-1 cells isolated from wild-type or CD5 knockout mice were stimulated with anti-IgM, anti-CD40, or LPS. As expected, B-1 cells from wild-type mice proliferated in response to CD40 or LPS but not anti-IgM. In contrast, B-1 cells from the knockout mice responded to all three. Calcium transients were sustained longer in the CD5− peritoneal B cells than in the wild type. Associated with these changes in signaling, CD5− B-1 cells showed reduced apoptosis and increased nuclear localization of NF-κB following BCR ligation. Therefore, in B-1a cells, as in T cells, CD5 is a negative regulator of antigen-induced signaling. The functional consequences of this negative regulatory activity of CD5 were examined by Hippen & Behrens (39). Close examination of anergic B cells generated in mice transgenic for antibody to hen’s egg lysozyme (HEL) as well as for the anergizing antigen soluble HEL (40) revealed that they expressed a very low level of CD5, even lower than seen on typical B-1a cells. This suggested that the induction of CD5 by autoantigen might be a mechanism by which the production of autoantibodies is avoided. This hypothesis was tested by breeding to produce CD5 knockout mice expressing the anti-HEL and soluble HEL transgenes. Consistent with this hypothesis, a fraction of the knockout, but none of the CD5+ wild-type mice, produced antibody to HEL.

Mice Transgenic for a B Cell Targeted CD5 Construct Chen, Matsura, & Kearney inserted CD5 cDNA into an expression construct such that it was under control of an immunoglobulin heavy chain promoter, the

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intronic enhancer, and portions of the switch region. With this construct they created transgenic mice that expressed high levels of CD5 on all B cells (249). In fact, CD5 was found on sIgM-negative, bone marrow cells, indicating that even pre-B and possibly pro-B cells might be capable of expressing CD5 on their surfaces. Additional preliminary experiments revealed that mature splenic CD5 transgenic mice did not differ from littermates in numbers of B cells or in the level of expression of B cell molecules mIgM, mIgD, MHC class II, CD38, or CD23. These B cells responded normally to a variety of mitogenic agents including antiIgM and CD40 ligand. There were no detectable differences in serum Ig levels in nonimmunized animals nor in responses to TI-2 or TD antigens. Notably, when immunized with NP-chicken gamma globulin, the transgenic mice produced anti-NP antibody. A limited number of these were sequenced and found to have somatic mutations at a frequency no less than seen in wild-type mice. This is evidence against the hypothesis that the infrequency of somatic mutation in the immunoglobulins produced by B-1a cells is a consequence of the expression of CD5 per se. The mutated sequences found in these NP-immunized transgenic mice were notable in that they were particularly enriched in a mutation, W33L, known to raise the binding affinity for NP tenfold (250). As CD5 is a negative regulator of BCRmediated activation, it may well be that only this higher affinity antibody can be selected in the presence of the transgenic CD5. That is, CD5 sets a higher threshold for BCR-mediated activation. Yet, in mice that were six months or older, there was a striking splenomegaly that could be attributed to a hyperplasia of marginal zone B cells and plasma cells. This was not a simple founder effect, as it was seen in animals derived from four founders. It could be that CD5 negatively regulates tolerogenic signaling in the MZ or that it acts positively by an unknown mechanism. ACKNOWLEDGMENT This work was supported by NIH grant AI R0115803. Visit the Annual Reviews home page at www.annualreviews.org

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E PROTEIN FUNCTION IN LYMPHOCYTE DEVELOPMENT Melanie W. Quong∗ , William J. Romanow∗ and Cornelis Murreˆ University of California, San Diego, Division of Biology, 9500 Gilman Drive, MC 0366, La Jolla, California 92093-0366; e-mail: [email protected], [email protected], [email protected]

Key Words E2A, Id, thymocyte, B lymphocyte, helix-loop-helix (HLH) ■ Abstract Lymphocytes arise from hematopoietic stem cells through the coordinated action of transcription factors. The E proteins (E12, E47, HEB and E2-2) have emerged as key regulators of both B and T lymphocyte differentiation. This review summarizes the current data and examines the various functions of E proteins and their antagonists, Id2 and Id3, throughout lymphoid maturation. Beyond an established role in B and T lineage commitment, E proteins continue to be essential at subsequent stages of development. E protein activity regulates the expression of surrogate and antigen receptor genes, promotes Ig and TCR rearrangements, and coordinates cell survival and proliferation with developmental progression in response to TCR signaling. Finally, this review also discusses the role of E47 as a tumor suppressor.

INTRODUCTION Self-renewing, hematopoietic stem cells (HSC) are multipotent, capable of generating erythroid, myeloid, dendritic, and lymphoid cell lineages. Lymphocyte development occurs through a common lymphoid progenitor (CLP), which has restricted lineage potential (1). CLPs develop into three distinct cell types: B and T lymphocytes and NK cells. Beginning with lineage commitment and continuing throughout maturation, both B and T lymphocytes develop through stages that can be defined by the rearrangement of antigen receptor genes, the acquisition or loss of cell surface and intracellular proteins, and responses to growth and survival factors. Gene expression that occurs during lymphocyte development is controlled, at least in part, by a class of transcription factors known as the helix-loop-helix ∗

These authors contributed equally to this manuscript. ˆ To whom correspondence should be addressed.

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proteins (HLH). This review focuses on the roles of the class I HLH proteins and their antagonists in lymphoid development. Class I HLH proteins, also known as E proteins, were first identified based on their ability to bind with relative high affinity to the palindromic DNA sequence CANNTG, referred to as an E box site (2–4). E boxes have since been found in the promoter and enhancer regions of a wide variety of B and T lineagespecific genes. These include the enhancers in the immunoglobulin (Ig) loci and the T cell receptor (TCR) α and β loci, the promoters of the mb-1, λ5, and pre-Tα genes, and the CD4 silencer and enhancer elements (5–10). The E protein family includes five members, designated as E12, E47, HEB, and E2-2, in vertebrates, and the Drosophila gene product, daughterless (for review, see 11). They share a highly conserved motif termed the helix-loop-helix (HLH), which consists of two amphipathic α-helices separated by a loop structure (12). This domain imparts dimerization capabilities between HLH members (12). Immediately amino-terminal to the HLH is a conserved basic region that allows HLH dimers to bind to DNA. In addition to the HLH motif, the mammalian E proteins share two conserved transcriptional activation domains referred to as the AD1 and loop-helix (LH) domains (13–15). The AD1 domain forms an acidic helical region and is also present in Rtg3p, an HLH protein in S. cerevisiae (15, 16). The LH domain contains a potential loop located adjacent to a putative amphipathic helical structure and is conserved among all E protein members, including daughterless (13). The conservation of the activation domains suggests that the target molecules with which E proteins interact are conserved throughout evolution (11). Ablation of either activation region, through deletions or structural mutations, severely affects the transcriptional capabilities of the E proteins (13, 15). The E2A gene encodes for two E proteins, E12 and E47, which arise through differential splicing of the exon encoding for the HLH domain (4, 17). HEB and E2-2 are encoded by separate genes (3, 18). All of the E proteins have the ability to bind DNA as homo- or heterodimers (19). Although not ubiquitous, E proteins are broadly expressed; however, certain E protein complexes are restricted to specific cell types. In B lineage cells, the predominant E box binding complex is comprised of E47 homodimers, whereas in thymocytes, E47/HEB heterodimers predominate (9, 20–23). Additional control of E protein activity occurs through interaction with the Id HLH proteins (24). Members of this group lack the basic region and thus do not have the ability to bind to DNA (25). Id proteins efficiently heterodimerize with E protein members and effectively act as dominant-negative HLH proteins. Four mammalian members have been identified: Id1, Id2, Id3 and Id4, as well as one Drosophila homologue, extramacrochaete (emc) (25–31). Of the four Id proteins, only Id2 and Id3 are abundantly expressed in B and T lymphocytes (32, 33). Since the Id proteins can act as dominant-negative HLH proteins, the ratio of E proteins to Id proteins ultimately determines the level of E protein activity.

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B Lineage Commitment and Early Stages Commitment and progression through early stages of B lineage development is dependent on specific gene expression programs as well as environmental cues (Figure 1). The study of gene transcription has included regulatory elements of the Ig genes, as well as genes encoding the structural components of the B cell receptor (BCR), including Igα, Igβ, λ5, and VpreB. Additionally, components of the recombination machinery, namely RAG1 and RAG2, have been examined in great detail (34, 35). The analysis of genetically altered mice has led to the identification of a number of transcription factors, including E2A, EBF, and Pax5, that regulate many of these B lineage specific genes (36–39). E2A, EBF, and Pax5 act in a cascade and in synergy to establish and maintain the expression of target genes that promote the B lymphocyte maturation program (10). E2A-deficient mice display a complete block in B lineage development at a very early stage prior to the onset of IgH DJ rearrangement (Figure 2a) (36, 37).

Figure 1 Schematic diagram of murine B cell development. Successive developmental stages are shown together with their characteristic cell surface markers. Transcription factors indicated above the arrows denote the approximate developmental stages at which they have been described to function. The appropriate stages when Ig rearrangements are incurred are indicated by the horizontal lines. Vertical dashed lines delineate checkpoints requiring pre-BCR and BCR-mediated signals for further developmental progression.

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Figure 2 Summary of the stages at which E proteins affect B and T lymphocyte development. (a) B cell development. E proteins have been shown to be required at several stages of development as indicated by horizontal arrows on the left side of the figure. The corresponding functions are described on the right side (see text for details). Cell surface expression of the antigen receptors are indicated where appropriate. At the commitment stage, E2-2 and HEB are required for generating wild-type levels of pro-B cells. (b) T cell development. Horizontal arrows on the left indicate where E proteins are required for progression. Blunt-ended arrows denote those stages at which E protein activity must be decreased in order for progression to occur. The function at each stage is listed on the right (see text for details). Surface expression of the pre-TCR and TCR during development is shown. HEB is required for expression of the pre-Tα chain gene and the rearrangement and expression of the TCR α locus.

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Early B lineage specific transcripts, including Igα, Iµ, RAG1, and Pax5, are lacking in E2A-null mutant bone marrow cells. In contrast, myeloid development is normal. A similar phenotype is observed in mice expressing a transgene for Id1, an inhibitor of E protein activity (40). These mice display the same block in B cell development, and its severity is dependent on the expression level of the transgene. In human bone marrow cells, ectopic expression of Id3 causes an analogous block, which demonstrates the conserved function of E proteins in murine and human lymphopoiesis (41). Two other E protein members, E2-2 and HEB, also have roles in B lineage development (Figure 2a). Mice deficient for HEB or E2-2 generate mature B lineage cells but contain 50% fewer pro-B cells in the fetal liver (42). Interestingly that mice that are transheterozygous for any two of the four E proteins display fewer pro-B cells than mice that are heterozygous for any E protein alone (42). Restoration of E protein expression in an E2A null background allows for B cell development albeit at varying degrees. Expression of both E12 and E47 transgenes in E2A-deficient mice promotes B lineage development better than either transgene alone (43). Furthermore, expression of two (but not one) copies of the HEB gene introduced into the E2A locus results in a partial rescue of B lineage development (44). Mature B cells are generated, but wild-type numbers are not achieved (44). Therefore, the overall timing and dosage of E protein activity may be the key determinant in B lineage progression rather than the activity of a specific E protein. Absence of the EBF transcription factor results in an early B lineage block similar to that observed in E2A-deficient mice (Figure 1) (38). E2A and EBF genetically interact to promote B lineage maturation, as mice that are heterozygous for each of these genes show a more severe B cell phenotype than mice that are heterozygous for either gene alone (45). However, on a per cell basis, the level of VpreB transcripts in double heterozygous mice was shown to be similar to that of wild-type pro-B cells, indicating that the synergy involving E2A and EBF is not at the level of gene expression, but perhaps at the level of cell survival or cellular expansion (45). In addition to E2A and EBF promoting B lineage progression, a key function for Pax5 in establishing B cell commitment was recently demonstrated (46). Pax5deficient B cells arrest during the pro-B cell stage after IgH DJ rearrangement (Figure 1) (39, 47). Thus the absence of Pax5 leads to a block in B cell development at a stage subsequent to that observed in E2A- or EBF-deficient mice. However, pro-B lineage cells derived from Pax5-deficient bone marrow express non-B lineage genes such as pre-Tα, GATA-1, and macrophage colony-stimulating factor (46). Furthermore, B cell progenitors lacking Pax5 maintain the potential to be diverted to other lineages under the appropriate culture conditions (46). Thus, Pax5 expression is essential for commitment to the B cell lineage through its role in suppressing the transcription of genes normally associated with alternative hematopoietic lineages. Since E2A deficiency results in a complete lack of B lineage cells, other strategies have been employed to identify targets of the E2A proteins involved in

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initiating the B lineage program. Ectopic expression of E47 in NIH3T3 cells leads to the induction of Ig germline transcription and the expression of other early B cell genes, such as RAG1 and TdT (48). Additionally, overexpression of E12 in 70Z/3 macrophage cells, which carry rearranged IgH and Igκ genes but lack IgM expression, is sufficient to activate the transcription of many B lineage-specific genes including RAG1, λ5, EBF, and Pax5 (49). Interestingly, when EBF is expressed in the 70Z/3 macrophage cell line, transcription of a subset of the E2A-induced genes is upregulated, including Pax5 and λ5 (49). These observations suggest that the E2A proteins regulate the transcription of downstream targets such as Pax5 through its regulation of EBF. Consistent with these observations is the recent finding that the Pax5 promoter contains functional EBF binding sites (45). Moreover, studies suggest that E2A and EBF synergize to induce the expression of common target genes. For example, ectopic expression of both E47 and EBF in a mast cell line, Ba/F3, activates the transcription of the λ5 and VpreB genes (50). Dissection of the promoter of the λ5 gene identified EBF and E47 binding sites, both of which are required for its regulation (50, 51). Taken together, these data suggest that E2A and EBF play dual roles, establishing lineage commitment and promoting B lineage progression, through their ability to activate the expression of genes involved in both processes.

Initiation of V(D)J Recombination and the Pro-B to Pre-B Cell Transition Transition from the pro-B to the pre-B cell stage is dependent on the surface expression of a functionally rearranged IgH chain. Association of IgH chains with surrogate light chains, in conjunction with signaling components Igα and Igβ, constitutes the pre-B cell receptor (BCR). Pre-BCR signaling results in a transient burst of proliferation and allelic exclusion of the IgH chain, followed by initiation of IgL chain gene rearrangement (52–54). B cells from mice that are deficient for either the IgH chain itself or the expression of RAG1 or RAG2 are blocked at the pro-B cell stage (55–57). Similarly, B cell development in mice that are deficient for λ5, a component of the surrogate light chain, fails to progress efficiently to the pre-B cell stage (58, 59). Signaling in pro-B cells mediated through either Igα, Igβ, or an activated form of Ras is sufficient to promote maturation to the pre-B cell stage (60–62). Targets downstream of the pre-BCR remain to be identified, but they are likely to include the E proteins (see below). Recently, the E2A proteins were shown to be required during the IL-7-dependent expansion and survival of pro-B lymphocytes (Figure 2a) (63). In addition to promoting cell survival, E2A proteins are intimately involved with the expression and rearrangement of the IgH and IgL chain genes (Figure 2a). Although it remains to be demonstrated in primary cells, expression of the RAG1 and RAG2 genes has been shown to be regulated by the E2A proteins (45, 49). E2A proteins have the ability to promote IgH and IgL chain gene rearrangement in various cell types. Ectopic expression of E47 in the pre-T cell line, 2017, causes the induction of

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IgH D to J rearrangement, accompanied by an increase in the level of Iµ germline transcripts (64). Furthermore, expression of E47, in addition to RAG1 and RAG2, in an embryonic kidney cell line promotes both IgH DJ and Igκ VJ rearrangements (65). Rearrangements of the Igκ locus predominantly used Vκ1-family segments, which contain a conserved E box in their promoter regions. Ig rearrangements in this system were similar to those seen in endogenous B cells: distinct V segments were utilized and the coding joints were diverse and often contained deletions (65). These data suggest that E2A proteins play a key role in the regulation of both IgH and Igκ recombination. Since, in vivo, these rearrangements typically occur sequentially rather than concurrently, it is likely that additional transcriptional regulators act together with the E2A proteins to promote the ordered rearrangement of the IgH and IgL genes. One such candidate is EBF, which has been shown to promote IgH DJ but not Vκ-J joining (65, 65a). Additionally, it is possible that E protein levels are differentially regulated in pro-B versus pre-B cells. It will be important to examine carefully the relative levels of the individual E proteins in pro-B and pre-B cells and to determine whether E protein activity is regulated by signals mediated by the pre-BCR similar to that described for TCR-mediated signaling (see below).

Antigen Dependent Maturation of B Lineage Cells Antigen engagement of the mature B cell receptor initiates B cell activation characterized by cell cycle entry and upregulation of specific surface markers. Activated B cells receiving co-stimulatory T cell derived signals differentiate to become low affinity plasma cells or migrate to form germinal centers. There they undergo clonal expansion, affinity maturation and immunoglobulin isotype switching, resulting in high-affinity antibody-secreting effector cells and memory B cells (66–69). In the peripheral lymphoid organs, the E2A proteins are present at low but detectable levels in resting na¨ıve B cells (70). In contrast, E2A protein expression is high in activated, mature B cells and in cells present in the dark zone of germinal centers (70, 71). In vitro activation of B lymphocytes through co-stimulation with T cells and antigen or through various mitogenic stimuli results in higher levels of E2A proteins and DNA binding activity (70). Thus, induction of E2A activity appears to be a common feature during B cell activation. Id3 transcripts have been shown to be rapidly induced upon BCR engagement, suggesting a role in promoting cell cycle entry (72, 73). Consistent with this model, Id3-deficient mice display a proliferation defect in response to anti-IgM stimulation (72). The increase in Id3 levels is transient and occurs prior to the induction of E2A activity (70, 73). Whether Id3 regulates subsequent events during antigen-induced differentiation remains to be elucidated, but it is plausible that Id3 functions, at least in part, by regulating the activity of the E proteins. In contrast to pro-B lymphocyte expansion, loss of E2A activity in peripheral activated B cells does not interfere with proliferation and survival (70). In addition, the induction and regulation of early and late activation markers, including CD69,

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CD44, IgD, and IgM, is not impaired. However, activated B lymphocytes do not undergo isotype switching in response to appropriate stimuli in the absence of E2A activity (Figure 2a) (70). Similarly, ectopic expression of Id1 in two human B cell lines undergoing isotype switching significantly interferes with IgA surface expression (71). Activated primary B lymphocytes lacking E2A activity are blocked in class switching at the level of genomic recombination (70). Since the appropriate switch region germline transcripts are present, it is unlikely that the primary defect in switching is caused by a lack of chromatin accessibility (70, 71). Rather, we propose that the E2A proteins are required for the expression of components of the switch recombinase machinery.

THE ROLES OF E PROTEINS IN T LYMPHOCYTE DEVELOPMENT Thymocyte Maturation T cell development can be characterized based on the rearrangement status of the TCR loci and the expression of the CD4 and CD8 co-receptors (Figure 3). The earliest T cell progenitors in the thymus are present within the CD4 and

Figure 3 Schematic representation of murine T lymphocyte development. The developmental stages are shown together with their characteristic cell surface markers. Transcription factors listed above the arrows denote the approximate developmental stages at which they have been shown to function. The progression of β selection deficient DP cells in the absence of E2A to the DP stage is denoted by the long arrow from the DN3 to DP stage (see text). The appropriate stages when TCR rearrangements are incurred are indicated by the horizontal lines. Vertical dashed lines delineate pre-TCR and TCR mediated checkpoints required for developmental progression.

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CD8 double negative (DN) population. These cells are characterized as being CD44+ and CD25− and still have the ability to mature into NK and T lineage cells. Commitment to the T lineage is largely associated with the activation of CD25 expression and is followed by initiation of gene rearrangements at the TCR γ , δ, and β loci. The formation of a functional TCR β gene product allows for the expression of the pre-TCR complex, which includes the TCR β chain as well as the pre-Tα protein and the CD3 assembly of signaling molecules. Signaling mediated by the pre-TCR complex results in developmental progression, also referred to as β selection. This transition is characterized by inhibition of gene rearrangement, initiation of cellular expansion, and the maturation into CD4 and CD8 double positive (DP) thymocytes. DP cells then exit the cell cycle and begin TCR α gene rearrangement. The expression of an αβ TCR allows DP cells to undergo major histocompatibility complex (MHC)-mediated positive or negative selection. Positively selected DP thymocytes downregulate either CD4 or CD8 expression to become single positive (SP) mature T lineage cells (74–77).

The T versus NK Lineage Decision NK and T lineage cells develop from a bipotent T/NK precursor cell (78–80). Recent evidence has indicated a role for HLH proteins in both NK and T lineage commitment (Figure 3). Mice that are deficient for Id2 display a block in NK cell development at the T/NK precursor cell stage (81). In contrast, αβ T cell development is normal. E2A-deficient mice display a complimentary phenotype characterized by a partial block in T cell development and normal NK cell development (23). Similarly, forced expression of Id3, another E2A antagonist, in precursor T/NK cells or in committed progenitor T cells, blocks T lineage progression but not NK cell development (82, 83). Taken together, these observations indicate a key role for HLH proteins at this stage and demonstrate that the ratio of E2A and Id2 likely regulates the NK versus T cell lineage decision.

Expression of E2A during T Lineage Development The expression of E47 in γ δ and αβ T lineage cells has been examined in great detail. The majority of CD44+ CD25– (DN1) thymocytes represents cells that have not yet committed to the T cell lineage and express little or no E47 (84). The transition to the CD44+ CD25+ (DN2) stage is closely correlated with a bias towards T cell development. Interestingly, E47 expression is induced at the DN2 stage. In addition, γ δ T cells, which diverge from the αβ T lineage, express elevated levels of E47 (I. Engel, C. Murre, unpublished observations). E47 levels remain high throughout DN thymocyte maturation; however, upon pre-TCR mediated signaling, E47 DNA-binding activity is significantly downregulated (84). As compared to DN thymocytes, DP cells express lower levels of E47 proteins, which are decreased further during the transition from DP to SP cells. These

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Figure 4 Model of T lymphocyte progression and E protein expression. Relative E47 protein levels in DN, DP and SP thymocytes (as described in text) are indicated as the plateaus. Progression from one stage to the next is initiated by pre-TCR/TCR signaling (vertical arrows) resulting in increases in Id3 transcripts and decreased E protein DNA binding activity (as described in text). T lineage developmental progression is represented by the x axis. The rate of decrease of E2A at each transition is conjectured and remains to be determined.

observations indicate that a gradient of E47 expression is present during thymocyte development: E47 levels are highest in DN cells and lowest in SP cells (Figure 4) (84).

Early T Lineage Development Consistent with the expression pattern of E47 in thymocytes, loss of E2A affects both the αβ and γ δ T cell lineages (Figures 2b and 3). In E2A-deficient mice, not only are γ δ T cell numbers reduced, but the temporal regulation of TCR γ and δ recombination is disrupted (85). E2A is required to repress fetal-specific γ and δ rearrangements in adult thymocytes, while promoting the rearrangement of the appropriate adult V segments (85). Furthermore, E2A and HEB, in conjunction

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with RAG1 and RAG2, have the ability to induce γ δ TCR rearrangements in an embryonic kidney cell line (86, 87). αβ T cell development in E2A-deficient mice is also affected. Thymocyte cell numbers are decreased five- to ten-fold, and thymocyte development is partially blocked at the DN1 to DN2 transition (23). During the DN stages of αβ T cell development, rearrangement of the TCR β locus occurs. In addition to the induction of the RAG recombination machinery by E2A, evidence suggests that ablation of E protein activity, through expression of a dominant-negative form of HEB or an Id1 transgene, results in a decrease in Vβ to DJβ rearrangements (88, 89). Taken together, these data suggest that E protein activity is necessary for efficient progression toward the T cell lineage as well as directing early events such as V(D)J recombination in T cell development.

The DN to DP Transition Upon successful rearrangement of the TCR β locus, the TCR β chain pairs with pre-Tα and the CD3 subunits to form the pre-TCR complex. Signaling through this complex is essential for thymocytes to mature from the DN to the DP stage, a process referred to as β selection (90). HEB is necessary for the expression of the pre-Tα gene as demonstrated in HEB-deficient and SCL/LMO transgenic mice (91). Similarly, transduction of human pre-T cells with Id3 results in the suppression of pre-Tα transcription and an analogous block (83). E2A also functions to regulate β selection (Figure 2b). Genetic data have recently indicated that E47 deficiency is sufficient to promote the maturation of DN cells to the DP cell stage in the absence of a functional TCR β chain (84). This implies that pre-TCR signaling functions to promote progression, in part, by inhibiting E2A activity. Consistent with this model, E47 expression remains high during DN thymocyte maturation; however, upon pre-TCR-mediated signaling, E47 DNA binding activity is downregulated. In conjunction, Id3 RNA levels are increased upon pre-TCR-mediated signaling in DN thymocytes through a pathway dependent upon the ERK MAPK cascade (84). Taken together, these observations provide both genetic and biochemical evidence that E protein activity is downregulated by pre-TCR signaling to promote β selection (Figure 4). Once the DN T cells pass β selection, CD8 expression is upregulated, resulting in intermediate single positive (ISP) cells, followed by progression to the DP stage. Thymii of HEB-deficient mice display an increased proportion of ISP cells due to a block in the transition to the DP stage (92). How HEB functions during this transition remains to be clarified; however, regulation of the CD4 gene by HEB may play a role (9).

Thymocyte Selection Upon reaching the DP cell stage, thymocytes initiate TCRα rearrangement. Once a functional αβ TCR has formed, DP cells interact with peptide-MHC complexes

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to undergo selection. DP cells receiving the appropriate signal mediated by the TCR are positively selected. In contrast, cells expressing a TCR with high affinity for self-peptide-MHC complexes undergo negative selection. Failure to receive a TCR-mediated signal results in death by neglect (93–95). E2A-deficient mice have decreased proportions of DP cells and elevated percentages of SP T cells with a particular skewing toward the CD8 SP population (23). The decrease in DP cells is due in part to reduced viability (96). In fact, massive apoptosis is observed in thymocytes from Id1 transgenic mice, attributable to the fact that both E2A and HEB are inhibited (88). In addition, enhancement of both MHC class I- and class II-mediated positive selection is observed in E2Adeficient mice (96). In particular, E47-deficient, H-Y TCR transgenic female mice have increased percentages of CD8 SP cells in both the thymus and peripheral tissues. The acceleration of selection to the CD8 SP lineage in the thymus is evident in mice heterozygous for E47 (96). Thus, selection to the CD8 lineage appears sensitive to E protein dosage. Recently, studies analyzing E47 deficiency in mice with blocks in TCR β rearrangement have demonstrated that not only is progression to the DP stage restored, but development of a small number of CD8 SP thymocytes can be detected (84). It is interesting that CD4 SP cells are consistently absent, which indicates that a loss of E2A is sufficient to drive CD8 SP positive selection, whereas additional signals appear to be required for maturation to the CD4 lineage. Although it remains to be determined if the effects in positive selection are T cell intrinsic, these data suggest an important role for E2A in attenuating thymocyte selection (Figure 2b). Id3 has been shown to interact genetically with E2A in the thymus. Mice deficient for Id3 display a thymic phenotype complimentary to that of E2A-deficient mice (33, 96). Moreover, mice lacking both E2A and Id3 display a relieved thymic phenotype with respect to either deficiency alone (33). Total thymic cellularity in Id3-deficient mice is normal; however, decreased percentages and numbers of CD4 SP cells are observed in the thymus, and decreases of both CD4 and CD8 SP cells are observed in the spleen (33). When analyzed in the AND and H-Y TCR transgenic backgrounds, both MHC class II- and MHC class I-restricted positive selection, respectively, are inhibited in Id3-deficient mice. In particular, CD4 positive selection in AND TCR transgenic mice is almost completely blocked in the absence of Id3. Furthermore, the effect of Id3 on positive selection is intrinsic to the T cell lineage (Figure 3) (33). A role for Id3 in the negative selection process has also been demonstrated (33). Thymocytes from male H-Y TCR transgenic mice recognize the male specific antigen as self and are deleted. Loss of Id3 in these mice results in increased thymic cellularity, considerably more DP thymocytes and increases in CD4 SP cells in the spleen. In contrast, superantigen-mediated deletion appears normal in Id3−/− mice in the MHC class II I-Ed BALB/c background (33). Preliminary results indicate that the defect in negative selection observed in H-Y TCR transgenic male Id3-deficient mice is not intrinsic to the T cell lineage and remains to be further examined (R. Rivera, C. Murre, unpublished results).

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SIGNALING PATHWAYS REGULATING E PROTEIN FUNCTION


Regulation of E Protein Activity by the Ras/ERK MAP Kinase Cascade Thymocytes expressing a dominant-negative form of MAPKK1 (MEK1) fail to progress from the DN to the DP stage in fetal thymic organ culture (97). Furthermore, mice lacking Ras activity or the downstream effectors MEK1 or ERK1 display defective thymocyte maturation at the DP to SP transition reminiscent of the block in Id3-deficient mice (33, 98, 99). As mentioned above, E proteins appear to be functioning at these checkpoints (Figure 4) (84, 100). From these observations, it was postulated that TCR-mediated signals promote developmental progression through the modulation of E protein DNA binding and/or transcriptional activation (Figure 4). In fact, recent observations have demonstrated that signals mediated by both the pre-TCR and the αβ TCR act to downmodulate E2A/HEB DNA binding activity (84, 100). This decrease is caused, in part, by a decrease in E2A protein levels during thymocyte maturation. Additionally, Id3 levels are increased in response to CD3 mediated signals at both the DN and DP stages (84, 100). Whereas it remains to be determined how varying levels of E47 protein are established, the regulation of Id3 has been characterized in great detail. Mice carrying a dominant negative form of Ras do not induce Id3 transcription in response to TCR crosslinking (100). Inhibition of the downstream target c-Raf, using the pharmacological drug PD98059, blocks the induction of Id3 transcripts in a dosedependent manner. Additional evidence indicates that the transcription of Id3 is regulated, at least in part, by the ERK MAPK cascade and is mediated by the immediate early transcription factor, EGR-1 (100). The dose-dependent induction of Id3 transcripts in response to ERK MAPK signaling raises the interesting possibility that the magnitude of the signal determines the level of E protein activity (100). Although more evidence is required, the observed bias toward the CD8 lineage in the absence of E2A suggests a role for E proteins in the CD4 versus CD8 lineage decision (23). Thus, it is conceivable that a strong signal mediated by the ERK MAP kinase pathway leads to high levels of Id3, favoring CD4 lineage commitment, whereas a weak signal would only modestly affect Id3 levels, promoting CD8 development (101). It will be particularly interesting to modulate the relative levels of E2A, HEB, and Id3 and to examine how these ratios affect cell fate.

Regulation of Id Expression by TGF-β and c-myc In addition to its regulation by the Ras-ERK MAP kinase pathway, Id gene expression has also been shown to be regulated by members of the TGF-β family (63). For example, bone morphogenetic protein 4 (BMP4) has been shown to activate both Id2 and Id3 transcription in embryonic stem cells (102). Furthermore, in

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primary B lymphocyte progenitors (BLP) TGF-β induces cell cycle growth arrest through the transient induction of Id3 transcription (63). Ectopic expression of Id3 alone is sufficient for growth arrest, and Id3-deficient BLPs respond more slowly to TGF-β signaling. Unlike in DP thymocytes, the mechanism of Id3 induction by TGF-β appears to involve the activation of Smad transcription factors and not the Ras-ERK module (63). Thus, distinct signaling pathways are utilized to modulate E protein activity by targeting Id gene expression. Recent observations have suggested a role for c-myc in controlling Id2 gene transcription (103). Enforced expression of N- or c-myc induced Id2 expression in a number of cell lines. It was postulated that myc induction of Id2 leads to the inactivation of Rb, thereby driving cellular proliferation (103). However, we note that it is plausible that increases in Id2 levels promote cellular transformation through direct inhibition of E protein activity (discussed below).

E2A and Notch-Mediated Signaling Recent data has implicated Notch-mediated signaling in the decision between B and T lineage determination (Figures 1 and 3). Mice conditionally deficient for Notch1 are blocked at the progenitor T cell stage (104). Conversely, mice that are transgenic for a constitutively activated form of Notch share many phenotypes in common with E2A-deficient mice: (a) B cell development is blocked prior to the onset of Ig gene rearrangement; (b) T cell lymphomas develop with similar kinetics; and (c) abnormalities are observed in both the αβ and γ δ T cell lineages (105, 106). These observations have led to the suggestion that E2A activity is regulated in part by the Notch signaling pathway. In support of this, an activated form of Notch has been shown to repress E2A activity in transient transfection assays (107). Further in vivo studies will be required to determine if Notch and E2A act in a linear pathway or, alternatively, act in parallel.

E PROTEIN ACTIVITY, LYMPHOCYTE SURVIVAL, PROLIFERATION AND MALIGNANCY The E2A proteins have been implicated in cellular proliferation and apoptosis. Several studies have shown that both high and low levels of E2A activity promote rapid cell death, which suggest that lymphocytes can only survive within a limited range of E2A activity (88, 108, 109). Mice deficient for E2A rapidly develop highly malignant T cell lymphoma (23, 110). Similarly, ectopic expression of Id1 or Id2 in transgenic mice promotes lymphomagenesis (88, 111). Restoration of E2A activity into cells derived from E2A-deficient lymphomas leads to apoptosis (108). Likewise, ectopic expression of E47 in human T cell acute lymphoblastic leukemia (T-ALL) cell lines results in a block in cell cycle progression and activated programmed cell death (109). These observations have led to the proposal that E2A functions as a tumor suppressor and its inactivation is a key factor in the development of human T-ALL (108, 109).

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E2A is also involved in a significant percentage of childhood pro-B and preB cell leukemias resulting from chromosomal translocations. In pre-B cell ALL resulting from a t(1;19) translocation, the 50 portion of the E2A gene is fused to the homeodomain-containing region of the pbx1 gene (112). This translocation event results in the expression of a chimeric E2A-pbx fusion protein. In pro-B cell leukemias containing a t(17;19) translocation, the E2A gene is fused to HLF, a gene encoding a bZIP protein (113). The exact mechanism by which these translocations cause leukemia has not yet been elucidated. It has been postulated that the targetting of the transactivation domains of E2A to alternative loci by a heterologous DNAbinding domain results in the aberrant expression of genes associated with cell growth and/or survival ultimately leading to cellular transformation. However, these translocation events also effectively reduce the copy number and expression of the E2A gene by half. Although it remains to be proven, reduction of E2A activity may, in fact, be an essential event in the transformation of B cell progenitors in the same way that E2A functions as a tumor suppressor in the T cell lineage. Various signaling molecules, including p56Lck, TPL2/Cot, MEK1, and most notably, activated forms of Ras, have been implicated in the development of T cell lymphomas (114–116). For example, overexpression of p56Lck in developing thymocytes results in the rapid development of T cell tumors (114). The oncogenic protein TPL2/Cot is a serine kinase with the ability to phosphorylate MEK1 (116–118). It is conceivable that its expression inappropriately activates Id gene expression in T lineage cells, ultimately resulting in lymphoma. Interestingly, activated forms of Ras and high levels of Id gene expression have been detected in pancreatic cancers (119–121). It will be important to determine if malignancies that possess activated Ras have become transformed through the induction of Id gene expression.

CONCLUSION The development of B and T lymphocytes is dependent on the temporal activation of lineage-specific genes, productive antigen receptor rearrangements, as well as the coordination of survival and proliferation with developmental progression. E proteins have now been shown to function in all of these processes (Figure 2). A number of target genes of E proteins have already been identified in lymphocytes. The E proteins regulate the expression of the RAG genes and invariant receptor chains λ5, VpreB and pre-Tα, and promote Ig and TCR gene rearrangements. Thus, the target genes of these proteins perform similar functions in both B and T lymphocytes. However, it is in the T cell lineage that the role of E proteins as downstream effectors of signaling has been elucidated. The E proteins have been demonstrated to be downstream of TCR signaling via the ERK MAP kinase pathway, acting to regulate lineage progression between the DN to DP and DP to SP cell stages (Figure 4). Furthermore, E proteins function to inhibit both proliferation and developmental progression in the absence of rearrangements. In B cells, a role for E proteins in pre-BCR signaling has not yet been demonstrated.

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However, we note that in mature B lineage cells, E2A protein levels are regulated by signals emanating from the BCR. Given that Ras signaling is essential at the pro-B to pre-B cell transition, it is conceivable that the E proteins are regulated similarly to promote B lineage progression. A key question that requires further analysis is how these and other target genes of E proteins are regulated in a tissue- and temporal-specific fashion. Ultimately, this will require a precise and detailed analysis of the regulatory elements that control the transcription of these genes. A comprehensive understanding of how HLH proteins function in lymphocyte development will have to include additional studies of the mechanisms by which E proteins are regulated. For example, it will be important to determine how differing levels of E47 expression are formed during thymocyte development, whether similar gradients are formed during B lineage differentiation and whether they are regulated by antigen receptor mediated signaling. In addition, modifications to the E proteins and their effects on transcriptional activity will need to be further investigated (122, 123). Finally, the identification of novel targets involved in promoting survival and proliferation will give insights into how the E proteins coordinate cell survival and developmental progression. ACKNOWLEDGMENTS We would like to thank Dr. Eben Massari for critical reading of the manuscript and members of the Murre Lab for helpful discussions. This work was supported by grants from the National Institutes of Health (NIH). W.J.R is supported by a Developmental Biology NIH Training Grant. Visit the Annual Reviews home page at www.annualreviews.org

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LYMPHOCYTE-MEDIATED CYTOTOXICITY John H. Russell1 and Timothy J. Ley2 Department of Molecular Biology and Pharmacology, 2Departments of Medicine and Genetics, Siteman Cancer Center, Washington University School of Medicine, St. Louis, Missouri 63110; e-mail: [email protected], [email protected]


1

Key Words granule exocytosis, Fas, apoptosis, perforin, granzymes ■ Abstract Virtually all of the measurable cell-mediated cytotoxicity delivered by cytotoxic T lymphocytes and natural killer cells comes from either the granule exocytosis pathway or the Fas pathway. The granule exocytosis pathway utilizes perforin to traffic the granzymes to appropriate locations in target cells, where they cleave critical substrates that initiate DNA fragmentation and apoptosis; granzymes A and B induce death via alternate, nonoverlapping pathways. The Fas/FasL system is responsible for activation-induced cell death but also plays an important role in lymphocyte-mediated killing under certain circumstances. The interplay between these two cytotoxic systems provides opportunities for therapeutic interventions to control autoimmune diseases and graft vs. host disease, but oversuppression of these pathways may also lead to increased viral susceptibility and/or decreased tumor cell killing.

INTRODUCTION Mechanisms of lymphocyte-mediated cytotoxicity were last reviewed in this series by Kagi et al. (1). Much of our understanding of cytotoxic pathways has come from experiments in cultured cells. At the time of the previous review, it had been established that the two dominant mechanisms of contact-dependent, lymphocytemediated cytotoxicity were the granule exocytosis and the Fas pathways (2), and animals deficient in each of these pathways had just recently been identified or produced (3–5). Here we focus on two elements: the biological significance of these pathways in various in vivo settings, and the molecular components of these pathways. A better understanding of these two elements is necessary to provide targets for rational therapeutic intervention in pathological conditions.

LYMPHOCYTE-MEDIATED KILLING AS AN INTERNAL EVENT IN THE TARGET CELL As early as the 1950s, morphological distinctions had been made between targets attacked by lymphocytes vs. targets attacked by antibody and complement, especially when one compared effects on nuclear structure (6). These differences 0732-0582/02/0407-0323$14.00

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were subsequently confirmed using isotope release assays that distinguished between events affecting the plasma membrane and those affecting the nucleus. These experiments demonstrated that antibody and complement damage was restricted to the plasma membrane, while lymphocytes produced a more general internal disintegration of the target cell, including the nucleus (7, 8). Such experiments suggested that target cells may play an active role in their own destruction, a notion strengthened by the observation that the severity of the nuclear lesion inflicted by the same cytotoxic lymphocyte (CTL) varied with the lineage of the target cell (9). It is likely that these differences reflect differences in the levels of effector caspases and caspase and/or of granzyme substrates in cells of different lineages.

TWO PATHWAYS OF LYMPHOCYTE-MEDIATED KILLING In the earlier review, Kagi et al. suggested that lymphocyte-mediated killing could be confined to two pathways, the perforin/granzyme-mediated and the Fasmediated pathways (1). Emerging evidence suggests that, although this interpretation is true to a first approximation, it may be more useful to consider the two mechanisms as those initiated by FADD through a target cell receptor and those that require perforin. Based on genetically deficient animals, Fas is the most physiologically important receptor initiating death through the recruitment of FADD and caspase 8, but other members of the TNF receptor pathway, including TNFR1 and TRAILR, converge on FADD as well (10–13). The emerging evidence discussed below suggests that the perforin/granzyme pathway is not a single pathway, but rather a series of parallel pathways that depend on the particular granzyme or spectrum of granzymes expressed in a given effector lineage or activation state. Several important similarities and differences appear between the two pathways. The perforin-dependent pathway is dominant in CD8+ CTL and natural killer (NK) cells (2). In NK cells, granules are preformed, although NK activity can be increased by cytokines like IL-2 and IFN-γ . NK cells are thus constitutively armed and can kill within minutes of the first stimulation of activating receptors, but they generally do not proliferate significantly in response to this stimulation. Thus, NK cells are part of the innate immune system and have a rapid response to challenge, but limited capacity for antigen/viral loads. In contrast, naive CD8+ CTL precursors have no cytotoxic activity and must undergo an activation process requiring 1–3 days for maximal activity. This activation process requires TCR-stimulated induction of cytokine receptors (e.g., IL-2 and IL-6), which then induce the expression of granule components, including perforin and granzymes. The same signals that activate CD8+ cells also stimulate their proliferation. As recently reviewed (14), virus-specific CD8+ can expand several orders of magnitude in response to viral infection. Thus, CD8+ cells are

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part of the adaptive immune system with a slow response, but a high capacity for viral load. The activated CD8+ cells are then armed for their next encounter with antigenbearing target cells. When granules are present, the killer cell reorients its granules to the region of receptor activation (NK or TCR) and releases the granule components into the region of contact between the killer and the target. This region of contact is initiated by the receptor but maintained by adhesion molecules (e.g., LFA-1 and ICAM-1). CD8+ cells can kill multiple cells by reorienting their granules to another region of contact, but there is evidence that NK cells must rearm themselves in response to IL-2 before they are effective against new targets (15). The vectorial release of granules to the region of contact between the killer and target cells provides one mechanism of exquisite specificity of the perforin-initiated pathway (16). A second mechanism is that perforin reacts nonspecifically with lipids in the presence of Ca++ (17) and therefore is rapidly inactivated by Ca++ and lipids in the extracellular space. The mechanism of killing by perforin and the granzymes is discussed in more detail below. The FADD pathway (see below) is similar in that maximal expression of the TNF family of ligands requires an initial activation of the effector cell (18). This pathway appears to be active in all killer cell lineages but most important for CD4+ cells, especially those of the Th1 phenotype (19). An important difference between the FADD and perforin-initiated pathways is the speed of the cytotoxic event. Once formed, granules can be reoriented and released within minutes of TCR stimulation (16). In contrast, very little ligand (e.g., FasL) is stored, even in activated cells. Therefore, maximal activity requires the induction of new ligand over a 1–2 h period after TCR stimulation. Induction of new ligand continues as long as there is TCR stimulation and FasL is cleared from the surface either by proteolysis or endocytosis, depending on the effector cell. This clearance of ligand from the surface occurs with a half-life of 2–3 h (20). The long half-life of ligand on the surface allows effector cells to continue to display cytotoxic activity in the absence of TCR stimulation and leads to the phenomenon of bystander killing, which means that cells in the area that express the appropriate receptor (e.g., Fas) can be killed even though they do not express the antigen recognized by the TCR (21). This can be especially important for CD4+ cells whose class II restricting element is not widely expressed on a variety of cellular lineages. Thus, the FADD pathway can be much more promiscuous than the perforin-initiated pathway. It is clear that cultured human and mouse CD4+ and CD8+ cell lines can use both the Fas and perforin/granzyme pathways (19, 22, 23). However, in vivo transplantation and graft vs. host disease (GvHD) experiments across MHC class I or II (CD8+ and CD4+ effectors, respectively) suggest that the perforin/granzyme pathway dominates the class I elimination pathway and that Fas/FasL dominates class II elimination (24, 25). Perforin deficiency has the major phenotype in an in vivo model of NK-mediated tumor rejection (26).

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APOPTOTIC PATHWAYS The last 15 years of research in numerous laboratories have demonstrated that vertebrate cell populations have two ways of maintaining homeostasis (see Figure 1). The first is a pathway that is evolutionarily conserved in multicellular organisms as distinct as C. elegans, D. melanogaster, and H. sapiens. This pathway is known as the intrinsic pathway and is important for the appropriate development of a variety of organ systems (especially the nervous system) and the elimination of cells that have developed abnormally or with genetic errors. The other pathway, known as the extrinsic pathway (the FADD pathway discussed above), may be unique to vertebrates. Both of these pathways have recently been reviewed (27, 28). Both pathways are based on protein-protein interaction domains that lead to the activation of a cascade of proteases with the unusual properties of a cysteine residue in the active site, and a specificity for cleavage at aspartic acid residues (caspases). The activation of these caspases is modulated physiologically by functional dominant

Figure 1 Extrinsic and intrinsic signaling pathways of apoptosis. Two pathways exist for the correct development and homeostasis of organs and cell populations. The extrinsic pathway begins with ligation of a membrane death receptor (e.g., Fas) and is transduced through a series of protein-protein interaction domains culminating in the activation of unique proteases (caspases). The intrinsic pathway is triggered by a lack of trophic receptor stimulation, DNA damage, glucocorticoids, or inappropriate loss of contact with neighboring cells. This ultimately leads to a loss of mitochondrial function and cytochrome c release, which in turn activates the Apaf-1/caspase 9 complex (“apoptosome”), causing further caspase activation.

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negative caspases (e.g., FLICE-like inhibitory protein, c-FLIP/Casper) or caspase binding proteins (inhibitors of apoptosis, IAPs). The substrates of the various caspases (29) are responsible for the morphological (cytoskeletal, nuclear membrane breakdown) and biochemical (DNA laddering) changes associated with apoptosis. As is discussed in more detail below, the best understood granzyme (B) can act on both caspases and caspase substrates in both the intrinsic and extrinsic pathway.


PROTECTION FROM INTRACELLULAR PARASITES CTL of the MHC class I–restricted, CD8+ lineage are uniquely selected to control intracellular pathogens because of the loading of the MHC class I molecule with peptides derived from intracellular proteins. Early experiments by Zinkernagel demonstrated that CTL could kill viral-infected cells before new viral antigens could be detected on the surface by antibodies (30). The availability of null or functional null mutations in the Fas and perforin/granzyme pathways has allowed a first approximation analysis of the importance of these pathways in the control of viral infections (see Table 1). The results of these experiments indicate that the perforin/granzyme pathway is absolutely essential for the control of some viral infections, has a partial role in some, and appears to play no role in others. In contrast, the Fas pathway plays little role in the clearance of viruses unless the perforin pathway is also inactivated (31, 32); Fas may also play a role in clearing some reservoirs of persistent viral infection (see below) (33). The situation with a persistent, neurotropic variant of murine hepatitis virus is particularly interesting. Perforin deficiency significantly delays viral clearance from the CNS (34). Fas deficiency had no effect on the role of viral clearance, but elimination of both Fas and perforin led to uncontrolled infection (32). However, the virus also could not be cleared from mice deficient in IFN-γ . Rather, these mice developed increased CD8+ infiltrates, and virus was cleared from microglia and astrocytes, but not from oligodendrocytes (33). The role of IFN-γ in selectively limiting viral replication in oligodendrocytes has not been established, but one might speculate that IFN-γ could affect the sensitivity of the oligodendrocytes to CTL by increasing either viral peptide MHC class I or Fas expression, rendering this normally resistant cell sensitive to the action of CTL. The initial experiments with perforin-deficient mice suggested that perforin was important in controlling noncytopathic viral infections, but not cytopathic ones (35). However, this is clearly an oversimplification because the perforin/granzyme pathway is involved in protection from some cytopathic viruses (36). With other viruses, the type of viral pathology may depend upon the cell type infected (34). Most of the viral infection experiments have been performed only with perforindeficient mice, so it is unclear whether different granzyme pathways may be more important in some viral infections than others. Similarly, only a few of the experiments have attempted to identify the relative importance of different perforin-expressing effectors essential for controlling the virus (e.g., CTL vs. NK). Analysis of the role of specific granzymes in viral protection may point to

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TABLE 1 Analysis of apoptotic pathways in protection from viral infection using null mutations Virus

Perforin

Fas

Notes (with references)

Lymphocytic choriomeningitis virus

+

−

Complete protection by perforin deficiency (35)

Vesicular stomatitis virus

−

−

(35)

Semliki forest virus

−

−

(35)

Vaccinia

−

−

(35)

Ectromelia

+

NT

(36)

Cowpox

−

NT

(36)

Theiler’s virus

+

−

Acute infection leads to death (261) Also MHC class I-dependent and decreased neurologic symptoms (262)

MAIDS

+

NT

Also MHC class I dependent (263)

−

No effect of perforin deficiency on viral clearance from cornea, but decreased keratitis in the absence of perforin (38) or perforin protection from lethal infection, but no difference in viral clearance from the eye (37)

+

Herpes simplex-1

−/

Coxsackie B3

−

NT

No role of perforin for viral clearance, but decreased myocarditis in the absence of perforin (79)

Neurotropic hepatitis virus

Partial

Only in the absence of perforin

Absence of perforin delays viral clearance (34) IFN-γ required for complete clearance (33)

Influenza

Partial

“

Role for perforin or Fas is only evident when CD4 helper cells are depleted (31)

Adenovirus vectors

−

+

Primarily dependent on TNFR1, which is required for monocytic infiltrate (39)

Murine cytomegalovirus

+

−

Perforin (264) but not Fas is required for viral clearance, but Fas is important in limiting chronic inflammatory disease (265)

γ Herpes

−

NT

(266)

/−

specific effector populations. Using mice deficient for specific granzymes to analyze protection against specific viruses will also lead to a better understanding of the interactions between viruses and the immune system that lead to the selection of viral inhibitors of apoptosis, and the parallel pathways in the immune system that exist to bypass those inhibitors.

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There are several difficulties with the viral experiments performed to date. The principal difficulty is that different viruses have specific viral-resistance genes scattered throughout the genome. In addition, many of the mutant mice are on mixed backgrounds between B6 and different 129 substrains; the viral resistance genetics of the 129 strains are poorly defined. Therefore, it is critically important to use large numbers of deficient animals (with littermates as controls) to eliminate possible effects of unknown background resistance genes. Many of the deficiency mutations are becoming available on the B6/B10 background, which minimizes the effects of resistance genes but limits the number of viruses that can be analyzed. Even when similar viruses and animal strains are used, results can vary, perhaps because of differences in viral dose or strain-specific pathogenicity (37, 38). Another difficulty, especially with deficiencies of the TNF receptor family, is that the mutations are pleiotropic and have profound effects on lymphocyte homeostasis and trafficking, as well as effector function. A good example is the finding that the TNFR1 mutation has the most dramatic effect on the clearance of adenoviral vectors from the liver through its effects on monocytic infiltration (39). TNFR1 has previously been demonstrated to play an important role in this process, through its effects on the induction of addressins and adhesion molecules (40). Intracellular bacteria have not been studied as extensively as viruses. Mice deficient in perforin (41) have a modestly attenuated primary response to Listeria infection and a somewhat more dramatic deficit in the secondary response. Mice deficient in Fas have a small defect, but the combination of Fas and perforin deficiency greatly prolongs the clearance time (42). However, all these deficient mice recover from severe infection. It appears that TNF (43) from a non-CD8 source (44) is crucial in limiting Listeriosis. Recent experiments suggest that CTL and NK granules from some species, including humans, contain proteins that can be used as defense against both intracellular and extracellular bacteria. These proteins have structural similarity to antimicrobial virulence factors of amoebae (45). The best characterized of these molecules is granulysin (46). This protein, originally identified as a T cell activation product, has the ability to kill tumor cells by caspase-dependent and caspase-independent mechanisms (47). However, it may be more important for its broad spectrum antimicrobial activity (48–50). Murine orthologues have not been found, so it has not been possible to test the physiology of granulysin through loss-of-function mutations.

CELL-MEDIATED CYTOTOXICITY IN IMMUNE REGULATION AND AUTOIMMUNE DISEASE Immune Regulation The role of Fas/FasL in lymphocyte homeostasis was clearly established with the recognition that functional null mutations in these proteins (lpr and gld, respectively) were associated with exacerbated autoimmune disease (4, 51). Evidence

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that cell-mediated cytotoxicity played a role in lymphocyte homeostasis was provided by Goodnow and colleagues in adoptive transfer experiments utilizing a transgenic model of B cell tolerance (52). These experiments implicated Fasdependent killing of anergized B cells by Th1 T cells. It was demonstrated that B cell sensitivity to Fas-dependent killing was regulated by signals from the BCR and CD40, another member of the TNF receptor family (28, 53). The Fas pathway is also important in T cell homeostasis, especially in chronic exposure to self-antigens, while the intrinsic pathway is more important after acute immunization (54). It is unclear whether Fas/FasL-dependent elimination of chronically activated cells is accomplished by one T cell killing another (fratricide) or through a specialized suicide process. There is evidence for both mechanisms in culture, but whether only one or both occur in vivo is difficult to determine. The Fas system plays little, if any, role in negative selection in the thymus (55–57). A number of reports regarding a potential role of the granule exocytosis pathway for lymphocyte homeostasis were recently reviewed by de Saint Basile and Fischer, who suggest that defects in this pathway are primarily responsible for the hemophagocytic syndromes (58). Clearly, some mutations that affect granule formation and trafficking in many cell lineages have similar phenotypes in both mice and humans (59–61). However, animals deficient for perforin have lymphocyte populations that are phenotypically normal. Perforin-deficient animals do develop a syndrome with hematophagocytosis when they are infected with viruses that they cannot clear (62, 63), but it is unclear whether the increase in the number of activated CD8+ cells and macrophages is a failure of lymphocyte homeostasis or the consequence of chronic inflammation due to persistent viral infection. A secondary role for the granule exocytosis pathway in lymphocyte hemostasis is unmasked by an exacerbation of the lymphoproliferative phenotype when a null mutation in perforin is added to the lpr or gld mutations (64, 65). Similarly, deficiency in TNFR1 dramatically exacerbates both the lymphoproliferative disease and autoantibody production when combined with the lpr mutation (66). A selective role of an interaction between TNFR1, TNFR2, and Fas in mature CD8+ cell homeostasis has also been suggested (67–69). This hypothesis has produced modest results when tested in vivo (70, 71). Thus, the Fas system has a major role in lymphocyte homeostasis, along with other cytotoxic ligands, working through TNF receptors. The granule exocytosis pathway can also contribute. How much of this activity is mediated by classical killing of one cell by another, vs. cytokineassisted suicide, remains to be determined.

Autoimmune Disease The potential roles of the granule exocytosis and Fas pathways in autoimmune disease have been most extensively studied in induced or spontaneous diabetes and induced demyelinating disease (EAE). The clearest demonstration of a role for the perforin/granzyme pathway is in induced diabetes. In these models, viral proteins expressed as transgenes in the β cells of the islets target the β cells

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for attack upon viral infection. In both models where the disease is dependent upon CD8+ effectors, perforin deficiency eliminates or dramatically reduces the severity of the induced disease without affecting lymphocytic infiltration of the islets (72, 73). In the NOD model of spontaneous diabetes, there is evidence that both the perforin/granzyme and Fas pathways (as well as others) are important in pathogenesis (74–78). An interesting role for the perforin/granzyme pathway in virally induced autoimmune disease comes from studies on coxsackie virus-induced myocarditis (79) and HSV-1-induced keratitis (38). In both studies, perforin deficiency had no effect on viral clearance, but dramatically ameliorated the autoimmune sequelae. In the latter study, the role of lymphocytes for initiating the disease was confirmed by adoptive transfer. However, in both instances, it appears that the actual effectors in the pathogenesis are not the CTL, but other cells in the inflammatory infiltrate. It has been known for some time that the process of apoptosis, including that induced by CTL, can be important in the release of proinflammatory mediators (80). In these two viral models, the apoptosis caused by the CTL in response to the viral infection is not important in controlling the virus but may be important for recruiting a pathogenic inflammatory infiltrate. Perforin deficiency did not ameliorate the development of induced demyelinating disease (EAE) in the model where it was tested. If anything, it appeared to slightly exacerbate disease, perhaps reflecting its role in lymphocyte homeostasis (81). In contrast, Fas/FasL deficiency dramatically limits disease incidence and severity in some models (82–84), but it has little or no effect in others (81, 85, 86). Some of these differences in the dominant effector mechanism appear to be caused by genetic influences that are distinct from MHC regulation of disease susceptibility (87). Because the Fas system is also important for lymphocyte homeostasis, null mutations in Fas or FasL can also exacerbate demyelinating disease in models where Fas is not a dominant mechanism of pathogenesis (86, 88). The evidence that cytotoxic activity of lymphocytes is involved in each of the above instances comes from adoptive transfer experiments using lymphocytes that cannot initiate a specific apoptotic pathway (68, 73, 77, 84, 89). These models indicate that CTL can use their apoptotic mechanisms to initiate autoimmune disease. The potential role of perforin in human disease is difficult to address. Perforinand granzyme-containing cells have been identified in patients at sites of autoimmune inflammation (90, 91), but their role in pathogenesis has not been established. There is similar circumstantial evidence for an involvement of Fas in several human autoimmune pathologies (92, 93), including multiple sclerosis (94, 95). Whether FasL from lymphocytes is always the initiating factor is somewhat controversial, in part because many of the FasL reagents used for immunohistochemistry have not been specific (96). The experiments in mice provide proof of the concept that the FADD pathway and the perforin/granzyme pathways are effectors not only in protection from pathogens, but also in the pathogenesis of autoimmune disease. As discussed below, the biochemical pathways utilized by the different granzymes are only

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now being elucidated. A better understanding of these pathways, coupled with the continued analysis of the FADD pathway, may provide unique targets for therapeutic intervention in specific pathological situations.


AN OVERVIEW OF THE GRANULE EXOCYTOSIS PATHWAY AND HOW IT CAUSES CELL DEATH The granule exocytosis pathway was first proposed in this journal in 1985 (97). When T cells receive specific signals to activate and proliferate via the T cell receptor, transcriptional mechanisms are activated that lead to the production of cytotoxic granules and their constituent proteins (98). In the case of NK cells, these granules are preformed during NK cell development, perhaps at an NK precursor stage that is not morphologically identifiable. Within a day after T cell activation, granules begin to be synthesized, along with perforin and granzymes and other granule components. These granules then reside in the cytoplasm of the cell, where they await further instructions. Upon target cell identification and conjugation, specific signals are generated in the effector lymphocyte that cause the granules to migrate by vector to the site of contact. At the cell surface, the granule fuses with the plasma cell membrane, and its contents are secreted into the tight intracellular junction formed between the two cells. There, in the presence of calcium, perforin polymerizes and enters the target cell membrane. In 1996, when this review was written (1), perforin was thought to form a channel through which the other granule proteins pass into the target cell cytoplasm. Perforin was therefore thought to act as an important gateway for granule proteins into target cells, and it was also thought that perforin could induce membrane damage that could ultimately lead to the death of the target cell. However, several early experiments suggested that perforin itself was not capable of causing target cell apoptosis unless molecules like the granzymes were also added (99–105). By 1996, it was clear that granzyme B was required for the rapid induction of target cell apoptosis by cytotoxic T cells and NK cells (106, 107). The roles of granzyme A and the many other granzymes found in cytotoxic granules were completely unknown. Granzyme B was known to prefer to cleave substrate proteins after an aspartic acid residue in the P1 position (108), and its similarity to the caspases was therefore well recognized. In fact, it was known that granzyme B could cleave procaspase 3 in vitro (109, 110) and that it was required for the cleavage of caspase 3 in target cells after CTLs delivered their granule load (111). It was widely believed that granzyme B killed by activating caspases in target cells, which then led to the cleavage of additional apoptotic substrates that led to DNA fragmentation and apoptosis. Because cytotoxic granules are self-contained death machines, these organelles have continued to be intensively studied over the past five years. A great deal of new information has come to light regarding every step in the pathway described above. Many of the notions described in the central dogma of 1996 have been

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shown to be flawed or simply incorrect. The roles of additional granule proteins in regulating the death process are beginning to be understood. Regardless, one thing has not changed. It is clear that activated CD8+ T cells and NK cells absolutely rely on the granule exocytosis pathway for their ability to induce target cell death and that this pathway is crucially important for the ability of CTL to induce target cell death in many biologically important scenarios, including allogeneic rejection, tumor cell killing, and the clearance of some viruses. In addition, a potential role for granule-mediated cytotoxicity in lymphocyte homeostasis has also evolved, even though this role was not recognized in 1996 (58, 112). The recent developments in our understanding of this complex pathway are outlined in the sections below.

GRANULE FORMATION AND CONSTITUENT GRANULE PROTEINS As noted above, the cytotoxic granules of activated cytotoxic T cells are synthesized de novo when the cell receives signals to proliferate and activate. Over the course of one to two days, the genes for perforin, granzyme A, granzyme B, and other granzymes are transcriptionally activated, and these newly synthesized proteins are then appropriately trafficked and assembled into the functional granule. After synthesis of the granzymes, important posttranslational modifications occur. First, the granzymes must be processed to assume an active conformation. The signal peptide is first removed by a signal peptidase, and then a short prosequence is removed by a second enzyme. In the case of granzymes A and B, the second enzyme is clearly dipeptidyl peptidase I (DPPI) (113–116). Mice deficient for DPPI synthesize normal amounts of granzymes A and B, but the enzymes are completely inactive since they are not processed normally at the N terminus (117). DPPI-deficient CTL are identical to granzyme A × B–deficient mice in their inability to induce apoptosis in allogeneic target cells. The precise compartment in which DPPI processes the granzymes into their mature forms is not known, but the granzymes are thought to exist in the granules as the active processed enzymes. The granzymes are also glycosylated and sorted by the Man-6-P receptor in the Golgi apparatus on their way to the specialized granules (118, 119). This modification of the granzymes may be important for their entry into target cells (see the section on the trafficking of perforin and granzymes below). Perforin is also thought to be modified posttranslationally to render it active. Uellner et al. (120) demonstrated that perforin is synthesized as a 70-kDa precursor that is cleaved at the carboxyl terminus to yield the active 60-kDa form. The enzyme that mediates this cleavage event has not yet been identified, but it may require an acidic environment (121). Accordingly, chloroquine, a lysosomatropic agent that raises the pH of the acidic compartment in which perforin is processed, may inhibit its maturation. Taylor et al. (122) have shown that chloroquine does indeed inhibit perforin activity in LAK cells, and that chloroquine-treated mice have defects in

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their ability to clear YAC-1 tumor cells from the lungs or to reject incompatible bone marrow transplants via traditional NK pathways. It is not yet known whether chloroquine acts solely to inhibit NK cell function by inhibiting perforin activation, or whether other effects may also contribute to the actions of the drug. Glycosaminoglycan complexes are also found in cytotoxic granules, including one proteoglycan called serglycin, which contains chondroitin 4-sulfate glycosaminoglycans. This molecule is thought to act as a scaffold for packaging the highly positively charged granzymes, and it may also act as a chaperone for the secreted proteases. Galvin et al. (123) have shown that granzyme B complexed with serglycin is proteolytically active and able to induce target cell apoptosis; they have also shown that granzyme B is actually secreted in a high-molecular-weight complex, presumably containing serglycin. The requirement of serglycin for the induction of target cell death is not yet understood, because recombinant granzyme B is fully capable of causing target cell death in reconstituted systems (124). Direct comparisons of the cytotoxic potential of serglycin-complexed granzymes and their recombinant counterparts have not yet been described. Calreticulin is also stored in cytotoxic granules. This molecule is a chaperone protein of the endoplasmic reticulum (ER) and is the only resident ER protein known to exist in CTL granules. Fraser et al. (125) have recently shown that calreticulin inhibits perforin-mediated target cell damage, but it does not do so by directly interacting with perforin, by sequestering calcium, or by inhibiting granzymes. The possibility therefore exists that calreticulin acts as a regulatory molecule that dampens the effect of perforin by stabilizing membranes to prevent excessive damage. Bossi & Griffiths (126) have shown that Fas ligand appears to be stored in the same cytotoxic granules that contain perforin and granzymes, and that Fas ligand may in fact be delivered to target cell Fas receptor via the same granules that deliver other granule components. This mechanism could be important for sequestering Fas ligand in the effector cell, to prevent it from causing suicide as it interacts with the receptor intracellularly. The relative importance of Fas ligand in the cytotoxic granules, as opposed to Fas ligand that is newly synthesized and directly transported to the cell surface, has not yet been fully explored. Finally, another granule protein called granulysin has been identified in human CTL, and its role in antimicrobial clearance was described above (48–50, 127). Granulysin, like perforin and the granzymes, is synthesized during T cell activation and is thought to be packaged in the same cytotoxic granule compartment. Granulysin can cause target cell membrane damage directly and can cause mitochondrial depolarization and the release of cytochrome c (47, 128). Despite causing cytochrome c release, granulysin-induced mitochondrial damage does not cause procaspase 9 activation via the classical apoptosome, but it still manages to activate caspase 3. The importance of granulysin for CTL function in vivo has been difficult to assess because the murine orthologue of this molecule has not yet been identified. Importantly, experiments with loss-of-function mice have

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strongly suggested that mice deficient for perforin have cytotoxic defects similar to those of mice deficient for granzymes A and B. This would suggest that if a murine orthologue of granulysin exists, it must cause target cell damage by a perforin-independent mechanism. Since most of the perforin-independent cytotoxicity delivered by allogeneic CTL is due to Fas (2, 129, 130), the cytotoxic role of granulysin in this setting must be limited. The cytotoxic granules are specialized lysosomes. A defect in the genes that control a crucial process in lysosomal trafficking causes the Chediak-Higashi syndrome in humans and the beige defect in mice. This trafficking regulator, called CHS1 in humans and LYST in mice, is a large cytosolic protein that is not clearly related to other known proteins (61, 131–133). This gene product affects the sorting of many lysosomal proteins in lymphocytes (and other cell types), including CTLA-4, granzymes, perforin, and MHC class II molecules in B cells (58). With the loss of function of CHS1/LYST, the granule enzymes accumulate in giant intracellular granules that apparently are unable to release the enzymes in response to appropriate stimuli, which accounts for the decreased cytotoxic activity of CTL and NK cells from patients with this syndrome.

SECRETION OF CYTOTOXIC GRANULES When an activated T cell recognizes its target, a tight junction is formed between the effector and target, and a signal is generated in the effector cell that causes its granules to migrate vectorally to the site of contact. Recently, several studies by Djeu and colleagues (134–136) have elucidated the signal transduction pathway required for granule mobilization and secretion in NK cells. These authors have shown that phosphoinositide-3 (PI-3) kinase is activated by ligation of NK cells to their targets. PI-3 kinase subsequently activates RAC-1, which activates P21activated kinase 1 (PAC-1), which in turn activates MAPK kinase and finally the extracellular signal regulated kinase (ERK). Inhibition of RAC-1 or PAC-1 mimics the suppressive activity of PI-3 kinase inhibitors, as does inhibition of MAPK-ERK kinases. Since the granule exocytosis pathway is required for NK cell cytotoxicity, inhibitors of this signal transduction pathway dramatically reduce the ability of NK cells to kill their targets. Presumably, similar signal transduction pathways are utilized to cause granule secretion in activated T cells after conjugation. Another important component of granule exocytosis has recently been discovered in association with Griscelli syndrome and the Ashen mouse (58). Mutations in RAB27A have previously been linked to immune defects in humans. Ashen mice were known to contain a mutation in RAB27A that alters its splicing and creates loss of function for this protein. Haddad et al. (60) showed that CTL and NK cells derived from Ashen mice have profoundly decreased cytotoxicity, even though they have normal Fas ligand expression and Fas-induced cytotoxicity. Ashen CTL have normal appearing granules containing normal levels of perforin and granzymes A and B, and normal polarization of granules. However, granule

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secretion itself is defective in CTL-derived from Ashen mice (137), even though other secretory pathways are normal (60). Patients with Griscelli syndrome, a rare autosomal recessive disease associated with hematophagocytic syndrome, also have mutations in RAB27A (59). Therefore, there is strong evidence that RAB27A is required for cytotoxic granule secretion. It is interesting to note, however, that Fas ligand–mediated killing is normal in CTLs derived from Ashen mice (60), which suggests that the Fas ligand found in granules is not required for Fas ligand– mediated cytotoxicity.

THE ESSENTIAL ROLE OF PERFORIN FOR GRANULE-MEDIATED KILLING In the original paradigm of perforin function, perforin was thought to directly induce target cell death by damaging target cell membranes, causing cell lysis similar to that induced by complement. The similarity of perforin to that of complement component 9 further suggested that this molecule was the key granule component that causes target cell death. However, the discovery that perforin-induced membrane damage was not sufficient to cause apoptosis (100), the hallmark of CTL-induced target cell death, suggested that it may instead act as a portal of entry for other cytotoxic molecules that induced the apoptotic hits. Upon release of perforin from cytotoxic granules, the molecule rapidly polymerizes in the presence of calcium to form a ring-like structure that apparently contains a central pore when it is inserted into the target cell membrane. However, the size of this pore is probably too small to permit large molecules like granzymes to enter target cells (138). Therefore, in the last several years, the idea has been questioned that perforin simply creates a channel through which other critical death-inducing enzymes pass. The insertion of polymerized perforin into target cell membranes may not be a random process, but instead may be mediated by “receptors.” This idea was first advanced by Tschopp et al. (139), who noticed that perforin and complement component 9 are distinct in their modes of target cell recognition. While complement 9 insertion is dependent on a receptor assembled from upstream complement molecules in the pathway, no components for a perforin receptor are present in the cytotoxic granules. Tschopp et al. (139) demonstrated that phosphocholine on target cell membranes acted as a specific, calcium-dependent receptor molecule for perforin. Müller & Tschopp (140) later described evidence for a lymphocyte membrane protein that interacted with perforin, providing a mechanism for CTL to protect themselves from inadvertent perforin membrane damage. This idea was further examined by Rochel et al. (141), who explored the role of lipid inhibition of perforin activity as a mechanism for protecting CTL from perforin-induced membrane damage. The concept of a perforin receptor has been further explored in two recent publications. Berthou et al. (142) have suggested that NK cells can release the

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lysolipid platelet activating factor (PAF), which may act as a chaperone for perforin, synergizing with it to produce membrane damage. PAF receptors were found on perforin-sensitive cell lines, but not on perforin-resistant lines; interferon-γ induced PAF receptor expression correlated with the induction of perforin sensitivity. The authors went on to propose that if interferon-γ were to fail to induce PAF receptor expression, it could make tumor cells resistant to perforin-mediated attack. A similar idea was independently explored by Lehmann et al. (143) who showed that a human leukemia cell line (ML-2) demonstrated resistance to NKmediated killing by virtue of the fact that perforin was not able to bind to the surface of these cells. They went on to examine perforin binding to leukemic cells derived from several patients and found a correlation between the failure to bind perforin and resistance to NK cell–mediated cytotoxicity. Resistance to perforin-mediated membrane damage may represent a novel mechanism of tumor cell resistance to immune killing; it certainly merits additional study.

THE TRAFFICKING OF GRANZYMES IN TARGET CELLS As noted above, the original ideas about the collaboration between perforin and granzymes for the induction of cell death have evolved considerably (105, 144). The idea that perforin created a channel through which granzymes could pass into target cells was widely held in 1996 (Figure 2). However, the channels created by polyperforin range in size up to 16 nM, which are not large enough to permit diffusion of even small proteins (i.e., 8 kDa) into cells (138). The granzymes range in size from approximately 30 to 65 kDa, and they are probably complexed with serglycin upon secretion. These observations suggested that the granzymes cannot enter target cells directly via a perforin pore. A second hypothesis for granzyme entry, namely reparative endocytosis, was offered to explain this paradox (105). In this model, perforin entry into the target cell membrane creates a signal for the target cell to repair the damage by endocytosing the perforin and surrounding plasma cell membrane; granzymes in the vicinity of the lesion are also endocytosed and ultimately delivered to the target cell cytoplasm and nucleus, where they deliver the apoptotic hits. Most of the studies described below utilize in vitro systems where target cells are bathed in solutions of perforin and granzymes. It is not yet known whether the models provided by these studies are physiologically relevant; in vivo, cytotoxic lymphocytes secrete large amounts of granule proteins onto a very small patch of target cell membrane defined by the conjugation site. These conditions cannot currently be reproduced in vitro, but they may be very important for defining perforin pore size and/or mechanisms of granzyme entry in vivo. Several groups have now demonstrated that purified granzyme B can enter target cells without any chaperones or perforin (145–147). These studies demonstrated that the internalized granzyme B requires an additional signal to cause cell death (that can be provided by either perforin, adenovirus, or bacterial toxins)

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Figure 2 Mechanisms of granzyme entry into target cells. The effector cell is shown at the top, and the target cell on the bottom. On the left side, the traditionally held view of perforin as a pore-forming molecule is shown. In this model, perforin secreted by the effector cell polymerizes in the presence of calcium and forms a channel through which the granzymes and other constituent granule proteins pass into the target cells. On the right side of the panel, a more recent model for granzyme entry is presented. In this model, perforin enters the target cell membrane and creates a stimulus for repair. Perforin enters the target cell via reparative endocytosis. In one model of granule entry, the granzymes would be inadvertently taken into the target cell with the perforin during this process. In a more recent model, granzyme B enters the target cell by virtue of its binding to the cation-independent mannose-6-P receptor (CI-MPR). After the receptor is internalized, the granzyme B is released into an endolysosomal compartment where it is rendered harmless. Internalized perforin provides a signal for granzyme B to leave this compartment, where it can then cleave cytoplasmic and nuclear apoptotic substrates. See text for details.

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(138). This second signal is responsible for trafficking the intracellular granzyme B from an endolysosomal compartment (where it is harmless to the cell) into the nucleus. Granzyme B delivered into target cells by microinjection can directly cause apoptosis (147), suggesting that nuclear entry of granzyme B (148–150) does not require a second signal. Similar results have been described for granzyme A (151). Even though these studies clearly demonstrate that granzymes can enter target cells autonomously, they do not demonstrate whether this pathway is minor or major for CTL-mediated granzyme delivery. The notion that granzymes could enter target cells independently of perforin led to a search for granzyme receptors, which were first described by Motyka et al. (152). Granzymes A and B were previously known to be targeted to the cytotoxic granules via the mannose-6 phosphate receptor (MPR) (118). Motyka et al. therefore explored the possibility that granzyme B could enter target cells via the MPR, and they discovered that native granzyme B binds to both the cationindependent (CI) and cation-dependent (CD) MPR (Figure 2). However, they found that only the CI-MPR (also known as the insulin-like growth factor 2 receptor) was required for the induction of granzyme B–mediated apoptosis in an in vitro system. In addition, the expression of the CI-MPR was required for the killing of allogeneic cells in an in vivo model system. Clearly, these results suggest that granzyme entry into target cells could be modified by CI-MPR receptor blockade. However, the relative utilization of this pathway for granzyme entry into target cells has not yet been clearly defined. Does a large percentage of the granzyme B delivered by a CTL enter the target cell via this mechanism, or is it only a small fraction? Importantly, active recombinant granzyme A and B have been produced in systems (yeast and baculovirus) that do not attach Man-6-P to proteins. Recombinant granzyme B is active against peptide and protein substrates (116, 153) and can cause the induction of apoptosis when it is delivered to target cells in the presence of perforin (124). However, the relative apoptotic activities of Man-6-P modified granzymes and their recombinant counterparts have not yet been reported.

THE STRUCTURE AND FUNCTION OF GRANZYME B Granzyme B Substrate Specificity Granzyme B, a serine protease, was originally defined as an aspase because of its preference to cleave after aspartic acid in the P1 position (108). In this regard, it is similar to the caspases, which also prefer to cleave with aspartic acid at P1. However, purified human granzyme B has a unique specificity, different in the P2, P3, and P4 positions, so that the preferred recognition motif for granzyme B is (I/V) EPD (154). The substrate specificity for recombinant rat granzyme B was further characterized by Harris et al. (155), who discovered that the enzyme has an extended substrate specificity, which includes any amino acid in the P10 position and Gly in the P20 position. The activity of the enzyme was highly dependent on the length and sequence of substrate peptides, implying that the targets for this

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enzyme are highly restricted. The extended substrate specificity correlated well with the presence of granzyme B cleavage sites in many of the caspases and other substrates known to be cleaved by granzyme B upon target cell entry.


Granzyme B Structure The crystal structures of both rat and human granzyme B have recently been elucidated (156–158). The structure of rat granzyme B in complex with a macromolecular inhibitor revealed the structural determinants that explain its extended substrate specificity. The primary specificity for Asp in the P1 position occurred through a buried side chain of arginine at position 226. Nine additional amino acids were shown to make contact with the substrate, and they defined the extended substrate specificity profile. The structure of the human enzyme revealed that the S1 subsite of this enzyme was larger and less charged than the corresponding Asp-specific site of the caspases or of rat granzyme B. These differences are significant enough to be relevant for substrate specificity, and they point out the importance of species of origin when granzymes are being compared. Subtle differences in peptide substrate specificities have been detected for rat, mouse, and human granzyme B (N. Thornberry, personal communication), and they will need to be taken into account as inhibitors are prepared, as cellular substrates are examined, and as purified or recombinant enzymes are used in the killing of cells of a different species.

The Death Pathways of Granzyme B After granzyme B is released from its endolysosomal compartment, it is apparently rapidly trafficked to the nucleus by a mechanism that does not require perforin or alterations in nuclear pore size (148–150). Presumably, free granzyme B in the target cell initiates cell death by cleaving a variety of protein substrates that are either directly or indirectly linked to the induction of DNA fragmentation and cell death (Figure 3). Over the past several years, many studies have clearly shown that a number of procaspases (including caspases 2, 3, 7, 8, 9, and 10) are substrates of granzyme B both in vitro and in vivo (109, 159–163). A large number of studies have also evaluated the requirement of caspases in the target cell for granzyme B’s ability to induce target cell apoptosis (111, 124, 162, 164–169). These results have been somewhat variable from group to group, but ultimately, a fairly clear consensus has emerged. Even though granzyme B can clearly induce target cell DNA fragmentation and apoptosis in cells that contain broad spectrum inhibitors of caspases, or that lack caspase 3 (124), the induction of death occurs more rapidly in cells that contain active caspases, suggesting that they amplify or feed forward the granzyme B death signal. The ability of granzyme B to cause DNA fragmentation in cells that lack functional caspases suggested that granzyme B could directly activate apoptotic nucleases. Wang & Nagata and their colleagues (170–173) independently identified such a nuclease, known as caspase-activated DNAse (CAD, also known as DFF40). CAD exists as a heterodimer with ICAD (Inhibitor of CAD, also known as DFF45)

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Figure 3 Currently identified pathways of granzyme B action. After granzyme B enters target cells, it cleaves and activates procaspase 3 (and several other procaspases), Bid, and ICAD. The granzyme B proapoptotic signal is amplified by caspase activation and/or Bid cleavage and translocation to the mitochondrial membrane, where Bax and Bak then form a channel that permits cytochrome c release. Granzyme B is also capable of directly acting on mitochondrial membranes to cause depolarization in the absence of cytochrome c release. “X” refers to a putative cellular cofactor for granzyme B-induced mitochondrial depolarization (186). All of these pathways initiate either DNA fragmentation or apoptosis in the target cell.

in the nucleus, where CAD is retained in its inactive form. After a cell receives a death signal, caspase 3 is activated by one of a variety of pathways, and it can then cleave ICAD/DFF40, a process that facilitates the assembly of CAD into its active form (170, 174). Thomas et al. (124) showed that ICAD is a direct substrate of granzyme B and that CAD can be directly activated in vitro and in target cells by granzyme B. Furthermore, ICAD is not cleaved during the induction of apoptosis by granzyme B–deficient CTL. Finally, ICAD-deficient cells (175, 176) are partially resistant to granzyme B–mediated killing, which demonstrated the biological significance of the ICAD pathway for granzyme B-induced death (124). Similar studies by Sharif-Askari et al. (177) confirmed these results. However, since a large

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percentage of cells that contain neither ICAD nor functional caspases are susceptible to granzyme B–mediated death, additional death pathways must also exist. A number of observations suggested that one of these pathways may involve mitochondria, since Bcl-2 variably blocks death caused by perforin and granzymes (178–183). Barry et al. (167), Sutton et al. (169), Heibein et al. (184), and Alimonti et al. (185) all went on to show that granzyme B is capable of directly cleaving Bid upon target cell entry. Bid cleavage resulted in translocation of tBid to the mitochondria, where it interacted with its receptors Bax and Bak to cause cytochrome c release. Cytochrome c then activates the apoptosome, which activates procaspase 9, and ultimately caspase 3 (Figure 3). Although granzyme B can clearly cleave and activate Bid, and cause cytochrome c release, Thomas et al. (186) have recently shown that neither Bid-deficient nor Bax/Bak doubly deficient mouse embryo fibroblasts are resistant to granzyme B–induced death. Granzyme B can cause mitochondrial depolarization even in the absence of cytochrome c release, a process that requires neither the permeability transition pore or the Bax/Bak receptors. These data all suggest that granzyme B amplifies its death signal upon target cell entry by cleaving Bid and causing mitochondrial dysfunction, but it also suggests that these pathways are not required for induction of death. Commitment to death probably proceeds more slowly in the absence of Bid, but granzyme B can persistently attack alternative targets, like ICAD, to cause the ultimate death of the cell. Granzyme B also cleaves a variety of other substrates upon target cell entry, but the relationship of these events to the induction of apoptosis is not yet clear. For example, granzyme B cleaves poly (ADP-ribose) polymerase (PARP) (187), DNA-PKcs and NuMA (188), Filimin (189), cartilage proteoglycan (190), nuclear lamins (191), and a variety of autoantigens (192) during the induction of target cell death. The significance of these cleavage events for cellular homeostasis and/or the production of autoimmune diseases are not yet clear.

GRANZYME A Granzyme A was the first serine proteinase activity to be discovered in cytotoxic granules (193) but far less is known about its mechanism of action in inducing cell death than that of granzyme B. The structure of granzyme A has not yet been solved. Granzyme A is a tryptase and prefers to cleave synthetic substrates with Arg or Lys at the P1 position (108). Granzyme A exists in granules as a disulfidelinked homodimer, which is probably complexed with serglycin, like granzyme B (123). After granzyme A is released from activated CTL, it can circulate in an active form, bound to proteoglycans that protect it from inactivation by proteins like anti-thrombin III or α2-macroglobulin (194). Shi et al. (101) and Shiver et al. (103) independently showed that granzyme A is a pro-apoptotic enzyme, using different approaches. When delivered to target cells with perforin, purified granzyme A can clearly induce target cell apoptosis (101).

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When cotransfected with perforin into rat basophilic leukemia cells, granzyme A can cause target cell death with DNA breakdown (103). The timing of cell death induced by granzyme A, however, is much slower than that of granzyme B; granzyme A requires many hours to induce apoptosis within in vitro–reconstituted systems. The reasons why granzyme A induces death so slowly are unknown. They could include: (a) a proteolytic cascade that involves a large number of enzymes and substrates, and/or (b) a very slow catalytic rate for a rate-limiting step in death induction, or (c) a specialized compartment of granzyme A–expressing CTL that delivers its death signals more slowly than the cells that deliver granzyme B. None of these potential mechanisms has yet been tested. The completely different specificities of granzymes A and B suggested either that these enzymes could be synergistic or that they may act through nonoverlapping, alternative pathways as fail-safe mechanisms for the induction of target cell death. Although some evidence has been gathered for a synergistic interaction of granzymes A and B (104), experiments from knockout mice (195) strongly suggest that these pathways are independent. The protein targets of granzyme A have been extensively investigated. Granzyme A is known to bind to and cleave nucleolin (196), activate the thrombin receptor on neuronal cells and astrocytes (197), cleave and activate interleukin-1β (198), and cleave PHAP II, a ubiquitous, putative HLA-associated protein (199). Granzyme A binds to heat shock protein 27 in target cells but does not cleave it (200), and it enhances DNA accessibility to exogenous endonucleases by degrading histone-H1 (201). Finally, granzyme A directly cleaves lamins, thereby disrupting the nuclear lamina and causing nuclear breakdown during the induction of apoptosis (191). Some of these substrates are highly likely to be involved in granzyme A–mediated death, but definitive experiments to assign the requirement of each of these substrates for cell death have not yet been performed. The cellular pathways used by granzyme A to induce death appear to be independent from those utilized by granzyme B. Granzyme A does not appear to activate caspases in target cells (202, 203), nor does it induce cleavage of many granzyme B substrates, like caspase-3, DNA-PK, or PARP. Furthermore, granzyme A–induced death does not cause oligonucleosomal DNA degradation, but rather it appears to cause single-stranded DNA breaks, suggesting that it activates a different nuclease than granzyme B or the caspases (203). The uniqueness and alternative features for this pathway were further demonstrated by studies of the granzyme A knockout mouse (see below).

ORPHAN GRANZYMES We refer to all of the granzymes of unknown function as the orphans. A catalog of these orphan enzymes and their specificities has recently been assembled by Kam et al. (204); they include granzymes C, D, E, F, G, and K in the mouse (205), granzymes H, K, and M in the human, and granzymes C, I, J, K, and M in the rat. The

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specificities of these enzymes are largely unknown, except for human granzyme H, which is a chymase (206), granzyme K, which is a tryptase, and granzyme M, which is a metase (204). The roles of these enzymes for CTL-mediated death are all essentially unknown at this time. The granule genes are organized into three independent clusters in the genome. Granzymes A and K are tightly linked on chromosome 13 in the mouse (207) and on chromosome 5 in humans. Granzymes B, C, D, E, F, and G are tightly linked in a gene cluster that also includes mouse cathepsin G and several mast cell chymases on chromosome 14 (208). Human granzymes B and H are tightly linked with the human cathepsin G and mast cell chymase gene on chromosome 14 (209, 210). Finally, human Metase is linked to the neutrophil elastase, proteinase-3, and azurocidin genes on chromosome 19 pTer (211, 212). In vitro, granzymes A and B are highly expressed, along with perforin, in activated T cells and NK cells. In human NK cell lines, granzyme H is also expressed, but at levels considerably lower than granzyme B (210). In the mouse, granzymes A and B are abundantly expressed in cytotoxic T cells and NK cells; granzymes C, D, F, and K are expressed at high levels in NK cells and LAK cell preparations (129, 208). However, the pattern of expression of these orphan granzymes in CTL activated in vivo has not yet been reported; the biologic significance of these restricted patterns of expression from in vitro activations is therefore unknown. Human granzyme H is a chymase that is predominantly expressed in the NK cell compartment; the gene that encodes it lies between granzyme B and cathepsin G (210). Murine granzymes C-G are expressed in in vitro–generated LAK and NK cells, and their location between granzyme B and cathepsin G suggests that they may serve similar functions in the mouse and the human. Indeed, human granzyme H 50 flanking sequences can target the expression of SV40 T-antigen to murine NK-T progenitors and activated LAK cells (213). All of these data suggest that the murine granzymes downstream from granzyme B may indeed subserve functional roles for murine cytotoxic lymphocytes. Although there is no data to support this hypothesis as yet, it is tempting to speculate that mice may have evolved a more divergent set of granzymes to protect them from a more diverse set of microbial pathogens than that encountered by humans (it seems unlikely that selection pressure was provided by tumors, because the reproductive cycle of mice is very short, and the life span of feral mice is measured in months). Regardless, the broad array of granzymes expressed in CTL, and the different specificities of these enzymes, suggest that they may play specialized roles in the induction of target cell death in specific physiologic situations.

LESSONS FROM THE KNOCKOUTS Perforin Deficiency in Mice and Humans Four independent groups created perforin-deficient mice (5, 214–216), and the phenotypes of these mice have been extensively described here and elsewhere (1). Because perforin is required for generating trafficking signals for granzymes

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after they are delivered to target cells, perforin deficiency causes functional pangranzyme deficiency. CD8+ T cells and NK cells are dramatically altered in their ability to cause the membrane changes associated with the induction of target cell apoptosis. Perforin deficiency results in increased susceptibility to a variety of viral infections and is associated with delayed tumor clearance in a variety of whole animal systems (1). It is also associated with the mitigation of GvHD (GvHD) severity in some model systems, especially those that are heavily dependent on the CD8+ compartment for disease (217–224). However, a role for the perforin pathway can be detected in graft vs. host models that are dependent on CD4+ cells as well (25, 225, 226). CD8+-dependent engraftment also requires perforin (227). Although perforin-deficient mice were initially thought to have no defects in lymphocyte homeostasis (1), a series of subsequent studies has suggested otherwise (112). In some model systems, perforin is involved in downregulating peripheral T cells after activation (65, 228). Activated CD8+ cells accumulate in perforindeficient mice after LCMV infection (62, 65), and perforin x Fas ligand double deficient mice die early of pancreatitis associated with infiltration of macrophages, MAC-1+ T cells, and CD8+ cells in the pancreas (229). Importantly, these defects in lymphocyte homeostasis do not occur spontaneously (as they do in FasL or FasR deficient mice); a second mutation or an infection is required to elicit the phenotype. Recently, Stepp et al. (230) described homozygous loss-of-function defects in the perforin gene in human patients with familial hemophagocytic lymphohistiocytosis (FHL). This disease is characterized by uncontrolled activation of T cells and macrophages and overproduction of inflammatory cytokines. FHL mapped to 10q21-22, the location of the perforin gene, in a fraction of families with this disorder. A more extensive analysis of additional families revealed perforin mutations in about 20% of all FHL patients investigated; the nature of the mutations for the other patients is not yet known (231). Interestingly, mice deficient for perforin do not spontaneously develop anything that resembles FHL. The addition of a viral infection or a second defect in a pathway that also controls lymphocyte homeostasis (i.e., Fas) is required for the development of the hemophagocytic syndrome. The nature of this striking difference between perforin-deficient humans and mice is not yet clear.

Granzyme B Cluster Deficiency Heusel et al. (106) first described a mouse with a targeted null mutation of the granzyme B gene; the mutant allele contained a PGK-neo cassette in the granzyme B gene. This mouse was null for granzyme B expression in CTL, but on careful analysis, it was also profoundly deficient for the expression of granzymes C, D, and F in the LAK cell compartment, suggesting that the PGK-neo cassette located at the 50 end of the granzyme gene cluster caused downregulation of all the genes in the domain (208). All of the published results for granzyme B deficiency have utilized these mice, and therefore all of the defects described could be due to granzyme B and/or one or more of the orphan genes that lies downstream. Recently, Thomas

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et al. (D. Thomas, R. Behl, T. J. Ley, unpublished data) have removed the PGK-neo cassette from the granzyme B locus using LoxP-Cre technology and showed that the in vitro cytotoxicity defect present for the granzyme B cluster–deficient CTL is identical for CTL deficient for granzyme B only. Therefore, granzyme B itself clearly plays a major role for the in vitro phenotype previously described. In vivo experiments comparing these mice are in progress. Granzyme B–deficient CTL are profoundly deficient in their ability to induce DNA fragmentation in target cells, even though perforin-induced membrane damage caused by these CTL is normal. Prolonged incubation of granzyme B–deficient effectors with target cells results in restoration of nearly all of the cytotoxic potential of the effectors (107, 130). Several groups showed that the perforinindependent component of this cytotoxicity could be accounted for the by the Fas pathway (2, 129, 130); the second component is a perforin-dependent mechanism, which is due to the activity of granzyme A (see below). The early pathway of cytotoxicity that requires granzyme B is important in vivo. Effectors deficient for granzyme B have an attenuated acute GvHD phenotype in partially and fully allogeneic model systems (232). This attenuation is not nearly as prominent as that of perforin deficiency, since granzyme B represents only one of the granzymes delivered to target cells by perforin. Granzyme B–deficient mice have not yet been systematically challenged with a variety of viruses because a minimal phenotype is predicted, due to the fact that these mice contain alternative pathways for clearing viruses. Granzyme B–deficient humans have not yet been described.

Granzyme A Deficiency Ebnet et al. (233) and Shresta et al. (207) both generated granzyme A–deficient mice. Granzyme A lies in a cluster that also contains granzyme K in the mouse. Retention of the PGK-neo cassette in granzyme A knockout mice did not affect granzyme K expression, and these mice do indeed have residual tryptase activity in their CTL (207, 234). Granzyme A–deficient mice exhibited no detectable abnormality of cytotoxicity in vitro, probably because these effector cells had normal amounts of perforin and granzyme B. However, the residual tryptase activity provided by granzyme K in these mice may also partially account for the minimal phenotype. Granzyme A–deficient mice fail to clear the poxvirus ectromelia as efficiently as wild-type mice (36, 235, 236). Ectromelia contains within its genome a CrmA orthologue, a serpin that may be capable of inactivating granzyme B. With this infection, therefore, granzyme B may be inactivated by the virus-produced CrmA, so that granzyme A–deficient cells exhibit the delayed-clearance phenotype. Granzyme A–deficient mice also fail to restrict the spread of herpes simplex virus in the peripheral nervous system of mice (237), but the mechanism of this effect is not yet clear. Granzyme A–deficient humans have not yet been described.

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Granzyme A × B Cluster Deficient Mice The granzyme B cluster–deficient mouse was intercrossed with the granzyme A mouse generated by Ebnet et al. (233) and also with the one generated by Shresta et al. (207); these two phenotypes were similar (195, 238). Granzyme A and B doubly deficient CTL can release chromium from target cells, and they have normal abundance and activity of perforin. Regardless, cytotoxic effectors from these mice had an apoptotic defect that was virtually equivalent to perforin-deficient effectors at all time points tested, strongly suggesting that granzyme A represents the perforin-dependent, late pathway in granzyme B-deficient mice. In an allogeneic bone marrow–transplant model that was dependent on CD8+ T cells, granzyme A × B cluster–deficient mice had a markedly attenuated incidence of acute GvHD (that was virtually equivalent to that of perforin-deficient mice) (195). That result suggested that perforin itself does not cause target cell death in this in vivo system and that the granzyme A and B cluster enzymes provided the lethal hits. Granzyme A × B double knockout mice have also been evaluated in a viral clearance model. Müllbacher et al. showed that granzyme A × B cluster-deficient mice were as susceptible to ectromelia as perforin-deficient mice, again suggesting that perforin itself was not the direct cause of death of the ectromelia-infected cells; the lethal hits are provided by granzyme A and B cluster enzymes. However, Davis et al. (239) have recently suggested that granzyme A × B cluster-deficient mice resisted RMAS tumor cells (cleared by NK cells) as efficiently as wild-type mice, while perforin-deficient mice are highly susceptible to these tumors. Perforin-deficient mice were also more susceptible to tumor initiation by methylcholanthrene than were granzyme A × B mice. These results suggest that perforin itself, or some other granule component delivered by perforin, is most critical for the in vivo anti-tumor effector functions of NK cells and cytotoxic T cells. In vitro, the defect in the killing of many tumor cells lines has been compared for perforin-deficient vs. granzyme A × B cluster-deficient mice, and this difference has not been appreciated. These results have not yet been reproduced by other laboratories, but they are clearly provocative and require additional experimentation with other model systems.

DPPI (Cathepsin C) Deficiency in Mice and Humans The study of DPPI-deficient mice (117) confirmed that this enzyme is responsible for the activation of granzymes A and B in CTL in vivo. As expected, DPPIdeficient CTL had an in vitro cytotoxicity defect that was virtually equivalent to that of mice deficient for granzymes A and B. Virtually all of granzymes A and B produced in DPPI-deficient CTL had retention of the N-terminal dipeptide or abnormal processing of the N terminus of the molecule, and no associated enzymatic activity. Recent studies have clearly shown that DPPI is also required for the processing and activation of mast cell chymases, (but not tryptases) (240), and it is responsible for activation of murine neutrophil elastase, cathepsin G,

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and proteinase 3 (117, 241). Therefore, DPPI-deficient mice have broad defects in serine proteinase activities in several hematopoietic lineages. Shortly after the description of DPPI-deficient mice, human patients with lossof-function DPPI mutations were described by Toomes et al. (242) and Hart et al. (243); these patients had Papillon-Lefèvre syndrome, also known as keratosis palmoplanterus with periodontopathia. This autosomal recessive disorder is associated with premature tooth loss due to periodontal disease, and with thickening of the skin. Homozygous loss-of-function mutations for DPPI/cathepsin C were identified in several consanguineous Papillon-Lefèvre kindreds (244) and also for additional kindreds with the very similar Haim-Munk syndrome (245) and prepuberal periodontitis (246). The natural history of these patients is not well understood at this time (i.e., do they have excessive viral infections or high cancer incidence?). Furthermore, cytotoxic lymphocytes from these individuals have not yet been examined to see whether they contain active granzymes. Based on the hypothesis that the granule exocytosis pathway is essential for lymphocyte homeostasis, these patients would be expected to have an hematophagocytic syndrome if their granzymes are nonfunctional. However, descriptions of these patients from the literature suggests that this is not the case (247). DPPI-deficient mice do not reproducibly develop skin thickening. Since mouse dentition is fundamentally different from human dentition, no periodontal disease has been observed in these animals. Importantly, DPPI-deficient mice can process some granzyme C to its fully mature form, suggesting that mice contain at least one other enzyme that is capable of processing some of the orphan granzymes (117). Whether humans possess this activity is not known. Mice and humans with DPPI deficiency therefore have somewhat different phenotypes, with the effect of the mouse loss-of-function mutation again being milder than that of the human mutations. Again, however, it remains to be seen whether the cytotoxic lymphocytes of human DPPI–deficient patients lack functional granzymes A and B.

ARE GRAFT VS. HOST AND GRAFT VS. LEUKEMIA (GvL) EFFECTS MEDIATED BY THE SAME OR DIFFERENT CYTOTOXIC MECHANISMS? It has been clear for many years that much of the benefit enjoyed by leukemia patients after allogeneic transplantation is a potent GvL effect that is provided by the T cells in the donor graft. This potential has been further exploited in recent years using donor lymphocyte infusions (DLI), which have been used successfully in patients with chronic myeloid leukemia in relapse after allogeneic stem cell transplantation. A small dose of donor T cells can cause potent GvL effects and clear the residual leukemia clone, but these T cells usually cause the development of GvHD. It is crucial, therefore, to understand whether the graft vs. host and GvL effects can be separated. The question, simply put, is whether the granule

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exocytosis pathway or the Fas pathway contributes more heavily to one or the other. Three recent studies, summarized below, have addressed this issue. Before reviewing these studies, some background information is required. Since the development of perforin-deficient and Fas ligand–deficient mice in the mid1990s, a large number of bone marrow transplantation experiments have been performed that evaluated each of these effector pathways for their contributions to GvHD in a variety of model systems. These systems have many variables that differ from study to study, including strain pairs, the dose and purity of T cells used in the donor product, the dose of bone marrow cells and purity of hematopoietic stem cells in that product, the conditioning regimen, the housing and feeding conditions, and even the evaluation of GvHD severity. In each study, these variables are different, and although controlled for in each study, it is very difficult to compare two studies of this kind and draw absolute conclusions. Nonetheless, the overall results support the notion that both the Fas and perforin pathways contribute significantly to acute GvHD in some settings (25, 217–219, 248). The relative contribution seems to be determined by the model system. Importantly, few (if any) of the mouse transplantation systems used to evaluate GvHD highly resemble chronic human GvHD, which remains the rate-limiting obstacle for human transplantation. Human chronic GvHD is usually caused by minor histocompatibility differences, and it develops weeks to months after transplantation occurs. Tsukuda et al. (222) evaluated leukemia development and GvHD in lethally irradiated F1 recipient mice (B6 × DBA/2) transplanted with bone marrow and spleen cells from C57 Bl/6 donors (WT, Fas ligand-deficient, or perforin-deficient). Just before transplant, the mice were inoculated with L1210 leukemia cells or P815 mastocytoma cells, and these mice were compared with nonleukemic controls. Deficiency of either perforin or Fas in this model system reduced lethal GvHD, but the GvL effect was apparent only in the mice that received their grafts from wild-type mice or Fas ligand–deficient mice; the use of perforin-deficient cells for donor engraftment resulted in early death from leukemia. These results suggested that loss of the Fas pathway reduced GvHD without impairing the GvL effect, which was perforin dependent. However, these results could have been influenced by the fact that both L1210 and P815 cells express class I but not class II. Pan et al. (223) used a model system that involved G-CSF mobilized allogeneic peripheral stem cells, which cause a reduced severity of acute GvHD in murine models, possibly because of immunomodulation of cells in the donor graft (i.e., enhanced mobilization of Tc2 cells over Tc1 cells). These authors determined that G-CSFmobilized allogeneic stem cell products caused less GvHD, but maintained potent GvL effects that were perforin dependent. The model system used was similar to that of the previous study (B6→B6D2F1), and leukemia was again induced with P815 mastocytoma cells (H-2d). Finally, Schmaltz et al. (224), using a parent→F1 model system like that previously described (and leukemia induction with P815 mastocytoma cells or 32DP210 cells), similarly concluded that GvHD could be mitigated by Fas ligand deficiency without an increase in the incidence of leukemia, but that perforin deficiency caused mice to prematurely develop leukemia.

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All of these studies concluded that the allogeneic GvL effect was predominantly due to the perforin pathway. Even though the perforin and Fas systems both contributed to the induction of acute GvHD in these experimental models, inhibition of only the perforin pathway was detrimental to the outcome of allogeneic transplantation that requires GvL for curative intent. In a parallel study that examined the cytotoxic mechanisms essential for killing a syngeneic myeloid leukemia cell line that is a target for both CD4+ and CD8+ cells, Hsieh & Korngold (249) again found that the perforin pathway was required. The perforin pathway may therefore be required for both GvL effects and antileukemia surveillance. Similarly, Smyth et al. (249a) have recently shown that perforin-deficient mice have a high incidence of spontaneous lymphomas (of T, B, or NK lineages) that develop after long latent periods, suggesting that the perforin pathway is indeed important for the immune surveillance of some tumor types.

GRANZYME INHIBITORS For many years, investigators have questioned how CTL can avoid suicide after granule exocytosis. Presumably, some granule contents must inadvertently re-enter the effector cell, which should cause effector cell death. Differential insertion of perforin into target cell membranes vs. effector cell membranes had been postulated previously. More recently, Bird and his colleagues (250–253) have provided a series of observations that suggest that effector cells contain a potent inhibitor of granzyme B, known as proteinase inhibitor 9 (PI-9). This human serpin is found in both the cytoplasm and nucleus of CTL, and it can form a tight complex with granzyme B via a classical serpin-proteinase interaction. When PI-9 is overexpressed in target cells, it can inhibit the induction of apoptosis either by whole CTL or by the addition of purified perforin and granzyme B. PI-9 does not inhibit most caspases, strongly suggesting that the inhibition of granzyme B alone is sufficient to block apoptosis even by whole CTL (which can kill either by perforin or Fas-based mechanisms). The true importance of this molecule for protecting CTL against accidental suicide has not yet been evaluated because the true murine orthologue of PI-9 has not yet been identified. A murine homologue, SPI-6 (with 68% identity to PI-9), and at least six other highly related murine serpins exist, but the true orthologue remains to be defined (P. Bird, personal communication). The studies of Bird and colleagues have provided strong evidence that CTL take significant precautions to protect themselves from granzyme B that is misdirected to the effector cell after granule secretion. The overexpression of PI-9 or its close relatives in tumor cells or virus-infected cells might be expected to protect these cells from death as well. Indeed, Madema et al. (253a) have recently shown that SPI-6 and PI-9 are expressed in a variety of murine and human tumors, respectively, and that overexpression of SPI-6 in tumor cells can protect them from CTLmediated clearance in vivo. However, the relative importance of this mechanism for tumor “escape” from immune surveillance remains to be defined.

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Similar studies for inhibitors of granzyme A and effector cells have not yet been reported. However, two molecules are known to interact with granzyme A, antithrombin III and α-2 macroglobulin. Both of these molecules can stably interact with granzyme A and neutralize it; complexes of the inhibitors and granzyme A have been detected in the circulation (194). It is not clear whether these molecules are present in activated CTL or NK cells, or whether they can inhibit granzyme A-induced death.

VIRUS-ENCODED INHIBITORS OF GRANZYME B If viruses are targeted for immune destruction by CD8+ T cells, it would be highly likely that the virus-infected cells could survive for longer periods of time if they were able to inhibit the action of perforin or granzyme B, based on the lossof-function models presented above. It does not seem likely that viruses would gain anything by creating inhibitors to granzyme A. However, it is possible that specific granzymes, especially C-G in the mouse, may have roles that are restricted to specific classes of viruses, although there are no data as yet to support this hypothesis. Accordingly, several different viruses have now been reported to encode inhibitors of granzyme B, and several different mechanisms of granzyme B inhibition have been implicated by these viral processes. The first, and best described, is that of the poxvirus-encoded cytokine response modifier A gene (CrmA). Quan et al. (254) showed that CrmA does associate with granzyme B in vitro and inhibit it; Tewari (255) showed that overexpression of CrmA in target cells could inhibit CTL-mediated killing, but predominantly through a Fas-mediated pathway. Macen et al. (256) also suggested that CrmA (Spi2) could inhibit both Fas- and granzyme B-mediated killing. Subsequently, Zhou et al. (257) indeed showed that CrmA was capable of interacting with several caspases and inhibiting them; the most prominent inhibition was to caspase 1 and caspase 8. The presence of CrmA in a poxvirus-infected cell therefore would be expected to reduce CTL-mediated killing by inhibiting both granzyme B and caspases activated by the Fas pathway. A second described mechanism of granzyme B inhibition is a loss of granzyme B mRNA in virus-infected T cells. Parainfluenza virus type 3 can directly reduce granzyme B mRNA abundance in virus-infected CTL (258). The mechanism of granzyme B mRNA loss caused by the virus was not elucidated (i.e., transcriptional downregulation vs. increased RNA turnover), but it was specific for granzyme B (and not granzyme A) mRNA. No additional reports of direct effects of viruses on the RNA abundance of cytotoxic granule genes have subsequently been reported. Finally, Andrade et al. (259) have reported that the adenovirus assembly protein L4-100K is a granzyme B substrate and that it also potently inhibits the enzyme. The inhibition is dependent on the presence of specific aspartic acid residue within this target protein, found within a classical granzyme B consensus motif. Interestingly, the 100K protein inhibited granzyme B specifically and had no activity

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against caspases 1-13 (in contrast to CrmA, which interacts with both granzyme B and several caspases). This is therefore the first description of a viral inhibitor of granzyme B that specifically targets this enzyme by acting as a decoy substrate. The idea of specifically targeting granzyme B, as opposed to a broader, pan-caspase inhibition, is supported by the many studies described above that have made it clear that granzyme B does not require caspases, or even mitochondrial pathways that release cytochrome c, to induce target cell death. It is interesting that the 100K protein, which has important functions in the adenovirus life cycle, inhibits granzyme B by acting not as a serpin, but as a complex between a large amount of the inhibitor and a small amount of protease, which results in a slow rate of cleavage of the substrate. 100K protein is produced in adenovirus-infected cells at levels likely to be vastly in excess of the amounts of granzyme B that enter target cells during CTL-induced killing. The presence of this inhibitor probably allows adenovirusinfected cells to live several extra hours after CTL-mediated attack, which may buy the virus time to replicate. With additional time, granzyme A (and/or other mechanisms) still cause apoptosis of the target cell by using other biochemical pathways and substrates, which explains why CD8+ T cells ultimately have no difficulty in eliminating virtually all adenovirus infections. Nonetheless, this is an important demonstration of a virus that specifically targets granzyme B, implicating granzyme B as a clear and immediate threat to the adenovirus-infected cell.

THE DEVELOPMENT OF THERAPEUTIC INHIBITORS OF THE GRANULE EXOCYTOSIS PATHWAY The granule exocytosis pathway contains an enormous number of potential steps where inhibitors could be targeted. Granule formation, granule secretion, perforin insertion into the target cell membrane, the entry of granzymes, the intracellular trafficking of granzymes, or the granzyme molecules themselves could all be viewed as targets for inhibitors. The two clinical circumstances where inhibitors of this pathway could be utilized would be in GvHD and in autoimmune diseases like rheumatoid arthritis, where the granule exocytosis pathway has been implicated as a contributor to disease pathogenesis. Mouse models suggest that it would probably be very unwise to inhibit the granule exocytosis pathway when allogeneic transplants are being performed to provide a potent GvL effect. Under these circumstances, any benefit that could be realized by the reduction of GvHD could conceivably be lost by a higher relapse rate in the patients. However, the abrogation of GvHD for the treatment of nonmalignant disease would seem a more reasonable setting to test inhibitors of this pathway, if they could be developed. One newly identified risk of long-term inhibition of the granule exocytosis pathway, however, may be the development of hematophagocytic syndromes. Regardless, if inhibitors of this pathway were to be developed, it would seem prudent to target a position in the pathway where multiple granzymes could be

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inhibited. The loss-of-function models revealed that granzyme B cluster–deficiency resulted in some attenuation of CD8+-mediated GvHD (232), while granzyme A deficiency resulted in none (195). Combined deficiency was similar to that of perforin deficiency, and therefore, both granzymes probably would need to be inhibited in order for significant clinical benefit to occur (195). Specifically, inhibiting either granzyme A or B with a small molecule is still an extraordinarily difficult task (204); highly efficient inhibitors of these enzymes do not yet exist. This limits the possibilities for therapeutic intervention to altering the granule environment, inhibiting granule secretion, inhibiting perforin function (either directly, or via inhibition of perforin insertion into target cell membranes), inhibition of granzyme entry or trafficking, or pan-granzyme inhibition. Because the granzymes have different active sites and specificities, a limited number of options exist for broadly inhibiting granzyme function. One option is inhibition of DPPI, which is known to be required for the processing and activation of granzyme A and B (117). Clearly, a good DPPI inhibitor would also reduce the activity of mast cell chymases and several neutrophil azurophil granule proteinases (i.e., neutrophil elastase, cathepsin G, and proteinase 3). Remarkably, however, loss of function of this enzyme results in a relatively mild phenotype in human patients, and inhibition of this enzyme could therefore be tolerated. One small molecule inhibitor of DPPI has been developed, but it has not yet been tested in vivo (260). Finally, inhibition of the CI-MPR, suggested by Bleakley and colleagues, remains another possibility for exploration. This receptor can internalize granzyme B but is also expected to mediate internalization of granzyme A (and possibly the other granzymes as well). The CI-MPR receptor is widely expressed. It is not clear what would be required to develop an effective inhibitor of this enzyme, since the receptor is efficiently recycled after releasing ligand upon internalization. Although mice require this receptor during development, it is not required for adult life, and systemic inhibition could therefore presumably be tolerated. As noted above, however, the efficiency of CI-MPR blockade for granzyme inhibition will ultimately depend on whether this is a minor or a major pathway for granzyme uptake in vivo. The granule exocytosis pathway, first proposed by Henkart in this Annual Review in 1985 (97), has come a long way in 16 years. This pathway is extraordinarily intriguing and complex, and it has yielded tremendous biological information important for a variety of systems. The basic science that has led to the explosion of new facts about this pathway is still robust, and the translational potential of these discoveries will, we hope, be realized soon.

ACKNOWLEDGMENTS The authors dedicate this review to the memory of Arnold Greenberg. His many seminal contributions to this field are evident throughout this review. The authors thank Nancy Reidelberger for expert editorial assistance. This work was supported

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by NIH AI45861 (JHR), National Multiple Sclerosis Society Grant RG2835 (JHR), NIH DK49786 (TJL), and the Alan and Edith L. Wolff Professorship (TJL). Visit the Annual Reviews home page at www.annualreviews.org


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Annu. Rev. Immunol. 2002. 20:371–94 DOI: 10.1146/annurev.immunol.20.092601.111357

SIGNAL TRANSDUCTION MEDIATED BY THE T CELL ANTIGEN RECEPTOR: The Role of Annu. Rev. Immunol. 2002.20:371-394. Downloaded from arjournals.annualreviews.org by HINARI on 09/01/07. For personal use only.

Adapter Proteins∗

Lawrence E. Samelson Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, 37 Convent Drive, Building 37, Room 1E24, Bethesda, Maryland, 20892-4255; e-mail: [email protected]

Key Words signaling pathways, linker for activation of T cells (LAT), plasma membrane microdomains, cytoskeleton, protein tyrosine kinases, tyrosine kinase substrates ■ Abstract Engagement of the T cell antigen receptor (TCR) leads to a complex series of molecular changes at the plasma membrane, in the cytoplasm, and at the nucleus that lead ultimately to T cell effector function. Activation at the TCR of a set of protein tyrosine kinases (PTKs) is an early event in this process. This chapter reviews some of the critical substrates of these PTKs, the adapter proteins that, following phosphorylation on tyrosine residues, serve as binding sites for many of the critical effector enzymes and other adapter proteins required for T cell activation. The role of these adapters in binding various proteins, the interaction of adapters with plasma membrane microdomains, and the function of adapter proteins in control of the cytoskeleton are discussed.

INTRODUCTION Signal transduction refers to the process by which extracellular events or cues are transmitted via a receptor or multiple receptors to the interior of the cell. Many of the current principles in the study of signal transduction have arisen from the study of various growth factor receptors. In these systems a transmembrane receptor binds ligand and then undergoes a conformational change or aggregation that has intracellular consequences. In many systems the receptor either is itself a protein kinase or is linked to one (1). The change in the receptor induced by binding results in kinase activation. The protein kinase(s) then phosphorylates a number ∗ 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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of proteins, some of which may be effector enzymes, and this phosphorylation may result in their activation. In many cases these critical kinase substrates also include adapter molecules (2, 3). Adapter or linker molecules are proteins lacking enzymatic activity and are comprised of multiple binding domains and sequence motifs to which such domains bind. Phosphorylation of adapter molecules alters the surface of these molecules and allows additional enzymes or adapters to bind, which results in formation of multiprotein complexes. The associated and activated enzymes can be at the proximal end of a series of subsequent activation events, and multiple enzymes and enzyme pathways can be involved. The general theme of these processes is the transfer of information (4), that is, the transmission of an event on the exterior of the cell (receptor engagement) to activation and regulation of multiple intracellular events occurring at the plasma membrane, cytosol, and nucleus. Several principles leading to development of the above scheme represent some of the greatest advances in the field of signal transduction over the past decades. First, receptor molecules have both extracellular and intracellular functions. Ligands engage specific extracellular domains, thereby inducing conformational changes or aggregation that are transmitted intracellularly. The cytoplasmic component of growth factor receptors was recognized to contain multiple sites to which various signaling molecules could bind. In many cases the cytosolic tails of these receptors contain tyrosine residues, which upon phosphorylation serve as docking sites for proteins containing specific phosphotyrosine recognition domains such as SH2 domains. The second and related theme is that most signaling molecules are modular. Many enzymes and a large variety of adapter proteins contain domains for phosphotyrosine, polyproline, or lipid interaction as well as motifs to which some of these domains bind (Figure 1). There is great variety in the way in which these domains are brought together in a form of combinatorial diversity. Over the past decade adapters have been increasingly recognized throughout biology as critical to cell function and, in particular, cellular signaling. Investigation of adapter molecules in cells of the immune system has also been intense. Numerous review articles contain surveys of the multiple adapter proteins found in lymphocytes (5–11). Many of these molecules are well studied, while others have only been recently described. Instead of providing a catalog of the multiple adapter molecules isolated from lymphocytes, the goal of this article is a combination of the general and the specific. The first intent is to put the study of adapter molecules in a broad context by reviewing certain issues common to various signaling systems and to relate these to the signaling pathways coupled to the T cell antigen receptor (TCR). The bulk of the discussion, in the second part of the review, focuses on certain topics now actively studied by those interested in the role of adapters in TCR-mediated activation. One particular adapter, LAT (linker for activation of T cells), is extensively discussed throughout the review.

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SIGNALING VIA THE T CELL ANTIGEN RECEPTOR—BACKGROUND The most obvious difference between growth factor receptors and the TCR is the added complexity of the latter both in terms of receptor structure and in the molecules most closely coupled to the receptor. The ligand recognition component of the TCR, the α and β chains, which engage the complex of peptide and MHC molecule, have been extensively studied for over two decades (12, 13). Their complexity lies in the genetic and cellular mechanisms involved in creating millions of different clonotypic receptors in any individual. A discussion of these processes— genetic rearrangement, the pairing of chains to create stable dimers, and then the intricate intrathymic selection of receptors—is outside the scope of this paper. The recognition components interact with the nonpolymorphic CD3, γ , δ, and ε, and the TCRζ chain dimer (14). These molecules are integral TCR components and as such are required for TCR expression. The cytosolic components of these molecules contain a unique motif, the ITAM (immunoreceptor-based tyrosine activation motif), which has a consensus sequence of YxxI/L x(6-8)YxxI/L (15, 16). Each of the CD3 chains contains one such motif, whereas each TCRζ chain contains three. The actual arrangement and stoichiometry of CD3 and TCRζ chains within a TCR are unknown. However, for a working model of the TCR, one can consider each αβ to associate with a δε dimer, a γ ε dimer, and a TCRζ dimer. Each TCR in this configuration would thus contain ten ITAMs. ITAMs are necessary and sufficient for TCR-mediated activation (17, 18). The tyrosine residues within each ITAM become rapidly phosphorylated upon optimal TCR engagement. The phosphorylated ITAMs become subject to binding by additional molecules. In this fashion the CD3 and TCRζ chains themselves behave like adapter proteins containing motifs that are modifiable by phosphorylation and serve as binding sites for critical proteins. ITAM phosphorylation is mediated by two members of the Src family of PTKs found in T cells. Lck is the predominant enzyme involved in ITAM phosphorylation, while Fyn also has this capacity (19, 20). The most important consequence of ITAM tyrosine phosphorylation is the binding of ZAP-70, a member of a second family of PTKs involved in TCR signaling (21). The two phosphorylated tyrosines of each ITAM are bound by the tandem SH2 domains of ZAP-70 in a highly specific and cooperative fashion (22, 23). ZAP-70, once bound to the TCR in this fashion is activated by phosphorylation of the kinase domain activation loop mediated by the Src PTKs (24, 25). Other phosphorylations of ZAP-70 allow additional proteins to bind, giving ZAP-70 itself the role of a scaffold (26). The activated TCR is thus characterized by phosphorylated ITAMs associated with phosphorylated, activated ZAP-70. These activated PTKs then phosphorylate a large number of protein substrates. Over the past decade these proteins were first identified by detection of the subset of proteins phosphorylated on tyrosine residues after TCR engagement. Some of these proteins, such as the enzyme phospholipase Cγ 1 (PLC-γ 1), were known from

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other systems and were rapidly identified (27, 28). Others were isolated because of their phosphorylation. These include other enzymes and, importantly, several of the adapter molecules such as LAT and SLP-76, discussed extensively in this review. The recognition that adapter molecules, both those subject to phosphorylation and others that are not, are critical in TCR signaling creates a number of analogies to other signaling systems in which adapters are of central importance in the signal transduction process (Figure 2). Among the most prominent PTK substrates detected after TCR engagement is a protein of 36–38 kDa (29). Early studies demonstrated that this protein could be detected in a complex with a number of other PTK substrates including PLC-γ 1 and the small linker molecule Grb2 (see below) (30–32). Despite both the ease of detection and its association with known proteins, the protein known then as pp36 proved difficult to isolate. Ultimately modified protein purification conditions enabled Zhang et al. to obtain amino acid sequence and clone the cDNA encoding this protein, which was named LAT (linker for activation of T cells) (33). Sequence analysis demonstrated that LAT is a member of a relatively unusual class of transmembrane adapter molecules. It is a class III–type protein, lacking a signal sequence. It contains a short extracellular sequence, a transmembrane domain, and a long cytosolic component containing nine tyrosine residues conserved between mouse and human LAT. Early studies also revealed that two cysteine residues (C26 and C29) are subject to posttranslational palmitoylation, which is responsible for specific localization within the plasma membrane (see below) (34). The central role of LAT in TCR-mediated signaling has been revealed in studies of the Jurkat T cell and Jurkat variants that lack LAT (35, 36). Jurkat cells, activated by cross-linking the TCR with monoclonal antibodies directed at either TCRβ or CD3ε show activation of multiple intracellular biochemical pathways and transcriptional elements leading to induction of interleukin 2 synthesis. These events include calcium elevation and ERK, AP-1, and NFAT activation. They do not occur in the Jurkat variants that lack LAT, but all activation events can be observed if LAT expression is restored following transfection of LAT cDNA. LAT function is also required for intrathymic development of normal T cells (37). In animals that are genetically modified to lack the LAT gene, intrathymic development of T cells is blocked at an early stage, and thus no T cells are found in the lymph nodes or spleens. LAT is rapidly phosphorylated on tyrosine residues following TCR engagement. Overexpression studies in fibroblastoid lines revealed that ZAP-70 and Syk are the PTKs most likely to be responsible for these phosphorylations. This conclusion is supported by the observation that LAT is very poorly phosphorylated in T cells lacking ZAP-70 (38). Nonetheless low levels of LAT phosphorylation are induced by activated forms of Lck, and it is possible that some of the multiple tyrosines in the LAT can be phosphorylated by PTKs other than ZAP-70. The consequence of these multiple phosphorylations on LAT in T cells is that a number of signaling proteins bind at these sites following TCR engagement.

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The proteins that interact with LAT fit into two categories, enzymes and adapter proteins. A full description of all these proteins is outside the scope of this manuscript. It is also likely that additional proteins that bind LAT remain to be described. One set of LAT-binding adapter proteins, the Grb2 family, contains Grb2, Grap, and Gads. All are of very simple design, consisting of a central SH2 domain flanked by two SH3 domains. These domains show considerable sequence homology between the three proteins. The Gads protein, known also by a series of other names [Grpl, MONA, Grf40, reviewed in (9)] also contains an internal proline-rich region of unknown function between the SH2 and the C-terminal SH3 domains (39). These simple linker proteins bind to tyrosine phosphorylated LAT via their SH2 domains. LAT structure-function studies have shown that three distal LAT tyrosine residues, Tyr 171, 191, and 226 (human sequence), following phosphorylation bind Grb2, while Gads binds only phosphorylated 171 and 191 (40). Grb2, Grap, and Gads are bifunctional molecules in the sense that their SH2 domains bind one protein, in this case phosphorylated LAT, while their SH3 domains bind other proteins, which contain the relevant proline-rich sequences. Grb2 and the related proteins are cytosolic and their binding to phosphorylated LAT translocates them and their SH3-associated proteins to a different cellular compartment, the plasma membrane. Thus, a consequence of LAT phosphorylation is the association of a number of Grb2-binding proteins at the membrane. Grb2 is a ubiquitously expressed protein, and its function in many systems is to shuttle associated signaling proteins to tyrosine phosphorylated receptors or adapter molecules following ligand activation (41). The list of proteins that bind Grb2 SH3 domains is vast. In T cells several proteins have been prominently described (42). SOS is well known from many studies in nonlymphoid tissues as a critical activator of the small G protein Ras (43). Grb2-mediated translocation of SOS from the cytosol to the plasma membrane brings it to the site of Ras localization. The Grb2-SOS complex has been detected in T cells, and SOS has been found in association with LAT following TCR engagement (33, 44). Recently there has been the identification of another Ras activator, in T cells, Ras-GRP, and thus the relative importance and function of each of these effector molecules remains unclear (45, 46). Cbl is another Grb2-associated protein found in T cells, bound to phosphorylated LAT. Cbl too has many domains and interactions. Recently Cbl has been shown to be part of a ubiquitin ligase assembly, and its function in that capacity in T cells also needs much additional investigation (47). Another small linker molecule, Shc, was originally demonstrated to bind to growth factor receptors and simultaneously to Grb2, which in turn in these studies was bound to SOS. Thus, in these systems an additional molecule was interposed between the receptor and the effector enzyme, SOS. Shc interactions with Grb2-SOS complexes have been studied in T cells (48). Another member of this simple adapter family is Grap, which also binds LAT following LAT phosphorylation (49). Grap-associated proteins include SOS, dynamin, and Sam68. Currently there is no good explanation for why T cells need two

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adapter proteins, Grb2 and Grap, with similar SH3 binding specificities. The third member of the family is Gads, which differs from Grb2 in its tissue distribution. While Grb2 is ubiquitously expressed, Gads is found only in hematopoietic cells. Gads also has unique SH3 specificity. While Grb2 does not bind to SLP-76 in T cells, Gads specifically interacts with SLP-76. Thus, Gads brings another critical linker molecule to phosphorylated LAT (50). Recently Gads has been shown to interact with the serine-threonine kinase HPK (51). Certainly, the full inventory of Gads-associated proteins remains to be described. SLP-76 was first identified as a tyrosine kinase substrate that could be affinity purified in vitro by binding to Grb2 (52). It is a 76-kDa protein expressed exclusively in hematopoietic cells. SLP-76 lacks enzymatic activity and structurally can be divided into three domains. At the amino-terminal end, multiple tyrosines become phosphorylated on TCR engagement. The central domain is rich in proline residues including those that interact with the SH3 domains of Gads. The carboxy-terminal end of the protein contains an SH2 domain. Overexpression of SLP-76 in Jurkat cells led to an increase in TCR-mediated activation of NFAT and IL-2 promoters (53). No increase in calcium mobilization was seen in such experiments, though ERK activation was enhanced. All three SLP-76 domains are required for this augmentation of function (54). Study of a Jurkat mutant lacking SLP-76 revealed that following TCR engagement these cells show decreased calcium flux and no ERK activation (55). Not unexpectedly in these cells, IL-2 promoter activity was not increased in response to TCR cross-linking. SLP-76 also has a critical role in normal thymocyte development. Mice lacking SLP-76 fail to generate normal, peripheral T cells (56, 57). An intrathymic block occurs in T cell development at an early stage. The T cell phenotype of these mice resembles that of mice that lack the LAT gene, as described above. Both of these mice demonstrate the same developmental block and absence of mature T cells. SLP-76 functions as an adapter protein that binds multiple effector molecules, which can be brought to LAT via its association with Gads (50). A recent study demonstrated that a LAT-SLP-76 chimera containing only the transmembrane domain and palmitoylation sites of LAT, i.e., the raft targeting region (see below), suffices to reconsitute LAT-deficient variants of Jurkat. Although overexpression of such constructs in Jurkat variants does not fully mimic all the complex interactions that might occur in more physiologic T cell systems, the result does emphasize the significance of SLP-76 recruitment (58). Upon TCR engagement, activated ZAP70 phosphorylates multiple tyrosine residues in the amino-terminal end of SLP76. These phosphorylated residues serve as binding sites for a series of proteins containing SH2 domains. These include Vav, a guanine nucleotide exchange factor for the G-proteins of the Rac family; Nck, itself an adapter protein that interacts with the serine-threonine kinase Pak1; and the PTK Itk (59–61). These associated molecules have been implicated in the regulation of a number of pathways critical to T cell activation. Vav and Nck may integrate the activation of a number of pathways involved in both gene transcription and cytoskeletal rearrangement as discussed below. The association of Itk with SLP-76 brings this PTK into close contact with

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PLC-γ . Recent studies suggest that Itk activation is required for optimal PLC-γ 1 phosphorylation and activation (62). SLP-76 SH2 domain interacts with a 130kDa protein named both SLAP (SLP-76-associated phosphoprotein) and Fyb (Fyn binding protein). This protein is also a multidomain adapter protein containing a proline-rich region, a tyrosine-rich region, and an SH3-like domain. The gene encoding this molecule has been genetically deleted by two groups (62a, 62b). The new proposed name for the protein is ADAP (adhesion and degranulation promoting adaptor protein).

LAT AND THE RAFT MODEL OF T CELL ACTIVATION The classic lipid bilayer model describing the molecular organization of the plasma membrane has been modified over the past decade because of the realization that the plasma membrane is not a homogenous array of glycerophospholipids (63–65). Instead there is considerable heterogeneity of lipids in the membrane. Glycosphingolipids and cholesterol were shown to self-associate in model membrane systems, and similar phenomena were then observed in plasma membranes isolated from cells. The aggregation of glycosphingolipids and cholesterol is thought to induce the formation of microdomains in the membrane that are distinct from the more abundant and diffuse glycerolipids. These domains are known by a large number of acronyms: GEMs (glycolipid enriched microdomains), DIGs (detergent insoluble glycolipid-enriched membranes, DRMs (detergent-resistant membranes), or rafts. Many of these names reflect the standard method used to isolate such domains, which is the inability of non-ionic detergents such as Triton X-100 to solubilize these domains from plasma membranes in the cold. Such insoluble material can be separated from solubilized cellular material by sucrose gradient centrifugation. These microdomains or rafts are also enriched in a number of molecules relevant to receptor-mediated signaling. These include the lipid substrates of PLC-γ and GPI (glycosylphosphatidylinositol)-anchored proteins, including such molecules expressed on T cells as Thy1 and Ly6. Additionally Ras, various G proteins, and members of the Src PTK family are enriched in these domains (66). For the proteins in rafts that are not GPI-anchored, one shared characteristic is posttranslational modification by several lipids. For example, Ras is both palmitoylated and farnesylated. Most of the Src PTKs (except for Src itself) are modified by myristoylation and palmitoylation (67, 68). Evidence obtained from study of the T cell–specific Src family PTK, Lck indicates that both of these lipid modifications are necessary for raft localization targeting and phosphorylation of the TCRζ chain (69). Studies on the FcεRI receptor were the first to demonstrate that immunoreceptor activation involves interactions with rafts. These investigators showed that this receptor associated rapidly with rafts upon activation (70, 71). The very early tyrosine phosphorylation of receptor subunits by the Src family PTK, Lyn depended on this localization. Several groups subsequently made similar observations about TCR activation in cell lines and thymocytes (72, 73). Receptor engagement increased

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the level of TCR association with rafts, and TCR subunits demonstrated enhanced tyrosine phosphorylation. Phosphorylated, and thus activated, ZAP-70 was found associated with these activated receptors. All of these studies depend essentially on a negative result, the inability of detergent extraction of the receptor at certain conditions of activation, cell number, temperature, and detergent type. Despite these results, several investigators at the time did not observe enhanced TCR association with membrane rafts. A possible explanation for the conflicting results was proposed by investigators who took an independent approach to the question (74). They visualized rafts using fluorescent cholera toxin B subunit, which binds glycosphingolipids. Antibodies to the subunit induced cross-linking of these lipids forming patches that are visible microscopically. With this technique they were able to observe colocalization in rafts of Lck and TCR subunits. Interestingly TCR subunits patched in this fashion were more sensitive to detergent extraction than were other raft-associated proteins. The authors speculated that this result indicated that the TCR is more weakly associated with rafts than are other molecules, which might constitutively localize in these domains. The study of the LAT protein contributed to an understanding of raft localization and T cell activation. Examination of the LAT amino acid sequence revealed the presence of two cysteine residues (positions 26 and 29) adjacent to the putative transmembrane domain of the protein. Since juxtamembrane cysteines are likely to be targets of the membrane-associated palmitoylation machinery, T cells were labeled with [3H]-palmitate and incorporation was demonstrated (34). Mutation of these two cysteines to alanine blocked this incorporation. These mutations and especially mutation of Cys 26 alone also had dramatic effect on LAT localization to rafts, as determined by the standard biochemical extraction assay. Though mutation of the cysteine residues did not affect membrane localization of LAT, mutation of Cys 29, partially, and Cys 26 fully inhibited LAT localization to rafts. LAT with these mutations were also expressed in T cells and examined following TCR engagement. Mutation of cysteine 26 had a dramatic effect, and no LAT tyrosine phosphorylation was detected. From these studies it was concluded that LAT had to be in rafts for it to be phosphorylated. The majority of LAT molecules localize in rafts as determined by the sucrose gradient analysis. As expected in samples from nonactivated T cells, essentially none of the LAT substrates are detected in these fractions. However, upon activation and LAT phosphorylation, one observes a translocation of LAT binding proteins to the raft fraction. The fraction of such LAT binding proteins as PLC-γ , Cbl, or Grb2 that shifts in this way is small, but this is presumably the fraction that is functionally active. The consequences of the absence of LAT localization to rafts and the failure of LAT tyrosine phosphorylation were thought likely to be dramatic, and this prediction was confirmed in two subsequent studies (36, 75). In both, independently isolated Jurkat mutants lacking the LAT molecule were used. The absence of LAT had no effect on TCR-induced receptor phosphorylation or ZAP-70 activation, but all steps distal to this were inhibited. There was no activation of PLC-γ 1, and thus there was minimal calcium flux or ERK activation. Several

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transcription factors were not activated. In both of these studies, reintroduction of wild-type LAT restored all function. Introduction of LAT deficient in one residue, Cys 26, had the same effect as though no LAT was present in the cell. Thus, without LAT localization to rafts, there is no LAT tyrosine phosphorylation, no translocation of critical signaling molecules to the rafts, and no T cell activation. If LAT is in membrane rafts and the TCR in the resting state is not, how do these structures interact, and how does the TCR become raft-associated, albeit weakly? It is possible that receptor engagement alters some biophysical properties of the TCR. A common model for TCR activation requires some level of TCR-TCR interaction, which at the most extreme would be TCR oligomerization or aggregation (76). Hypothetically this process could expose different regions of TCR subunits, which might enhance interactions with different membrane components. Similarly, receptor aggregates might also be more likely to trap rafts. Interactions of other TCR and LAT associated proteins have been described. Some or all of these interactions might be involved in bringing the activated TCR and associated PTKs to LAT molecules. The Lck PTK is located in rafts, and its SH2 domain can bind a phosphorylated tyrosine residue in the activated ZAP-70 PTK (77, 78). This intermolecular bridge may bring TCRs bearing activated ZAP-70 to rafts. Additionally, a subset of CD4 interacts with LAT (79). Other CD4 molecules may interact with Lck. Since CD4 interacts with MHC class II molecules and since some CD4 molecules exist as dimers (80), one can construct a model by which TCR and CD4 engage the same MHC, and the CD4-associated Lck and LAT, both in rafts, are brought to the TCR. Several additional molecules have been proposed to bind both TCR and LAT, thus potentially linking these molecules. PLC-γ 1 contains two SH2 domains, and it is well documented that the N-SH2 domain interacts with LAT. Williams et al., studying SH2 fusion proteins, proposed that the C-SH2 domain interacts with phosphorylated residues on activated ZAP-70 in a fashion similar to that described above for Lck (81). Deckert et al. have proposed that a molecule originally isolated as an Abl-SH3 interacting protein, 3BP2, can interact via its SH2 domain with both ZAP-70 and LAT (82). Though this interaction cannot be simultaneous because 3BP2 has only one SH2 domain, perhaps the molecule could multimerize. The authors of this study suggest that a functional coupling could occur, leading to the observed enhancement in T cell activation. Finally a small adapter protein known as Shb does contain two separate phosphotyrosine-binding domains (83). A classic SH2 domain in Shb was shown to bind TCRζ chain on phosphorylated tyrosine residues. A non-SH2 phosphotyrosine binding domain bound phosphorylated LAT. Expression of a mutant form of Shb with a defective SH2 domain inhibited LAT phosphorylation and distal signaling events. These data suggest that Shb links between the TCR and LAT have major functional significance. A recent study provides further insight into TCR-LAT interactions. Harder & Kuhn incubated T cells with anti-TCR antibodies coupled to beads (84). Activation via the TCR was induced with warming to 37◦ C, and the cells were subjected to nitrogen cavitation. The material that associated with the TCR on the bead over

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different times of warming was then analyzed biochemically. No detergent was used in these preparations, and what was isolated, presumably, were membrane patches. In addition to the TCR subunits and the associated ZAP-70, the investigators showed that over time LAT was detected in these complexes. Some of the proteins that bind activated LAT such as PLC-γ 1, Grb2, and Cbl were also found in these patches. In contrast what they did not see associated with the TCR were the raft-associated PTKs, Lck or Fyn. The association of TCR and LAT in these membrane fragments depended on tyrosine kinase activity, and mutant LAT, which lacked the cysteines required for palmitoylation, did not co-isolate with the TCR. The conclusion from these studies is that TCR-LAT interactions do not represent an interaction of the receptor with LAT via large lipid aggregations in which Lck and Fyn would also be expected to be found. Instead the study supports the idea that protein-protein interactions induced by TCR activation and dependent on tyrosine phosphorylation control the critical TCR-LAT association. Interest in the raft model of immunoreceptor signaling has been intense, and numerous studies have expanded an understanding of the interactions between signaling receptors and the plasma membrane. However, it is wise to recognize that there are still a number of problems with this model that investigators in this field must acknowledge. The main concern is methodologic. Nearly all studies of raft function rely on the crudest of preparations, material that fails to be solubilized by certain detergents. Moreover, the preparations are made from cells that have been chilled to near freezing temperature. In so doing one may force interactions of proteins and lipids that might not normally occur under physiologic conditions. Efforts to reproduce raft isolation without detergent or chilling are rare, but, as described above, some investigators have begun this process. A similar criticism can be made about methodologies in which raft components are visualized after heavy cross-linking induced by multivalent toxins and antibodies. These studies demonstrate clustering of molecules shown to colocalize by biochemical analysis, but clearly the system is being forced. Ideally, imaging techniques could be used to demonstrate membrane heterogeneity. Investigators have attempted to use fluorescence energy transfer (FRET) techniques to visually demonstrate clustering of GPI-linked proteins. Two groups have reported contradictory results, with one obtaining results consistent with microdomains of less than 70 nM containing just a few molecules, whereas the other group saw no such structures (85, 86). The discrepancy could be resolved if only a few molecules were clustered over a minor fraction of the surface, or if such structures were short-lived. There is certainly strong evidence that membrane heterogeneity exists in model systems and in cells. There is much evidence that membrane microdomains are relevant for signaling in lymphocytes. The dramatic effect on T cell signaling of the cysteine LAT mutations is an example of such an experiment. Frequently however one senses that rafts have come to imply long-lived, well-defined membrane substructures. The conclusion that membrane heterogeneity is a dynamic process and that transient interactions of lipids and proteins are likely is much more reasonable and cautious.

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SIGNALING COMPLEXES: THE LAT-PHOSPHOLIPASE Cγ 1 EXAMPLE PLC-γ 1 was one of the first enzymes demonstrated to be a PTK substrate in T lymphocytes, following its identification as a PTK substrate in growth factor receptor tyrosine kinase systems. Phosphorylation of PLC-γ 1 on multiple tyrosine residues is required for its activation (87). PLC-γ 1 is a central signaling molecule in T cells as well as other cells activated by PTKs. Activation of this enzyme leads to hydrolysis of phosphatidyl inositol (4,5)-bisphosphate to inositol (1,4,5)-trisphosphate and diacylglycerol (88). The former regulates intracellular calcium mobilization, and the latter regulates protein kinase C activation. Recent studies in T cells demonstrate that calcium and diacylglycerol regulate RasGRP, a newly described activator of Ras. By contributing to Ras activation, PLC-γ 1 thus indirectly can control PI3 kinase and MAP kinase cascades (45, 46). The multiple binding interactions that engage PLC-γ 1 and molecules that regulate this enzyme are now under intensive scrutiny, and the results of these studies serve as an excellent example of signaling complexes containing adapter molecules involved in TCR activation. An interaction of PLC-γ 1 and a 36–38 kDa protein was described long before LAT was isolated and characterized. The association was seen after T cell activation and was dependent on the two PLC-γ 1 SH2 domains (30, 89). This and a subsequent study showed that the N-terminal SH2 domain was more specific for phospho-LAT, but in the later study the C-terminal SH2 domain was shown to bind LAT as well as other proteins (90). Mutation of the N-SH2 resulted in depressed tyrosine phosphorylation of PLC-γ 1 following TCR binding. The sites of PLCγ 1 interaction with LAT were addressed in a LAT structure-function study. TCR engagement of LAT-deficient variants of the Jurkat cell line (J.CaM2) failed to activate PLC-γ 1 and thus failed to elevate intracellular calcium or activate ERK. Reconstitution with wild-type LAT restored these pathways. Zhang et al. created a series of stable lines in which J.CaM2 was reconstituted with LAT mutants containing one or more tyrosine-to-phenylalanine mutations (40). The residues adjacent to Tyr 132, YLVV, form a consensus binding sequence for PLC-γ 1 SH2 domains. Mutation of tyrosine at this site abrogated PLC-γ 1-LAT association and PLC-γ phosphorylation on tyrosine residues. Cell lines expressing this mutant showed altered calcium flux following TCR engagement. The rapid onset of calcium elevation was observed, but the sustained influx, normally seen following TCR engagement, did not occur. Two additional reports showed subtly different results. In one, the 132 mutations inhibited PLC-γ 1 phosphorylation and calcium flux (91). In the other, phosphorylation of PLC-γ 1 persisted, but calcium flux was sharply curtailed (92). The differences are likely due to slight variations in technique; nonetheless, all three studies point to the critical role of this site on LAT for PLC-γ 1 function. The three distal tyrosine residues of LAT (Tyr 171, 191, and 226) all are found within YXNX motifs. Phosphorylation of this motif defines sites for binding via the SH2 domains of Grb2 or related adapters. Interestingly, mutation of all three

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of these sites also had a strong impact on PLC-γ 1 (40). The PLC-γ 1-LAT association was not detected; PLC-γ 1 tyrosine phosphorylation was nearly completely inhibited, and calcium flux was partially inhibited. These sites might bind the C-terminal SH2 domain of PLC-γ 1 directly or might bind PLC-γ 1 indirectly as described below. These three sites were also tested for binding by Grb2 and the related protein Gads. Mutation of any one of the three distal tyrosine residues (171, 191, or 226) had no effect on Grb2 or Gads binding, which suggests a degree of redundancy in the system. Loss of both 171 and 191 decreased Grb2 binding, and only mutation of all three of these tyrosines blocked Grb2 binding. Gads binding proved more restricted because mutation of both 171 and 191 inhibited interaction. Since a major binding partner of Gads is SLP-76, which in turn interacts with PLC-γ 1, these results account for the loss of SLP-76 binding in the double Tyr 171, 191 mutant. The multidomain adapter SLP-76 is critical to T cell activation, and as mentioned above, cell lines deficient in this molecule have a significant defect in PLC-γ 1 activation (55), a result that now can be explained. As noted above, many molecules bind to SLP-76 following phosphorylation of its N-tyrosines or via its C-terminal SH2 domain. Yablonski et al. demonstrated the significance of a proline rich region (157–223) that interacts with the PLC-γ 1 SH3 domain (93). This stretch of prolines is distinct from the residues involved in Gads binding (224– 265). An SLP-76-deficient variant of Jurkat was used as the recipient for SLP-76 mutants in a structure-function study. Constitutive association between PLC-γ 1 and SLP-76 was dependent on the PLC-γ 1 SH3 domain and an inducible increase in association that they attributed to direct and indirect interactions via LAT. They proposed that two previously defined complexes, LAT-Gads-SLP-76 and LATPLC-γ 1, in fact interact via the binding of SLP-76 to PLC-γ 1. They suggest, in other words, that a multiprotein complex nucleated at LAT contains Gads, SLP-76, and PLC-γ 1, and in this complex both Gads and PLC-γ 1 bind LAT. This conclusion is bolstered by another study in which they demonstrate that the functional complex of these molecules must be bound to the same LAT molecule (92). How the individual phosphorylation sites on all PTK substrates are targeted by various PTKs in T cells is still under investigation. Nonetheless, it is clear that members of the Tec PTK family are required for full PLC-γ 1 phosphorylation and activation. In support of this conclusion is the observation that deletion of the Tec PTK Itk or deletion of two Tec PTKs, Itk and Txk/Rlk, produces defects in sustained calcium elevation following TCR engagement (62, 94). Overexpression of Txk/Rlk in transgenic mice also showed enhanced PLC-γ 1 phosphorylation and calcium flux (95). If regulation of PLC-γ 1 activation at LAT is likely to involve Tec family PTKs, the next question is how these enzymes are targeted to this site. A full answer is not yet in hand, though the multiple domains of Itk interact with many molecules, and one or more of these interactions might be relevant to this question of targeting (61). The N-terminal PH domain of Itk or the palmitoylation of Txk/Rlk are likely to control plasma membrane localization. The TH (Tec homology) region contains a proline-rich region that interacts with a Grb2 SH3 domain. The Itk SH3 domain interacts with proline-rich regions

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of PLC-γ , though the authors of the study on PLC-γ 1-SLP76 interactions think that this interaction is not physiologically relevant (93). The Itk SH2 domain has been shown, by several investigators, to interact with SLP-76 (60, 61). Finally, Itk interactions with LAT have been reported, though it is not known whether this is a direct Itk SH2-mediated interaction or whether it is indirect (96). Thus, it is not now clear whether one predominant mode of Itk interaction with LATassociated molecules predominates, or whether there are multiple mechanisms of interaction. The consensus derived from many investigators is that a complex of LAT, Gads, SLP-76, PLC-γ 1, and a Tec PTK, usually Itk, regulates PLC-γ 1 activation in response to TCR signaling. These conclusions follow from characterization of cell lines and mice lacking expression of one of these proteins and from an extensive analysis of the fine specificity of multiple protein-protein interactions. This model has certain strikingly positive features, but it remains incomplete in a number of ways. Its greatest strength is the manner in which the strength of multiple protein-protein interactions is likely to be far greater than the sum of individual interactions. Ladbury and Arold have noted that the difference in affinity between specific and nonspecific SH2 and SH3-mediated individual interactions is usually less than two orders of magnitude (97). These authors note that the assembly of multimolecular complexes involving many such interactions ensures that a proper assembly must occur before signaling transpires. Such interactions can be defined as highly cooperative. Interactions of this sort are central to the generation of the multiprotein complex regulating PLC-γ 1. The multiple individual interactions might zip together the functional PLC-γ 1 machine. In addition such multistage events also offer great potential for regulation, as inhibition of any of the multiple steps might block assembly. However, before defining such a putative entity as a signalosome, a term that might imply a far more stable structure of defined stoichiometry, a number of caveats and concerns must be discussed. The major issue is that LAT has been shown to bind a large number of different signaling molecules. Gads, SLP-76, and PLC-γ 1, described above, are just a subset. Moreover, even these molecules have additional binding partners. Gads also binds the serine-threonine protein kinase HPK, and thus a different LAT-Gads complex may exist (51, 98). Similarly SLP-76, as described in detail below, has many additional partners. Competition for LAT can also occur. Grb2 binding to LAT is well defined, and Grb2 is capable of coordinating a number of LAT-based complexes including interactions with SOS and Cbl. The SH2 domains of Gads and Grb2 have similar binding characteristics, and both were shown to bind to two of the three distal tyrosine residues of LAT (171 and 191) following activation. It is not clear what the relative binding affinities between Gads and Grb2 are for these sites, nor has the relative stoichiometry of binding between LAT, Gads, and Grb2 been determined. Clearly, though, the issue of whether and how much Grb2 or Gads is bound to LAT would have a great impact on which other molecules are brought to LAT. It is unlikely that all possible interactions can occur at one LAT molecule at the same time because there are too many possibilities leading to competition at the same site. The competition for

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interaction might direct formation of a particular complex via multiple cooperative interactions, so that the binding of Gad-SLP-76 might enhance the probability of PLC-γ 1 binding. However, the presence of any particular complex might also depend on local concentrations of proteins, and these factors might depend as well on the state of the cell. To date there has been little analysis of the heterogeneity of LAT-based complexes to address the sort of questions raised above. It should be possible to determine by immunodepletion which molecules can coexist in the same complex. The possibility that multiple, different LAT-based complexes exist is real. Tremendous variety in time and space might be observed. Such different LAT-based complexes might come together so that the sum of complexes would be the critical factor determining progression of a signal for activation. The role of complexes not mediated by LAT will likely receive much attention (99). Signaling events may require the generation of a variety of structures or complexes of complexes to coordinate the various events that occur following TCR engagement.

LAT AND THE CYTOSKELETON To date, most of the studies of adapter molecules involved in lymphocyte signaling have focused on characterizing associated proteins and demonstrating how these interactions regulate classic biochemical signaling cascades. One such pathway involves Grb2, which brings the Ras activator SOS to receptors or to other adapter molecules such as LAT, which have been phosphorylated on tyrosine residues. Activation of Ras leads to subsequent activation in sequence of several serinethreonine kinases, which in turn are responsible for enhanced transcription of a number of genes. (4). Recently there has been increasing attention to another consequence of receptor-mediated signaling, the regulation of the cytoskeleton. Cytoskeletal changes are required for lymphocyte movement, and they accompany and control adhesive interactions that regulate cell-cell interactions (100). These issues have relevance to the early events leading to T cell activation. Of more immediate relevance for this review is the realization that T cell activation involves significant rearrangement of a number of receptors and intracellular molecules over a prolonged period following interaction of the T cell and the ligand-bearing antigen presenting cell (APC). These molecular movements create a supramolecular arrangement of receptors known as the synapse (101, 102). The dynamics of these molecular rearrangements are in part regulated by the cytoskeleton. Another significant and relevant recent breakthrough comes from the basic cell biological study of the cytoskeleton (103–106). Investigators studying several model systems have made a number of conceptual advances in understanding the dynamics of actin polymerization. Many of the molecules involved in this process are either identical to or related to molecules known to interact directly or indirectly with critical lymphocyte adapters during T cell activation. A detailed description of the immune synapse is outside the scope of this manuscript, and the topic has been extensively reviewed. In brief, several groups

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observed, using fluorescence microscopy, that TCR engagement induces a series of molecular rearrangements at the contact zone between a T cell and an antigen presenting cell (APC). Upon T cell contact with an APC bearing peptide-MHC, a central region containing T cell integrin receptors and APC integrin receptor ligands is surrounded by a ring of MHC-peptide complexes. Over minutes this pattern reverses such that the TCR-MHC contacts move to the center. This region is known as the cSMAC (the central supramolecular activation cluster). It is surrounded by the integrin receptors that define the pSMAC or peripheral supramolecular activation cluster (101, 102, 107). Additional studies have located a number of molecules within the context of the SMAC architecture. Thus, for example PKC2 is found in the cSMAC, talin is found in the pSMAC, whereas CD43 is excluded entirely from both cSMAC and pSMAC (101, 108). More recently individual molecules have been shown to migrate in relation to the SMAC over time. The integral membrane tyrosine phosphatase CD45 is initially excluded from the cSMAC and later migrates back to it (109). These supramolecular structures are stable over hours. The importance of actin polymerization to the generation of these structures is confirmed by their disruption after blockade with cytochalasin D. Actin polymerization can be observed using fluorescent phalloidin binding in a microscopic or flow cytometric assay. A ring of polymerized actin can be detected in T cell-APC conjugates or at the interface between T cells and beads coated with stimulatory anti-TCR antibodies. Actin polymerization has recently been observed in live cells using Jurkat T cells stably expressing EGFP-actin (110). In this assay the cells are dropped on to cover slips coated with stimulatory anti-TCR antibodies. Upon contact lamellipodial projections from the cells engage the cover slip and merge into a circumferential ring tightly adherent to the coverslip. This ring spreads outward over 3–5 min as the cell spreads on to the coverslip. The ring is formed of polymerized actin, and rapid polymerization-depolymerization reactions can be observed with fast microscopy systems. This system is also amenable to quantitation. An index of spreading can be calculated by measuring the ratio of actin clustering at the cover slip and the cell body. With this assay actin polymerization appears to be biphasic with an early peak at 3–5 min followed by a prolonged shoulder lasting 15–20 min. The assay can be used to test the effects of various inhibitors on actin polymerization. The role of the LAT molecule can be demonstrated using this assay. Jurkat cells lacking LAT (J.CaM.2 cells) spread very poorly on coverslips coated with stimulatory antibodies, and the little actin polymerization that is seen is short lived. In parallel the assay was also used to evaluate the function of various LAT tyrosine residues and thereby the role of different pathways coupled to LAT. Interestingly no difference appeared in response by these various mutants. The same degree of inhibition of actin polymerization was observed regardless of whether LAT was absent or whether LAT lacked a PLC-γ binding site or lacked all the Grb2 and Gads binding sites. Inhibitors were used to demonstrate a calcium-sensitive component to the regulation of actin polymerization; the inhibition of actin polymerization due to lack of PLC-γ 1 binding may be thus explained. The loss of Grb2 and Gads binding sites may have many consequences leading to problems with actin

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polymerization. These include loss of optimal PLC-γ 1 binding, as mentioned above, or failure of SLP-76 association, among many possibilities. As described above SLP-76 is a multidomain adapter protein. The central region of SLP-76 contains a proline-rich region that mediates Gads binding. In addition to that interaction and its interaction with PLC-γ 1, SLP-76 also makes contact with a number of proteins that have an impact on the cytoskeleton. The SLP-76 aminoterminus contains tyrosine residues, which after phosphorylation bind Vav and Nck (59, 111, 112). Vav too is a PTK substrate and multidomain protein consisting of PH, SH2, and SH3 binding domains and a Dbl-homology domain required for activation of Rac or cdc42, small G proteins of the Rho family (113–115). Targeted disruption of Vav produces a complex T cell deficit, including a partial block in calcium mobilization and a defect in IL-2 production. Two recent studies demonstrated that T cells from these mice also demonstrate a defect in cytoskeletal function (116, 117). Antibody cross-linking of T cells from these mice failed to produce antigen-receptor caps or patches. A failure of actin polymerization was also demonstrated in a phalloidin-binding assay. The pattern of inhibition was mimicked in these studies by treatment with cytochalasin D. More recently SMAC formation was also shown to be impaired in T cells deficient in Vav (118). The absence of Vav or, in the case of the LAT-deficient cells, the failure of Vav recruitment via Gads and SLP-76 would decrease the amount of activated Rac and cdc42 in the vicinity of the TCR and LAT. Consequences could include inadequate activation of phosphatidylinositol 4-phosphate 5-kinase, which is responsible for generating the PLC-γ 1 substrate phosphatidylinositol 4,5-bisphosphate (PIP2) (119). More importantly for this discussion, lack of activated Rac could result in inadequate WASP activation. WASP is known to bind the Nck adapter molecule, which in turn binds SLP-76 (120). WASP was first identified as the defective protein in patients with WiskottAldrich syndrome (121). T cells from patients with that disease, and murine cells from animals with targeted deletion of the WAS gene, have a phenotype similar to that observed in the Vav −/− animals, showing decreased calcium flux, IL-2 production, and notably, defective actin polymerization (122, 123). An explanation for this phenotype is now clear with an increased understanding of the protein WASP (104, 124). This multidomain protein contains regions capable of binding activated Rac, phospholipids such as PIP2, soluble actin-profilin complexes, and at the C-terminus, the Arp2/3 complex responsible for actin polymerization. In the resting state WASP exists in an autoinhibited state in which the GTPase binding domain interacts with the C-terminal region. Activated G proteins and phospholipids synergistically activate WASP, thus allowing the Arp2/3 complex to mediate actin polymerization. Thus LAT, by recruiting the Gads-SLP-76 complex, may bring together WASP and Vav, the enzyme needed to produce activators of WASP. A third protein bound to SLP-76 and relevant to actin polymerization is SLAP130/Fyb (62a, 62b, 125, 126). This protein was independently isolated as a protein that binds the SLP-76 SH2 domain and as a Fyn-binding protein. A clue to the function of this protein was recently presented (127). These investigators were studying a family of WASP-related proteins and in particular were interested

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in proteins that bound to a particular domain in these proteins, the EVH1 domain. Their studies revealed that SLAP-130/Fyb is one such protein. They then demonstrated that this protein colocalized to polymerized actin in the assay in which Jurkat was activated by anti-CD3 coated beads. In addition they found colocalization with WASP, Arp2/3, Vav, and the EVH1-bearing protein, Evl. Microinjection of peptides capable of blocking the interaction of SLAP130/Fyb with Evl blocked actin remodeling at the bead interface. Similarly, disruption of Arp2/3 localization blocked actin polymerization. The actual function of Evl and other VASP-related proteins in actin polymerization in T cells is not clear. However, these authors demonstrate in this study that SLAP130/Fyb is an additional required component of the actin polymerization machinery in T cells. SLP-76 thus binds the central molecular machinery involved in actin polymerization. Phosphorylation of LAT might bring these SLP-76-bound molecules together with other enzymes such as PLC-γ 1 and PI3K, which may bind LAT directly (91) to the site of TCR engagement and PTK activation. Many of these molecules themselves integrate multiple inputs as described above for WASP and previously for PLC-γ 1. In turn colocalization of these molecules at LAT ensures a highly ordered process of activation. Thus a staggering amount of molecular integration is occurring at multiple levels. In this context it is worth re-asking the question of whether one LAT molecule or one SLP-76 molecule can itself bind all the possible proteins to which it could bind. At the level of the individual protein molecule this seems sterically unlikely. However, mixed populations of molecules and complexes may very well be colocalized at sites of activation. In this view, T cell activation represents the assembly of multiple and varied complexes over time and in particular locations. The challenge for future studies of adapters and T cell activation is to develop approaches and techniques capable of defining these complicated molecular interactions and dynamics.

ACKNOWLEDGMENTS Thanks to Drs. Oreste Acuto, Stephen Bunnel, Claudette Fuller, Jon Houtman, and Connie Sommers for reading the manuscript and making numerous constructive suggestions. Visit the Annual Reviews home page at www.annualreviews.org

LITERATURE CITED 1. Schlessinger J. 2000. Cell signaling by receptor tyrosine kinases. Cell 103:211–25 2. Pawson T. 1995. Protein modules and signalling networks. Nature 373:573–80 3. Pawson T, Scott JD. 1997. Signaling

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Figure 1 A selection of signaling proteins found in T lymphocytes depicted to highlight their modular structures. SH2, SH3 and PH domains are in red, blue and green respectively. A Tec homology domain is in pink, and a transmembrane domain is in light green. Sites of tyrosine phosphorylation are indicated with Y and proline-rich sites are indicated Pro. Domains with enzymatic function are in yellow.


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Figure 2 Signaling complexes at the T cell antigen receptor and at the LAT molecule. The TCR is depicted with its associated protein tyrosine kinases. Tyrosine phosphates are indicated with a black ball and the modular components of other linkers and enzymes are color coded with SH2 domains in red, SH3 in light blue, PH in green, proline-rich regions in dark blue, and enzymatic domains in yellow.


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INTERACTION OF HEAT SHOCK PROTEINS WITH PEPTIDES AND ANTIGEN PRESENTING CELLS: Annu. Rev. Immunol. 2002.20:395-425. Downloaded from arjournals.annualreviews.org by HINARI on 09/01/07. For personal use only.

Chaperoning of the Innate and Adaptive Immune Responses Pramod Srivastava Center for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut School of Medicine, Farmington, Connecticut 06030-1601; e-mail: [email protected]

Key Words dendritic cells, cross-priming, indirect presentation, cancer, infectious diseases ■ Abstract Heat shock proteins are abundant soluble intracellular proteins, present in all cells. Members of the heat shock protein family bind peptides including antigenic peptides generated within cells. Heat shock proteins also interact with antigen presenting cells through CD91 and other receptors, eliciting a cascade of events including re-presentation of heat shock protein-chaperoned peptides by MHC, translocation of NFκB into the nuclei and maturation of dendritic cells. These consequences point to a key role of heat shock proteins in fundamental immunological phenomena such as activation of antigen presenting cells, indirect presentation (or cross-priming), and chaperoning of peptides during antigen presentation. Heat shock proteins appear to have been involved in innate immune responses since the emergence of phagocytes in early multicellular organisms and to have been commandeered for adaptive immune responses with the advent of specificity. These properties of heat shock proteins also allow them to be used for immunotherapy of cancers and infections in novel ways.

THE MHC AND THE HEAT SHOCK PROTEINS, A COMMON PEDIGREE Transplantation of tissues and tumors among mice led to the identification of fundamental immunological roles for two major groups of molecules, the MHC and the heat shock proteins (HSPs). At first sight, the HSPs and the MHC proteins appear quite dissimilar. The MHC proteins are of very recent evolutionary vintage, while the HSPs appeared at the very dawn of life. The MHC are among the most polymorphic (poly-allelic) proteins, while the HSPs are typically monoallelic. The MHC are cell surface proteins, whereas the HSPs are essentially intracellular. The MHC are expressed at modest levels while the HSPs are embarrassingly abundant. However, 0732-0582/02/0407-0395$14.00

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these significant differences hide powerful similarities and convergence of functions. In fact, the HSPs have been among the key players in host defense for billenia and appear to have been laying the groundwork for many of the latter day functions of the MHC molecules. I aim to tell here the story of the primordial functions of the HSPs in innate immunity and to describe the many paths where the functions of the MHC proteins and the HSPs converge to create the symphony of adaptive immunity. While the MHC molecules are familiar to immunologists, a brief introduction to HSPs may not be out of place (1). Approximately 40 years ago, somebody inadvertently turned up the temperature of an incubator full of fruit flies, and the salivary gland chromosomes of the fruit flies, thus heat-shocked, showed the characteristic puffs indicative of transcriptional activity at discrete loci (2). These loci came to be known to encode HSPs, which were gradually identified in all species tested. They are expressed in all cells in all forms of life and in a variety of intracellular locations: in the cytosol of prokaryotes and in the cytosol, nuclei, endoplasmic reticulum, mitochondria, and chloroplasts of eukaryotes. In addition to their ubiquity, the HSPs constitute the single most abundant group of proteins inside cells. They are expressed in vast quantities under normal non–heat shocked conditions, and their expression can be powerfully induced to much higher levels as a result of heat shock or other forms of stress, including exposure to toxins, oxidative stress, glucose deprivation, etc. Approximately ten families of HSPs are known, and each family consists of anywhere from 1 to 5 closely related proteins. There is little or no obvious homology among the individual HSP families even as members within a family are closely related. All HSP families are represented in all organisms although individual members may show variety in distribution. Since their discovery, an increasing array of functions such as folding and unfolding of proteins (3), degradation of proteins (4), assembly of multi-subunit complexes (5), thermotolerance (6), buffering of expression of mutations (7), and others have been attributed to HSPs. In addition, they have become absorbing models for the study of transcriptional regulation (8), stress response (6), and evolution (9). In order to tell the story of the common experimental pedigree of the MHC and the HSPs, I begin with a quote from a recent article by George Klein that summarizes the beginnings of the connection between the MHC molecules and tumor immunity (10): During the first part of the 20th century, cancer researchers spoke about transplantable and non-transplantable tumors. The mice and rats were not inbred and transplantability meant therefore transgression of histocompatibility barriers, but most researchers were unaware of this . . . tumor immunology was an optimistic field due to this artifact. In spite of this clear, definitive evidence that was available already in the early 50s, the artifactual, allograft-based “tumor immunology” continued to flourish during at least one more decade. Meanwhile Ludwik Gross performed some not too well controlled experiments suggesting that chemically induced mouse sarcomas could be immunogenic in syngeneic mice. Subsequently Prehn and Main confirmed this in critically controlled experiments . . . Their data also indicated that the chemically

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induced tumors did not cross-react with each other . . . We suspected that even the experiments of Prehn and Main may have been flawed. Obviously, the ultimate evidence had to be based on experiments with the primary, autochthonous tumor host. We did these rather laborious experiments and published them in 1960 in Cancer Research. Yes, it was all true.


Figure 1 shows the general outline of the experiments of Klein et al. (11). They demonstrated the extraordinary phenomenon that one could immunize against

Figure 1 A cartoon showing the design of the experiments of Klein et al. (11) that established formally that methylcholanthrene-induced tumors are immunogenic in the primary and in the syngeneic hosts, and that immunity is individually tumor-specific.

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syngeneic tumors in the same manner as one could against smallpox and polio viruses. The extraordinariness derived from the fact that in contrast to viruses, the tumors were of self origin and yet were immunogenic. Inherent in this observation was the prediction that the tumors express tumor-specific antigens. Further, as the individual tumors were not cross-reactive, the tumor-specific antigens were individually rather than commonly tumor-specific. Speaking with respect to the identity of these antigens, referred to at the time as tumor-specific transplantation antigens (TSTAs), Klein ends his article with the accurate remark “The TSTAs of the chemically induced tumors are still a mystery.” I began to look for the cancer-specific antigens by their ability to elicit protective immunity to cancer challenges, i.e., by the very assay that pointed to their existence (see 12). This approach typically involved fractionation of cancer homogenates into various protein components by conventional chromatographic methods. The fractions thus obtained were used to immunize animals that were then challenged with live cancer cells. The fractions that elicited protection against the cancer were then re-fractionated and the cancer rejection assay repeated until apparently homogeneous preparations were obtained. This approach, with variations, led to identification of cancer-rejection molecules from cancers of diverse histological origins, induced in mice and rats of different haplotypes by chemicals or UV-radiation, or they were of spontaneous origin (Table 1). The cancers ranged in immunogenicity from the nonimmunogenic (e.g., the Lewis lung carcinoma) to the highly immunogenic regressor cancers induced by UV-radiation. Surprisingly, all the well-characterized molecules identified by this method, by us and then by others, turned out to be HSPs of the hsp90, hsp70, calreticulin, or the grp170 family (Table 1). The phenomenon of graft and tumor rejection among histo-incompatble mice played a key role in the discovery of histocompatibility and the molecules that mediate it, i.e., the MHC molecules. The same phenomenon in histocompatible (syngeneic) mice led to the discovery of the molecules that mediate such rejection, i.e., the HSPs.

THE STRANGE IMMUNOGENETICITY OF VERY COMMON MOLECULES: DISCOVERY OF HSP-ASSOCIATED PEPTIDES Consistent with the experiments with intact tumors, the HSPs purified from a given cancer were observed to elicit protective immunity specific to that particular cancer. HSPs derived from normal tissues did not elicit protective immunity to any cancers tested (13). The observed specificity of immunogenicity of cancer-derived HSPs suggested that HSPs ought to harbor somatic polymorphisms, such that HSPs would differ between cancers and normal tissues and from one cancer to another. However, extensive sequencing studies of HSP cDNAs of cancers and normal tissues did not support that idea (14). What then was the basis of the specificity of immunogenicity of these very common HSP molecules? The first

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TABLE 1 Representative studies that demonstrate the tumor-specific immunoprotective activity of tumor-derived HSP-peptide complexes, or infectious disease-specific immunoprotective activity of HSP-peptide complexes derived from infected cells. Immunogenicity of tumors is graded as −,+,++ or +++ by subjective criteria. Asterisks refer to models of therapy of pre-existing cancers, as opposed to models of prophylaxis. Cancer or infectious agent

Induced by

Immunogenicity

Host

Molecule

Ref.

Zajdela hepatoma

Chemical

++

Rat

gp96

39

Meth A fibrosarcoma

Chemical

++

BALB/c mice

gp96 hsp90 hsp70 hsp110 grp170

50, 68*, 94* 98 18 99 99

CMS5

Chemical

+

BALB/c mice

gp96

50

CMS13

Chemical

++

BALB/c mice

gp96

100

Lewis lung ca.

Spontaneous

−

C57BL/6 mice

gp96 hsp70

68*, 94* 68*

B16 melanoma

Spontaneous

−

C57BL/6 mice

gp96

68*, 101*

CT26 colon ca.

Chemical

++

BALB/c mice

gp96

68*

BALB/c mice

hsp110 grp170

99* 99

Colon 26 Ca. UV6138

UV

+++

C3H mice

gp96

102

UV6139SJ

UV

++

C3H mice

gp96

68*, 102

Dunning G prostate ca.

+

Rat

gp96

103*

A20 B cell lymphoma

+

BALB/c mice

gp96 hsp70 hsp90 Calreticulin

104 104 104 104

+

Xenopus

Gp96 Hsp70

90 90

M. tuberculosis

BALB/c mice

Gp96

22

Listeria

BALB/c mice

Gp96

22

LCMV

BALB/c mice

Hsp70

105

L15/0 lymphoma

Spontaneous

obvious answer lay in the possibility that the homogeneous HSP preparations were not so homogeneous after all, but contained unexamined contaminants that were responsible for the immunogenicity. This possibility, as sensible as it was depressing, did not turn out to be true: The immunogenic HSP preparations were certifiably free of other protein contaminants as determined by all structural criteria tested, and the immunogenicity did not derive from associated carbohydrates, lipids, or nuclei acids (P. Srivastava, unpublished observations). The possibility was then envisaged that low molecular weight substances, not detectable by polyacrylamide gel electrophoresis, are associated with HSPs and are responsible for the specificity of immunogenicity of HSP preparations (15, 16). This idea was tested and

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derived a modicum of support when a large collection of peptides could be shown to elute from a homogeneous gp96 preparation as it was treated with trifluoroacetic acid (17). Strong support for the idea came when treatment of an immunogenic (tumor-protective) hsp70 preparation with ATP had two consequences: It resulted in elution of a wide array of peptide peaks from the hsp70 polypeptide, leaving the polypeptide intact, and it rendered the hsp70 preparation nonimmunogenic, i.e., ineffective in immunizing against cancer cells, even though the hsp70 polypeptide was present in equivalent amounts in untreated and ATP-treated hsp70 preparations (18). This was the first demonstration that hsp70, as isolated from tumors, was associated with peptides, that dissociation of peptides from hsp70 resulted in abrogation of the immunogenicity, and that the hsp70 polypeptide was not immunogenic in and of itself. It was shown subsequently that the hsp70 and gp96 are associated with peptides in vivo, and the observed association of hsp70 and gp96 with peptides is not the result of an artifact occurring after cell lysis and during purification of the HSPs (19). Considerable immunological and structural evidence now supports the notion that certain HSP molecules (gp96, hsp90, hsp70, calreticulin, hsp110, and grp170) are peptide-binding proteins and are associated with antigenic epitopes (Tables 1, 2).

The Immunological Evidence The immunological evidence for association of HSPs and antigenic peptides has continued to accumulate at an impressive pace. The large number of studies that TABLE 2 Selected structural or immunological studies that have shown that specific, defined antigenic peptides are associated with HSPs Epitope/antigen

MHC I

Ref.

TUMOR ANTIGENS PRL1a mouse leukemia Human melanoma MART-1 Human melanoma tyrosinase Human melanoma gp100

Ld A2 A2 A2

28 26 26 26

Kb d Kb Db, Kb

23, 27 25 20 106 30

MODEL ANTIGENS β galactosidase Ovalbumin

Ld Kb

24 29

NORMAL CELLULAR ANTIGENS Minor H

Kd, Kb

24

VIRAL ANTIGENS Vescicular stomatitis Herpes simplex-2 Influenza SV40 Hepatitis B ag

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show the individually tumor-specific immunogenicity of tumor-derived gp96, hsp70, hsp90, calreticulin, hsp110, and grp170 preparations has been referred to earlier (Table 1). When the hypothesis that HSPs must associate with various cellular antigens was first proposed (15, 16), it was argued broadly that HSP preparations purified from cells infected with viruses or other infectious agents must be associated with the antigenic epitopes of those agents and that such HSP-peptide complexes should be found to be immunoprotective against the cognate infectious agents (Table 1). That prediction has been amply fulfilled. Gp96 preparations isolated from influenza virus–infected cells have been shown to be protective against a challenge with the influenza virus (20, 21). Gp96 preparations isolated from mouse tissues infected with Mycobacterium tuberculosis and Listeria are protective specifically against those agents (22). A dramatic demonstration of the binding of endogenously generated antigenic peptides to gp96 came from the work of Podack and colleagues (22a), who constructed a gene encoding a gp96 molecule fused with the Fc portion of murine IgG1, generating a secretory gp96-Ig. Transfection of gp96-Ig into tumor cells decreased their tumorigenicity and increased their specific immunogenicity. The tumors were rejected after initial growth. In addition to demonstrating the binding of peptides to gp96 in vivo, these studies provide a common tool for easy generation of gp96-peptide complexes for any tumor, and possibly also for immunotherapy of human cancer. Zheng et al. (22b) have shown the broader applicability of this idea and have further developed it mechanistically, as discussed in another section. These studies do not provide definition of the antigenic epitopes associated with the HSPs, but they provide compelling circumstantial evidence by virtue of the fact that HSPs purified from antigen-negative control cells did not immunize against the particular tumor, virus, or parasite. A number of other studies provide direct evidence of association of defined antigenic epitopes with HSP molecules (Table 2). Gp96 preparations isolated from vescicular stomatitis virus (VSV)–infected or SV40-transformed cells elicit classical MHC I–restricted, antigen-specific cytotoxic T lymphocytes (CTLs) against defined antigenic epitopes of the two viruses (20, 23). In experiments with VSV, the gp96 preparations were able to cross-prime; preparations from VSV-infected cells of the b or the d haplotypes could immunize mice of the b haplotype and elicit b-specific CTLs, thus showing that gp96 was associated with peptides regardless of the MHC I haplotype of the cells from which it was purified. Arnold et al. (24) showed that immunization with gp96 preparations isolated from cells transfected with the gene encoding β-galactosidase elicited CTLs specific for an Ld-restricted epitope of β-galactosidase; similarly, immunization with gp96 preparations purified from cells expressing selected minor histocompatibility antigens was able to prime (as well as cross-prime) CTL responses against the particular minor antigens. More recently, Navaratnam et al. (25) immunized mice with gp96 isolated from cells transfected with the gD antigen of Herpes simplex virus-2. The first evidence of association of antigenic peptides with human HSPs comes from a recent study by Castelli et al. (26) who showed that human

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melanoma-derived hsp70, but not hsp70 from other human sources, was associated with peptides corresponding to antigenic epitopes derived from gp100, Mart 1, and tyrosinase, but not Trp2. Issels and colleagues have obtained similar results with the chaperoning of tyrosinase epitopes associated with hsp70 isolated from a human melanoma (personal communication). Corresponding studies with gp96 from human melanoma cells are now in progress (C. Castelli, G. Parmiani, personal communication).


The Structural Evidence What are the structural characteristics of the HSP-associated peptides? Immunological evidence indicates that HSPs appear to bind all peptides tested. In 13/14 instances in which the association of a given antigenic epitope with HSPs has been sought, such association has been found. (Trp2 is the sole exception thus far, and the basis of its absence in human melanoma-derived hsp70 preparations is unclear.) Based on the ability of HSP-peptide complexes to cross-prime, HSPs associate with peptides regardless of the MHC haplotype of the cells from which they are isolated. These lines of evidence point to a promiscuous ability of HSPs to bind peptides. Such promiscuity is consistent with the primordial roles of HSPs in folding and assembly of proteins, and it requires structural definition. In spite of an impressive number of studies reporting the presence of HSP-associated peptides as detected immunologically in a diverse array of systems (Table 1, 2), structural scrutiny of HSP-associated peptides has been more limited (Table 2). Four studies to date have analyzed HSP-associated peptides structurally (27–30), and each has done so with respect to a single peptide. Nieland et al. (27) first identified a known Kb-restricted viral epitope to be associated with gp96 purified from virus-infected cells; such peptides could not be detected in gp96 preparations from uninfected cells. Consistent with the cross-priming studies described earlier, the epitope was detected in VSV-infected cells of the b or the d haplotype. In a study with a mouse leukemia (28), whose Ld-restricted epitope has been defined, Ishii et al. isolated gp96, hsp90, and hsp70 from the leukemic cells. They eluted peptides from each of the three preparations and fractionated them by column chromatography. Each column fraction was tested for the presence of the antigenic epitope by pulsing Ldexpressing antigen-negative cells with it and testing the ability of specific CTLs to lyse them. Antigen-positive factions were identified among peptides eluted from each of the HSP preparations and were analyzed by mass spectroscopy. Interestingly, while each of the HSPs was associated with the precise epitope, hsp90 and gp96 were found to be associated in addition with longer precursor peptides of it. Breoler et al. (29) identified the ovalbumin-derived SIINFEKL epitope associated with gp96 and hsp70 isolated from ovalbumin-transfected cells. Meng and colleagues (30) have provided the first structural evidence for association of antigenic peptides with a human HSP. They isolated gp96 from human livers infected with hepatitis B virus and showed, by mass spectroscopy, the presence of a virus-encoded peptide with it.

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HSP-associated peptides have not been examined thus far with the tools that have been so effective in corresponding analyses of MHC-associated peptides. Attempts in this direction have often floundered partly because the HSP molecules themselves have often degraded during elution of peptides, as a result of trace amounts of proteases present in gp96 preparations used (H-J. Schild, personal communication). However, improvements in the methods of purification used as well as the use of increasingly sophisticated mass spectroscopy tools is beginning to address this lacuna (C. Liu, P.K. Srivastava, unpublished observations). Such studies must be distinguished from others that have characterized the peptides that may be made to associate with HSPs in vitro (31, 32). Using such assays in vitro, Flynn et al. (31) suggested that “the peptide-binding site of hsp70 selects for aliphatic residues and accommodates them in an environment energetically equivalent to the interior of a folded protein.” Blond-Elguindi et al. (32) have suggested a sequence motif for peptides that may bind BiP in vitro. However, this motif is at variance with other studies that have shown hsp70-peptide binding. Blachere et al. (33) and Basu & Srivastava (34) have also analyzed a number of peptides for binding gp96, hsp70, and calreticulin in vitro and have observed considerable variation among peptides with respect to their ability to bind the HSPs. Clearly, more studies, modeled on corresponding studies with the MHC molecules, are needed to resolve the questions. The evidence for structural features of HSPs that allow them to bind peptides may be described as following. Zhu et al. (35) crystallized a ligand-binding fragment of the bacterial hsp70 known as DnaK and identified a definite peptidebinding pocket in it. “The structure consists of a beta-sandwich subdomain followed by alpha-helical segments. The peptide is bound to DnaK in an extended conformation through a channel defined by loops from the beta sandwich.” The peptide-binding activity of gp96, hsp70, and calreticulin has been demonstrated independently by Blachere et al. (33), Wearsch & Nicchitta (36), Basu & Srivastava (34) and Sastry et al. (37, 38). Wearsch et al. (36) have used fluorescent probes to identify the presence of a possible hydrophobic peptide-binding pocket in gp96. Pursuing a similar theme, Sastry et al. (37, 38) have used peptides tagged with fluorescent probes to explore the molecular environment of the peptide-binding site of gp96. Based on these studies, they have identified the amino acid position 624-630 in a highly conserved region of gp96 as the peptide-binding site (38). While these results are potentially illuminating, the not-too-well-controlled use of a large bulky probe that may alter the physicochemical properties of gp96peptide interaction places some doubt on their general validity. Interestingly, Nicchitta and colleagues as well as Sastry suggest that gp96 molecules exist as dimers and that the dimeric state is the true peptide-binding state. These observations recapitulate the original observations of Srivastava & Das (39), who demonstrated that gp96 (then called p100) molecules eluted from size exclusion columns exclusively as dimers and tetramers. The cytosolic homologue of gp96, hsp90, is also being studied structurally. Scheibel et al. (40) suggest the existence of two substrate-binding sites in hsp90, while ongoing crystal structure studies with hsp90

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are in the process of clarifying the identity and structure of the peptide-binding pocket of hsp90 (41–43). Buchner and colleagues (44) suggest that the hsp90 contains two and perhaps more distinct peptide-binding sites. The thesis that HSP molecules are associated with peptides including antigenic peptides was advanced solely to explain the specific immunogenicity of tumorderived homogeneous HSP preparations. Ten years later, there is overwhelming evidence for that proposition. Tumor antigens, viral antigens, antigens of intracellular parasites, mouse antigens, human antigens, cytosolic antigens, nuclear antigens, and secreted antigens have all been shown to be associated with the HSPs, and the peptide-binding pocket of at least one of the HSPs has been defined through crystallographic analysis. However, similar studies with other HSPs are yet to be carried out. The rules through which apparently any peptides are able to bind the HSPs have yet to be defined. These are decidedly rewarding avenues for future structural analyses, and the elegant and extensive work carried out with MHC-associated peptides provides a powerful precedent.

“The TSTAs of the Chemically Induced Tumors Are Still a Mystery” Let us return briefly to the initial question regarding the identity of the antigenic peptides that confer individually specific immunogenicity upon tumors. Such immunoprotective peptides have been identified in a small number of instances (see 45), and in each instance, they are mutations of normal proteins. There is no common pattern among the mutations identified, and I believe that the immunogenicity is a consequence of the random mutations that are an inevitable part of cell division (14). The individually unique antigenicity of tumors suggests a lack of relationship between the transforming and the immunogenic mutations. Unique, individually tumor-specific antigens resulting from random mutations are being increasingly identified in human cancers as well (46). With respect to the present context, the immunogenic antigens of tumors are associated with HSPs purified from the tumors in the two instances tested, i.e., a mouse leukemia (26) and a fibrosarcoma (T. Matsutake, P.K. Srivastava, submitted). Should the proposal (14) that random mutations not associated with malignant transformation are the basis of immunogenicity of mouse (and human) cancers continue to be substantiated, the mystery of TSTAs will have been resolved not in favor of an instantly gratifying molecule or family of molecules but as a myriad mutations, random but specific, in the common molecules of the cancer proteome.

HSPs Are Adjuvants: HSP-Peptide Complexes Elicit CD8+ T Cell Responses in Spite of Exogenous Administration The unequivocal demonstration that the specific immunogenicity of tumor-derived HSP preparations is elicited by HSP-associated peptides leads inevitably to the question of whether immunogenic HSP-peptide complexes could be generated in vitro. Blachere et al. (33) did just that. They reconstituted gp96-peptide and hsp70-peptide complexes in vitro using a panel of 7 peptides, and they showed

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that while HSPs alone and peptides alone were nonimmunogenic, HSP-peptide complexes elicited MHC I–restricted antigen-specific CD8+ CTLs. These results were reproduced by Houghton (Moroi, et al. 47) who demonstrated that BiPpeptide complexes were similarly immunogenic. These authors modified the original approach of Blachere et al. (33) by using a peptide that contained the antigenic epitope and another sequence selected for a higher affinity binding to BiP. There was little evidence that the higher affinity interaction resulted in significantly higher immunogenicity over that of the unmodified peptide; however, this is an interesting question that will no doubt be resolved through future experiments. In another variation of the original approach, Suzue et al. (48) fused a mycobacterial hsp70 gene with a fragment of the ovalbumin gene and purified the fusion product. Immunization with the fusion product led to potent ovalbumin-specific CTLs and rejection of an ovalbumin-expressing tumor. A number of other genes, such as those encoding papilloma virus or malarial parasite antigens have since been fused with hsp70, and the immunogenicity of such fusion products has been demonstrated (49, 49a). In addition to settling unequivocally the questions for which they were intended, the experiments of Blachere et al. (33) led to a number of other significant findings and implications. First, they showed that HSPs could be loaded in vitro with synthetic peptides. For this, the HSPs could first be denuded of associated peptides (as by ATP treatment in case of hsp70), or they could be gently denatured in the presence of a higher temperature (50◦ C) or of guanidium hydrochloride (as in case of gp96) and then renatured in the presence of exogenous peptides. Either treatment led to association of the exogenous peptides with the HSPs. The extent of reconstitution was variable, and depending upon the HSP and the conditions used, between 1% and 10% of gp96 molecules could be loaded with peptides. Second, they showed, remarkably, that the HSP-peptide complexes were stable under conditions of denaturing polyacrylamide gel electrophoresis. Unlabeled HSPs complexed with labeled peptides migrated as radioactive bands of the size expected of the HSP-peptide complexes. This observation is consistent with our results in 1986 where a gp96 band eluted from denaturing gels was used to immunize mice successfully against the tumor from which gp96 was isolated (50). There is little precedent for this kind of noncovalent interaction, and it highlights the need for close structural examination of HSP-peptide interaction as discussed in the previous section. Third, the results of Blachere et al. show that HSP-peptide complexes elicit CD8+ T cell responses in spite of exogenous administration. Exogenous antigens are typically routed through the MHC II–presentation pathway and elicit CD4+ responses, whereas endogenously synthesized antigens are presented through MHC I molecules and stimulate CD8+ cells (51). In only a small number of instances have exogenous antigens been shown to enter the MHC I–presentation pathway (52). This demonstration makes HSPs powerful adjuvants for generation of CD8+ responses and makes them the first adjuvants of mammalian origin. (See a later section for discussion of adjuvanticity of α2 macroglobulin or α2M.) In this

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regard, we have recently made the not-too-surprising observation that immunization with HSP-peptide complexes also elicits antigen-specific MHC II–restricted CD4+ response (T. Matsutake and P. Srivastava, submitted for publication). Fourth, Blachere et al. (33) demonstrated that the quantity of peptide complexed to HSP molecules required for successful immunization is extremely small. As little as a few hundred picograms to a nanogram of peptide, if complexed to an HSP, was found to be sufficient to immunize. This observation, which is also germane for the adjuvanticity of the HSPs, brought into sharp focus the novelty of the mechanism of specific immunogenicity of HSP-peptide complexes (discussed in the next section). Finally, Blachere et al. (33) showed that immunogenicity did not result when peptides were complexed with mouse serum albumin that binds peptides just as effectively as the HSPs do. This suggested that the HSPs were doing something more than simply protecting the peptides from degradation or other such physical dangers. This observation too had a powerful role in our imagining of the mechanism of immunogenicity of HSP-peptide complexes.

MECHANISMS OF IMMUNOGENICITY OF HSP-PEPTIDE COMPLEXES: THE EIGHT-FOLD PATH THROUGH THE HSP RECEPTORS The Two Paths (CD8+ and CD4+) to Adaptive Immunity We were impressed with the fact that immunization with femtomole quantities of antigenic peptides chaperoned by HSPs (but not other proteins) was effective in eliciting such potent T cell responses (33). At the same time we had learned that priming of immune response by HSP-peptide complexes was exquisitely sensitive to abrogation of function of antigen presenting cells (APCs) (53). Putting these ideas together with the general biological principle that extraordinary efficiencies are often achieved through specific receptors, we proposed that HSPs interact with APCs through specific receptors and that such interaction results in endocytosis of HSP-peptide complexes followed by processing of peptides and their presentation by MHC I molecules (54). The first step in validation of this idea came from experiments that showed that macrophage, but not B cells or fibroblasts, take up gp96-peptide complexes (isolated from cells or reconstituted in vitro) and re-present the gp96-chaperoned peptides on the MHC I molecules of the macrophages; re-presentation does not occur by transfer of peptides from the gp96 molecules to MHC I on the cell surface but does require internal processing (23). Singh-Jasuja et al. (55) further demonstrated that receptor-mediated endocytosis of the gp96-chaperoned peptides is essential for re-presentation of these peptides by MHC I; nonspecific endocytosis of the gp96-peptides does not result in re-presentation. Essentially similar data for re-presentation of hsp70-chaperoned peptides were shown recently by Castellino et al. (56). The extraordinary efficiency of the process observed earlier through immunization experiments became evident again through such re-presentation

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assays. Gp96-chaperoned peptides were re-presented by the MHC I molecules of the APC several hundred-fold more efficiently than unchaperoned peptides. Subsequently, we and others demonstrated specific, saturable, and competitive binding of various HSPs to APCs (57–59) as further evidence for the existence of an HSP receptor on APCs. In attempting to identify the long-proposed HSP receptor (54), Binder et al. (60) applied solubilized membranes of APCs on gp96 affinity columns and eluted and sequenced a gp96-binding protein. This turned out to be the previously known α2 macroglobulin (α2M) receptor CD91. α2M as well as antibodies to CD91 were shown to inhibit completely the re-presentation of gp96-chaperoned peptides by APCs. Soon thereafter, Basu et al. (61) demonstrated that CD91 acted as the receptor not only for gp96 but also for hsp90, hsp70, and calreticulin. These data were interpreted to suggest CD91 as a global HSP receptor on APCs. The wider significance of this observation is discussed later under its own heading, and we shall leave the subject of HSP receptors for now. Basu et al. (61) also shed some light on the pathway of intracellular processing of gp96-chaperoned peptides (Figure 2). It was clear from the work of Arnold et al. that the endocytosed gp96-peptide complexes enter an endosomal compartment (57) and from the work of Suto & Srivastava (23) that these compartments were not acidic. Basu et al. (61) showed that further processing of peptides required a functional proteasome and transport of the peptides through transporter associated with antigen processing (TAP), followed by the classical secretory pathway. This picture appears reasonable and straightforward. However, the mechanism of transport of peptide from the endosome to the cytosol is unclear. Further, Castellino et al. (56), who have shown an essentially similar pathway for re-presentation of hsp70-chaperoned peptides, have made the additional and remarkable observation that the structure of the peptide can dictate if the transport of the peptide into the ER is TAP-dependent or not. Thus, while the broad outlines of the internal trafficking of HSP-chaperoned peptides from the APC surface to binding to the MHC I molecules of the APCs are clear, a number of interesting and important details remain to be characterized. We have demonstrated recently that the HSP-chaperoned peptides are represented by the MHC II molecules of the APCs, in addition to re-presentation by MHC I molecules discussed thus far. Presentation of the exogenous antigens through the MHC II molecules of APCs is not surprising in and of itself. What is surprising here is the observation that the re-presentation by MHC II molecules also occurs through the CD91 receptor and that in quantitative terms, it is significantly more efficient than re-presentation through phagocytosis. This observation indicates that once an HSP-peptide complex is taken up through CD91, it may enter one or more of several trafficking and processing pathways. The factors that contribute to such molecular decisions would make for important discoveries not only for the biology of the APCs, but for cell biology in general. They will also have significant implications for strategies of vaccination. Some number crunching will illustrate how such a small quantity of antigenic peptide complexed with a small quantity of a HSP becomes powerfully immunogenic. Typically, a mouse is immunized with 1 µg of HSP-peptide

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Figure 2 The pathways of re-presentation of HSP-chaperoned peptides by MHC I and MHC II molecules of the APCs (macrophages and DCs) after uptake of the HSPpeptide complexes through CD91 molecules. All HSPs tested use the CD91 receptor for both pathways. The re-presentation of peptides by MHC I requires proteasomal activity and TAP (61), although TAP-independent mechanisms may also operate (56).

complexes intradermally in order to get full tumor protection. In case of gp96, this is equivalent to ∼6 × 1012 gp96 molecules chaperoning perhaps half as many peptides (assuming that the dimer is the minimum peptide-binding unit). The proportion of specific peptides among these may be estimated conservatively as 1/100,000 (in absolute numbers, 3 × 107 specific peptides in the immunizing dose). The immunization is carried out intradermally in an area of 1 mb) block of satellite AAGAG DNA inserted into the coding region of the locus. In heterozygotes (bw+/bwD), silencing of the wild-type allele is promoted by the somatic pairing of the bw+ allele with its dominant allele and the recruitment of both alleles to constitutive heterochromatin bearing the same AAGAG repeat. In this example, the normal bw+ allele is silenced by virtue of its physical recruitment to pericentric heterochromatin (93). Modifiers of PEV that increase the proximity of this interaction also enhance the silencing of the bw+ allele (94), providing direct evidence that these components can influence gene recruitment within the nucleus.

Ikaros and Gene Silencing in Lymphocytes The observation that the DNA-binding protein Ikaros is associated with several transcriptionally inactive genes, and that these loci are recruited to centromeric DNA in cycling lymphocytes, has prompted a series of speculations about its role (95, 96). Ikaros was originally identified as a protein that binds the D’ element within the murine terminal transferase (TdT ) gene promoter and an element within the murine CD3δ enhancer (97, 98). Although we do not yet know how Ikaros regulates gene expression or even why it is so vital for the normal development of T, B, and NK cells (99), it is likely that Ikaros has a role beyond that of a conventional transcriptional activator. For example, Ikaros proteins compete with the activator protein Elf-1 (an Ets family member) for binding to the D’ region of the TdT promoter (Figure 5); mutations that disrupt Ikaros binding prevent downregulation of the TdT gene during T cell differentiation (100). Parallel studies examining the contribution of Ikaros binding motifs in the promoter for the λ5 gene have also shown the importance of Ikaros for gene silencing (101). In this study employing transgenic mice, mutation of an Ikaros binding site (Figure 5) resulted in the failure of the transgene to be silenced appropriately in mature B cells. Interestingly, integration of this mutant transgene directly into pericentric heterochromatin did not restore normal silencing. These data provide evidence that Ikaros proteins can initiate the gene silencing process. The establishment of heritable gene silencing may require several sequential steps, which may also require the functions of Ikaros proteins. It is conceivable that the recruitment of inactive genes close to centromeric heterochromatin serves to stabilize gene repression, either on the basis of energetic considerations or because this compartment contains components (such as histone deactylases and methyltransferases) that are involved in propagating the inactive state. In this respect, it is intriguing that Ikaros binds directly to major satellite DNA (102) and associates with the NuRD complex, which contains histone deacetylases and nucleosome remodeling factors (103). Since Ikaros multimers appear to interact with target genes, centromeric DNA, and chromatin modifiers, it is conceivable that

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Figure 5 Working model for the downregulation and heritable inactivation of candidate Ikaros target genes in developing lymphocytes.

Ikaros mediates the conversion of candidate genes from transiently to permanently repressed states. Although such a role would be consistent with the observation that the rag-1 and TdT genes are recruited to Ikaros-containing domains in thymocytes during heritable, but not transient, silencing (96), no formal proof for this hypothesis has yet been shown.

Heritable Silencing and the Formation of Stable Epigenetic Imprints Although most of the details of how cellular memory operates are still unknown, several conceptual models have been proposed (36). These models have assumed that intrinsic, self-templating mechanisms underlie the propagation of chromatin states and that active and inactive chromatin structures are “tagged” by a variety of means. Accordingly, as described above, hypotheses have been proposed in which active and inactive chromatin regions are functionally segregated by modification of the DNA itself (by methylation) or of associated proteins such as histones (by methylation, acetylation, or other posttranslational modifications). DNA methylation can be propagated through DNA replication by DNMT1, which, as described earlier, selectively methylates hemimethylated DNA strands during S phase. Histone modifications or other protein tags could be propagated by differences in the timing or location of DNA synthesis. The idea that the replication timing and nuclear location of active and inactive chromatin might be distinct has been supported by correlative evidence. However, it remains unclear whether such a generalized model could reasonably accommodate the patchwork of active

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and inactive genes resident along the autosomal chromosomes of differentiated cells. Although progress in this area has been slow, complementary research in understanding the extent and diversity of histone modifications has led to the proposal that covalent modifications to these proteins could form the basis of a novel epigenetic code (4). A string of related reports has shown that the acetylation, phosporylation, and methylation of histone tails can profoundly affect the recruitment of chromatin-associated proteins and transcriptional components (Figure 6). For example the lysine acetylation of histone H4 induces an increased affinity for bromodomain proteins such as TAFII250 and P/CAF (104). Because these proteins have intrinsic istone acetyltransferase activities, this recruitment may serve to enhance transcriptional activity at a histone tagged site. Conversely, methylation of histone H3 at lysine 9 by the methyltransferase Suv39H1 targets binding of the heterochromatin component HP-1, a protein that is associated with transcriptional repression (Figure 6) (105). Interestingly, whereas methylation of histone H3 at lysine 9 is a characteristic of inactive heterochromatin (such as that surrounding mouse centromeres), methylation of lysine at position 4 is a selective feature of active domains (Figure 6). This observation underscores the repertoire and complexity of this emerging epigenetic code. Our future challenges will be to clarify the links between these molecular tags and upstream mediators such as Pc-G and

Figure 6 Summary of events likely to contribute to the establishment of stable epigenetic imprints.

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Trx-G, and also to examine in greater detail their relevance to the coordinate regulation of complex loci.


SUMMARY AND FUTURE GOALS We have described examples in which chromatin structure appears to make important contributions to gene regulation in the immune system. Instead of listing the many instances in which transcription factors have been reported to interact with chromatin remodeling and histone modification complexes, we have focused on specific genes that are being subjected to systematic chromatin analyses. Although a considerable amount of effort was required to develop these types of model systems, similar approaches will need to be applied to other genes to uncover the mechanisms by which they are regulated by key transcription factors. Although a long-term goal will be to elucidate the functions of specific trans-acting factors in modulating chromatin structure, an analysis of cis-acting sequences will be of equal importance for the next series of advances. For example, to determine how the IL-4 locus is decondensed during Th2 development, it will be necessary to identify the control regions and specific DNA elements required for this process. An exclusive focus on the characterization of trans-acting factors would be insufficient because of the inherent difficulty distinguishing direct from indirect effects upon overexpression or underexpression of a factor. Similarly, at the IL-12 p40 locus, the specific DNA elements required for nucleosome remodeling will need to be identified before the critical trans-acting factors can be pursued. The future goals of the IFN-β chromatin analysis appear to be quite different because all of the components of the stable enhanceosome appear to act in concert to stimulate remodeling of the downstream nucleosome. It is possible that an analogous situation will be found at the IL-4 locus, such that every DNA element within one or more control regions will be necessary for chromatin decondensation. With this scenario, the functions of specific transcription factors would be much less significant than the concerted functions of a specific combination of factors. In addition to uncovering mechanisms responsible for chromatin decondensation and nucleosome remodeling at specific loci, a major goal will be to elucidate the mechanisms that contribute to the heritable propagation of transcriptionally competent and silent states. As described above, recent advances in dissecting the histone modification code and the regulation of DNA methylation have generated considerable optimism that this goal will be realized in the foreseeable future. The current studies of gene regulation in the immune system are likely to make important contributions to this effort. The Th1/Th2 differentiation system is particularly well-suited for studies of the events involved in heritable lineage decisions: The culture conditions and molecular events required for the in vitro differentiation of T helper cells have been relatively well established, the differentiation process is amenable to analysis using gene disruption technologies, and it has received, quite appropriately, the attention of a large number of laboratories that are

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consistently performing elegant and ambitious experiments. The groundbreaking studies of allelic bias nicely exemplify the power of this system and the potential for contributions that greatly advance our broader understanding of gene regulation mechanisms in mammalian cells. ACKNOWLEDGMENTS


We thank Jane Grogan, Richard Locksley, Matthias Merkenschlager, and Anjana Rao for valuable comments and critical reading of the text. Visit the Annual Reviews home page at www.annualreviews.org

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PRODUCING NATURE’S GENE-CHIPS:


The Generation of Peptides for Display by MHC Class I Molecules Nilabh Shastri, Susan Schwab, and Thomas Serwold Division of Immunology, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200; e-mail: [email protected], [email protected], [email protected]

Key Words antigen processing, antigen presentation, proteasome, ER proteolysis ■ Abstract Gene-chips contain thousands of nucleotide sequences that allow simultaneous analysis of the complex mixture of RNAs transcribed in cells. Like these gene-chips, major histocompatibility complex (MHC) class I molecules display a large array of peptides on the cell surface for probing by the CD8+ T cell repertoire. The peptide mixture represents fragments of most, if not all, intracellular proteins. The antigen processing machinery accomplishes the daunting task of sampling these proteins and cleaving them into the precise set of peptides displayed by MHC I molecules. It has long been believed that antigenic peptides arose as by-products of normal protein turnover. Recent evidence, however, suggests that the primary source of peptides is newly synthesized proteins that arise from conventional as well as cryptic translational reading frames. It is increasingly clear that for many peptides the C-terminus is generated in the cytoplasm, and N-terminal trimming occurs in the endoplasmic reticulum in an MHC I–dependent manner. Nature’s gene-chips are thus both parsimonious and elegant.

THE PEPTIDE/MHC CLASS I DISPLAY Immune surveillance by CD8+ T cells is one of the key mechanisms for detecting and eliminating abnormal cells, including those infected with viruses or bacteria and tumor cells. CD8+ T cells probe the repertoire of peptide/MHC class I (p/MHC I) complexes on the target cell surface for novel peptides that indicate expression of foreign or abnormal gene products. Because cells cannot distinguish their normal proteins from nonself or mutant proteins, they constitutively display peptides derived from all proteins. For effective immune surveillance it is essential that the major histocompatibility complex (MHC) class I (MHC I) molecules display as large a peptide repertoire as possible to include those originating from the novel genes. Analogous to gene-chips, which contain thousands of nucleotide sequences to allow simultaneous detection of a large number of, if not all, transcripts in a 0732-0582/02/0407-0463$14.00

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cell, the peptides displayed by MHC molecules represent the entire ensemble of polypeptides expressed within a cell. The MHC molecules and the sets of peptides they display can therefore be considered nature’s gene-chips that are probed by the CD8+ T cell repertoire. The essence of the MHC I antigen processing pathway is to load MHC I molecules with thousands of different peptides for display on the cell surface. This goal is accomplished by merging two distinct pathways: one for generating peptide receptive MHC I molecules in the endoplasmic reticulum (ER) and another for generating the pool of antigenic peptides in the cytoplasm. The two pathways merge when the cytoplasmic peptides are translocated into the ER by the transporter associated with antigen processing (TAP) and are made available to peptidereceptive MHC I molecules. The pathway that generates the peptide-receptive MHC I involves several steps and key components. MHC I molecules are associated with TAP, and tapasin acts as a bridge between the two molecules. Also present in the TAP/tapasin/MHC complex are the chaperones calreticulin and calnexin, as well as the thiol oxidoreductase ERp57. This configuration places the peptide receptive MHC I molecules in the immediate vicinity of transported peptides and also retains empty MHC I molecules until they are loaded with appropriate peptides. Tapasin may also play a role in editing and/or loading the peptides. After MHC I molecules are loaded with the peptides, they are released from this complex and exit through check-points in the ER and make their way out to the cell surface. The loading complex, and the roles of the individual components in this process, have been the subject of several excellent reviews and are not considered here (1–4). We have chosen to focus on the less well understood aspects of the pathway that generates antigenic peptides. The peptides are enormously diverse and yet precisely tailored to fit the highly polymorphic MHC I molecules present in the cell. Furthermore, the topological separation between the cytoplasm, the major site of proteolysis, and the ER, the site of MHC loading, poses a daunting challenge to the antigen processing machinery. We begin with a brief description of the strengths and limitations of the methods most commonly used to dissect the antigen processing pathway. Next, we start at the beginning of the pathway to address key unanswered questions about the source of antigenic peptides (Figure 1). Are these precursors proteins undergoing normal turnover, or are they newly synthesized gene products? We limit ourselves to endogenously synthesized precursors, leaving the question of crosspriming, in which MHC I molecules are loaded with peptides derived from exogneous antigens, to other excellent reviews (5–8). Third, we summarize our understanding of the endpoint of the pathway: the peptide/MHC complexes on the cell surface. Fourth, we examine the intermediate steps in the pathway, following the precursors as they are transformed from polypeptide chains into the precise peptides that are presented by the MHC I molecules. We focus on the proteolytic events required to generate the peptides. Where and how are the precursors degraded to generate the antigenic peptides? Is the precisely cleaved peptide

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Figure 1 Overview of the MHC I antigen processing pathway. See text for details. Question marks indicate key outstanding issues.

product generated during antigen processing in the cytoplasm, or does the cytoplasmic product contain additional flanking residues that require trimming in the ER? What is the nature of the proteases involved and what are their proteolytic products? And finally, what is the role of the MHC I molecules in antigen processing?

METHODS FOR STUDYING THE MHC I ANTIGEN PROCESSING PATHWAY Processing of polypeptides in the MHC I pathway results in surface expression of the p/MHC I complex, whose raison d’ eˆ tre is to serve as a potential ligand for CD8+ T cells. T cell assays have therefore remained the most convenient method to detect the existence of a specific p/MHC complex in intact antigen presenting cells (APC). Conventional assays measure the activation of cytotoxic CD8+ T cells (CTL) by specific lysis of APC or by cytokine secretion. The presence of the p/MHC can also be assayed using T cell hybridomas that secrete IL-2 (9) or accumulate β-galactosidase (lacZ) (10, 11). These assays are often used to compare p/MHC levels on various APC. However, such comparisons can be difficult because even low copy numbers can often maximally activate T cells. Many CD8+ T cells can detect peptides at low picomolar concentrations; nanomolar concentrations are generally more than

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saturating. In this case increasing p/MHC copy number will not increase the activation readout. Two apparently equivalent APC may in fact have very different p/MHC levels. A technically demanding but more informative method for measuring processed peptides is to fractionate cell extracts by high performance liquid chromatography (HPLC) and to detect the naturally processed peptides in an exogenous assay using appropriate APC and T cells (12, 13). The high resolution of HPLC permits the identification of extracted peptides as well as their relative amounts by comparison with synthetic peptide standards. MHC–bound peptides have also been detected and identified by massspectrometry (14, 15). This technique has been particularly successful in revealing posttranslational modifications in the antigenic peptides (16–18). Another method to detect the p/MHC I complex in the APC using a monoclonal antibody has recently emerged. This antibody was generated in the Germain laboratory, and it recognizes the ovalbumin (OVA)-derived SIINFEKL (SL8) peptide bound to the mouse Kb MHC I molecule (19). A final method measures the total p/MHC levels on the cell surface using antibodies specific for peptide loaded MHC molecules (20). This provides a window on the overall efficiency of the antigen processing machinery without requiring knowledge of specific peptides or the T cells to detect them (21–23). With assays to measure the final p/MHC, the study of antigen processing involves experimentally manipulating the precursors and the cells in various ways and correlating changes in the p/MHC expression with the intervening steps.

ANTIGENIC PEPTIDES ARE DERIVED FROM A WIDE RANGE OF PRECURSORS With the notable exceptions of the Epstein Barr virus–derived EBNA I protein and the cytomegalovirus (CMV)-derived 72K principal immediate-early protein, no endogenously synthesized precursor appears to be excluded from entry into the antigen processing pathway (24–26). Rammensee et al. have compiled a comprehensive list of hundreds of self and nonself peptides known to be presented by MHC I molecules (27). This list includes peptides derived from proteins with widely varying functions, cellular locations, and abundance. This impressive diversity is important and suggests that omissions, which would constitute a blind spot for the immune system, are rare; the antigen processing mechanism operates on most, if not all, proteins available in the cell. The original list of peptides bound to human MHC I molecules also included many peptides of unknown origin. Using the recently completed draft of the human genome sequence, we have identified putative precursors of some of these peptides (Table 1). Even this short list of 21 precursors contains a strikingly broad spectrum of proteins.

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LOADING NATURE’S GENE-CHIPS TABLE 1 Potential sources for unknown MHC binding peptidesa


MHC ligand

MHC molecule

Protein/gene

GenBank accession number/celera protein number (hCP)

AA position

APRQPGLMA

HLA-B∗ 5501

16.7-kD protein

NP 057223

49–57

HPKYKTEL

HLA-B8

Butyrate response factor 2; TIS11B protein; zinc finger protein homologous to Zfp-36 in mouse

NP 008818; Q07352; NP 003398

189–196; 150–157; 139–146

DRHERITKL

HLA-B14

Chromosome 11 open reading frame 2

NP 037397

159–167 125–133

SIRDGVRAY

HLA-B∗ 4601

Crystallin, zeta-like 1

NP 005102

KIKSFEVVF

HLA-A3

Dihydroxyvitamin D3induced protein

1090504

TYYGSFVTR

HLA-A∗ 3302

Eukaryotic translation initiation factor 3, subunit 3

NP 003747

123–131

STYYGSFVTR

HLA-A∗ 1101


NP 003747

122–131

SQFGGGSQY

HLA-B∗ 1501


NP 003744

61–69

FIKDGSSTY

HLA-B∗ 4601

hCP37886

hCP37886.1

489–497

ALSNLEVKL

HLA-A∗ 0201

hCP38933

hCP38933.1

155–163

EHAGVISVL

HLA-B∗ 3801

Hepatitis B virus xinteracting protein

NP 006393

40–48 443–451

6–14

KRFEGLTQR

HLA-B∗ 2705

KIAA0965 protein

BAA76809

RRFTRPEH

HLA-B∗ 2705

KIAA1063 protein

BAA83015

435–442

EVAPPEYHR

HLA-A∗ 6801

MORF-related gene 15

NP 006782

312–320

EVAPPEYHRK

HLA-A∗ 6801

MORF-related gene 15

NP 006782

312–321

RRISGVDRY

HLA-B∗ 2705

NADH dehydrogenase 1, alpha subcomplex 1

NP 004532

52–60

HLPETKFSEL

HLA-Cw∗ 0102

Non-lens beta gammacrystallin-like protein

AAB53791

856–865

DAYALNHTL

HLA-B∗ 5101

POU domain class 2, associating factor 1 Ribosomal protein L15

Q16633

243–251

EVILIDPFHK

HLA-A∗ 6801

NP 002939

131–140

IAPTGHSL

HLA-Cw∗ 0102

Septin 6; hypothetical protein FLJ10849

Q14141; NP 060713

152–159; 154–161

AYVHMVTHF

HLA-A24

Testis enhanced gene transcript protein

AAB87479

45–53

a

The peptide sequences are from the list of sequences marked as “unknown origin” in Reference (27). Potential sources were identified by searching the Genbank and Celera human genome databases. The AA position indicates where in the open reading frame the peptide sequence is located.

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ARE THE PRECURSORS OLD OR NEWLY SYNTHESIZED PROTEINS? Antigenic peptides clearly represent a wide range of gene products. The nature of the precursors, however, remains an intriguing and open question. A key issue is, when does a protein enter the antigen processing pathway? One can envision four stages in the life of an endogenous protein: The polypeptide is translated by the ribosomes, the polypeptide folds, it carries out its biological function, and it is subsequently degraded. For antigen processing, using only those proteins that are being turned over after their biological function has been completed is economical and politically correct. Why not re-use parts of a protein at the end of its useful life? Contrary to this idea, recent data indicate that the bulk of antigenic peptides come from proteins degraded immediately after synthesis. Obtaining antigenic peptides from newly synthesized proteins alleviates the theoretically difficult requirement that proteins or their fragments be retrieved from different cellular compartments such as the nucleus for entry into the antigen processing pathway. Furthermore, because proteins are sampled concomitantly with their synthesis, this provides for early detection of novel peptides derived from intracellular pathogens or mutations. If polypeptides are marked for rapid degradation because they failed to fold properly or were incorrectly translated to begin with, obtaining antigenic peptides from these precursors retains the same advantage of economy as using protein turnover. Historically it was generally believed that antigenic peptides were derived from the turnover of mature proteins. In support of this model, mature proteins artificially introduced into the cell (28) or secreted into the cytoplasm by the intracellular bacterium Listeria monocytogenes (29) were processed into antigenic peptides. Additionally, proteins can be retrieved from several subcellular compartments for entry into the pathway, indicating that there is no obligate link between protein synthesis and peptide generation. The nonclassical MHC I molecule H2-M3 presents N-formylated peptides derived from proteins synthesized in the mitochondria (30, 31). In two instances peptides with Asn to Asp modifications were found; this is consistent with glycosylation in the ER followed by retrieval into the cytosol for entry into the antigen processing pathway (17, 32). Several additional cases of peptides with posttranslational modifications have been reported (16, 18). Furthermore, as predicted, when exogenous proteins were introduced into cells, the rate of peptide generation mirrored the protein half-life. Two studies demonstrating this relationship compared peptide generation from stable and unstable proteins that were constructed taking advantage of the N-end rule: Proteins with certain N-terminal amino acids are rapidly degraded by the 26S proteasome in a ubiquitin-dependent manner (33–35). These studies unequivocally demonstrated that proteasome-mediated turnover of mature proteins can produce antigenic peptides, but they did not address the question of whether this turnover contributes a significant supply of peptides derived from endogenously synthesized precursors.

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One prediction of the model in which newly synthesized proteins are the main entrants into the antigen processing pathway is that the time course of peptide generation from an endogenous precursor will correlate with its synthesis rather than degradation of the mature protein. In the first study of an endogenous precursor, Townsend and colleagues compared peptide generation from two vaccinia constructs that encoded the influenza nucleoprotein (NP) protein and differed only by the N-terminal amino acid (36). Using CTL lysis as a measure of p/MHC expression, they found that cells expressing the unstable NP protein were more sensitive to killing by NP/Db-specific CTL than those expressing the stable protein. However, lysis by an NP/Kk-specific CTL did not correlate with the difference in stability of the same precursors. In a similar experiment, cells were infected with vaccinia encoding two forms of the influenza virus matrix protein (37). Cells expressing the stable or unstable proteins were equally sensitive to CTL lysis. One difficulty with both studies is that CTL lysis is not a quantitative measure of peptide abundance; it is possible that in the cases in which killing was equivalent, peptide abundance may have differed, but the CTL response was maximal even at the lower peptide amount. A third study from our laboratory found no difference in the amounts of naturally processed peptides generated in cells expressing pairs of DNA constructs that differed in the N-terminus of the encoded proteins (38). Although the precursor half-lives differed by six- to ninefold, the amounts of naturally processed peptides extracted from transfected cells were comparable. Unlike the CTL lysis assays, this experiment was quantitative; a difficulty was that peptides were detectable only after both proteins had gone through multiple halflives. Nevertheless, these studies questioned the notion that peptides are generated from mature protein turnover, and they suggested a link between protein synthesis and antigen processing. Reits et al. took an entirely different and elegant approach to the study of antigen processing and found a striking correlation between protein synthesis and peptide supply. They monitored the availability of peptides for MHC I presentation by measuring TAP activity in living cells, using the fluorescence bleaching/recovery technique (39). TAP moves peptides from the cytoplasm into the ER, a key stage in the journey of more than 90% of antigenic peptides. After the addition of cycloheximide, a protein-synthesis inhibitor, TAP activity rapidly ceased, within 25 min in uninfected and 40 min in influenza-infected cells, suggesting a paucity of peptides. The simplest interpretation of these results is that the majority of antigenic peptides comes from newly synthesized proteins. Schubert et al. took another novel approach (40). They argued that if antigenic peptides came primarily from newly synthesized proteins, then inhibiting the proteasome would cause these proteins to accumulate quickly. They treated cells with and without a proteasome inhibitor, pulse-labeled them with [35S]methionine, and measured the total amount of radioactivity in fractionated proteins larger than 14 kDa. Addition of the proteasome inhibitor increased the total amount of radioactivity recovered immediately after labeling, and the increase occurred preferentially in high molecular weight proteins, a fraction of which were ubiqitinated. They

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calculated that 30% of newly synthesized proteins were degraded within 10 min. They referred to these proteins as defective ribosomal products (DRiPs) and suggested that they represent a major source of processed peptides. Together these studies indicate that newly synthesized proteins are a major source of precursors for antigen processing. In fact, they raise the question of whether proteins at the end of their natural lives enter the antigen processing pathway at any significant rate. Nevertheless, the links between protein synthesis and protein degradation remain to be disentangled. Notably, both of these studies relied heavily on the use of inhibitors of central cellular processes, and secondary effects of these inhibitors were not ruled out. If antigenic peptides are in fact derived primarily from newly synthesized proteins, the question arises of which new proteins are targeted for the processing pathway. Proteins that are defective in some way would naturally be degraded rapidly. Yewdell et al. proposed that DRiPs consist of prematurely terminated polypeptides and misfolded polypeptides produced from translation of bona fide mRNAs in the proper reading frame (41). Experiments have yet to verify the importance of misfolding and premature termination in generating antigenic peptides. C-terminally truncated polypeptides are probably not a major source of antigenic peptides because an N-terminal preference is not evident in the list of known peptides compiled by Rammensee (27) or in the new list (Table 1).

CRYPTIC TRANSLATION AS A SOURCE OF ANTIGENIC PEPTIDES In addition to misfolded and prematurely terminated proteins, it is increasingly clear that antigenic peptides can come from sources other than translation of the primary open reading frame. Evidence comes from several experiments in which a T cell clone was used to define its cognate p/MHC I complex (Table 2). In one case, a murine leukemia, the antigenic peptide was tracked to the 50 untranslated region of an mRNA transcript. In nine instances—six human cancers and three virus-infected mouse models—antigenic peptides were traced to alternative open reading frames of mRNAs encoding longer proteins. In four cases of human melanomas, antigenic peptides were tracked to introns or to the intron-exon junction. These examples indicate that unconventional peptides can elicit cytotoxic T lymphocyte responses. We suspect that such peptides may in fact play an important role in immune surveillance. Fundamentally the immune system cares about only one attribute of a peptide: whether it is self or nonself. Whether the peptide came from a functional protein or an unconventional source is irrelevant. DNA contains a wealth of information that is not captured by translation of the primary open reading frame; translation of alternate reading frames, intronic sequences, and 50 and 30 “untranslated” regions of mRNA can make this information available. The resulting polypeptides could be particularly valuable in cases in which the

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TABLE 2 Examples of cryptic translation Cell type

Peptide source

Reference

Stably transfected murine cell line (Lmtk−)

Region downstream of stop codon introduced into the SV40 gene

(115)

Stably transfected murine tumor cell line (P1.HTR tk−)

Promoterless fragment of the P91A gene

(116)

Murine cell lines (BALB/3T3, L929) infected with MMLVderived retrovirus

Region downstream of stop codon introduced into the NP gene

(117)

Murine cell line (P815) infected with vaccinia

Region downstream of stop codon introduced into the HA gene

(118)

Transiently transfected simian cell line (COS)

Region of the ovalbumin gene placed out of frame with the primary AUG, region of the NP gene placed out of frame with the primary AUG. Neither gene had an alternative AUG start.

(119)

BALB C radiationinduced leukemia

50 untranslated region of c-act oncogene

(120)

Human melanoma

Region of the MUM-1 gene spanning intronexon junction

(121)

Transiently transfected simian cell line (COS), stably transfected murine cell line (Hepa1)

Region of gene out-of-frame with the primary AUG, and without an alternative AUG start

(122)

Transiently transfected simian cell line (COS), stably transfected murine cell line (Lmtk−)

Region of gene without upstream AUG

(48)

Cells from vacciniainfected mouse

Region downstream of stop codon introduced into the NP gene. Mice infected with vaccinia encoding the mutant NP generated CTL against downstream epitopes.

(47)

Cells from vacciniainfected mouse

Region downstream of stop codon introduced into the NP gene. Mice infected with vaccinia encoding the mutant NP generated CTL against downstream epitopes.

(123)

Human melanoma

Intron of the N-acetyl glucosaminyltransferase V gene

(124) (Continued)

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TABLE 2 (Continued) Cell type

Peptide source

Reference

Human melanoma, normal cultured melanocyte cell line

Alternate ORF of gp75

(125)

Murine cell line (L929) infected with vaccinia

Alternate ORF of mutant influenza NP. The authors found that out-of-frame epitopes were expressed even when the AUG for the primary ORF was in an excellent context for translation initiation. The level of expression of out-of-frame epitopes increased as the context of the primary AUG worsened.

(126)

Human melanoma, normal cultured melanocyte cell lines

Intron of gp100 gene

(127)

Human melanoma

Intron of TRP-2 gene

(128)

Cells from mouse infected with LP-BM5 MAIDS retroviral complex

Alternate ORF of MAIDS gag gene. Mice infected with MAIDS generated CTL against this epitope.

(129)

Human squamous cell carcinoma, lung adenocarcinoma

Alternate ORF of SARC-1

(130)

Human melanoma, breast cancer cells

Alternate ORF of NY-ESO-1

(131)

Human melanoma

Alternate ORF of CAMEL

(132)

Human B cell acute lymphoblastic leukemia, Epstein-Barr virus–transformed B cells

HB-1, initiated at CUG codon

(133)

Transiently transfected simian cell line (COS)

A minigene that was (a) initiated with a nonAUG codon (CUG) and (b) 30 of a stop codon terminating another minigene in a conventional translational context. The CUG initiation codon was translated as leucine rather than the canonical methionine.

(49)

Renal cell carcinoma

Alternate ORF of the intestinal carboxyl esterase gene, initiated at ACG

(134)

Human renal cell carcinoma, normal kidney and liver cells

Alternate ORF of the M-CSF gene

(135)

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conventional protein had few epitopes that could squeeze through the bottlenecks in the antigen processing pathway. These bottlenecks include the ability of cellular proteases to produce the processed peptide, the ability of TAP to transport the peptide into the ER, and the ability of the peptide to bind an available MHC I molecule (42–44). Furthermore, the precursors of these peptides, which presumably have no function in the cell, are likely to be shuttled immediately to the antigen processing pathway. They would thus be early sentinels of an infection or mutation and perfect candidates for the newly synthesized proteins that are degraded by the proteasome and transported by TAP as discussed above. These peptides are often termed cryptic, because the mechanism of their translation is unknown. Thierry Boon and Aline Van Pel were the first to offer an explanation in the form of the pepton hypothesis (45). They argued that antigenic peptides were not degradation products of cellular proteins at all but instead were generated directly by the “autonomous transcription and translation of short subgenic regions” called “peptons” using a novel RNA polymerase. Although there is no evidence to date for this specialized transcriptional machinery, Boon & Van Pel deserve credit for directing attention toward unconventional sources of antigenic peptides. A second mechanism that could account for these peptides is mis-splicing of primary transcripts. Mis-splicing could explain translation of intronic sequences; it could also lead to transcripts missing the primary AUG codon, allowing translation to begin at an out-of-frame initiation codon. In this context a recent report of coupled transcription and translation in the nucleus is intriguing; the authors suggest that this accounts for 10–15% of protein synthesis in the cells and provides a mechanism for nonsense-mediated decay (46). The proximity of the proteasome to the nuclear translational site could make these polypeptides available to the antigen processing pathway as well. Cryptic peptides could also be generated by the ribosome translating a conventional mRNA transcript in an unconventional way. The accepted model of translation is that the ribosome binds mRNA at the 50 cap, scans in the 30 direction for the first AUG, and translates nucleotides in triplets until a stop codon is reached. Cryptic peptides would be generated if the ribosome (a) bound the mRNA at the 50 cap but scanned through the primary AUG to start at a downstream initiation codon, (b) bound the mRNA at an internal site and initiated translation at a codon downstream of the primary AUG, or (c) initiated translation at the primary AUG and frameshifted during elongation. Bullock & Eisenlohr demonstrated that ribosomal scan-through of the primary AUG is a potent mechanism for translation of alternate reading frames (47). The pool of potential precursors was widened by studies showing that initiation of antigenic peptides can occur not only at alternate AUGs but at several additional codons (48, 49). Malarkannan also implicated a novel initiation mechanism for generation of antigenic peptides: When initiation occurred at the CUG codon (one of the non-AUG codons), it was decoded as leucine rather than the canonical methionine. The underlying molecular

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mechanisms that allow the translation of these polypeptides and target them into the antigen processing pathway are unknown. Cryptic peptides could enhance immune surveillance by adding to the diversity of peptides displayed on the cell surface. However, many open questions remain; the most basic issue requiring resolution is whether cryptic translation is a rare anomaly, seen only in virally infected, transformed, or transfected cells, or whether it occurs systematically in normal cells as well. A second important question is the relative contribution of cryptic translation to the total peptide pool, in terms of numerical abundance, diversity, and immunological significance.

THE ANATOMY, DIVERSITY, AND ABUNDANCE OF NATURALLY PROCESSED PEPTIDES Whereas our knowledge of the pool of precursors for antigen processing is still rather murky, the picture of the final product is crystal clear. The final products are short, usually 8–10 residue, peptides bound to MHC I molecules on the cell surface. A cell from a heterozygous individual expresses several hundred thousand copies of up to six different MHC I molecules. Each MHC I molecule contains a single peptide. Thus, it is possible for a single cell to express thousands of distinct p/MHC I complexes. Most peptides displayed by a given MHC I molecule share a consensus motif defined strictly by size and the presence of conserved residues at defined positions. The consensus motifs for MHC I–bound peptides were first discovered by sequencing the mixture of peptides eluted from purified MHC I molecules (44, 50). These “simple” yet elegant experiments yielded remarkably profound insights into the mechanism that permits MHC I to bind a diverse set of peptides that is essential for effective immunesurveillance. As expected, the MHC I–bound peptide pool was heterogenous, but conserved amino acids were clearly present at one or two internal, usually the p2, p3, or p5, positions and at the C-terminus. In contrast, residues at other positions were highly variable. The variability of these six or seven residues in octamer or nonamer peptides allows up to 206–7 (∼1 × 108) different peptides to be presented by any one MHC I molecule. The crystal structures of many peptide/MHC I complexes subsequently showed that the peptides were primarily tethered to the MHC molecule by their N- and C-termini, the side chains of the conserved carboxyl and internal residues, and the peptide backbone (51). Notable exceptions to the peptide length, consensus motifs, and mode of binding have been found among the peptides presented by MHC I molecules. For example, the murine Kb MHC I, which usually presents octamers with the consensus sequence XXXX[F,Y]XX[I,L,M] (amino acid residues are in single letter code; X = any amino acid), can accommodate a nonamer peptide, FAPGNYPAL, with a central bulge (52). Likewise the human HLA-A2, which usually presents X[L]XXXXXX[L,V] nonapeptides, can also bind to a MLLSVPLLLG decamer with the C-terminal glycine residue extending out of the MHC I antigen-binding

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groove (53). In general these and other exceptions are a result of amino acid combinations in the antigenic peptide that alter the peptide conformation or can serve as secondary anchor residues to provide requisite stability to the p/MHC I complex (54). Together the variable residues of the peptide as well as the surface of the MHC molecule interact with the TCR to provide highly specific recognition of even very subtle changes in the antigenic peptides (55). The number of peptides that can be presented by a given MHC I molecule is constrained by the consensus motifs required for binding. Even this constraint, however, is alleviated because a heterozygous individual expresses up to six different MHC I molecules; as of July 2001, almost 800 MHC I alleles had been discovered in the human population as a whole (http://www3.ebi.ac.uk/Services/imgt/hla/cgibin/statistics.cgi). The highly polymorphic nature of the MHC I loci generates amino acid substitutions within the antigen-binding groove of the MHC molecules. As a result the complementary pockets of the MHC I that accommodate the primary and secondary anchor residues of the antigenic peptide vary extensively. Accordingly, the consensus motifs are remarkably variable among the peptides bound to different MHC I molecules. This is illustrated by the presence of over 48 different consensus motifs among the 69 MHC I molecules surveyed by Rammensee and his colleagues (27). The “X-Proline(P)-Xn” motif represents a notable exception to the high variability found among the MHC-bound peptide motifs. The “X-P-Xn” motif accounts for over 20% of all known motifs in the human and other species (27, 56). However, the predominance of this motif is paradoxical because TAP excludes peptides with this motif from transport into the ER (43, 57). We discuss a possible explanation for this paradox later. Nevertheless, the high overall variability of consensus motifs makes it possible that any one individual can present six entirely distinct sets of peptides. This peptide diversity provides the basis for efficient immunesurveillance by CD8+ T cells but also poses special challenges for the antigen processing mechanisms responsible for generating these peptides for loading the MHC I molecules. The diversity in the sets of peptides presented by the MHC I molecules also implies that the overall abundance of individual peptide/MHC I complexes detected by antigen-specific CD8+ T cells must be quite low. CD8+ T cells possess an impressive capacity to recognize and respond to as few as one p/MHC on the target cell surface (58). Compelling evidence directly demonstrating the complexity of the peptide pool at the level of individual peptides has been obtained using sensitive mass spectrometric analysis (59). The authors estimated that although some peptides were presented at several hundred copies/cell, others, possibly thousands, could be presented at far lower levels. Most important, the low abundance of specific p/MHC complexes displayed on the cell surface has been confirmed in experiments in which the naturally processed peptides were quantitated in antigen presenting cell (APC) extracts and were in many cases present at fewer than 10 copies/cell (60–62). The antigen processing machinery faces an extraordinary challenge: It must produce a set of peptides that are enormously diverse and yet are precisely cleaved

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to conform to the consensus motifs of the MHC I molecules expressed in the cell. The remainder of this review addresses some of the key questions about how this is achieved.


THE RAMMENSEE PARADOX: MHC I MOLECULES INFLUENCE ANTIGEN PROCESSING An absolutely critical issue in understanding the transformation of precursor polypeptides to the exactly cleaved peptides is the role of the MHC I molecules. Are MHC I molecules passive recipients of the peptides made available to them, or do they affect the antigen processing events? The important role of MHC I molecules in influencing the outcome of the antigen processing reactions was first underscored a dozen years ago by the Rammensee laboratory. Soon after the recognition that naturally processed peptides could be detected in cell extracts fractioned by high performance liquid chromatography (HPLC) (12, 13, 63), Rammensee and his colleagues compared cells expressing the antigen and the appropriate MHC I molecule with cells that expressed the antigen alone (64, 65). Most surprisingly, they found that each of three minor histocompatibility and two influenza virus–specific CTLs could detect their cognate peptides only in extracts of cells that expressed the appropriate MHC I molecule. With rare exceptions (66), these observations of the ability to detect the final processed peptide only in the presence of the appropriate MHC I have been consistently reproducible in different model systems and laboratories (67–71). How the MHC I molecules in the ER could influence the naturally processed peptide pool believed to be generated in the cytoplasm has remained an enduring paradox with profound implications for understanding the antigen processing pathway. To explain the Rammensee paradox, one hypothesis, termed here the “protection” model, was advanced by Elliott and colleagues (72). The protection model suggested that the processed peptides were generated in the cytoplasm but were extraordinarily labile and rapidly degraded. The processed peptides were therefore not detectable in cell extracts unless they were protected by binding to MHC I. An alternative hypothesis proposed by Falk et al. suggested that the MHC I molecules themselves were essential for generating the final version of the naturally processed peptides by acting as “templates” (64). Most importantly these two hypotheses had very different implications for the nature of the cytoplasmic peptide pool. The protection model implied that cytoplasmic proteolysis generated the final processed peptide, which was rapidly transported by TAP and immediately loaded onto the MHC I molecules in the ER (72). However, the “template” model did not require that the final peptide be generated in the cytoplasm or even be labile. The cytoplasmic precursors could have additional flanking residues, but ER proteases would be essential for trimming the extra flanking residues under the influence of the MHC I molecules (64).

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Recent studies discussed below provide plausible explanations for the resolution of this paradox and insights into the intermediate steps between the precursors and their final peptide products.


MAKING THE CUTS Regardless of the source and the sampling criteria used, all normal precursors must be fragmented to generate the precisely cleaved peptides presented by MHC I molecules. Whereas passage through the proteolytic steps is not obligatory— antigenic precursors that exactly match the final peptide product are efficiently presented by MHC I (49, 70)—all known naturally processed peptides are embedded within a polypeptide chain. This internal location dictates that precise N- and C-terminal cuts must be made to remove all flanking residues. It is conceivable that a single endopeptidase, such as the proteasome, could cleave the antigenic precursors in a concerted manner to release the final peptide and satisfy the expectation of the protection model (73). However, the recent discovery of proteolytic intermediates and related evidence indicate that different proteolytic mechanisms account for generating the precise C- and N-termini of the final peptide products. Proteolytic intermediates longer than the final peptide cannot normally be detected because they are inactive in exogenous T cell assays. This was a major limitation that prevented analysis of such intermediates earlier (64, 69). Paz et al. devised a new method that combined HPLC fractionation of cell extracts to separate the different intermediates with protease treatments to release the optimally active antigenic peptides from inactive precursors, allowing their detection by T cell activation (74, 75). This new method for analysis of processed peptides revealed that cells do not generate the exact peptide in a single step. Instead the cells generated a number of proteolytic intermediates containing the antigenic peptide with one to four or more flanking residues. The composition of intermediates in the cytosol was distinct from that of the ER, and only the shorter set of peptides was transported by TAP. The Paz analysis provides a key insight into the Rammensee paradox. Antigen processing in the cytoplasm did not generate exactly cleaved peptides as envisaged in the protection model (72). Next we discuss our understanding of the proteolytic mechanisms that generate the final peptides with the exact “start,” the N-terminus, and the “end,” the Cterminus.

STARTING AT THE “END”: GENERATING THE C-TERMINUS OF ANTIGENIC PEPTIDES The Paz study discussed in the previous section was limited by the precursor design in which the antigenic peptide was located at the C-terminus (74). Unlike natural precursors, generation of the intermediates that yielded the final SL8 peptide from

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this precursor required only N-terminal and no C-terminal cleavages. How do cells generate the precise ends, the C-termini of antigenic peptides? Several lines of evidence indicate that the C-terminal ends of antigenic peptides are generated in the cytoplasm and are made by the proteasome.


PROTEASOMES IN THE ANTIGEN PROCESSING PATHWAY Antigen processing begins in the cytoplasm, and the proteasome plays a central role. Following the clue that two MHC linked genes, LMP2 and LMP7, were subunits of the multicatalytic proteasomes (76), the Rock and Goldberg laboratories provided the first key insight into the proteolytic mechanism for generating antigenic peptides (77). They showed that inhibiting proteasome activity caused a profound block in the turnover of short- and long-lived intracellular proteins and also abrogated the generation of p/MHC I complexes in cells. Numerous subsequent studies have confirmed that the proteasome is the key protease in the generation of p/MHC I complexes (78). For most antigenic peptides, specifically inhibiting the proteasome causes loss of presentation, but for reasons not yet clear the presentation of some peptides actually increases in cells treated with proteasome inhibitors (21, 79, 80). Recognizing the importance of the proteasomes in generating the processed peptides, many studies have focused on defining its structure and role in antigen processing. These studies have been recently reviewed (78, 81, 82). The proteasome is a complex and heterogenous structure with several distinct catalytic activities. In addition to the constitutive 20S proteasome expressed in most cells, the “immunoproteasome” is the predominant species in lymphoid tissues. The latter includes the γ -interferon–inducible subunits LMP2, LMP7, and MECL-1 that replace the constitutive subunits (83). These 20S core particles can be further modified by the addition of a 19S cap structure as well as by the γ -interferon–inducible PA28α and PA28β. The proteasome, owing to its central role in many distinct biological processes such as signal transduction pathways and the cell cycle, is essential for cell viability. Notwithstanding reports that other proteases can replace proteasome function (84, 85), knock-outs of the delta or X subunit of the constitutive proteasome (replaced by LMP2 and LMP7, respectively, in the immunoproteasome) are lethal at the very early pre-implantation state of development (J. Monaco, personal communication). However, the immunoproteasome is not essential for viability, and its absence caused by knock-out of LMP2 (86), LMP7 (87), and the MECL-1 genes (J. Monaco, personal communication) correlates with subtle defects in antigen presentation. Likewise PA28 knockout mice are impaired in their ability to process some endogenous and exogenous antigens and to generate CTL responses (88). Note that the loss of TAP1 (89) or tapasin (90, 91) causes severe five- to tenfold reduction in the overall presentation of antigenic peptides by MHC I molecules.

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In contrast, the loss of LMP2, LMP7, MECL-1, or PA28 causes far less severe defects. It is therefore very likely that the constitutive proteasome and/or other proteases are sufficient for generating most antigenic peptides for MHC I molecules, and the immunoproteasome and other modifiers influence the quality or quantity of the processed peptide pool under some circumstances. Delineating the role of the proteasome subunits and modifications in antigen processing is an active area of research. In vitro assays using colorimetric and peptide substrates have consistently identified three major cleavage specificities of the proteasome (83). These specificities promote cleavage after hydrophobic, basic, and negatively charged amino acids. Notably, introduction of the IFN γ –inducible subunits enhances cleavages after the basic and hydrophobic residues, consistent with peptide C-terminal preferences MHC I.

PROTEASOMES MAKE THE C-TERMINAL CUT Several lines of evidence indicate that the proteasome is required to generate the C but not the N terminus of the final peptide. Craiu et al. used proteasome inhibitors to block presentation of the ovalbumin-derived SL8/Kb complex in cells expressing precursors that were extended at the N- or C-termini (92). They noticed that the inhibitors could block presentation of the SL8 peptide when it contained even one C-terminal flanking residue but not with up to 25 N-terminal flanking residues. Similar observations with two other antigenic peptides (93) suggested that proteasomal activity was essential only for generating the precise C-terminus and was not required for generating the precise N-terminus. Independently another study used the HPLC/enzymatic method to detect the SL8 peptide as well as the Cterminal extended SL8-I peptide in cells expressing Kb and a precursor with SL8-I at its C-terminus (75). Again in this study, the generation of naturally processed SL8 but not the SL8-I peptide required active proteasomes. A third study examined the generation of the SL8 peptide analogs by the constitutive and immunoproteasome in vitro (94). In a significant advance over other similar studies, the authors were able to use whole ovalbumin rather than synthetic peptides as a substrate. They found that proteasomes, particularly the immunoproteasome, preferentially generated N-terminally rather than C-terminally extended analogs of the SL8 peptide. This study supports the in vivo studies discussed above and directly implicates the proteasomes in generating the C-termini of antigenic peptides. It is important to note that biochemical proof that the C-termini of antigenic peptides are generated in the cytosol based upon analysis of proteolytic intermediates in the cytosol is not yet available. Furthermore, the requirement that the C terminus be made in the cytoplasm does not appear to be absolute. This is illustrated by the discovery of MHC I–bound peptides that were derived from signal sequences (14, 95) and by the presentation of three different peptides from the HIV env protein in TAP deficient T2 cells (96, 97). The location of these peptides

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requires liberation of both N- and C-terminal flanking residues, indicating that both N- and C-terminal trimming could occur in the secretory pathway.


OTHER CYTOPLASMIC PROTEASES Cytoplasmic proteases other than proteasomes may also be involved in generating peptides for MHC I molecules. This evidence comes from mass-spectrometric analysis of peptide generation in proteasome-inhibited cells and from a number of T cell epitopes whose expression increases in the absence of the proteasome (21, 79, 80). Based upon its ability to cleave peptides in vitro and its upregulation in cells surviving partial proteasome inhibition, tripeptidylpeptidase II (TPPII) was suggested to be a peptide supplier (85). In addition, several proteases with aminopeptidase activity have been proposed to take part in the antigen processing pathway. Leucine aminopeptidase was found to generate actual antigenic peptides when potential N-terminally extended precusors were treated in vitro (98). Notably, the expression of leucine aminopeptidase is upregulated in γ -interferon treated cells. Recently two other aminopeptidases were identified by purification from the cytosol (99). Each of these proteases, puromycin sensitive aminopeptidase and bleomycin hydrolase, was able to trim the N-termini of synthetic peptide substrates. However, the role of these proteases in the antigen processing pathway has not yet been established and will require protease-deficient cells or highly specific inhibitors.

ARGUMENTS FAVORING THE GENERATION OF C-TERMINI IN THE CYTOSPLASM Regardless of the mechanism, it appears that the C-terminus of the antigenic peptide must be made in the cytoplasm because proteases in the ER do not seem to be capable of C-terminal trimming. This was evident in an early study by Eisenlohr et al., who found that an antigenic precursor with two extra C-terminal flanking residues could not be presented by MHC I unless the cells also co-expressed a carboxypeptidase in the ER (100). Subsequent analyses with several different antigenic peptides have shown that N-terminal but not C-terminal extensions can be efficiently trimmed from ER-targeted antigenic precursors (101–103). Independently the lack of carboxypeptidase activity in the ER was also inferred from the analysis of the rat cim effect (104). The rat RT1.Aa MHC molecule prefers the arginine residue at the peptide’s C-terminus. RT1.Aa could not be loaded with such peptides in cells that expressed the TAP2B allele because it transports peptides with hydrophobic C-termini, and these could not be trimmed back to arginine in the ER. Finally, direct quantitative analysis of naturally processed peptides showed that the ER is about 300-fold less efficient in trimming C- rather than N-terminal flanking residues from an antigenic peptide (23). Together these studies strongly support the idea that C-termini are made in the cytoplasm because they are inefficiently generated in the ER.

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The second attractive feature of generating the C-termini of antigenic peptides in the cytoplasm is that it could explain the intriguing link between TAP specificity and the conserved C-termini of MHC binding motifs. In the mouse and rat cimb strain, for example, TAP prefers to transport peptides with hydrophobic C-termini, mirroring the C-terminal preference of the available MHC I molecules. Whereas the C-terminal bias of TAP is consistent with MHC I–binding motifs, the length preference of TAP is variable. Peptides significantly longer than binding motifs allowed are efficiently transported, suggesting that further trimming, possibly at the N-terminus, might be required in the ER (105, 106). Analysis of TAP transport specificity in vivo is also consistent with this analysis (107). Therefore, a general correspondence between the C-terminus specificity of the proteasome TAP and MHC binding underscores the importance of cytoplasmic events in generating the C-termini of peptides presented by MHC I.

SHUTTLING THE INTERMEDIATES How the cytoplasmic intermediates en route to TAP get there is not known. Peptides may simply diffuse or may be specifically shuttled assisted by chaperones. Several independent studies have shown that cytosolic and ER heat shock proteins hsp70, hsc73, hsp90, and gp96 are associated with antigenic peptides and can elicit donor APC–specific CTL responses (reviewed in 108). These studies have unequivocally shown that these chaperones are associated with antigenic peptides, but the functional role of these chaperones in the antigen processing pathway is not clear. Recently Binder et al. showed that cells presented specific p/MHC I with a high efficiency when peptide-loaded hsp70 or hsp90 were introduced into the cytoplasm (109). Furthermore, cells treated with deoxyspergualin, which binds these heat-shock proteins, disrupted antigen presentation. Similar findings were also reported by T. Torigoe and N. Sato (personal communication). In addition they determined that hsc73 can physically associate with TAP, providing a potential role for peptide-loaded heat-shock proteins in peptide delivery to TAP. This may increase the p/MHC I presentation efficiency by decreasing nonspecific degradation in the cytosol. Because peptide association with the chaperones can serve as an important check-point in the antigen processing pathway and influence the overall efficiency of p/MHC I expression, this is an important area of research and is reviewed by Srivastava in this volume.

THE “START” AT THE END: AMINOPEPTIDASES GENERATE THE N-TERMINUS IN THE ER There is now good evidence indicating that for most peptides, the C-terminus is generated in the cytoplasm. The N-terminal trimming necessary to generate the start of the antigenic peptide appears to be less constrained and may occur in either the cytoplasm or the ER. Several recent studies have established the

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general importance of ER aminopeptidases for the generation of many antigenic peptides. Whereas we refer to these events as occurring in the ER, we emphasize that this location has not been firmly established. The studies most pertinent to this issue have been carried out either in isolated microsomes (as representative of the ER) or in cells lacking TAP, in which antigenic precursors are introduced into the ER via the translocon. It is conceivable that microsomes may be contaminated with other organelles and that antigenic precursors introduced into the ER may have been processed in post-ER compartments and returned into the ER by retrograde transport (110, 111). Therefore, strictly speaking the site of processing in these studies is actually a post-TAP compartment that we refer to here as the ER because MHC I are located and loaded in this compartment, as well as for the sake of brevity. As reviewed above, studies using ER-targeted precursors have shown that Nterminal but not C-terminal flanking residues can be efficiently cleaved from antigenic peptides in the ER. However, because the precursors used in these studies were targeted into the ER necessarily bypassing TAP, it was unclear whether these precursors were trimmed by proteases that were an integral part of the normal antigen processing pathway. Three recent studies have addressed this issue. Paz et al. performed a direct biochemical analysis of the intermediates to the SL8 peptide using their novel HPLC/enzymatic method. They found that N-terminally extended intermediates, and not the minimal peptide, were transported into the ER in living cells. In the presence of Kb, these precursors were trimmed and high levels of the Kb/SL8 complex were generated (74). In a second study, Lavau et al. showed that two antigenic peptides presented by HLA-A2 were poor substrates for TAP in vitro, and when expressed as minimal precursors in the cytoplasm they were not presented (112). Interestingly, extending these precursors to include either one or two N-terminal amino acids greatly enhanced the ability of these peptides to be transported by TAP, and similar extensions of the minimal precursors enabled in vivo presentation. Finally, Serwold et al. treated cells with the aminopeptidase inhibitor leucinethiol, which blocked peptide trimming in the ER, and they found a 30–70% reduction of surface MHC I expression, clearly showing that aminopeptidase trimming in the ER is an essential component of the antigen processing pathway (23). Serwold et al. also systematically defined the specificity of the ER aminopeptidase using a series of ER targeted precursors with N-terminal extensions (23). All N-terminal amino acids, with the important exception of proline, could be efficiently trimmed from the precursors. Instead of being trimmed to the minimal peptide, proline-flanked precursors were trimmed to yield products with the “X-Pro” sequence at their N-terminus (X-P-Xn). Interestingly, the expression of Ld, which prefers X-P-Xn peptides, was severely reduced in cells treated with leucinethiol. This is consistent with the idea that the generation of the X-P-Xn requires aminopeptidase activity and that these peptides are used by MHC I. Aminopeptidase trimming of precursors in the ER to preferentially generate X-P-Xn peptides provided a plausible solution to another paradox in the antigen

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processing pathway. TAP translocation assays have consistently shown that peptides containing proline in position 2 are the least preferred substrates for TAP (43, 57). Therefore, it was rather surprising that this “untransportable” sequence should account for approximately 20% of known MHC I-binding motifs (27, 56). Serwold’s observation that aminopeptidase trimming leads to the preferential accumulation of X-P-Xn peptides explained the source of these peptides and also suggested that MHC I molecules that bind to the X-P-Xn motif may have evolved to take advantage of this abundant pool of peptides. An interesting corollary is the possibility that TAP evolved to avoid transporting X-P-Xn peptides specifically because these could not be further tailored. Interestingly, the “X-P” sequence is found not only among peptides loaded onto MHC I, but also among a number of other ER resident proteins in the antigen processing pathway. Tapasin, calreticulin, and β2M, as well as some MHC I alleles share the same X-P residues at their N-termini, suggesting that they too might need protection from a highly active aminopeptidase in the vicinity of the peptide loading complex. Together these studies provide compelling arguments for aminopeptidase trimming in the ER as a key event in the antigen processing pathway. However, the molecular mechanism of ER trimming is obscure. An interesting candidate was recently suggested by the Srivastava group, who discovered that gp96, an ERresident chaperone, possesses aminopeptidase activity (113). This is an intriguing observation because gp96 is well known for its ability to bind antigenic peptides (108). The authors note, however, that the gp96 aminopeptidase activity is extremely weak, a million–fold lower than that of the benchmark aminopeptidase M. Whether low enzymatic activity rules out gp96 as the putative ER aminopeptidase or whether its high abundance compensates for its low activity remains to be determined.

MHC MOLECULES AS TEMPLATES FOR N-TERMINAL TRIMMING IN THE ER The notion that the N-terminal flanking residues of many peptides are trimmed in the ER raises the question of how this trimming is accomplished. In the context of the Rammensee paradox, the MHC I molecules could define the peptide pool in the ER by two different mechanisms (64). The proteolytic intermediates could be processively trimmed at the N-terminus independently of the MHC I. The product of each cleavage cycle could be tested for binding to the MHC I molecules so that once the optimal peptide is made it could be firmly bound and remain protected from further trimming. Alternatively, the extended intermediates could associate with the peptide receptive MHC I molecules, which could then serve as a template for the aminopeptidase until the optimal N-terminus was achieved. Whereas the influence of MHC I on the peptide pool is clear, distinguishing these two models has remained difficult. In one study a nonapeptide (M-SL8) and an octapeptide (SL8) were shown to bind Kb on the cell surface (69). In

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addition, when M-SL8 was expressed as a precursor in cells, it was presented by Db, showing that it was present in the ER. Nevertheless, only SL8, but not MSL8, was recovered from Kb expressing cells, suggesting the possibility that the M-SL8 nonapeptide was first bound to Kb in the ER and was then trimmed to the SL8 peptide. Alternatively, it was possible that a quality control mechanism prevented M-SL8/Kb binding in the ER but not on the cell surface. Stronger evidence favoring a role of MHC I molecules in trimming peptides was noted by Paz et al. (74). In cells without Kb Paz et al. found an N-terminally extended K-SL8 peptide in ER extracts. Remarkably, in cells expressing Kb this precursor decreased dramatically with a concomitant increase in the accumulation of the Kb-bound SL8 peptide. The simplest interpretation of this result is that the N-terminally extended K-SL8 peptide was a precursor for SL8 and that its conversion required the Kb MHC molecule. A prediction of the template model of antigen processing is that MHC I molecules should be bound to extended peptides before the final peptide product is generated. Brouwenstijn in our laboratory recently discovered such complexes in isolated murine microsomes pulsed with N-terminally extended precursors (114). These extended peptide/MHC I complexes were rapidly converted to the optimal peptide/MHC I complexes by an aminopeptidase activity. The discovery of this intermediate together with the Paz and Serwold studies discussed above provide a satisfactory resolution to the Rammensee paradox. They explain why the naturally processed peptides are not found in cells expressing the antigen without the MHC I. The antigen processing mechanism provides only N-terminally extended peptides to the ER that cannot be normally detected owing to the sensitivity limits of the methods. Whether similar extended peptide-bound MHC I complexes exist in vivo is not known. Also, the mechanism of template-driven trimming of extended peptides and the identity of the aminopeptidase have not yet been defined.

A GENERAL MODEL FOR THE P/MHC I ANTIGEN PROCESSING PATHWAY In conclusion, based upon the evidence discussed above, we suggest a model for the MHC I antigen processing pathway. Newly synthesized polypeptides including cryptic translation products serve as the primary source of antigenic peptides. The precursors are cleaved by the proteasome and other proteases to generate a mixture of proteolytic intermediates that have the correct C-terminal end of the antigenic peptide. The intermediates, perhaps assisted by cytoplasmic chaperones, are transported by TAP into the ER. After transport into the ER, the extra Nterminal residues are processively removed by an aminopeptidase, likely using the MHC as a template, until the product fits the antigen-binding groove of the MHC I molecule with high affinity. This final peptide/MHC I complex exits the ER and serves as a potential ligand for CD8+ T cells on the APC surface.

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Several features of this model make it attractive for explaining the efficiency of the antigen processing pathway. First, using newly synthesized precursors rather than protein turnover as the primary source of antigenic peptides allows the antigen processing mechanism to tap into the total range of polypeptides synthesized in the cells, regardless of when and where the mature proteins are degraded. This mechanism would also accelerate the recognition of abnormal cells. Second, the stepwise proteolysis of precursors has important implications for the efficiency of peptide generation. Cytoplasmic processing in this view yields a rough-draft of the final peptide with the correct C-terminus. Cytoplasmic processing and transport, by making the C-termini, essentially define which peptides can be presented. Nonetheless, the proteolytic mechanisms do not bear the burden of generating the exact peptides that the MHC I molecules need in the ER. ER trimming of N-terminal flanking residues, under the guidance of MHC I molecules, enables the mechanism to be flexible, and at the same time, maximizes precision. ACKNOWLEDGMENTS We acknowledge our colleagues, past and present, who contributed methods, key observations, and ideas discussed here. We thank Natalie Brouwenstijn for help with the artwork. We are also grateful to colleagues who shared their findings prior to publication. Research in this laboratory is supported by grants from the NIH to NS. Visit the Annual Reviews home page at www.annualreviews.org

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peptides for processing in the endoplasmic reticulum and presentation to T cells. J. Exp. Med. 190:1227–40 Menoret A, Li Z, Niswonger ML, Altmeyer A, Srivastava PK. 2001. An endoplasmic reticulum protein implicated in chaperoning peptides to major histocompatibility of class I is an aminopeptidase. J. Biol. Chem. 276:33313–8 Brouwenstijn N, Serwold T, Shastri N. 2001. MHC class I molecules can direct proteolytic cleavage of antigenic precursors in the endoplasmic reticulum. Immunity 15:95–104 Gooding LR, O’Connell KA, Geib RW, Pipas JM. 1988. Cytotoxic T cell recognition of the SV40 tumor antigen: a note of caution. In Processing and Presentation of Antigens, ed. B Pernis, S Silverstein, H Vogel, pp. 87–95. New York: Academic Boon T, Van Pel A, De Plaen E, Chomez P, Lurquin C, Szikora J-P, Sibille C, Mariame B, Van Den Eynde B, Lethe B, Brichard V. 1989. Genes coding for Tcell-defined tum transplantation antigens: point mutations, antigenic peptides, and subgenic expression. Cold Spring Harbor Symp. Quant. Biol. 54:587–96 Fetten JV, Roy N, Gilboa E. 1991. A frameshift mutation at the NH2 terminus of the nucleoprotein gene does not affect generation of cytotoxic T lymphocyte epitopes. J. Immunol. 147:2697–705 Hahn YS, Braciale VL, Braciale TJ. 1991. Presentation of viral antigen to class I major histocompatibility complexrestricted cytotoxic T lymphocyte. Recognition of an immunodominant influenza hemagglutinin site by cytotoxic T lymphocyte is independent of the position of the site in the hemagglutinin translation product. J. Exp. Med. 174:733–36 Shastri N, Gonzalez F. 1993. Endogenous generation and presentation of the OVA peptide/Kb complex to T-cells. J. Immunol. 150:2724–36 Uenaka A, Ono T, Akisawa T, Wada H, Yasuda T, Nakayama E. 1994. Identifica-

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Annu. Rev. Immunol. 2002. 20:495–549 DOI: 10.1146/annurev.immunol.20.100301.064816

THE IMMUNOLOGY OF MUCOSAL MODELS OF INFLAMMATION1 Warren Strober,1 Ivan J. Fuss,1 and Richard S. Blumberg2 Annu. Rev. Immunol. 2002.20:495-549. Downloaded from arjournals.annualreviews.org by HINARI on 09/01/07. For personal use only.

1

Mucosal Immunity Section, Laboratory of Clinical Investigation, NIAID, NIH, Bethesda, Maryland 20892-1890; e-mail: [email protected] 2 Division of Gastroenterology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115

Key Words Crohn’s disease, ulcerative colitis, tolerance, cytokines, Th1/Th2 ■ Abstract In recent years the status of the inflammatory bowel diseases (IBDs) as canonical autoimmune diseases has risen steadily with the recognition that these diseases are, at their crux, abnormalities in mucosal responses to normally harmless antigens in the mucosal microflora and therefore responses to antigens that by their proximity and persistence are equivalent to self-antigens. This new paradigm is in no small measure traceable to the advent of multiple models of mucosal inflammation whose very existence is indicative of the fact that many types of immune imbalance can lead to loss of tolerance for mucosal antigens and thus inflammation centered in the gastrointestinal tract. We analyze the immunology of the IBDs through the lens of the murine models, first by drawing attention to their common features and then by considering individual models at a level of detail necessary to reveal their individual capacities to provide insight into IBD pathogenesis. What emerges is that murine models of mucosal inflammation have given us a road map that allows us to begin to define the immunology of the IBDs in all its complexity and to find unexpected ways to treat these diseases.

INTRODUCTION The study of animal models of mucosal inflammation as a means to probe the pathogenesis of inflammatory bowel disease (IBD) extends back almost a half century [for reviews of the older literature see Strober (1) and Kim & Berstad (2)], and it is fair to say that this kind of study embodied the first serious attempt to determine the immunologic basis of this category of disease. One class of early models is that devised by Kirsner and his colleagues in the early 1960s in which the mucosal immune system was manipulated in some way to cause a 1 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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mucosal (colonic) inflammation (3). Perhaps the most interesting of these models consisted of colonic inflammation that was induced in rabbits via the “Auer procedure,” wherein rabbits are first immunized with an antigen (such as OVA), then subjected to disruption of the colonic epithelial barrier with formalin, and finally are re-administered the original antigen by a local mucosal or system route (4). This procedure led to a colonic inflammation not unlike that in ulcerative colitis but was transient even when the procedure was repeated in the same animal. A more sustained inflammation, however, was obtained in the late 1970s by Mee et al., who modified the Auer procedure by sensitizing animals (rabbits) to an Escherichia coli–associated antigen (5). Similarly, in studies performed some 10 years earlier, now almost forgotten, Halpern et al. showed that immunization of rats with live or dead E. coli (in Freund’s adjuvant) led to chronic colitis even without introduction of a colonic irritant per rectum; in addition, feeding of E. coli prevented the development of colitis (6). These studies, together with early studies of dinitrochlorobenzene-induced colitis reported about the same time as the studies of Mee et al. and coworkers (5, 7, 8), clearly indicated that an initial immunologic assault of varying cause on the gastrointestinal tract can lead to more sustained inflammation as a result of a break in normal “tolerance” to antigens in the mucosal microflora. Another class of early models of mucosal inflammation were those produced by physical agents and included colitides produced by exposure to acetic acid, phorbol ester, F-met-leu-phe, and various sulfated polysaccharides such as carageenan, amylopectin sulfate, and dextran sulfate sodium (DSS) (9–20). One common feature of these agents appears to be their capacity to disrupt the epithelial cell barrier and therefore to promote increased cellular exposure to normal mucosal microflora. Evidence for this comes from studies of DSS-induced colitis in which it has been shown that DSS alters mucosal barrier function prior to the onset of colitis (19). In addition, colitis caused by exposure to F-met-leu-phe has also been shown to be associated with changes in barrier function, in this case mediated by neutrophils (14). One possible or even probable consequence of this change in barrier function is that mucosal phagocytes become subject to activation by substances in the mucosal flora and this, in turn, leads to antigen nonspecific release of pro-inflammatory cytokines (e.g., TNF-α) and inflammation. This scenario is supported by the observation that both DSS colitis and carageenan colitis can be effectively treated with antibiotics (20, 21). Disruption of barrier function(s) as a mechanism in physical agent–induced colitis fits with a second common feature of colitides caused by physical agents, namely their relative independence from lymphocyte-mediated responses. Thus, in DSS-induced colitis, it is evident that mice lacking T cells, B cells, and NK cells can still develop colitis in response to DSS (22). This being said, in the presence of an intact immune system containing these cellular elements, dextran sulfate leads to activation of lymphocytes and the induction of Th1 and/or Th2 responses. This leads to the conclusion that in physical agent–induced colitis a T cell–mediated inflammation can be superimposed on macrophage-induced inflammation.

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This brief survey of historically important models of mucosal inflammation leaves little doubt that on close reading they provide data that presaged many of the findings obtained with a set of newer models that had been extensively characterized over the past decade. Thus, as alluded to above, these models revealed the critical role of the mucosal microflora in the pathogenesis of mucosal inflammation and the related role of barrier function as a bulwark against extensive stimulation of the mucosal immune system by the microflora. In addition, they provide the first insights into the often overlooked role of antigen nonspecific factors in mucosal inflammation and even provide an early hint of the role of active tolerance in preventing such inflammation [the feeding studies of Halpern et al. (6)]. These considerations, of course, are by no means meant to belittle the new knowledge of mucosal inflammation (and by extension IBD) that have come from studies of newer models. Not only have the latter provided for the first time a detailed framework for the understanding of the various proinflammatory and antiinflammatory mechanisms at work in this type of inflammation, but they have also provided us with invaluable clues as to how the latter can be effectively treated in humans. In discussing these newer models of mucosal inflammation, we first survey their common features to derive basic principles of mucosal inflammation that are applicable to this area of study as a whole. We then discuss major individual models in depth, emphasizing the particular insights derivable from each model and how each model helps establish the basic principles and mechanisms of mucosal inflammation. This, the main body of the review, is subdivided into sections on Th1 models, Th2 models, and barrier function models.

BASIC (GENERAL) FEATURES OF MODELS OF MUCOSAL INFLAMMATION As is evident from the detailed review of individual models of mucosal inflammation that follows, certain recurrent principles emerge that relate to all models to a greater or lesser extent. These principles together define the basic immunology of both models of mucosal inflammation and of human IBD, and thus it is useful to discuss them first in an outline form that can later be fleshed out in the discussions of individual models to follow.

Final Common Pathways of Mucosal Inflammation As becomes amply evident below, models of mucosal inflammation reflect a remarkably wide variety of causes. Nevertheless, the resulting inflammation that develops is almost always channeled into a final common pathway of inflammation, mediated by either an excessive Th1 T-cell response associated with excessive IL-12/IFN-γ /TNF-α secretion or an excessive Th2 T-cell response associated with increased IL-4/IL-5 secretion (reviewed in 23). The great majority of models are in fact Th1 models, but why this is the case is far from clear (see Table 1). One factor may relate to the influence of strain on disease because the given model may

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TABLE 1 Models of mucosal inflammation classified by nature of T cell-mediated inflammation Th1 Models

Th2 Models

TNBS colitis (SJL/J mice)

TCR-α chain deficiency

SCID-transfer colitis

TNBS colitis in BALB/c mice**

TCR Tg mice with lymphopenia a

IL-10 deficiency colitis

Oxazalone colitis WASP deficiency


IL-10 signaling defects (CRF2-4 deficiency) Tgε26 mice TNF1ARE mice (TNF-α overproduction) C3H/HeJBir mice Gi2α-deficient mice Samp1/Yit mice T-bet Tg mice STAT4 Tg mice TGF-β RII dominant-negative Tg mice HLA-B27 Tg rats Mdr1a-deficient mice DSS colitis IL-7 Tg mice a

Abbreviations: SCID, severe combined immunodeficiency; TCR, T cell receptor; CRF2-4, cyto receptor family 2-4; TNF, tumor necrosis factor; STAT-4, signal transduction and activators of transcription-4; TGF, transforming growth factor; DSS, dextran sulfate sodium; WASP, Wiskott-Aldrich syndrome protein. **Mixed response but initially Th1, later Th2. Abbreviations: TNBS, trinitrobenzene sulfonic acid; SCID, xx; TCR, T cell receptor; CRF2-4, xx; TNF, xx; STAT4, xx; TGF, xx; HLA-B27, xx; DSS, xx; WASP, xx.

manifest a Th1 character in the SJL/J strain mouse but may manifest a Th2 character (or mixed Th1/Th2 character) in the BALB/c mice. A more likely explanation, however, relates to the fact that in most if not all models the inflammation is driven by antigens in the normal mucosal microflora, which in effect means that it will be influenced by mitogens [e.g., lipopolysaccharides (LPS), CpGs] and superantigens associated with these organisms that tend to induce IL-12 production and thus Th1 responses. This is nicely illustrated by trinitrobenzene sulfonic acid (TNBS)– colitis in SJL/J mice that manifest increased LPS-driven IL-12 responses, which are thought to play a key role in the Th1 response elicited by TNBS administration (see discussion below; G. Bouma & W. Strober, unpublished observations). A final possibility is that many of the models are due to a failure to regulate mucosal responses that are essentially normal responses to antigens in the mucosal milieu. As we see below, such regulation most likely involves the secretion of

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suppressor cytokines such as TGF-β (or IL-10), which is more closely related to the regulation of Th1 responses than Th2 responses (24, 25). In fact, there is considerable evidence that Th1 responses suppress both the expansion of TGF-β– secreting cells and TGF-β signaling (26, 27) and, contrariwise, TGF-β interferes with IL-12 signaling (28–30). Thus, Th1 and TGF-β responses have a reciprocal relationship to one another and appear to be mutually exclusive. On the other hand, there is some evidence that Th2 and TGF-β responses can co-exist and that it requires higher levels of TGF-β to suppress a Th2 response than a Th1 response (25, 31). These considerations suggest that Th1-mediated mucosal inflammations are more sensitive to defects of regulation (mediated by TGF-β) and thus, defects in regulation will more frequently lead to Th1-mediated inflammation than Th2mediated inflammation. The bias of the experimental models toward Th1-mediated inflammation raises the question as to when and how Th2-mediated inflammation ever occurs. One factor is again the nature of the antigen driving the inflammation or, alternatively, the specificity of the T cell receptor (TCR) on the reactive T cells. In this regard, certain antigens are “Th2-type antigens,” perhaps because the nature of the antigen dictates the type of antigen presenting cell that induces T cell differentiation in Th2 T cells. Evidence for this is inherent in the fact that one haptenating agent, TNBS, elicits a Th1 response in SJL/J mice, whereas another, oxazalone, elicits a Th2 response (31, 32). In addition, a Th2-oriented response may result in colitis associated with TCR-α chain deficiency because in this situation the T cells utilize a TCR (a ββ TCR) that may have the ability to recognize and expand in response to antigens only under conditions that allow Th2 responses (33, 34). One thing to keep in mind, however, is that the Th1-mediated inflammation may switch to a Th2 inflammation under some circumstances. This is seen in IL-10–deficient mice, perhaps because in the absence of IL-10, cells in which IL-4 signaling leads to GATA-3 suppression of IL-12 signaling gradually accumulate, and ultimately a Th2 T cell dominates the inflammation (35; A.D. Levine, personal communication). Whether a Th1 or a Th2 response is responsible for the mucosal inflammation has considerable impact on the nature of the inflammation because, as we see below, Th1 responses are marked by transmural cellular infiltration that in some cases is associated with granulomata (i.e., TNF1ARE model and SAMP1/Yit model) (36–38), and whereas epithelial cell layer changes are clearly present, they are not a dominant feature. A similar histopathologic picture is obtained in Crohn’s disease, and thus it is fair to say that, in general, Th1 models are related to this human disease (23). This presumed association between Th1 models and Crohn’s disease is also strengthened by the fact that Crohn’s disease is in fact a Th1-mediated inflammation (39–42). Th2-mediated inflammations are, on the contrary, marked by more superficial cellular infiltrates associated with a greater disruption of the epithelial layer and in some cases greater polymorphonuclear infiltration. This situation is more akin to ulcerative colitis, but this correlation is inexact because ulcerative colitis has not been clearly shown to be a Th2-mediated inflammation. Thus, whereas some authors have found high IL-5 levels in ulcerative colitis, IL-4

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levels are quite normal. Clearly, if a Th2 inflammation is present in ulcerative colitis, it is a highly atypical Th2 inflammation (39, 40).

Cellular Elements Involved in Mucosal Inflammation Antigen presenting cells (APCs) in mucosal tissues are probably key cells in the induction of both mucosal effector and regulatory cell responses. Hence, it is likely (but not yet proven) that defects of T cell responses arise either from defects in APC function or APC–T cell interactions. Alternatively, it is possible that APCs are the target of regulatory cells, a possibility proposed by Malmstrom et al. in relation to OX40-positive APCs present in the mesenteric nodes of mice with SCID-transfer colitis (43). Macrophages, a type of APC, are activated in mucosal inflammation and function mainly as effector cells. However, these cells may also be involved in regulatory interactions. This possibility is realized in mice with myeloid cell–specific STAT3 deficiency that have macrophages that cannot produce several STAT3dependent cytokines, such as the important regulatory cytokine, IL-10 (44). Thus, in vitro macrophages in this model of inflammation exhibit heightened effector activity characterized by increased LPS-induced production of IL-12, TNF-α, IL-6, and IL-1β; thus, in vivo these macrophages lead to LPS-induced mucosal inflammation.


ANTIGEN PRESENTING CELLS

T CELLS T cells play multiple roles in experimental mucosal inflammation both as effector cells and regulatory cells. The former are mainly CD4+ T cells because these cells make up the main cell populations that infiltrate mucosal tissues in all models so far studied and because in instances in which they are deleted in vivo, inflammation is ameliorated (45). CD8+ T cells are also present in tissues but do not appear to play a decisive pathologic role because in the few instances in which they were deleted in vivo no major effect on inflammation was obtained (46). This does not, however, rule out a supportive pathogenic role because increased cytotoxic T cell function has been observed in some of the models (47). Evidence has recently appeared that indicates that loss of epithelial cells in ulcerative colitis can be attributed to a T cell or NK cell–mediated cytotoxic event (48). The above information on the role of cytotoxicity in models would suggest, however, that even if cytotoxic elimination of epithelial cells occurs in ulcerative colitis, such cytotoxicity is not likely to be a major component of the overall immunopathologic process. γ δ T cells, i.e., T cells confined to the intra-epithelial compartment, do not play an important role as effector cells in any form of colitis, except perhaps in TCR-α chain–deficient mice, in which they are present in increased numbers (49). In recent studies it has been shown that whereas γ δ T cells cannot in themselves induce colitis (in lymphopenic TCR-α chain–deficient mice), injection of anti-TCRδ antibody into TCR-α chain–deficient mice prevented development of colitis (50). On this basis, γ δ T cells in this context appear to play an accessory

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role in the inflammation. γ δ T cells also do not have a major regulatory role in mucosal inflammation, as γ chain–deficient mice do not develop inflammation (51). Here too, however, one can find an exception, in that mice lacking γ δ T cells are reported to develop more severe TNBS-colitis (52). Finally, one very definite and in this case positive role of γ δ T cells in mucosal inflammation is their role in the healing of mucosal inflammation. This is shown by the fact that intra-epithelial γ δ T cells produce factors, notably keratinocyte growth factor, that may facilitate restoration of epithelial cell barrier integrity in DSS-colitis (53). Whereas, as indicated above, CD4+ T cells can function in the various models as either Th1 or Th2 effector cells, they can also function as regulatory cells. With regard to the latter, several different types of cells have been described, but it is very possible that these are in reality one cell that appears in different disguises. One type of regulatory cell is a TGF-β–secreting T cell (a so-called Th3 cell), which is the cell induced by antigen feeding during the development of oral tolerance. The mucosal cytokine milieu necessary for the induction of this cell is not well understood, although it is known that Th2 conditions favor induction and Th1 conditions inhibit induction (reviewed in 24, 26, 27). IL-10 has been seriously considered as a possible inductive cytokine for this cell, but in recent studies of oral tolerance induction as well as in in vitro studies of Th3 T cell development from naive cells, IL-10 has no direct inductive effect on the development of Th3 T cells and may enhance TGF-β production only through its capacity to down-regulate Th1 responses. However, in the same in vitro studies TGF-β itself had a positive autocrine effect on its own secretion (54). A second type of regulatory cell is an IL-10 secreting cell (a Tr1 cell), which may also secrete small amounts of TGF-β (55). This cell has poor proliferative capacities and in initial studies was induced by sequential antigenic restimulation in the presence of IL-10. More recently, however, it has been shown that both IL-10 and IFN-α are necessary for its induction (56). Yet another regulatory cell is the CD25+ T cell, which is a thymus-derived cell that inhibits effector T cells via cell-cell contact rather than secretion of an inhibitory cytokine (57, 58). Recently, however, Nakamura et al. have reported data that show that most CD25+ T cells bear surface TGF-β in the form of a latent TGF-β protein (TGF-β associated with latency-associated protein) and secrete TGF-β and IL-10 when activated in the presence of IL-2 and/or strong costimulatory signals (59). These authors suggest that under minimal stimulation conditions that might occur prior to inflammation, these cells inhibit via cellular contact and activation of the surface TGF-β at the cell-cell interface. In contrast, they suggest that under maximal stimulation conditions that occur in the presence of inflammation, CD25+ T cells inhibit via secretion of TGF-β and IL-10. Thus, the CD25+ T cells have qualities of both Th3 and Tr1 regulatory cells. A final type of suppressor cell is the NK cell or NK-T cell. The former has been shown to suppress inflammation in the SCID-transfer model of colitis (60), whereas the latter has been shown to suppress inflammation in DSS colitis (61). The NK-T cell preferentially recognizes glycolipid antigens presented via an atypical

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MHC class I molecule (CD1d), which is ordinarily expressed on dendritic cells, B cells, and epithelial cells; thus, this cell may be activated via antigens presented by both epithelial cells and more conventional APCs (62). The mechanism by which NK or NK-T cells suppress mucosal inflammation or other forms of inflammation is poorly understood, as their possible cellular targets of suppression are presently unknown. B CELLS Whereas autoantibodies are found in some models of colitis as well as in human IBD, it does not appear that B cells play a role either in induction of mucosal inflammation or its maintenance. In fact, in the one instance that B cells have been actively studied, in the Th2-mediated inflammation in TCR-α chain deficiency, they appear to play a protective role rather than a pathologic role (see below) (63). Whether such protection also occurs in human IBD is not known.

Epithelial cells form a barrier against exposure to mucosal microflora and other mucosal antigens and thus play a key role in the downregulation of mucosal immune response. As is evident from the discussion of individual models of mucosal inflammation below, in several models alterations in this barrier are the primary cause of colitis (64, 65), whereas in several other models a change in barrier function is a contributory (secondary) factor (66). Epithelial cells also function as sensors of the bacterial microenvironment and release chemokines in a programmed fashion when in contact with pathogens. Such chemokine release has the effect of drawing leukocytes into peri-epithelial sites, which then set up the first line of defense against invading organisms. Whether such chemokine release also plays a role in the initiation of mucosal inflammation is not yet clear. That it may is suggested by a recent study of Mdr1a-deficient mice, i.e., mice whose epithelial cell cannot expel proteins from within the cell including those proteins derived from infectious pathogens (65). As described below, such mice develop colitis that is likely to be due to prolonged secretion of chemokines and cytokines rather than a break in the epithelial cell barrier per se.

EPITHELIAL CELLS

Broad Categories of Mucosal Models of Inflammation As discussed in several previous reviews (24, 67–69), mucosal immune responses are fine-tuned by opposing immune mechanisms that on the one hand lead to effector cell responses addressing host defense at mucosal surfaces and on the other to tolerogenic responses preventing inflammatory reactions to the myriad of antigens in the mucosal environment. It is now apparent that the tolerogenic response has two major components: (a) processes by which mucosal antigens (in the form of unadjuvanted proteins) bring about “classical” tolerance via induction of T cell anergy or deletion either in the mucosal tissues per se or upon gaining entrance to the circulation and the central lymphoid tissues; and (b) processes by which mucosal antigen induces regulatory T cells, which secrete antigen-nonspecific suppressor

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cytokines such as IL-10 and TGF-β. These two tolerogenic processes operate in tandem and are probably both necessary to maintain mucosal homeostasis. Thus, whereas induction of anergy/deletion can greatly reduce the number of T cells that can respond to a mucosal antigen, it is probably not able to eliminate all such T cells, and the latter become memory cells potentially able to evoke inflammatory responses. These latter cells are held in check, however, by a cadre of regulatory T cells that respond to the same stimulating antigen. We must bear in mind that the main antigenic pool to which mucosal homeostasis must apply is the antigenic pool associated with the mucosal microflora, which by their persistence and proximity are formally equivalent to self antigens. Given the inevitable inefficiency of a deletion mechanism in relation to so large and mutable an antigenic pool and given the fact discussed above that a full scale effector cell response tends to obliterate a regulatory response (at least for a period of time), the burden (or the challenge) of the mucosal tolerogenic mechanism may fall disproportionately on regulatory cell function. This view is amply supported in the discussions of the individual models below in which the origin of the inflammation can be repeatedly traced to an inadequate regulatory response rather than to a hyperactive or excessive effector response to antigens in the mucosal microflora. The mechanisms governing the development of mucosal tolerance (also called oral tolerance) are not yet completely understood. One important mechanism probably relates to the special nature of the mucosal dendritic cell population, which may have an increased propensity to present antigen in a way that induces either anergic/deletional tolerance or suppressor cell tolerance. Recent work showing that subsets of mucosal dendritic cells have a substantially different cytokine secretion profiles than spleen dendritic cells, i.e., produce more IL-10, supports this concept (70, 71). Nevertheless, much remains to be learned about the origin of these cells and how they shape mucosal responses. In particular, it is not known how these cells are influenced in their development by the adjacent epithelium and how, in turn, these cells induce either the de novo development of regulatory cells or the expansion of a preexisting population of regulatory cells. In any case, the above considerations allow us to classify models of mucosal inflammation into two broad categories (Figure 1): “type 1 models,” wherein the defect lies with the effector mechanisms of the mucosal response and “type 2 models,” wherein the effector cell response is normal, but the regulatory cell response is impaired. One example of a type 1 model is the colitis seen in mice bearing a STAT4 transgene (72). These mice have an increased propensity to mount a Th1 T cell response because of excessive responsiveness to IL-12 signaling; thus, when T cells from these mice are exposed to autologous bacterial antigen in vitro and then transferred to a SCID recipient, they induce colitis in the recipient, whereas naive T cells from normal mice do not. A second example is TNBS colitis in SJL/J mice, wherein it is thought that the colitis is preceded by and is dependent on a genetically determined IL-12 hyperresponsiveness ignited by a disturbance of epithelial barrier function by ethanol followed by exposure of the mucosal APCs


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TABLE 2 Models of mucosal inflammationa Type I models

Type II models

TNFARE mice


TNBS colitis

IL-10 deficiency and IL-10 signaling defect colitis

?C3H/HeJBir mice

?C3H/HeJBir mice

Gi2α-deficient mice

IL-2-deficient mice

STAT4 Tg mice

TGF-β RII dominant-negative mice

N-cadherin dominant-negative mice

Tgε26 mice

IL-7 Tg mice DSS colitis Mice with NF-κB defects a

Unidentified: SAMP1/Yit mice; HLA-B27 Tg rats, mice with Wiskott-Aldrich syndrome protein deficiency.

Abbreviations: TNF, xx; TNBS, trinitrobenzene sulfonic acid; STAT4, xx; DSS, dextran sulfate sodium; SCID, xx; TGF, xx. For other abbreviations, see Table 1.

to antigens in the mucosal microflora (32). This response then conditions the mice to respond to TNBS with a massive Th1 response that rapidly inhibits a normal counter-regulatory response. Type II models, i.e., models that result from an inadequate regulatory response, are exemplified by the SCID-transfer model wherein transfer of naive CD45Rhi T cells leads to colitis, whereas transfer of both naive and memory (CD45RBlow) T cells does not (73). In this model, as described more fully below, the memory cell population contains regulatory cells so that transfer of only naive cells leads to an inadequate regulatory response and colitis. A second type II model that results from inadequate regulation is that seen in mice bearing a dominant-negative TGF-β RII chain (under a CD4+ promotor) that abrogates TGF-β signaling (74, 75). Here, regulatory cells are present, but they cannot function adequately because their intended targets are “blind” to their signals. In the following detailed review of various models, we characterize their basic mechanisms as a type I model (faulty effector cell function) or as a type II model (inadequate regulatory cell function), and in Table 2 we have categorized most of the models on this basis. ←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− − Figure 1 (upper panel) The normal mucosal immune system displays a balanced effector T cell response (Th1 or Th2) and regulatory T cell response (Th3 or Tr1). (lower panels) The abnormal mucosal immune system displays an unbalanced response consisting of either excessive effector cell response (type I models) or inadequate regulatory cell response (type II models).

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The Role of Mucosal Microflora in the Induction of Mucosal Inflammation Regardless of whether the experimental mucosal inflammation is type I or type II in character as defined above, the driving force of the inflammation is the nonpathogenic commensal organism resident on the mucosal surface, the mucosal microflora. This is supported by the data in Table 3, which shows that, with perhaps one or two exceptions, mice developing disease in a specific pathogen-free or conventional environment do not do so in a germ-free environment, and in most instances disease is ameliorated when the mice are treated with antibiotics that rid the mucosa of certain classes of organisms (reviewed in 21, 76–85). Exceptions to this consistent pattern are informative. The first exception is the IL-2–deficient mouse, which develops severe and aggressive gastrititis, duodenitis, and colitis under conventional conditions but only nonfatal, mild, focal, and nonproliferative gastrointestinal inflammation under germ-free conditions (80, 81, 86). In addition, these mice develop peri-portal hepatic inflammation, anemia, and generalized lymphoid hyperplasia, which is not ameliorated by the presence of a germ-free state. Thus, this exception to the rule can be explained if we assume that autoimmune inflammation against nonmucosal self-antigens is a component of IL-2 deficiency disease. The second exception is the induced colitis known as dextran sulfate colitis or DSS colitis. This model of colitis can also be observed under germ-free conditions (at least in some studies), although it is ameliorated by antibiotic treatment (21, 83, 84). This can be explained by the fact that this

TABLE 3 Colitis in models of mucosal inflammation in germ-free vs. specific pathogen-free (SPF) or conventional conditions SPF

Germ-free

Antibiotic treatment


+

0

c


++

Mild, focal


++

0

a

?

Tgε26

+

0

?

TCR-α chain colitis

+

0

? ?

SAMP-1/Yit mice

+

0

DSS colitis

+

0/+b

Carageenan colitis

+

0

Indomethacin colitis

+

0

a

See references 80, 81, 86.

b c

See references 21, 83, 84.

Decreased colitis.

Abbreviations: SCID, xx; TCR, T cell receptor; SAMP, xx; DSS, dextran sulfate sodium.

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is a colitis caused primarily by direct activation of macrophages by a physical agent (DSS), and T cell responses appear to be superimposed phenomena that can aggravate but are not essential to the inflammation. Thus, this exception to the rule is due to the fact that the colitis is at least in part driven by nonimmune factors. Additional and more direct evidence that mucosal microflora drive mucosal inflammation in models of mucosal inflammation comes from studies showing that mouse lamina propria T cells are usually unresponsive to their own microbial flora (with respect to either proliferation or cytokine production) but are responsive to the microflora of other individuals even if the other individual is a mouse of the same strain (87, 88). Thus, quite remarkably, oral tolerance to “self flora” appears to be every bit as specific as tolerance to “self antigens.” A related observation is that mice with TNBS colitis lose their nonresponsiveness to their own flora and regain it when the colitis resolves (88). This suggests that the colitis is at least in part driven by the antigen in the microflora, either as a result of cross-reactivity to TNBS or because with the onset of colitis, tolerance to many microflora antigens is lost. Those latter possibilities are supported by the finding that systemic immunization of IL-2–deficient mice with TNP-KLH or other TNP-substituted proteins produces rapid onset of colitis that is identical to the spontaneous colitis occurring in these mice (89). It is important to note that such loss of tolerance to self flora is also a feature of human inflammatory bowel disease (IBD), a fact suggesting that the human disease is also due to loss of tolerance to self microflora (87, 88, 90). A third kind of evidence supporting the fact that antigens in the normal microbial flora drive mucosal inflammation comes from an extensive series of studies of the spontaneous colitis occurring in an LPS-nonresponsive C3H/HeJ mouse substrain (called C3H/HeJBir mice), which is discussed more fully below. Suffice it to say here that CD4+ T cells stimulated in vitro by lysates of resident bacteria can transfer disease to naive disease-free recipients (91, 92). Similarly, studies of mice bearing a STAT4 transgene show that in vitro exposure of T cells from mice with an increased propensity to undergo Th1 T cell differentiation to autologous microfloral antigens induces in these T cells the capacity to cause a Th1 colitis in SCID recipients (72). Together, these studies provide a direct demonstration that T cells specific for mucosal microflora act as effector cells in models of mucosal inflammation. It should be noted, however, that whereas effector cells inducing colitis can be stimulated by antigens in the mucosal microflora, regulatory cells can also be so stimulated. This is shown in additional studies of C3H/HeJBir mice in which it was found that cell lines producing IL-10 could also be derived from these mice, which upon co-transfer with effector cells prevented development of colitis (reviewed in 93). From these studies and other studies below it is evident that the mucosal microflora can also induce regulatory cells, and it is really the loss of balance between induction of effector and regulatory cells that defines when disease occurs. The fact that the mucosal microflora is the major driving force in experimental inflammatory disease should not be taken to imply that all bacterial antigens take part in the disease process or even that the same antigens are necessarily implicated

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in a number of different models of inflammation. This is evident from the studies of the aforementioned C3H/HeJBir model showing that relatively few antigens within the large antigenic pool of the mucosal microflora are actually found to stimulate B cells or T cells during the course of the disease (92). One must hasten to add, however, that while the number of stimulating antigens was low relative to the total number of antigens present, it was still considerable (see below). A similar situation is observed in the SCID-transfer and TCR-α chain–deficient mouse models (34, 94). In the latter, T cells with aberrant T cell receptors (TCRs) exhibit restricted T cell clonality in conjunction with a common (public) motif in the CDR3 region of the TCR (33, 34). However, in this case the aberrant TCR probably dictates a limited ability to recognize the full set of antigens and thus, may exaggerate the narrowness of the antigenic repertoire recognized by the colitic mice. Similar considerations apply to human patients with IBD who also exhibit restricted T cell clonality and evidence of public motifs among the cells present in lesional tissues (95). Thus, in some patients with IBD there is evidence that the restricted T cell clonality reflecting the presence of a limited group of related stimulatory antigens may be involved in disease pathogenesis. However, in the majority of patients the data are more consistent with a broader T cell response that is characterized by the presence of private motifs that vary from individual to individual. The restricted yet variable nature of the antigens of the mucosal microflora capable of evoking mucosal inflammation does not conflict with the fact that monoassociation of HLA-B27 transgenic rats or mice with IL-10 deficiency and TCR-α chain deficiency with Bacteroides vulgatus can lead to colitis (76, 77, 96, 97). First, B. vulgatus is likely to be one among many organisms that can induce disease. Second, the effects of B. vulgatus mono-association may relate to the its ability to synergize with other Enterobacteriaceae in causing infection or to augment internalization of selected strains of bacteria (98). Similarly, Helicobacter hepaticus infection causes disease in IL-10–deficient mice under some animal room conditions but not others (99, 100). In addition, in one study microflora that included H. hepaticus caused colitis in Rag-2–deficient mice but not Rag-2–deficient mice also deficient in IL-7 or Rag-2–deficient mice treated with IL-10 (101). Because overproduction of IL-7 by epithelial cells is a cause of colitis in another model (102), these results suggest that the ability of an organism to cause colitis may depend on its ability to directly stimulate a particular cytokine pattern in the mouse host and thereby cause undue activation of certain cell populations, such as macrophage populations. Studies involving antibiotic treatment of murine models of inflammation with antibiotic also attest to the fact that many bacterial species are capable of promoting inflammation. Thus, whereas either ciprofloxcin or a combination of neomycin and metronizadole could prevent colitis in IL-10–deficient mice, only the combined antibiotic regimen was successful as a treatment of the colitis (85). Similarly, combinations of vancomycin plus imipenem were necessary to treat disease in IL-10–deficient mice, DSS-treated mice, and HLA-B27 transgenic rats, and other

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combinations of broad spectrum antibiotics were necessary to treat mice with TNBS colitis (21, 76, 77, 103). In line with the above discussion of effector and regulatory cells stimulated by mucosal microflora, certain organisms appear to be particularly involved in the induction of mucosal inflammation, presumably owing to their capacity to stimulate effector cells, and other organisms may have a special capacity to quell inflammation via an enhanced capacity to stimulate regulatory cells. Evidence in favor of this concept is that introduction of Lactobacillus species into the mucosal environment of IL-10–deficient mice prevents the development of colitis of the mice under specific pathogen-free conditions (104). Additional evidence comes from the observation that whereas colitis in mice with Mdr1a deficiency is worsened by infection with Helicobacter bili, it is ameliorated by infection with H. hepaticus (105). Finally, spontaneous colitis in the SAMP1/Yit mouse is more severe in a pathogen-free environment than in a conventional environment (37). This finding is relevant to human IBD because it is possible that the increase in the incidence of IBD observed in developed countries may be due to the fact that exposure to organisms that could ameliorate potential inflammation is decreased in these countries. This view is consistent with recent studies by Dalwadi et al. showing that a superantigen (called I2) derived from Pseudomonus species is associated with Crohn’s disease lesions and induces the regulatory cytokine IL-10 in vitro (106). Overall, these considerations make it likely that the presumed nonresponsiveness to mucosal flora may be more apparent than real in that normal organisms are responding to antigens in the mucosal microflora but only in the negative sense of inducing regulatory cells or the production of organism-specific IgA antibodies that regulate colonization and translocation. A final point of some interest is that the bacterial flora present in a particular niche in the intestine may have increased importance in eliciting inflammation in an animal model. Thus, creation of a cecal self-filling blind loop in HLA-B27 transgenic rats leads to proliferation of anaerobic bacteria, especially Bacteroides species and a more severe transmural cecal inflammation (103). Moreover, exclusion of the cecum leads to reduced gastric inflammation. Finally, in TCR-α chain–deficient mice, early removal of the tip of the cecum containing a large lymphoid aggregate leads to attenuation of subsequent colitis (107). Thus, it is possible that bacteria occupying a particular area of the intestine are of increased importance in generating effector cells that ultimately cause disease in all parts of the intestine. In summary, there can be no question from the foregoing discussion that the mucosal microflora play a critical role in models of mucosal inflammation by providing the major stimulus for the induction of effector T cells that cause the inflammation. This being said, it is also apparent that no single bacterial antigen has yet been shown to be responsible for this stimulation, although clearly some bacteria may be more important than others in this respect. Thus, whereas a continued search for a particular organism and/or antigen that causes IBD remains an important goal of some IBD investigators, the advent of models of mucosal inflammation that collectively show that mucosal inflammation is associated with

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inherent immune defects in the face of an unaltered flora indicates that this goal may prove futile.


Genetic Factors in Models of Mucosal Inflammation STUDIES IN MOUSE MODELS The possible genetic factors underlying models of mucosal inflammation have only recently received attention. Such factors could conceivably be operative both in models of spontaneous or induced colitis in mice strains with no known underlying genetic defects, or in mice with a known gene deletion or over-expression. In the latter case the genetic factor could conceivably influence the expression of the known gene defect. Evidence of such factors has been shown in relation to DSS colitis, in which it has been demonstrated that different mouse strains have different susceptibilities to disease (108, 109). Not surprisingly, strains in which mucosal inflammation has occurred spontaneously, such as the C3H/HeJBir mouse, have proved highly susceptible to DSS colitis, as did autoimmune-prone NOD mice of various types (108). Interestingly, Non-/LtJ mice were quite resistant to DSS colitis even though NON mice are congenic with NOD with respect to MHC. This lack of involvement of MHC genes in this form of colitis, if generalizable, is consistent with the view that no single antigen or set of antigens is involved in inducing experimental mucosal inflammation. In a second published study of genetic factors, a genome-wide search for quantitative trait loci (QTL) for susceptibility to DSS colitis in susceptible C3H/HeJ mice was conducted (110). In this study the C3H/HeJ mice were crossed with partially resistant C57BL/6 mice and strain-specific genetic areas associated with occurrence of colitis in their F2 progeny was determined. A number of QTLs were identified including those on chromosomes 1, 2, 5, and 18. In addition, several resistance loci were identified in susceptible NOD/Lt strain mice carrying resistance alleles from either B6 on chromosome 2 or from NON/Lt on chromosome 9. Thus, the genetic factors present in DSS colitis were highly complex. A third study of genetic factors in mouse models examined genetic factors controlling disease severity in IL-10–deficient mice (111). Here, IL-10–deficient mice on a C3H/HeJBir background manifested severe colitis when intercrossed with IL10–deficient mice on a C57BL/6 background that manifested mild colitis; this was done to determine inheritance of disease in the F2 generation and thus to identify QTLs. A C3H-derived colitogenic locus was found on chromosome 3 in two separate studies. This locus interacts in a complex fashion with other loci including a BL6-derived QTL on chromosome 18, a C3H-derived QTL on chromosome 8 for cecal lesions, and a C3H-derived disease QTL on chromosome 3, chromosome 9, and chromosome 19. These crosses thus found colitogenic susceptibility modifier genes that interact with IL-10 deficiency to cause more severe disease. Finally, an as yet unpublished study of genetic factors in TNBS colitis using susceptible SJL/J mice and resistant C57BL/6 mice subjected to a similar genomewide search revealed loci on chromosomes 9 and 11 (G. Bouma & W. Strober, unpublished observations). These findings parallel those derived from a recent

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study of SAMP1/Yit mice that also revealed the existence of a locus on chromosome 9 (112). Thus, a QTL in chromosome 9 may well harbor an important gene involved in susceptibility to colitis both in mouse models of inflammation and in humans. The QTL in chromosome 11 in the study of TNBS colitis is also of interest because the tendency of this strain of mice to manifest high IL-12 responses maps to the same region. Thus, the possibility emerges that a gene controlling IL-12 responses is an important susceptibility gene in a model of colitis as well as in IBD. STUDIES IN HUMANS WITH IBD One area of research into mucosal inflammation in which studies of humans with IBD can inform us about murine models of inflammation rather than vice versa is the area of genetic factors operating in these abnormalities. Two developments in the study of IBD are relevant. The first is that large-scale genome-wide searches conducted in families containing multiple members with IBD have led to the identification of 12 chromosomal loci associated with the occurrence of disease (113). In some but not all cases these loci have been confirmed by two or more independent studies and thus are genomic areas where disease genes can eventually be found. The finding that human IBD is a multigenic disease as implied in these human studies has relevance to the murine models, as it indicates that multiple genes are involved in the murine models even when the latter is due to a known genetic defect. This explains the fact that the expression of disease in, for example, Gi2a-deficient mice varies greatly with the strain of mouse bearing this defect. Finally, it is important to note that studies of susceptibility and resistance genes for murine mucosal inflammation, such as those discussed above, can greatly facilitate this search for disease genes in humans because the location of identified murine genes can ultimately be linked to syntenic genes in humans. The second development in the study of IBD is that the gene located in the most well established of the above loci, that in IBD-1, has recently been identified by two independent groups using two independent techniques (114, 115). These groups have shown that a gene encoding the protein present in macrophages and known as NOD-2 is a disease gene in Crohn’s disease; some 10–20% of individuals with the disease have mutations in NOD-2 and those that are homozygous for a mutated gene will invariably develop the disease. The function of the NOD-2 gene is poorly understand and thus its relation to the pathogenesis of IBD is essentially unknown. As reviewed by Beutler (116), some hints as to its function come from a knowledge of its structure: NOD-2 contains on one end a leucine-rich region where most of the mutations have been found and on the other end a caspase recruitment domain. Leucine-rich regions are thought to be binding regions and are found in toll-like receptors (TLRs). Thus, one possibility for the function of NOD-2 is that it interacts with ligands of TLRs (LPS and other bacterial products) that have gained entrance to the interior of the cell and then activate the NF-κB pathway through RICK, a protein known to bind to NOD-1, a close homologue of NOD-2. In this scenario, mutations of NOD-2 in Crohn’s disease are gain-of-function mutations that lead to increased NF-κB activation and inflammation. This would imply that

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processes involved in natural immunity (i.e., interactions of bacterial products with substances similar to those on TLRs) rather than adoptive immunity may play an important role in the initiation of Crohn’s disease. An alternative function of NOD-2 relates to the presence of the caspase recruitment domain and the possibility that NOD-2 is involved in caspase activation and apoptosis.One could propose that the mutations are loss-of-function mutations that lead to decreased apoptosis and thus to the persistence of cells that produce inflammatory cytokines. At the moment, neither of these possibilities is supported by either in vitro or in vivo evidence, and further studies are necessary to decide their validity or, indeed, the validity of other possibilities. Finally, with respect to the murine models of mucosal inflammation, none have yet been identified that appear to have NOD-2 mutations. Nevertheless, it is still possible that one or more of the models with no identifiable cause such as a SAMP1/Yit model can be due to a NOD-2 mutation, particularly because this model so closely resembles human Crohn’s disease (see discussion below). In addition, it is possible that, as implied above, a NOD-2 mutation acts as a contributing “background” abnormality determining susceptibility to mucosal inflammation primarily owing to another defect.

Th1 MODELS OF MUCOSAL INFLAMMATION By far the most common immunologic mechanisms leading to a model of mucosal inflammation are those involving a dysregulation of the Th1 T cell pathway. As already mentioned, the most important reason for this Th1 bias is that conditions in the mucosal environment, particularly the ubiquity of substances that induce IL-12, favor excessive Th1 response over excessive Th2 response if and when there is an imbalance in mucosal immune homeostasis. In general, the nature of the inflammation at both the macroscopic and microscopic levels in Th1 models is most closely related to Crohn’s disease, and indeed, this disease has quite clearly been shown to be due to a Th1 T cell disturbance (or a set of disturbances). Of the various Th1 models, two have been studied most intensively and have yielded insightful information. We discuss these models in some detail.

TNBS Colitis Hapten-induced colitis [trinitrobenzene sulfonic acid (TNBS)–colitis] is an important model of mucosal inflammation because it allows for the study of early or initiating events in the development of a mucosal inflammation and because it allows analysis of the relation of the response to a specific antigen (a hapten) to the overall mucosal immune response leading to colonic inflammation. Whereas this model has been the object of study for over two decades (117, 118), it was not until 1995 when Neurath et al. showed that TNBS administered per rectum (in the presence of ethanol) to SJL/J mice resulted in a transmural infiltrative

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disease limited to the colon and owing to an IL-12–driven, Th1-mediated response (32). The latter was definitively demonstrated by the fact that treatment of mice with only a single dose of anti–IL-12 antibody results in complete prevention of TNBS colitis or, in the case of mice with preexisting and ongoing disease, in complete and rapid disappearance of the inflammatory lesion (32). In addition, it was shown that, in common with the colonic inflammation seen in IL-2-deficient mice or in several other models of colonic inflammation, the disease could be prevented by administration of anti-CD40L (CD154), indicating that the Th1 response driving this Th1-mediated inflammation was based on CD40L-CD40 interactions (91, 119–121). In subsequent studies of the role of the various IL-12–induced Th1 cytokines participating in the pathogenesis of TNBS colitis, it has been shown that the role of TNF-α is surprisingly important. In particular, TNBS could not be induced in TNF-α–deficient mice and is far more severe in mice that over-express this inflammatory cytokine (122). One possible explanation of these findings is that TNF-α is necessary for both the initiation and persistence of the Th1 response, possibly by acting as a proximal cofactor for IL-12 or IL-18 production. The dramatic effect of anti–IL-12 antibody administration on TNBS colitis (and as subsequently shown, on other murine models of colitis) can be linked to the observation that such administration is associated with increased numbers of TUNEL-positive cells in lamina propria tissues and in dispersed cell populations (123). This, plus administration of Fas-Fc to mice undergoing treatment with anti–IL-12, strongly supports the idea that anti–IL-12 treatment leads to Fasmediated Th1 T cell apoptosis and that TNBS colitis is rapidly responsive to anti– IL-12 because the latter leads to the death of the Th1 T cells inducing the colitis. One of the major insights derived from the study of TNBS colitis is that regulatory mechanisms inherent in the mucosal immune responses can prevent the development of colitis. This was shown by the fact that whereas intrarectal administration of TNBS led to colonic disease, oral administration of TNBS in the form of TNP-haptenated colonic protein (TNP-CP) prevented colitis induced by intrarectal TNBS administration (124, 125). In addition, it was shown that the preventive effect was due to the induction of regulatory cells producing TGF-β, because TNP-CP feeding led to the appearance of TGF-β-producing cells in the lamina propria, and coadministration of anti–TGF-β antibody to mice fed TNP-CP abrogated the protective effect. This TGF-β -mediated protection is due to the induction of oral tolerance in the face of an induced mucosal inflammation and indicates that immune responses resulting in inflammation of the mucosa are as subject to mucosal regulatory effects as the response resulting from the feeding of protein antigens (24, 67–69). On this basis, it is reasonable to attribute the induction of TNBS colitis in SJL/J mice by intrarectal TNBS administration alone to the fact that such administration engenders a mucosal Th1 T cell response that is not balanced by the prompt appearance of a regulatory response. We return to the possible reason why this is so after we discuss the role of mucosal microflora in TNBS colitis.

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In other models of mucosal inflammation, most notably that seen in IL-10– deficient mice, IL-10 rather than TGF-β appears to be the major cytokine-mediating regulation. In recent, as yet unpublished, studies of the relation between TGF-β and IL-10 production in TNBS colitis involving administration of anti–TGF-β and anti–IL-10 as well as adoptive transfer of regulatory cell populations, it was shown that TGF-appears to be the major regulatory cytokine but that IL-10 is necessary for the maintenance and/or the effectiveness of the TGF-β response (125a). Thus, in the key experiment of these studies it was shown that whereas mice fed TNP-CP and then given TNBS per rectum to induce colitis were protected from colitis, administration of anti–IL-10 after feedings prevented protection and reduced both IL-10 and TGF-β responses; however, administration of anti–TGF-β prevented protection and reduced TGF-β responses but not IL-10 response. Thus, TGF-β levels were more closely associated with counter-regulation than were IL-10 levels. In recent studies taking advantage of the potent capacity of TGF-β regulatory cells to ameliorate TNBS colitis, DNA encoding active TGF-β was administered to mice intranasally to induce genetically engineered T cells producing TGF-β in vivo (29). Indeed, following such treatment, T cells and macrophages producing TGF-β were subsequently found in lamina propria and spleen, where they acted to prevent induction of and treat TNBS colitis. Interestingly, the induction of such regulatory cells was associated with production of high levels of IL-10, which also contributed to the regulatory effect. These studies open the door to the possibility that gene therapy with genes encoding regulatory cytokines will become a viable form of treatment of Th1 mucosal inflammation. Whereas TNBS (or more specifically the TNP epitope) may be the main antigenic stimulus that drives the Th1 responses in TNBS colitis, it is likely that other antigenic determinants present in the mucosal microflora also contribute to the immune response driving this disease. This view stems from evidence reviewed above, which shows that mice with TNBS colitis react to their own microflora and that such reactivity disappears with anti–IL-12 treatment (87, 88, 90). Reactivity to mucosal microflora also relates to TNBS colitis in a way that bears on genetic factors in this model. Recall that TNBS administered per rectum to induce colitis is administered with ethanol, a substance that disrupts the mucosal barrier and thus, as an initial event, causes increased exposure of the mucosal immune system to mucosal microflora. In SJL/J mice that are susceptible to colitis there is evidence that such exposure leads to a high IL-12 response, and it is reasonable to suppose that this sets in motion a massive Th1 response to TNBS that precludes a concomitant regulatory TGF-β response. In contrast, administration of TNP-CP by mouth (in the absence of ethanol) does not lead to an initial IL-12 response, and thus a normal mucosal response replete with a regulatory component ensues. Carrying this concept one step further, one might postulate that mouse strains susceptible to TNBS colitis are precisely those that mount high IL-12 when exposed to mucosal microflora. Evidence that this is the case comes from the study of susceptibility loci in SJL/J mice mentioned above, showing that two such loci controlling TNBS

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colitis can be identified, and one of these is similar if not identical to that associated with high IL-12 responsiveness (G. Bouma, W. Strober, submitted for publication). In summary, TNBS colitis in SJL/J mice is an immunologically mediated colitis that results from the rapid induction of an IL-12–driven, Th1-mediated response that precludes development of a counter-regulatory TGF-β response. As such, this is a type 1 model in that the major driving force is the overactivity of disease-causing effector cells. Emerging evidence suggests that this response is genetically controlled, most probably by genes that regulate the magnitude of an initial IL-12 response to substances in the mucosal microflora.

The SCID-Transfer Model of Colitis A second important model of mucosal inflammation is that produced by repletion of SCID or Rag2−/− with either CD45RBhi T cells (naive T cells) or with a combination of CD45RBhi T cells and CD45RBlo T cells (mature T cells) (73, 126, 127). In the former case repletion leads in 3–5 weeks to severe colitis, whereas in the latter case no inflammation occurs. Herein lies the power of the model: One can immediately identify two cell populations, one a source of effector cells and the other a source of regulatory cells, and one can conduct analyses of each population to identify the cells necessary for each type of function. In initial studies of this model it was found that the inflammation was due to a Th1-mediated T cell response, also driven by IL-12 and mediated by IFN-γ (128, 129). In this instance whereas the colitis was less effectively inhibited by anti–TNF-α treatment than TNBS colitis, cells from STAT4-deficient mice still gave rise to disease, perhaps because of their continued ability to produce TNF-α (130). Whereas CD45RBhi cells populate the small intestine as well as the large intestine of SCID recipients undergoing cell transfer, inflammation is limited to the colon. This immediately suggested that organisms endogenous to the colon provide the antigenic stimulus for the mucosal inflammation in this model. Evidence in support of this concept came from studies of the SCID-transfer model that showed that transfer of cells to mice reared in a “near gnotobiotic” environment (rather than the specific pathogen-free environment of the mice in the original studies) manifested greatly reduced levels of inflammation (94). In addition, CD4+ T cells from colitic mice proliferated and produced Th1 cytokines in response to antigen presenting cells pulsed with fecal extracts of normal but not germ-free mice (94). If indeed mucosal microflora drive effector cells in SCID-transfer colitis, the number of stimulating antigens in the microflora is circumscribed, because colitic mice contain populations of cells with restricted T cell receptor (TCR) diversity and expression of particular CDR3 sequences (131). It should be noted, however, that the selected TCRs differ from mouse to mouse despite MHC class II identity, indicating that the number of potentially stimulating antigens may be considerable. Finally, the expanded clones were widely dispersed in lymphoid tissue and were

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detected early (prior to development of overt colitis), indicating that stimulation of these clones was an early event in disease pathogenesis. Overall, these data are compatible with the view that cell activation in SCID-transfer colitis is an antigendriven event occurring as a result of inappropriate responsiveness to antigens in the mucosal microflora. As noted above, the SCID-transfer model is particularly useful for the study of regulatory cells in mucosal inflammation. In an early study of such regulation it was shown that the protective effect of CD45RBlo T cells was not due to the secretion of IL-4 because the cells that mediated the protection could be obtained from IL-4–deficient mice and protection occurred in spite of repeated administration of anti–IL-4; however, it was due to secretion of TGF-β because in this case repeated anti–TGF-β administration reversed protection (132). In more recent studies of the relationship of TGF-β and the regulation of SCID-transfer colitis it was shown that the regulatory cells were CD25+ T cells because CD45RBlo cells depleted of CD25+ T cells were unable to prevent colitis (133). In addition, evidence was presented that the protection afforded by CD25+ T cells was abolished by co-administration of anti–CTLA-4 and anti–TGF-β antibodies, indicating that this subset required stimulation via CTLA-4 and either produced TGF-β itself or induced such secretion in other cells. That the former possibility is correct is supported by recent studies by Nakamura et al., who showed that CD25+ T cells produce TGF-β when stimulated by anti-CD3 antibody under crosslinking conditions and costimulated with anti-CD28 or anti–CTLA-4 (59). Thus, the picture that emerges is that CD25+ T cells in the CD45RBlo T cell populations secrete TGF-β in a CTLA-4–dependent fashion to mediate suppression of colitis. Parenthetically, recent evidence suggests that CD25+ cells express surface TGF-β in the form of a latent (inactive) protein associated with latency-associated protein. This surface TGF-β may be responsible for CD25+ T cell suppression mediated by cell-cell contact, i.e., the form of suppression exerted under suboptimal stimulation and in the absence of overt inflammation. A final point to be made about the regulation of SCID-transfer colitis by CD25+ T cells relates to the fact that, as shown originally by Sakaguchi et al. (134), such cells develop in the thymus and, as recently shown by Bensinger et al. (135), are dependent on the presence of MHC class II–positive cortical epithelial cells for their intrathymic development. Thus, it is not surprising that CD25+ cells from MHC class II–deficient mice neither act as suppressor cells in in vitro assays nor suppress colitis when injected together with CD4+ CD45RBhi cells into Rag-2– deficient recipients. This evidence that CD25+ cells regulating colitis can originate in the thymus should not be taken to mean that this is the only site of development of these regulatory cells. It remains possible (albeit unproven) that such cells also develop, or at least undergo expansion, in the mucosal tissues. IL-10, no less than TGF-β, has also been implicated in the regulation of SCID colitis. Initial evidence for this came from a study showing that CD45RBhi T cells do not cause colitis if obtained from IL-10 transgenic mice (136). In further studies, Groux et al. showed first that T cell clones expanded in vitro in the presence

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of IL-10 produce high levels of IL-10 (and IL-5) and, in some cases, TGF-β as well (55). They then showed that these clones suppress T cell response in vitro and, more importantly, suppress SCID-transfer colitis when administered in place of CD45RBlo T cells. These studies thus defined a new class of regulatory cells (called Tr1 cells) that mediate immune suppression mainly via IL-10. It should be noted, however, that these cells also produce TGF-β and thus the relevant suppressor cytokine is uncertain. A final series of studies relative to IL-10 and regulation of SCID-transfer colitis showed that CD45RBlo T cells obtained from IL-10– deficient mice do not prevent colitis when administered with CD45RBhi T cells; similarly, treatment of SCID mice administered both CD45RBhi and CD45RBlo T cells with anti–IL-10 receptor led to the development of colitis (137). These studies thus show that IL-10 is necessary for protection against colitis even if it is not sufficient. Taken together, the above studies show quite definitively that IL-10 and TGF-β are important regulatory cytokines in SCID-transfer colitis. The question arises here, even more than in the case of TNBS colitis, as to how these regulatory cytokines interact to bring about regulation. To date, no studies addressing this question in the context of SCID-transfer colitis have appeared; however, based on the data derived from the TNBS colitis model we would suggest that TGF-β is the major suppressor cytokine and that IL-10 is needed to facilitate TGF-β secretion and/or activity. Yet another cell type contributing to regulation of SCID-transfer colitis is the NK cell. This is supported by the fact that transfer of CD45RBlo T cells into NK cell–depleted recipients results in more severe colitis (60). Such regulatory effects, as noted above, are also seen in other models of mucosal inflammation and occur via an unknown mechanism. However, it is know that this form of regulation is distinct from that mediated by CD45RBlo cells inasmuch as depletion of the latter of NK cells does not eliminate their regulatory effect. One possible but highly speculative explanation is that NK cells lyse activated effector cells or APCs inducing the effector cells. This concept is supported by the fact that NK cells from perforin-deficient mice have no regulatory effects (60). A final point to emerge from studies of the SCID-transfer model relates to the recent observation that colitic mice have greatly increased numbers of CD134+ (OX40+) dendritic cells in their mesenteric lymph nodes and that administration of anti-CD134L antibody leads to reversal of colitis (as it does in other models) (43, 138). It is likely that these mesenteric lymph node cells originate in the inflamed lamina propria and then migrate to the draining lymph node where they provide inductive signals to effector T cells about to migrate into lesional tissues. This would imply that much of the antigen presentation necessary for the development of mucosal lesions goes on in regional lymph nodes, rather than in the inflamed tissue itself. In summary, the SCID-transfer model is a model of mucosal inflammation that allows one to separate effector and regulatory T cell functions mediating the inflammatory process. Thus, by analysis of both effector cells in CD45RBhi

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T cell populations and regulatory cells in CD45RBlo T cell populations, it has been possible to clearly demonstrate the interplay between various cell types that determine whether mucosal inflammation will occur. In addition, this model demonstrates that abnormal reactivity to antigen in the mucosal microflora can develop in the absence of a genetic abnormality and does not require initial disruption of the epithelial cell barrier. Rather, the only precondition for the occurrence of colitis is a marked imbalance between effector and regulatory cell populations.

Type 1 Models of Colitis: Defects that Directly or Indirectly Affect the Synthesis of Key Cytokines in the Th1 Pathway of T Cell Differentiation Joining TNBS colitis as type 1 defects leading to mucosal inflammation are several models whose pathogenesis can be traced to abnormalities that lead to the overproduction of key cytokines in the Th1 T cell differentiation pathway, such as IFN-γ , TNF-α, and IL-12. COLITIS ASSOCIATED WITH DEFECTS IN THE PRODUCTION OF TRANSCRIPTION FACTORS CONTROLLING IFN-γ PRODUCTION Given the central role of IFN-γ in the

Th1 responses, it should come as no surprise that molecular defects resulting in IFN-γ overproduction can lead to colitis. Two such defects are now known to exist, one affecting T-bet and one affecting STAT4. As shown by Szabo et al., T-bet is a T-box protein that when over-expressed in T cells programs them for high IFN-γ responses and low IL-4 responses, even when the cell is a supposedly “committed” Th2 cell (139). This, plus recent data showing that over-expression of T-bet can lead to IFN-γ responses in cells lacking STAT4, has led to the concept that this factor is the molecular switch for Th1 differentiation (140). STAT4, on the other hand, has also been shown to be a necessary factor for Th1 differentiation that probably acts as both a transcription factor for IFN-γ and as a factor that maintains Th1 T cell survival (141, 142). In the relevant studies of colitis associated with a STAT4 abnormality, it has been shown that mice bearing a STAT4 transgene (under a CMV promotor) develop colitis when administered TNP-KLH in Freund’s adjuvant, an antigenic stimulus that has no colitogenic effect in normal mice (72). In addition, spleen cells from these mice proliferate when exposed to antigens in their autologous microflora in vitro, and T cells thus stimulated induce colitis in SCID recipients. Corresponding studies of T-bet abnormalities have shown that naive T cells from T-bet–deficient mice exhibit a reduced capacity to transfer colitis to SCID mice, whereas, conversely, naive T cells over-expressing T-bet (owing to infection with a T-bet-expressing retrovirus) induce accelerated colitis in SCID mice (M. Neurath, R. Blumberg, L. Glimcher, manuscript submitted). In addition, memory T cells from T-bet–deficient mice exhibit an enhanced capacity to protect SCID mice from colitis when cocultured with naive T cells.

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MUCOSAL INFLAMMATION IN MICE THAT OVER-EXPRESS TNF-α: TNF1ARE MICE Also in the category of models of inflammation owing to abnormalities of Th1 cytokine production is the model owing to over-expression of TNF-α, the TNF1ARE mouse (36). This model results from a targeted deletion of AU-rich elements located in the 30 untranslated region of the TNF-α gene, which gives rise to dysregulation of the processing of TNF-α mRNA and the overproduction of TNF-α protein. The phenotype of these mice is notable because the mucosal inflammation is mainly located in the terminal ileum and only occasionally in the proximal colon; in addition, it is remarkably similar to that in Crohn’s disease: It is a transmural infiltrative lesion that contains typical granulomata. Of considerable interest, TNF1ARE mice develop arthritis resembling rheumatoid arthritis, as well as mucosal inflammation. Studies of the immunopathogenesis of disease in TNF1ARE mice indicate that whereas the mucosal inflammation is dependent on the presence of T cells, the joint inflammation is not (36). This, plus the fact that the different TNF receptors are involved in the two kinds of inflammations, indicates that the pathogenesis of inflammation in the two areas is different. As far as the disease in the mucosa is concerned, it is likely that the inflammation is initiated and maintained by substances in the mucosal microflora that can induce TNF-α production (LPS, CpGs, etc.). However, it is important to mention that colitis does not occur in TNF1ARE mice that are also IL-12 p40–deficient (F. Cominelli, personal communication); thus, the inductive process does not appear to involve the direct stimulation of cells by microfloral stimuli, but rather their indirect stimulation via IL-12. It is important to mention here that IL-12 production may be enhanced in TNF1ARE mice because once TNF-α overexpression is initiated, a positive feedback loop between IL-12 and TNF-α is established.

COLITIS IN Gi2A MICE Mice deficient in the G protein Gi2α provide yet another type 1 model in which overproduction of a Th1 cytokine results in colitis. The inflammation in this model is a Th1-mediated colitis with an infiltrative histologic picture similar to other Th1 colitides (143). The basis of this colitis has been elucidated by studies showing that a stimulus that inhibits Gi protein signaling, such as pertussis toxin, enhances splenocyte production of IL-12, TNF-α, and IL10 in vitro upon culture with Staphylococcus aureus Cowan I and CD40L (144). In addition, pertussis toxin–treated BALB/c mice exhibit a healing phenotype when infected with Leishmania major, whereas untreated mice of this strain manifest progressive infection. That these findings are relevant to Gi2α mice was shown by the fact that these mice produce increased amounts of IL-12 and TNF-α when their CD8a+ (lymphoid) dendritic cells are appropriately stimulated. Thus, it is reasonable to conclude that the colitis in Gi2α-deficient mice is a type 1 colitis owing to the overproduction of IL-12. One can reasonably assume that antigens in the mucosal microflora are the driving force of such overproduction, but this needs to be verified experimentally.

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Type 2 Models of Colitis: Defects in the Production of Proteins that are Directly or Indirectly Involved in Regulation of Mucosal Responses


The mirror image of the previous category of models of mucosal inflammation are those in which there is an abnormality in the synthesis of a regulatory cytokine or a protein that affects the function of a regulatory cytokine. These are thus type 2 models and include colitis owing to IL-10 deficiency or abnormalities of IL-10 signaling and to defects in TGF-β function. COLITIS OWING TO IL-10 DEFICIENCY OR IL-10 SIGNALING DEFECTS As initially reported by Kuhn et al., IL-10–deficient mice raised in a specific pathogen-free (SPF) or a conventional environment develop colitis marked by epithelial cell hyperplasia and a transmural inflammation (145). Early on, the disease is due to a Th1 response and is completely ameliorated by anti–IL-12 treatment. Later, however, a Th2 response supervenes and the lesion is no longer treatable with anti–IL-12 (35; A.D. Levine, personal communication). The reason for this change is unclear, but it may relate to the fact that in the absence of IL-10 down-regulation, Th2 responses ultimately prevail. Further studies of the cytokines involved in the colitis of IL-10 deficiency have come from studies in which colitis was induced in SPF mice by infection with H. hepaticus. It was shown that whereas IL-12 had to be present for colitis to develop (99), IFN-γ or TNF-α did not, as treatment with anti–IFN-γ or anti–TNF-α had no effect on the colitis; it is thus apparent that each of these cytokines can mediate colitis in the absence of the other (146). Additional studies showed that treatment with both anti–IFN-γ and anti–TNF-α was also not effective in the treatment of colitis, suggesting that in IL-10 deficiency yet other cytokines induced by IL-12 may play effector roles in the absence of both IFN-γ and TNF-α. As in other models, the colitis of IL-10–deficient mice does not develop under germ-free conditions and is thus driven by antigens in the mucosal microflora (93, 99, 100). Recent studies of colitis in SPF mice infected with H. hepaticus underscore this fact. Thus, the study mentioned above showed that such infection led to greatly enhanced colitis, but another study showed that such infection led to no more colitis than that ordinarily seen under SPF conditions (99, 100, 146). This suggests that mucosal microflora (such as that present in some mouse holding areas) can influence the development of inflammation even when it is driven by a known pathogen. A final point concerning the role of the mucosal microflora in the colitis of IL-10–deficient mice is that these mice manifest increased intestinal permeability even prior to the development of overt colitis. This change in barrier function may lead to increased contact with or stimulation by antigens in the mucosal microflora and is thus a factor that facilitates the development of the inflammation (66). The basis of the immunoregulatory defect leading to colitis in IL-10–deficient mice undoubtedly lies in the fact that IL-10 has major suppressive effects on

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immune responses, and its absence in an area of the body constantly exposed to antigens leads to inflammation in that area. The negative effects of IL-10 on immune responses is well demonstrated in numerous in vitro studies showing that IL-10 inhibits IL-12 and TNF-α production, suppresses costimulatory molecules, and directly inhibits T cell proliferation and/or induces T cell apoptosis (147–150). In addition, in vivo studies of IL-10 transgenic mice and the SCID-transfer model discussed above have shown that mature CD45RBlo T cells from IL-10 deficient mice are incapable of preventing the development of colitis (137). This finding is underscored by the aforementioned studies in which Tr1 cells that produce IL-10 can substitute for CD45RBlo T cells in the prevention of SCID-transfer colitis (55). Taken together, these data provide ample reason to postulate that the colitis of IL-10 deficiency is a prototypic type 2 model of colitis owing to absence of a major regulatory cytokine. The only question that remains with respect to this conclusion is the one discussed previously in relation to TNBS-colitis concerning the relation of TGF-β and IL-10 in the regulation of mucosal inflammation. It was mentioned in that context that the evidence now available favors the view that TGF-β is the more proximal cytokine suppressor and that the main role of IL-10 is to maintain and facilitate the TGF-β suppressor effect. This leads to the supposition that in the colitis of IL-10 deficiency it is not the lack of IL-10 per se that leads to inflammation, but rather the lack of an adequate TGF-β response that occurs in the absence of IL-10. Finally, it is important to note that not only IL-10 deficiency can lead to colitis, but so can defects in IL-10 signaling that functionally are equivalent to IL-10 deficiency. This is seen in mice deficient in an “orphan” receptor termed CRF-2, which forms part of the IL-10 receptor, and in mice whose macrophages and neutrophils are deficient in STAT3 expression that exhibit a defect in IL-10 signaling (151, 152). COLITIS ASSOCIATED WITH TGF-β DEFECTS In light of the discussion above it is to be expected that deficiency in the production of TGF-β should also lead to type 2 models of mucosal inflammation. This was presaged by studies of mice with TGF-β1 deficiency owing to targeting of the TGF-β1 gene, in which it was shown that such mice exhibit widespread inflammation in multiple organs and early death (153, 154). It should be noted, however, that this inflammation is not more prominent in mucosal tissues than in other tissues, possibly because the mice die of widespread autoimmune disease before they have the chance to develop inflammation to “exogenous” mucosal antigens. A more particular relation of defects in TGF-β function to mucosal inflammation occurs in mice with defective TGF-β signaling, either only in T cells or only in epithelial cells owing to the presence of transgenes encoding dominant-negative TGF-β receptors (TGF-βRII) under T cell–specific or epithelial cell–specific promotors, respectively (75, 155). In the former case inflammation develops in the colon and lung and the mice develop autoantibodies and glomerular immune complex deposition. Interestingly, both Th1 and Th2 cytokine production is increased,

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probably because TGF-β regulates both Th1 and Th2 responses. In the latter case of the epithelial-specific expression of the dominant-negative transgene, the mice develop colitis under conventional conditions and manifest increased susceptibility to the development of dextran sulfate colitis. The development of colitis in these mice suggests that TGF-β also regulates epithelial cell function and in its absence the mucosal is more subject to stimulation by antigens in the mucosal flora.


Colitis in which the Presence of Type 2 Defects Are Traceable to Thymic Dysfunction Whereas mice with IL-2 deficiency (or IL-2R deficiency) obviously have a very different immunologic defect than bone marrow–reconstituted Tgε26 mice, the pathogenesis of the colitis associated with these defects is sufficiently similar to warrant their discussion under the same heading. Thus, in both models the inflammation is an IL-12–driven, Th1-mediated inflammation that depends on CD40L-CD40 interactions and is abrogated by the absence of IL-12 (89, 120, 121, 156). In addition, in both models there is evidence that the underlying abnormality is the defective generation of regulatory cells and that an intrathymic defect is probably responsible for this abnormality (157–159). COLITIS IN IL-2–DEFICIENT MICE Mice with IL-2 deficiency exhibit early development of lymphadenopathy, bone marrow infiltration, and hemolytic anemia indicative of a generalized autoimmune state, which are then followed in surviving mice by the development of a transmural colitis (86). The latter is a T cell–driven event that, as mentioned above, is due to a Th1 response (89). T cells in these mice bear markers of maturity and proliferation that may relate to the presence of an apoptosis defect, presumably occurring because in absence of IL-12 there is deficient activation-induced (Fas-mediated) apoptosis (86, 160). In the absence of IL-2, IL-15 may play a major role in this hyperproliferative state, but this is not supported by the relevant available in vitro studies (161). As in other models, it is clear that antigens in the mucosal microflora drive the T cells because disease does not develop under germ-free conditions (81, 86). In addition, there is some evidence that in the absence of IL-2, epithelial cells exert increased antigen-presenting function, which plays a role in T cell activation and cytokine secretion (162). The pathogenesis of colitis in IL-2–deficient mice has been successfully studied by inducing the rapid onset of colitis by intra-peritoneal injection of TNP-KLH in Freund’s adjuvant (89). This maneuver apparently stimulates T cells that crossreact with antigens in the mucosal microflora and mediate disease. Studies in this induced model of mucosal inflammation disclosed that whereas normal mice react to TNP-KLH stimulation with an IL-4/ TGF-β response, this response is absent in IL-2 deficient mice (89). Furthermore, they showed that concomitant administration of anti-CD3 does elicit an IL-4/TGF-β response and prevents development of disease. That such prevention is due to the TGF-β response and not the IL-4 response was shown by the fact that the protection obtained with anti-CD3 treatment was abrogated by simultaneous treatment with anti-TGF-β but not with

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anti–IL-4. Thus, these studies led to the conclusion that the underlying cause of colitis in IL-2 deficiency is a type 2 defect and an inadequate regulatory response (157). Further studies of immune function of IL-2–deficient mice as it relates to colitis focused on thymic function because it was known that thymic defects contribute to the autoimmunity seen in these mice (159). Again using the TNP-KLH to induce colitis in IL-2 deficient mice, it was shown that this stimulus leads to the appearance of increased numbers of single positive thymocytes in the thymus that displayed a Th1 cytokine secretion profile and transferred colitis to normal mice (159). These studies thus suggest that thymocyte development is defective in IL-2 deficiency and this defect leads to either increased numbers of effector cells capable of mediating either autoimmunity or colitis or to decreased numbers of regulatory cells capable of preventing these phenomena. Given the above role of regulatory cell dysfunction in IL-2–deficient mice, the latter rather than the former is likely to be the more important factor. Finally, it is important to mention that mice with IL-2R deficiency owing to either α or β chain gene targeting also develop autoimmunity and colitis (163–165). However, those with IL-2R deficiency owing to γ chain targeting do not, presumably because such mice cannot mount adequate T cell responses to support autoimmunity. IL-2Rβ chain–deficient mice differ somewhat from IL-2–deficient mice in that they display hypergranulopoiesis that crowds out normal marrow elements and leads to massive lymphoid infiltration with granulocytes (164, 165). In addition, they manifest poor responses to antigen, presumably because their cells respond poorly to both IL-2 and IL-15. This raises the question as to which cytokine is driving their autoimmune responses and leads to the possibility that in IL-2–deficient mice neither IL-2 nor IL-15 is necessary to support T cell responses. COLITIS IN BONE MARROW–RECONSTITUTED Tgε26 MICE Tgε26 mice are mice bearing a transgenic CD3-epsilon chain whose over-expression results in intrathymic T cell and NK cell death probably because of excessive signal transduction during thymic development (158, 166). In addition, they manifest a secondary defect in thymic stromal architecture because the development of the latter depends on the presence of normal thymocytes (167). Fetal mice bearing the transgene can be rescued by transplantation of T cell–depleted normal bone marrow because such transplantation preserves stromal architecture; in contrast, adult mice cannot be thus rescued because by this time the defect in architecture cannot be reversed (158, 167). Thus, whereas bone marrow reconstitution of adult mice leads to repair of lymphoid depletion, the reconstituted mice contain a cell population that has developed in a defective thymic micro-environment. Mice with the Tgε26 defect who are reconstituted with normal bone marrow (reconstituted Tge26 mice) develop an infiltrative colitis similar to that seen in IL-2 deficiency, which is due to an IL-12 driven Th1-mediated response driven by antigens in the mucosal microflora (158, 168). The IL-12 dependency of the inflammation is nicely shown by the fact that reconstitution of the mice with bone marrow from STAT4-deficient mice exhibit a greatly reduced level of disease, as do

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mice treated with anti–IL-12 (156). It should be noted, however, that reconstitution of mice with bone marrow from IFN-γ –deficient mice only slightly ameliorates the development of colitis, presumably because in this situation, as in the case of TNBS colitis and IL-10 deficiency colitis, TNF-α can induce inflammation in the absence of IFN-γ (156). In studies of the underlying factors leading to colitis in Tgε26 mice, cell transfer studies were performed using mice with and without transplantation of syngeneic normal fetal thymus (158). These studies showed first that transfer of nonmucosal cells from Tgε26 mice with colitis into untransplanted Tgε26 recipients resulted in colitis similar to that in the donor mice. It was then shown that Tgε26 mice reconstituted with normal bone marrow and transplanted with syngeneic fetal thymus (from Tgε26 mice) did not develop colitis, whereas those only reconstituted with bone marrow developed colitis. Because the transplanted fetal thymus maintained a normal architecture in mice reconstituted with normal marrow, the mice with the transplants could generate a cadre of normal cells that then intermixed with the abnormal cells arising from the abnormal thymus. Thus, these studies suggest that the abnormal cell population causes colitis because it lacks a regulatory cell population and that the colitis is a type 2 colitis. The above discussion makes it apparent that in IL-2 deficient mice and in bone marrow–reconstituted Tgε26 mice abnormal T cell development in the bone marrow is a major factor in the development of colitis. One theoretical difference, however, is that in the induced IL-2 deficiency model studied, the inducing antigen (TNP-KLH) is exogenous, whereas in reconstituted Tgε26 no exogenous antigens are introduced. This difference may be more apparent than real, however, because it is possible that mucosally derived antigens normally enter the thymus and affect thymic selection. Another difference is that in IL-2 deficiency the development of colitis may occur solely because of local mucosal dysregulation, whereas in reconstituted Tgε26 mice the thymus appears to be more intrinsic to the disease state.

Miscellaneous Models of Colitis: Th1 Responses Whose Underlying Immunopathogenesis is Not Understood Several models of mucosal inflammation have been described in which the basis of the mucosal inflammation has not yet been elucidated. In some cases these models may prove to be quite important because they may be due to one or more defects also present in humans with IBD. Two of these models are described in some detail below and the rest are summarized in Table 4. A substrain of LPS-unresponsive mice [lacking tolllike receptor 4 (TLR-4)] C3H/HeJ mice, termed C3H/HeJBir mice, have been noted to spontaneously develop a colitis centered in the cecum and proximal colon (169). The colitis consists of an transmural inflammation that begins at 3–6 weeks and gradually wanes. It is an IL-12–driven, Th1-mediated inflammation that can be transferred to SCID recipients by CD4+ T cells from the affected mice.

COLITIS IN C3H/HeJBir MICE

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INSIGHTS INTO IBD TABLE 4 Miscellaneous Th1 and Th2 models of mucosal inflammation Salient/unique immunopathologic features


Model

Type

Proposed mechanism

Reference

TCR transgenic mice with lymphopenia

Similar to SCID-transfer colitis; presence of Tg T cells that cross-react with mucosal antigens in most cases.

Th1, ? Type 2

Failure of intrathymic development of regulatory cells

204

IL-7 transgenic mice

Infiltrative lesion with crypt abscesses and loss of goblet cells; IL-7 over-expression only in involved areas.

Th1, type 1 Epithelial barrier defect?

Defective epithelial barrier function; activation of mucosal macrophages

102

Over-expression of HLA-B27

Inflammation of stomach as well as small and large intestine; joint inflammation

Th2, type 1;

Facilitated presentation of mucosal antigens to mucosal T cells; CD8+ T cell-mediated inflammation?

205, 206

NF-κB defects; “A20” mouse; Iκβα-deficiency

Inflammation in multiple organs including intestine

Type 1

Hypersensitivity to NF-κB activators; inability to regulate NF-κB response

207, 208

p50 deficiency

Typhlo-colitis; apparently normal T cell development

Type 2?

Defect in NF-κB pathway

209

Th2 TNBS-colitis

TNBS-colitis in Balb/c mice or C57BL/6 mice with IL-12 deficiency

Th2 colitis; Th1 component?; type 1

TNBS-induced, Th2 response to mucosal antigens in mice oriented to Th2 responses

210, 211

Colitis in WiskottAldrich syndrome protein (WASP) deficiency

Mild immunodeficiency; superficial inflammation reminiscent of UC rather than CD

Th2 colitis; Type 2?

Abnormality of regulatory cell development?

212

Abbreviations: TCR, T cell receptor; TNBS, trinitrobenzene sulfonic acid; UC, ulcerative colitis; CD, Crohn’s disease.

The focus of research utilizing this model has been to define the relation of bacterial flora to the induction of disease. A series of studies with this in mind showed that cells from C3H/HeJBir mice manifest increased B cell and T cell reactivity to mucosal antigens (92, 170). However, this reactivity was selective and was more or less limited to antigens associated with several species of facultative anaerobes. This corresponded to the fact that T cells in colitic C3H/HejBir mice displayed a skewed Vβ distribution (as do T cells in other models including the SCID-transfer model and the TCR-α chain–deficiency model) (92). It should be noted, however, that although the number of antigens implicated in the response is a small fraction of the total number of antigens, it is nevertheless a large number; thus, it is highly unlikely that the colitis is due to only a very limited number of antigens. A similar situation probably obtains in humans, in which a skewed expression of Vβ has also been seen.

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Recent cell transfer studies of the C3H/HeJBir model have disclosed that T cell lines driven by antigens in bacterial lysates also transfer colitis to SCID mice (91). This finding is a direct demonstration that antigens in the mucosal microflora can mediate colitis and corroborates data from studies of numerous other models that show this more indirectly. Whereas some of the lines were composed of effector cells secreting IFN-γ , others were slow-growing lines producing IL-10 and were presumed to be regulatory cells. Indeed, these cells inhibited Th1 responses both in vivo and in vitro. Thus, a reiteration of studies performed in the SCID-transfer model showed that these cell lines, when cotransferred with effector cells, could prevent the development of colitis in SCID recipients (93). Interestingly, this preventative effect was reversed by either IL-10 or TGF-β, again raising questions about the relationship of these regulatory cytokines. In any case, the presence of regulatory cells in the colitis of C3H/HeJBir mice suggests that the underlying defect in these mice is a partial block in the regulatory cells’ development and that the inflammation gradually subsides when these cells finally make their appearance. One issue raised by the occurrence of colitis in a substrain of mice that does not respond to LPS is the role of this stimulant in the regulation of mucosal immune responses. It appears paradoxical that a defect in the capacity to respond to a strong stimulator of the IL-12 response would be associated with colitis. This paradox, however, is resolved by the fact that numerous other substances associated with the mucosal microflora have this capacity as well. Perhaps a more cogent and specific role for LPS in colitis relates to the possibility that this stimulant is necessary for the normal induction of regulatory cells. COLITIS IN SAMP1/Yit MICE SAMP1/Yit mice were originally derived from AKR mice by extensive interbreeding, first to achieve accelerated senescence and then to enhance the development of intestinal inflammation (37). This model is important because the inflammation is remarkably similar to human Crohn’s disease in that it is mainly an ileitis rather than a colitis, and at the microscopic level ones sees typical granulomata and other features of Crohn’s inflammation. Once again the disease is driven by antigens in the mucosal microflora and is a Th1 event because it can be transferred to SCID recipients by T cells producing IFN-γ and TNF-α (37, 38). Interestingly, the transferred cells produce a disease similar to that in the donor mice, suggesting that they have a homing pattern governed by their site of origin or are expanded by antigens specifically present in the ileum. The underlying defect in SAMP1/Yit mice is not yet known. However, recent studies showing that epithelial cells in these mice produce increased amounts of chemokines suggest the presence of a type 1 defect (171).

MODELS OF COLITIS DUE TO EXCESSIVE Th2 T CELL RESPONSES Experimental colitis mediated by Th2 T cells forms a separate universe of colitides that, as discussed above, are associated with a form of inflammation that differs from that seen in the more predominant Th1 colitides in that it more closely

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resembles ulcerative colitis than Crohn’s disease (23). This has given rise to the idea that ulcerative colitis is in fact a Th2 T cell–mediated disease, but the evidence for this notion is ambiguous at best. Thus, whereas IL-12/IFN-γ production is not increased in ulcerative colitis, neither is IL-4 production, and the inflammation in lesional tissue is not usually characterized by Th2 inflammatory elements such as eosinophils and mast cells. In fact, the only Th2 cytokine reported as increased in ulcerative colitis is IL-5 (39–42), but this increase could be due to the presence of certain types of regulatory cells that produce IL-5 rather than to Th2 effector cells (i.e., Tr1 T cells). At the moment, therefore, it is premature to call ulcerative colitis a Th2 disease, despite its histopathologic relation to Th2 T cell–mediated experimental colitides. In the following discussion we review the most extensively studied Th2 models; pertinent data about several additional Th2 models is provided in Table 4.

Colitis in TCR-α Chain–Deficient Mice The first and perhaps best studied model of murine inflammation owing to a Th2mediated response was initially reported by Mombaerts et al., who noted that chronic colitis develops in gene targeted mice lacking TCR-α chains (51). These authors also found that TCR-β chain–deficient mice develop only very mild colitis, but recently it was shown that this colitis is more marked if CD5 deficiency is also present (172). Finally, they showed that mice with γ δ chain deficiency do not develop colitis. The colitis developing in TCR-α chain–deficient mice is relatively superficial and extends to the submucosa only occasionally. It is characterized by the presence of elongated and distorted crypts as well as by the presence of occasional crypt abscesses, but transmural fissures and granulomata are notably absent. Overall the lesion is different from that seen in Th1 models of colitis and resembles ulcerative colitis rather than Crohn’s disease. This fits with the fact that affected mice frequently develop circulating anti-neutrophil cytoplasmic antibodies (ANCA) and other antibodies found in ulcerative colitis patients (107, 173). Initial studies of the cell populations in TCR-α chain–deficient mice revealed that the main cells were γ δ TCR-bearing T cells, but these were admixed with a small population of alpha-beta+ (dim) TCR-bearing T cells (ββ TCR T cells), which later proved to be the effector cells responsible for the inflammation (49, 51). In subsequent studies involving treatment of TCR-α chain–deficient mice with anticytokine antibodies and cross-breeding of the mice with various cytokinedeficient mice, it was established that IL-4 and not IFN-γ was the effector cytokine, i.e., the colitis was a Th2 colitis (174–176). The ββ TCR T cells established as effector T cells in TCR-α chain colitis recognize antigens via a unique TCR composed of ββ homodimers, and it was therefore not too surprising that they display greatly restricted TCR diversity following the development of colitis (34). Thus, whereas T cells obtained from mice prior to the development of colitis or from mice on a non–disease-producing elemental diet display a wide range of Vβ family usage, those with colitis or on a regular diet

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display skewed Vβ usage marked by Vβ 8.2 predominance (34). In addition, it was found in single-strand conformation polymorphism (SSCP) analysis and CDR3 sequencing studies that T cells from colitic mice display mono- or oligoclonality in all T cell subsets, not just the Vβ 8.2 T cell subset. Finally, it was shown that T cell subsets expressing Vβ 8.2 exhibiting restricted diversity are characterized by a restricted CDR3 length and conservation of a single negatively charged amino acid in the second portion of the CDR3 sequence (33). This type of amino acid sequence is characteristic of clones specific for self-antigens and thus may represent a “germ-line” sequence that is cross-reactive with a variety of normally nonstimulatory environmental (mucosal) antigens, i.e., antigens that do not elicit effector cell responses in the mucosal immune system. This possibility is supported by the fact that cells with the stereotypic TCRs can be expanded by coculture with colonic epithelial cells, which presumably are presenting antigens derived from resident (nonpathogenic) microflora (33). It is also supported by the facts that ββ TCR T cells display vigorous responses to food antigens and that mice with TCR-α chain deficiency exhibit a heightened capacity to provide helper function for B cells that produce antibodies reacting to food antigens (34, 175). Finally, it is supported by the fact that TCR-α chain–deficient mice fed an elemental diet do not develop disease unless they are mono-infected with certain organisms (such as B. vulgatus) that are presumably among the organisms expressing cross-reacting antigens (97). The above data, considered as a whole, lead to the conclusion that whereas ββ TCR T cells may have a restricted ability to respond to antigens in general, they do respond to certain normally harmless antigens that then drives the cells to expand and exert effector cell activity causing disease. This raises the question of why these T cells have this propensity, but other T cells normally populating the mucosal tissues do not. One possibility already suggested by the TCR sequence data is that these cells have escaped negative selection in the thymus (or other selection areas exisiting in the mucosa) and thus represent a cadre of self-reactive cells that cross-react with mucosal antigens. This possibility finds strong support in independent studies showing that ββ TCR T cells have a tendency to escape negative selection in the thymus (177, 178). A second possibility relates to the fact that, as discussed above, mucosal responses are normally regulated by tolerogenic mechanisms, including the development of αβ TCR T cells that produce suppressive cytokines. Thus, it is reasonable to suggest that the abnormal reactivity of ββ TCR T cells to certain mucosal antigens is due to the fact that regulatory T cells cannot develop within the ββ TCR T cell population (nor in the accompanying γ δ TCR T cell population). A second, interrelated question concerns the reason why ββ TCR T cell stimulation leads to Th2 responses and not Th1 responses. One possibility alluded to in the general discussion of models is that the course of T cell differentiation depends largely on the nature of the antigen-presenting cell and the cytokine environment of the APC-T cell interaction. In this context it is already known that certain dendritic cells present in the Peyer’s patches (CD11c+ dendritic cells) preferentially

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secrete IL-10 rather than IL-12 and induce Th2 T cells rather than Th1 T cells during antigen presentation (70, 71). Thus, it is possible that the types of antigens that stimulate ββ TCR T cells are generally the types of antigens that are taken up by dendritic cells in Peyer’s patches that lead to Th2 T cell differentiation. Another, non–mutually exclusive, possibility is that ββ TCR T cells manifesting a Th2 phenotype have better survivability than their Th1 counterparts, again because of the cytokine milieu in which they develop. This possibility derives from the observation that stimulation of T cells from TCR-α chain–deficient mice with epithelial cells both under Th1 and Th2 conditions leads to poorer survival in the former instance than in the latter (33). Moreover, the surviving Th2 T cells display evidence of oligoclonality and can transfer disease, whereas the Th1 T cells do not. These data suggest not only that antigens stimulating ββ TCR T cells only do so under a Th2 condition but also that such stimulation under a Th1 condition leads to a different pattern of clonal stimulation and T cells that are subject to apoptosis. A final point to emerge from the study of TCR-α chain–deficient mice relates to the role of B cells in the pathogenesis of this model of inflammation and, by extension, in ulcerative colitis. In particular, it was found that double mutant TCR-α chain–deficient µIg-deficient mice somewhat paradoxically develop more severe colitis than single mutant TCR-α chain–deficient mice (63). Furthermore, transfer of mesenteric lymph nodes (MLN) cells from the double mutant to Rag-2– deficient mice produced colitis in the latter, which was abolished by the cotransfer of B cells and the coadministration of purified Ig or monoclonal antibodies reactive with colonic epithelial cells from the TCR-α chain–deficient mice. This decreased disease with B cells or B cell products (autoantibodies) was associated with decreased numbers of apoptotic cells in the epithelium and lamina propria and was attributed to decreased clearance of these cells mediated by the autoantibodies (63). Another explanation, however, is that the B cells produce regulatory cytokines that suppress disease (and also affect apoptosis). Indeed, preliminary studies suggest that B cells express high levels of CD1d and secrete IL-10 (A. Mizoguchi, R.J. Blumberg & A. Bhan, personal communication). In any case, these studies show that B cells or the autoantibodies they produce do not play a pathogenic role in TCR-α chain–deficient mice and may actually ameliorate disease. In addition, this suggests that autoantibodies in ulcerative colitis are likewise nonpathogenic.

Oxazalone Colitis Whereas administration of TNBS to SJL/J mice leads to colitis driven by polarized Th1 T cell responses, administration of another haptenating agent, oxazalone, leads to a colitis caused by a polarized Th2 T cell response. Oxazalone colitis, however, is a considerably different disease than its TNBS counterpart (31). First, when administered intrarectally without prior sensitization, it develops more quickly and resolves more quickly than TNBS colitis, usually within 4–5 days; in addition, it

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produces a more superficial inflammation that affects the distal half of the colon rather than the whole colon. Finally, rather than producing an intense infiltrative inflammation that obliterates villous architecture, it produces inflammation that generally maintains villous architecture but is associated with bowel wall edema and luminal exudates. Overall, the lesion is more reminiscent of ulcerative colitis than Crohn’s disease, but the examination of a more chronic oxazalone colitis would be necessary to verify this view. If mice are presensitized with subcutaneous oxazalone, a more chronic lesion ensues that lasts on the order of 1–2 weeks; this lesion retains the characteristics described above for the more acute lesion and lends credence to the idea that oxazalone colitis is indeed an ulcerative-colitis-like inflammation (F. Scheiffele, I. Fuss, W. Strober, unpublished observations). The cytokine response of oxazalone colitis is also very different from that in TNBS colitis (31). It is dominated by a high IL-4 and IL-5 response, but a normal or reduced IFN-γ response. This Th2 response is in fact the cause of the inflammation, as shown by the fact that anti–IL-4 administration abolishes disease, whereas an anti–IL-12 administration exacerbates the disease and causes a pancolitis. Another notable feature of the cytokine response in oxazalone colitis is a marked TGF-β response that is higher in the proximal colon than in the distal colon. In fact, the high TGF-β response may be responsible for the short duration of disease as well as its limitation to the distal colon. This is suggested by the fact that, as mentioned above, TGF-β production is higher in the proximal colon, as well as the fact that anti–TGF-β treatment leads to severe pancolitis. The reason lamina propria cells in SJL/J mice respond to oxazalone with a Th2 response rather than a Th1 response is unclear. One cannot invoke the idea that the presence of T cells with abnormal TCRs that only respond to antigens that induce Th2 differentiation because there is nothing to show that the T cell profiles of the responding mice are abnormal. A more likely possibility arises from emerging evidence that during the induction of oxazalone colitis, oxazalone is presented to T cells by APCs in the context of an atypical MHC class I molecule, CD1d, and that the interacting T cell is an NK T cell that is the effector cell causing the colitis (F. Scheiffele, I. Fuss, W. Strober, unpublished observations). Thus, one might postulate that this somewhat unique interaction preferentially results in Th2 T cell differentiation. Some evidence in support of this possibility is inherent in older studies showing that NK T cells have a propensity to produce IL-4, as well as newer studies showing that mice with a targeted deletion of the chemokine receptor, CCR5, when challenged with dextran sulfate sodium (DSS) to produce DSS colitis develop lesions containing T cells producing IL-4 (179). Whereas the reason mice with this deletion manifest this kind of response is not really known, one might postulate that the absence of CCR5 leads to decreased Th1 responses and thus the preferential expansion of NK T cells that inherently produces Th2 cytokines. It should be noted that in CCR5-deficient mice, DSS colitis is less severe than in normal mice, indicating that the NK T cells developing in this situation appear to be regulatory cells. This is in contrast to the situation in oxazalone colitis where, as mentioned, the NK T cells are effector cells. Whether NK T cells act as

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regulatory cells in the context of DSS colitis because they produce IL-4 or because of other factors remains to be determined. Aside from the nature and origin of the effector cell causing disease in oxazalone colitis is the question of the downstream inflammatory cytokines causing disease. One possibility that requires further study is that oxazalone colitis leads to production of IL-4, which in turn leads to the secretion of other cytokines, such as IL-9 and IL-13.

MODELS OF COLITIS RELATED TO DEFECTS IN EPITHELIAL CELL BARRIER FUNCTION A number of diverse models of colitis have been discovered that are due to defects in epithelial cell barrier function. It should be emphasized, however, that such defects in the present context are broadly defined to include both barrier function relating to permeability to macromolecules and barrier function involving processes that enable the intestinal epithelial cell to secrete immune mediators. The latter type of defect could take the form of inadequate secretion of mediators that thereby increases the exposure of the mucosal system to antigens in the mucosal microflora or to excessive secretion of mediators and the initiation of inflammation by the stimulation of “professional” cellular secretors of inflammatory cytokines (macrophages). In addition to the models described below, two models already discussed (colitis associated with IL-2 deficiency and with IL-10 deficiency) have been shown to have abnormal epithelial cell barrier function. In these cases it is likely that the latter is secondary to a more primary abnormality as it is known that both Th1 and Th2 cytokines can influence barrier function in various ways.

Colitis Associated with Dominant-Negative N-Cadherin Expression Cadherins are transmembrane glycoproteins that mediate adherence between many cell types including intestinal epithelial cells. On the cytoplasmic side they bind to the cytoskeleton via interactions with β-catenin and on the cell surface, and they enter homophilic interactions with cadherins on neighboring cells (180). Recently, Hermiston & Gordon created a model with disrupted epithelial cell cadherin function by expressing a dominant-negative N-cadherin in epithelial cells that interfere with normal expression of E-cadherin (64). In particular, they inserted embryonic stem cells with an N-cadherin gene lacking an extracellular domain under the control of a small intestinal epithelial cell promotor (the fatty acid binding protein promotor) into blastocysts to obtain chimeric mice that displayed patches of epithelial cells with poor cell-cell adhesion. The chimeric mice developed transmural cellular infiltration, cell crypt abscesses, goblet cell depletion, and both apthous and linear ulcers in lamina propria areas subjacent to the epithelial patches containing

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cells with defective adherence but not in areas subjacent to epithelial patches with normally adherent epithelial cells. The most reasonable explanation for this pattern of inflammation is that in areas of the mucosa where there is breakdown of the epithelial barrier (owing in this case to defective cell-cell adhesion) there is excessive exposure of mucosal lymphoid elements (normal mucosal microflora), which subsequently leads to a nonhomeostatic immune response and mucosal inflammation. This picture is thus not unlike that in TNBS colitis, in which the introduction of TNBS in the presence of the substance (ethanol) that disrupts the mucosal barrier leads to an unbalanced immune response and subsequent inflammation. As to the question of why such exposure leads to inflammation, it can be postulated that any exposure of the mucosal immune system to antigens in a manner that bypasses the Peyer’s patches leads to an inadequate regulatory T cell response because such cells preferentially develop in the organized lymphoid tissue of the mucosa. Finally, because the N-cadherin dominant-negative model of inflammation occurs in the vicinity of porous epithelial cells despite the fact that the microbial microflora are identical in both nonporous and porous areas, it is an exquisite demonstration of the fact that antigens in normal mucosal microflora are sufficient for the induction of responses that lead to disease.

Colitis in mdr1a-Deficient Mice A second and equally interesting model of colitis related to barrier function is a mouse model characterized by deficiency in the mdr1a gene (65). This gene is one of several “multiple drug-resistant” (mdr) genes expressed in many cells (including epithelial cells) that belong to a family of transporter proteins that pump small amphiphilic and hydrophobic molecules out of the cell and thus confer drug resistance (181, 182). This model was created because the gene encoding the mdr1a transporter is present in a region of the human genome that is thought to harbor a disease gene that leads to inflammatory bowel disease (183, 184). Bone marrow transfer studies involving wild-type donor cells into mdr1a-deficient recipients demonstrated that the colitis develops in mdr1a-deficient mice because of the deficiency of mdr1a in epithelial cells rather than in lymphoid or myeloid cells (65). Thus, the model allows one to focus on the role of epithelial cells in mucosal inflammation. Colitis developing in mdr1a-deficient mice is a spontaneous colitis consisting of a transmural T cell and B cell infiltration that is similar to that found in Crohn’s disease (despite the fact that it has been called a model of ulcerative colitis). It is important to mention, however, that the epithelial cells in mdr1a-deficient mice are arrayed in long, dysregulated crypts that are associated with crypt abscesses and surface ulcerations (65). The Crohn’s disease–like picture was borne out by the presence of an mRNA cytokine profile indicative of a Th1-mediated inflammation (J. Viney, personal communication). Recently, it has been shown that mucosal organisms can profoundly influence epithelial cell function with respect to cytokine/chemokine secretion. This influence can be negative in that epithelial cell interactions with certain nonpathologic

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organisms leads to a block in ubiquitination of Iκβα (necessary for Iκβα degradation) and thus a subsequent block in NF-κB translocation to the nucleus (185). This influence can also be positive because bacterial flagellin, signaling through TLR-5, leads to NF-κB expression (186). If these events are influenced or caused by bacterial products entering the cell, we can see how epithelial cell function can be negatively or positively impacted by a defect in a transporter mechanism. Furthermore, we can understand how such negative or positive perturbation could lead to increased epithelial cell production of chemokines and cytokines that lead to the influx of inflammatory elements into the epithelial layer. Thus, it seems possible that the mdr1a-deficient mouse does not develop inflammation because of increased epithelial layer permeability per se but because of increased bacterially induced activation of epithelial cells.

Dextran Sulfate Colitis Yet another model of colitis that is at least partially related to a change in epithelial cell barrier function is the colitis induced by the physical agent, dextran sulfate sodium. This is a relatively old model that has been frequently used to study the efficacy of potential therapeutic agents because of its ease to induce via administration of DSS in drinking water and because DSS induces a consistent level of colitis with a defined onset (18–22). As mentioned in the Introduction, DSS colitis can be induced in Rag-2–deficient or thymectomized mice (22). This argues that the mechanisms of inflammation in this form of colitis are, at least initially, the activation of nonlymphoid cells such as macrophages and the release of pro-inflammatory cytokines (187, 188). Changes in epithelial barrier function as measured by permeability of the intestinal wall to Evan’s blue can be found early (several days before the onset of frank inflammation) (19) and thus may set the stage for macrophage activation. The relation of DSS colitis to epithelial barrier function is further suggested by the fact that administration of DSS to mice with deficiency of intestinal trefoil factor, a factor important to maintenance and repair of the epithelial layer, leads to a far more severe colitis than observed in normal mice (189). However, this may be a result of the fact that epithelial cell layer integrity plays a role in the initiation of DSS colitis as indicated above, or because reestablishment of such integrity is a condition of colitis resolution. In the acute stages of DSS colitis the (secondary?) T cell response consists of a polarized Th1 response, but in later and more chronic phases of the inflammation, a mixed Th1/Th2 response occurs (188). In either case, DSS elicits the secretion of large amounts of TNF-α and IL-6, which are mainly responsible for the tissue damage in the disease. Whereas antigens in the mucosal microflora probably play a role in the production of DSS colitis, it has recently been shown that they also play a role in the suppression (and resolution) of the colitis. This is shown by the fact that mice administered α-galacocylceramide (a glycolipid antigen that activates NK T cells when presented to them in the context of CD1d, a nonclassical MHC class I antigen-presenting molecule expressed on epithelial cells and other APCs)

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manifest decreased DSS colitis as compared with mice administered a control glycolipid without these properties. This improvement is not seen in mice deficient in CD1d or in Rag−/− negative mice deficient in T cells (and NK T cells) (61). Finally, by confocal microscopy the administered α-galactocylceramide could be localized to the epithelium. These studies reveal that NK T cells stimulated by glycolipid antigen play a protective role in DSS colitis that is not unlike their role in the SCID colitis model mentioned above. The mechanism of this role is not yet defined, but given the localization of the stimulating antigen to the epithelium, the NK T cells may be secreting substances that reestablish epithelial integrity or regulate other aspects of the mucosal environment that normally drive the colitis in this model. As discussed in relation to NK T cells found in CCR5-deficient mice, this may involve the secretion of Th2 cytokines.

TREATMENT OF MODELS OF MUCOSAL INFLAMMATION The fact that models of mucosal inflammation, whatever their underlying cause, resolve themselves into either Th1 or Th2 T cell–mediated inflammation has led to the recognition that models of vastly different etiologies can be treated with agents that block these final common pathways at any of a variety of points. As shown in Figure 2, “points of attack” in the Th1 pathway can readily be identified and can be used to block the pathway in both models of inflammation and human Crohn’s disease. A similar diagram can be drawn with respect to the Th2 pathway, which can be applied to Th2 models of inflammation and perhaps human ulcerative colitis. Only the broad outline of such treatment approaches can be discussed in this review. With respect to Th1-mediated inflammation, the use of agents that block IL-12 secretion or IL-12 activity provides the most direct approach because, as we have seen, depriving Th1 cells of IL-12 leads to their apoptosis (32, 123). It should be noted that not only anti-IL-12, but also other agents that downregulate IL-12 secretion are possible therapeutic agents in this context (190, 191). A related kind of therapy involves the use of anti–TNF-α antibody and soluble TNF-R agents that, as discussed above, block the Th1 response at both the inductive and the effector phases of the response. This approach has already proven useful in the treatment of human Crohn’s disease (192). A parallel approach to Th2-mediated inflammation is more problematic in that although anti–IL-4 may be an effective treatment of Th2 models, it does not apply to human ulcerative colitis because this disease has not been shown to be caused by IL-4 dysregulation. A promising alternative approach is to target IL-6R with the use of an anti–IL-6 receptor antibody (193, 194). Such therapy blocks IL-6 “transsignaling” and leads to the apoptosis of both Th1 and Th2 T cells. Thus, it is theoretically applicable to both Th1- and Th2-mediated inflammation. It is also possible that models of inflammation can be treated with regulatory (suppressive) cytokines such as IL-10 or TGF-β. In studies conducted so far IL-10 has been applied with mixed success and, likewise, has been only marginally effective in human IBD (195, 196). The problem may be one of cytokine localization

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Figure 2 As shown in this diagram of Th1 T cell–mediated mucosal inflammation, the Th1 pathway can be “attacked” (i.e., inhibited or disrupted) at many different points, each representing a potential means of therapeutic intervention. These points are defined as follows: 1) inhibitors of IL-12/IL-18 (i.e., anti–IL-12, rCT-B, or β-agonists); 2) inhibitors of DC-T cell interaction (i.e., anti–CD40L or anti–CD134); 3) inhibitors of TNF-α (i.e., anti– TNF-α and TNF-αR); 4) IL-10 and TGF-β (i.e., TGF-β or IL-10 plasmids or administration of Th3 or Tr1 cells); 5) inhibition of IL-6 trans-signaling, anti–IL-6R; 6) NF-κB inhibitors; 7) inhibitors of homing or adhesion (i.e., anti-α4β7, anti-αEβ7, anti-CD44v7, or anti-sense oligos to ICAM1); 8) downstream inhibitors of TNF-α (i.e., phosphodiasterase inhibitor 4, pentoxyphylline, thalidomide, or metalloproteinase inhibitors).

because there is one report that IL-10 delivered by a Lactococcus lactis organisms was effective in two forms of experimental colitis (197). The use of TGF-β has been explored in studies in which plasmids encoding TGF-β are administered intranasally (29). As mentioned above, this leads to cells producing TGF-β that migrate to mucosal tissues that are capable of reversing established TNBS colitis. A possible objection to this approach is that TGF-β can induce fibrosis; however, the plasmid also induces IL-10 secretion, which appears to suppress TGF-β–induced fibrosis (198). Other approaches to the treatment of models of mucosal inflammation include the use of agents that target homing and localization of inflammatory cells. This

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includes antibodies to integrins (anti–MadCAM-1, anti-α Eβ 7 and anti-CD44v7 as well as anti-sense oligonucleotides that interfere with integrin synthesis (199– 201). Finally, attempts to control the mucosal inflammation by the use of agents that block the NF-κB pathway have been tested with some success in murine models (202–203). The question is whether such therapy will cause unacceptable toxicity when applied to humans.


CLOSING CONSIDERATIONS In this review of models of mucosal inflammation we have sought to emphasize recurrent characteristics of the models that allow them to be understood within a more or less consistent framework. This is perhaps best encapsulated by the fact that the models are invariably associated with one or another genetically determined or induced immune imbalance that ultimately expresses itself as a type 1 defect owing to excessive effector cell response or a type 2 defect owing to an inadequate regulatory cell response. Furthermore, in both the type 1 and type 2 defects, the response takes the form of either a Th1 or Th2 T cell-mediated inflammation that is driven not by antigens associated with exogenous pathogenic organisms but by antigens associated with the normal mucosal microflora. Given the fact that such antigens are the equivalent of self-antigens, the models may thus be visualized as a special type of autoimmunity that takes on a somewhat unique form because it involves effector and regulatory cell mechanisms that are characteristic of mucosal responses. The impact of the knowledge gained from the study of models of inflammation on the understanding of human IBDs is difficult to exaggerate. Thus, it is fair to say that the framework used to visualize the pathogenesis of these diseases is currently derived largely from the murine models and, in turn, new patient-oriented research is mainly motivated by one or another aspect of the models. This includes research on new treatments of the disease that are either suggested by the models or are tested in the models. Looking ahead to the emerging area of genetic research in IBDs, the models will be an essential tool in the identification of genes that determine susceptibility and resistance to these diseases and thus the genes that will enable their genetic manipulation. Visit the Annual Reviews home page at www.annualreviews.org

LITERATURE CITED 1. Strober W. 1985. Animal models of inflammatory bowel disease—an overview. Dig. Dis. Sci. 30:(Suppl.):3–8 2. Kim HS, Berstad A. 1992. Experimental colitis in animal models. Scand. J. Gastroenterol. 27:529–37

3. Kirsner JB. 1961. Experimental “colitis” with particular reference to hypersensitivity reactions in the colon. Gastroenterology 40:307–12 4. Kraft SC, Fitch FW, Kirsner JB. 1963. Histologic and immunohistochemical

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T CELL MEMORY Jonathan Sprent and Charles D. Surh Annu. Rev. Immunol. 2002.20:551-579. Downloaded from arjournals.annualreviews.org by HINARI on 09/01/07. For personal use only.

Department of Immunology, IMM4, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037; e-mail: [email protected], [email protected]

Key Words T cells, memory, activation, survival, homeostasis, IL-15, IFN-I ■ Abstract Typical immune responses lead to prominent clonal expansion of antigen-specific T and B cells followed by differentiation into effector cells. Most effector cells die at the end of the immune response but some of these cells survive and form long-lived memory cells. The factors controlling the formation and survival of memory T cells are reviewed.

INTRODUCTION Exposure to infectious agents usually culminates in a state of immunological memory where secondary responses are more intense than primary responses (1–6). Memory is carried by antigen-specific T and B cells and is often lifelong. Here, we review recent information on memory T cells. The origin and differentiation of memory T cells and the factors controlling the long-term survival of these cells are discussed.

T CELLS AND THE PRIMARY IMMUNE RESPONSE Before considering how memory T cells arise during the primary immune response, it is useful to discuss some of the salient features of naive T cells in unstimulated animals.

Naive T Cells and the Preimmune Phase Prior to contact with antigen, naive T cells congregate in the secondary lymphoid tissues (spleen, lymph nodes, and Peyer’s patches) and migrate continuously from one lymphoid organ to another via blood and lymph (7–11). T cell migration through spleen is distinctly different from migration through lymph nodes and Peyer’s patches. Initial entry of T cells to the spleen is nonspecific. Like other lymphohemopoietic cells, T cells are carried into the spleen by the splenic artery and deposited in the marginal zone at the border between the red and white pulp. T (and B) cells 0732-0582/02/0407-0551$14.00

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then move selectively to the white pulp where they accumulate around central arterioles; these areas, termed periarteriolar lymphocyte sheaths (PALS), are the main T cell zones in the spleen. How T cells move into PALS from the marginal zone is unclear, although it is notable that activated T cells lacking the chemokine receptor, CCR7, reach the red pulp but are excluded from the white pulp (12). Hence, T cell entry to the white pulp may be guided by CCR7 recognition of specific chemokines expressed on stromal cells. After about 12 h, naive T cells move from the PALS into the red pulp and leave the spleen via venous blood. In contrast to the spleen, entry to lymph nodes and Peyer’s patches from the blood is highly specific and occurs when cells enter high endothelial venules (HEV). For lymph nodes, the luminal surface of HEV expresses several ligands, including an addressin, PNAd (13), and a chemokine, SLC (14). These ligands are recognized by two lymph node homing receptors on T cells, namely CD62L and CCR7, respectively. Lymph node homing receptors enable T cells to bind to and penetrate the walls of HEV and reach the T cell zone (paracortex). Thereafter, T cells migrate out of the paracortex and leave lymph nodes via efferent lymphatic vessels, followed by re-entry into the bloodstream via the thoracic duct. This process of blood-to-lymph recirculation through lymph nodes takes about 12–18 hours (15). Continuous migration of T cells through the secondary lymphoid tissues is highly important for allowing T cells to make rapid contact with antigens released from pathogens. As discussed later, antigens are presented to T cells in the form of peptidic fragments bound to major histocompatibility complex (MHC) molecules (16). For naive T cells, these immunogenic peptide/MHC complexes have to be presented by specialized antigen-presenting cells (APC), especially by dendritic cells (17). These cells are strategically positioned as a dense network in the T cell zones and are continuously scrutinized by recirculating T cells for expression of foreign peptides. In unstimulated animals, presentation of peptide/MHC complexes by dendritic cells is limited to self peptides. Being largely tolerant of self components, naive T cells ignore the self-peptide/MHC complexes on normal dendritic cells, with the result that T cells are allowed to percolate slowly through the T cell zones and then re-enter the bloodstream for further recirculation. During their normal pattern of blood-to-lymph recirculation, naive T cells are metabolically quiescent and have a prolonged lifespan (18). Despite their inert appearance, however, the longevity of naive T cells is not innate but requires continuous contact with at least two external ligands, namely self-peptide/MHC complexes on dendritic cells (19–22) and a cytokine, IL-7 (23, 24). Recognition of these ligands presumably delivers low-level signals, which keep T cells sufficiently metabolically active to avoid passive death.

APC Activation and the Role of Adjuvants In considering how naive T cells first make contact with foreign antigens, it is important to stress that the migratory properties of naive T cells do not allow

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these cells to enter the initial site of infection, e.g., the lung for a respiratory infection. Naive T cells are programmed to recognize antigen only in the T cell zones of the secondary lymphoid tissues, and for this reason, initiation of the immune response depends upon transport of antigen to these areas from the site of infection. Antigens reach the T cell zones by two main routes: via afferent lymphatics for lymph nodes and via the bloodstream for the spleen. A priori, antigens could reach the T cell zones in soluble form and be degraded into peptides in situ by dendritic cells. This possibility is unlikely, however, because the mature dendritic cells found in the T cell zones are poorly equipped to ingest and process native proteins into peptides (25). By contrast, immature precursors of dendritic cells are highly efficient at antigen processing. In light of this finding, the current view is that, for the most part, antigens are conveyed to the T cell zones after phagocytosis by immature dendritic cell precursors (26–28). These cells are scattered throughout the body, including the skin (Langerhans cells), and ingest antigens at the site of infection. The antigen-laden cells then migrate to the secondary lymphoid tissues, differentiate into partly mature dendritic cells, and localize in the T cell zones; en route, the cells process antigen into immunogenic peptides and ferry these peptides to the cell surface on MHC molecules. This scheme is well documented for lymph nodes but is less clear for spleen. Since antigen-laden dendritic cell precursors entering the bloodstream are likely to be rapidly trapped by the liver and lungs, entry of these cells into the spleen is probably inefficient. A more likely scenario is that antigens enter the spleen in soluble form from the blood and are ingested by immature dendritic cell precursors situated in the marginal zone; these precursors then move into the white pulp and differentiate into mature dendritic cells in the PALS (29). In considering the movement of antigen-bearing cells into the T cell zones, it should be stressed that MHC-bound foreign peptides displayed on fully mature resting dendritic cells are poorly immunogenic (25). T cells do respond to these ligands, but the response is abortive and is often followed by the induction of tolerance, possibly because low expression of certain costimulatory molecules, e.g., B7 and/or CD40L, on resting dendritic cells prevents T cells from upregulating Bcl-2/Bcl-XL (30–32). For optimal immune responses, dendritic cells first need to be activated. This process is controlled by adjuvants. Most infectious microorganisms contain built-in adjuvants such as lipopolysaccharide (LPS), lipoproteins, unmethylated CpG DNA, and double-stranded viral RNA (33, 34). These products are recognized by cells of the innate immune system, including dendritic cells, by a spectrum of highly conserved, germ line–encoded Toll-like receptors (TLRs) (33–35). Through association with intracellular signaling molecules such as Myd88 (36, 37), ligation of TLRs by adjuvants then leads to cell activation. For immature dendritic cell precursors, such activation has several important consequences (26, 28, 38). First, antigen-laden immature dendritic cells are induced to leave mucosal sites and migrate to the T cell zones of draining lymph nodes; mobilization of dendritic cell precursors may be guided by a shift in the expression of chemokine receptors, CCR1 and CCR5 receptors for

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inflammatory chemokines being replaced by CCR7 and other receptors for lymphoid chemokines (38). Second, stimulation of dendritic cells causes upregulation of essential costimulatory molecules for T cells such as B7-1 and B7-2, the ligands for CD28 (26). Third, dendritic cell activation results in synthesis of proinflammatory chemokines and cytokines (26, 28, 38); chemokines, such as SLC (6Ckine) may guide movement of activated CCR7+ dendritic cell precursors into the T cell zone, whereas APC release of cytokines, notably TNFα and IL-6, may provide soluble second signals for T cell activation (39, 40), thereby complementing costimulation provided by CD28/B7 interaction. In addition to acting directly on dendritic cells, some pathogens, especially viruses, can activate dendritic cells through production of type I (α, β) interferons (IFN-I) (41, 42). APC activation can also be induced by inflammatory molecules (TNFα and heat shock proteins) released through contact with necrotic cells (43). It should be noted that adjuvants are not needed for T cell responses to certain particulate antigens, e.g., heterologous erythrocytes (44), presumably because phagocytosis of these large antigens is sufficient to cause APC activation. Although the origin of activated mature dendritic cells from immigrant antigenloaded dendritic cell precursors is well accepted, the precise origin of dendritic cells is controversial (26, 28, 45). Currently, dendritic cells appear to arise from both myeloid and lymphoid precursors in blood; however, it is unclear whether these precursors represent two distinct lineages or reflect different pathways of development from a common precursor. As discussed elsewhere, myeloid and lymphoid dendritic cell precursors appear to be positioned differently but are both capable of transporting antigens to the T cell zones (46). Myeloid and lymphoid dendritic cells also show distinct differences in their capacity to synthesize certain cytokines, notably IL-12 and IFN-I [the latter being produced by plasmacytoid precursors in humans (41, 47)], and in their expression of individual TLRs (26, 28). Hence, selective activation of myeloid vs. lymphoid dendritic cell precursors by different pathogens may explain the considerable heterogeneity of T effector function seen in one infection vs. another.

T Cell Trapping by APC As discussed above, in the absence of foreign antigen, T cells move freely through the T cell zones, contact with self-peptides on dendritic cells being nonimmunogenic. When antigen-loaded APC enter the T cell zone, specifically reactive T cells recognize the foreign peptide/MHC complexes on these cells and within minutes form a tight synapse at the point of T/APC interaction (48–50). As discussed later, synapse formation is a crucial prelude for initiating T cell triggering and proliferation. However, the immediate consequence of T/APC synapse formation is that T cells become immobilized (44, 51). Such trapping in the T cell zones prevents blood-to-lymph recirculation and leads to continuous recruitment of newly arriving naive T cells into the immune response. Trapping is antigen-specific, and nonreactive T cells maintain their normal pattern of recirculation.

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Trapping of T cells in the lymphoid organs by APC is highly efficient. Thus, following intravenous (iv) injection of particulate antigens, such as heterologous erythrocytes in high doses, selective localization of these antigens in the spleen causes virtually all specifically reactive T cells to disappear from lymph nodes and thoracic duct lymph, and the T cells become trapped in the spleen (51). Such extensive trapping of T cells by the spleen after i.v. injection of antigen reflects the fact that the spleen has a large blood supply, which allows the vast majority of recirculating T cells to migrate through the spleen at least once a day. The situation is different for typical immune response induced by pathogens infecting mucosal sites. Here, via movement of activated antigen-laden APC, antigen is deposited largely in lymph nodes draining the sites of infection rather than in spleen. Since lymph nodes are normally small structures with a limited blood supply, only a tiny fraction of the total pool of recirculating T cells passes through an individual lymph nodes over a period of 24 hours. One might conclude therefore that recruitment of antigen-specific T cells to lymph nodes draining the site of infection is inefficient and limited to the few T cells that randomly enter lymph nodes from the blood. This may not be the case in practice, however, because draining lymph nodes become conspicuously enlarged within 1–2 days of infection (8). This phenomenon, originally misnamed lymph nodes shutdown, is poorly understood but is probably a manifestation of a sharp increase in the blood supply to infected lymph nodes, thus considerably accentuating the rate of perfusion of lymph nodes by recirculating T cells. In light of current knowledge (see above), the enhanced blood flow through infected lymph nodes may be mediated by chemokines released by the influx of activated antigen-laden APC. Whatever the explanation, a nonspecific increase in traffic through infected lymph nodes may be an important device for amplifying recruitment of antigen-specific naive T cells, thus increasing the precursor frequency of T cells during the early stages of the immune response.

T Cell Proliferation and Differentiation Because pathogens often replicate at a prodigious rate, clonal expansion of naive T cells and differentiation of these cells into effector cells has to be as rapid and efficient as possible and has to continue until the pathogen is eliminated, which takes 7–10 days for a typical viral infection. As mentioned above, initial interaction of T cells with APC leads to the formation of synapses at the T/APC contact site (48–50). Synapse formation precedes T cell activation and is associated with rapid clustering of TCR molecules binding to peptide/MHC complexes on APC plus local accumulation of intracellular signaling molecules such as LCK, LAT, and PCKθ ; the latter associate with TCR/CD3 complexes in lipid rafts and, together with various other intracellular molecules, initiate the downstream signaling events that cause T cells to proliferate, synthesize cytokines, and differentiate into effector cells. TCR/CD3 triggering is aided by CD4 and CD8 coreceptors and also by a large number of costimulatory/adhesion molecules on T cells (52–54). These molecules,

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which include CD28, LFA-1, CD40L, ICOS, OX40, CD2, CD27, and 41BB, bind to complementary molecules on APC and, at least for CD28, CD2, and LFA-1, are drawn into the synapse along with TCR/CD3. Some costimulatory/adhesion molecules may provide essential second signals for T cell activation, but others may act largely by enhancing TCR triggering, e.g., by stabilizing synapse formation and/or recruiting intracellular signaling molecules (55, 56). Some costimulatory/adhesion molecules, notably CD28, are important for inducing cytokine (IL-2) synthesis by T cells, whereas others, e.g., CD40L, maintain or induce activation of APC and also stimulate B cells during T/B collaboration (57, 58). As mentioned earlier, optimal responses by naive T cells require APC activation by adjuvants. Through the release of stimulatory cytokines and upregulation of costimulating ligands, activated APC play a dual role in driving extensive T cell proliferation and promoting efficient differentiation into effector cells. However, the types of effector functions displayed by T cells following T/APC interaction appear to vary according to the stage of the immune response. During the early phase of the response, rapid replication of the pathogen ensures continuous entry of large numbers of activated APC into the T cell zones. Being highly stimulatory, these APC drive the responding T cells to proliferate rapidly (as often as every 8 h for CD8+ cells), synthesize a wide range of cytokines, and differentiate into cytotoxic cells (for CD8+ cells) and T helper cells (for CD4+ cells). Extensive clonal expansion applies to both CD4+ and CD8+ cells, although, at least in lymphocytic choriomeningitis virus (LCMV) infection, proliferation of CD4+ cells is somewhat slower and less prominent than for CD8+ cells (59–61). At later stages of the response, destruction of the pathogen at the site of infection by effector cells reduces inflow of antigen-laden APC into the T cell zones. Under these conditions, T cell interaction with diminishing numbers of antigenbearing APC exhausts the capacity of APC to produce stimulatory cytokines (62); the exhausted APC continue to elicit T cell proliferation but cytokine production by T cells and formation of fully differentiated effector cells are reduced. As discussed later, this stage of the immune response may be important for memory cell generation. The immune response presumably ceases when the influx of antigen-laden APC into the T cell zones is abolished. However, terminating the immune response may reflect the interplay of complex homeostatic mechanisms. For example, the recent finding that T cells can undergo up to seven rounds of cell division after being deprived of contact with APC (63, 64) may enable T cell proliferation and differentiation to continue for several days after the pathogen is cleared. Conversely, overexuberant T cell responses during the waning stages of the immune response may be limited by the action of inhibitory receptors on T cells, such as CTLA-4 and PD-1, binding to complementary ligands (B7 and PD-1L) on APC (65, 66). The notion that these receptors are inhibitory only when antigen becomes limiting is appealing—but unproven. With regard to effector cells, there is considerable interest in the range of cytokines produced by these cells and how cytokine synthesis is induced. Based on

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their patterns of cytokine production, T effector cells contain subsets of Th1 and Th2 cells (67). Th1 cells typically produce IFN-γ and TNFβ and protect against intracellular pathogens, whereas Th2 cells selectively synthesize IL-4, IL-5, IL-10, and IL-13 and counter extracellular pathogens through production of antibody by B cells. Other effector cells are uncommitted and either show limited cytokine synthesis or produce a nonpolarized spectrum of cytokines. The generation of Th1 vs. Th2 cells has been the topic of several recent reviews (28, 46, 68). For IFN-γ –producing Th1 cells, formation of these cells requires IL-12 production by APC. Since recently activated dendritic cells produce high levels of IL-12, IFN-γ production by T effector cells may be imprinted early in the immune response and continue until the response begins to wane. In viral infections, release of large amounts of IFN-I by APC may suppress IL-12 production (69). In this situation, IFN-I can substitute for IL-12 and lead to IFN-γ (and IL-10) synthesis by T cells via a STAT2/STAT4-dependent pathway (70); this pathway operates in humans but not in mouse because of a STAT2 defect (71). The generation of Th2 cells is less clear, but evidence is mounting that Th2 development may be a default pathway reflecting lack of exposure to IL-12 combined with contact with IL-4 (46). Th2 cells may be preferentially generated at the end of the immune response through contact with exhausted APC (62). During earlier stages of the response, high IL-12 synthesis by activated APC would be expected to preclude Th2 cell development. However, in this situation Th2 cells may arise through inhibition of IL-12 synthesis by suppressive cytokines, such as IL-10 and TGFβ (72, 73), and also by prostaglandin E2 (74).

FATE OF EFFECTOR CELLS In typical infections, prominent clonal expansion of specific T cells followed by differentiation into effector cells generally causes rapid elimination of pathogens. After elimination of the pathogen, the enormous numbers of effector cells become redundant and most of these cells rapidly disappear. The disappearance of activated T cells at the end of the primary response appears to reflect two distinct mechanisms, namely death and homing to nonlymphoid tissues. Early information on this issue came from studies on the fate of T cells responding to H2 alloantigens in vivo (75–77). Here, transferring parental-strain T cells to irradiated H2-heterozygous mice led to the appearance of large numbers of activated donor-derived effector T cells in thoracic duct lymph by 3–4 days posttransfer. When these effector cells were transferred to syngeneic (donor) mice, the cells homed initially to spleen and lymph nodes but then rapidly disappeared by two different mechanisms. First, many of the activated T cells left the spleen and localized in nonlymphoid tissues, notably the lungs, liver, and gut (75). Second, in both lymphoid and nonlymphoid tissues, the vast majority of the T cells died within 5 days (75), and only a small proportion of the cells survived to become

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long-lived functional memory cells (76, 77). Death was conspicuous in the liver— now regarded as a graveyard for dying T cells (78)–but was also apparent in the spleen; based on 51Cr-labeling studies, cells dying in the spleen were rapidly engulfed by phagocytic cells (75). Recent work on homing and death of effector T cells is discussed below.


Homing of Effector Cells Detailed information on the capacity of effector T cells to migrate to nonlymphoid tissues has come from the tetramer staining method for detecting antigen-specific T cells in cell suspensions (79) and also by histological staining of T cells in whole-mouse sections (80). In these studies, clonal expansion of CD8+ and CD4+ T cells responding to viruses and soluble peptides led to the prominent appearance of activated effector T cells in various nonlymphoid tissues, including the lung, liver, gut, kidney, and salivary glands; unlike naive T cells, activated effector T cells also migrated to the bone marrow and the thymic medulla. In both lymphoid and nonlymphoid tissues, most of the effector T cells disappeared within 2–3 weeks, which presumably indicated death; however, some of the cells survived to form a subset of activated memory cells. The properties of these effector memory cells are considered later. As discussed elsewhere (11, 81), the capacity of activated T cells to migrate to nonlymphoid tissues is a reflection of upregulation of several classes of homing receptors, including selectins/selectin ligands, chemokine receptors, and both β1 and β2 integrins, notably LFA-1. Increased expression of these receptors allows activated T cells to bind to and penetrate the walls of small blood vessels and thereby percolate into extravascular sites throughout the body. To some extent, such migration is tissue-specific and reflects the particular homing receptors expressed. Thus, activated T cell homing to inflamed skin depends on binding to E/P-selectins and also to chemokines, e.g., TARC on skin vascular epithelium (82) and CTACK on keratinocytes (83); these molecules are recognized by complementary receptors on T cells, i.e., by E/P-selectin ligands and CCR4 (for TARC), respectively. Likewise, homing to the gut is controlled by α4β7 integrins and CCR9 on T cells recognizing MAdCAM-1 and chemokines (TECK), respectively, on gut vascular endothelium (81, 84). Homing to the peritoneal cavity involves E/P-selectin ligands and CXCR3 on T cells binding to E/P-selectins and IP10 on blood vessels (85). Within the secondary lymphoid tissues, migration of CD4+ T helper cells into B-cell follicles and germinal centers is controlled by CXCR5/BCA-1 interaction (86). In general, homing to nonlymphoid tissues is skewed toward cytokine-polarized effector cells, especially Th1 cells, and is largely restricted to T cells that have downregulated the lymph nodes-homing receptors, CD62L and CCR7 (12, 85, 87). The lack of these receptors prevents the effector cells from entering lymph nodes, but migration through the spleen is maintained. In spleen, the cells are found in the red pulp but are largely excluded from the white pulp (12).

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Death of Effector Cells Following early studies with superantigens (88), it is now well accepted that termination of the primary response is known to be followed by wide-scale death of effector cells. Teleologically, removal of effector cells after the pathogen is cleared makes sense because allowing these cells to survive en masse would overburden the immune system with useless cells: lymphoid hypertrophy and traffic congestion would ensue and thus compromise the response of naive T cells to new pathogens. This situation arises naturally in mice lacking certain cell-death– inducing molecules (see below). Here, unrestricted responses to environmental antigens lead to marked overproduction and survival of activated T cells, massive splenomegaly/lymphadenopathy occurs, and the mice die, presumably from infection. The mechanisms responsible for eliminating effector cells are poorly understood, but are clearly complex. Many cell-surface molecules on T cells, such as Fas and receptors for IL-2, TNF, and other cytokines, are able to transduce death signals under defined conditions (89–91), but the precise role of these molecules in mediating elimination of effector cells in vivo has yet to be resolved. For CD8+ cells, expansion/deletion of T cells responding to viral antigens is near normal in Bcl-2 and Bcl-XL transgenic mice (30, 92) and in mice lacking CTLA4 (93), Fas, or TNFR, or both Fas and TNFR (94, 95), which implies that these molecules are not needed for deletion. By contrast, IFN-γ seems to play a major role in the deletion of CD8+ cells. Thus, IFN-γ −/− mice show strong clonal expansion of CD8+ cells in response to viruses but very poor subsequent elimination of effector cells (96). With regard to other mediators, perforin appears to limit the intensity of the primary response of CD8+ cells (96, 97), perhaps through killing of APC, and may also control exhaustion of CD8+ cells responding to high doses of virus (96, and see below). However, perforin does not affect the deletion phase of the normal primary response (96). As for CD8+ cells, Fas/FasL interaction does not seem to be crucial for the elimination of effector CD4+ cells in normal immune responses (98, 99). However, Fas does control the deletion of CD4+ cells involved in chronic immune responses to self-antigens and perhaps also to continuously encountered environmental antigens (99). Unrestrained responses to these antigens are presumed to explain the prominent T cell hyperplasia seen in Fas (and FasL)-deficient mice (89). However, this syndrome is also conspicuous in a strikingly wide variety of gene-knockout mice, e.g., mice lacking PD-1 (66), CTLA4 (100, 101), NFAT (102), IL-2 (103), CD25 (104, 105), CD122 (106), CD45 (107), and TGFβ (108). Here, a key issue is whether the T cell hyperplasia in these knockout mice reflects defective immunoregulation of both CD4+ and CD8+ cells or only of CD4+ cells. For Fas deficiency, the effects of selectively depleting either CD4+ or CD8+ cells suggest that both subsets contribute to T cell hyperplasia (109, 110). For CTLA4−/− (111) and PD-1−/− mice (112), by contrast, disease onset is prevented by removal of CD4+ cells, which suggests that T hyperplasia is primarily under the control of

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CD4+ cells; in these mice, recruitment of proliferating CD8+ cells could be a secondary event, perhaps reflecting enhanced exposure to stimulatory cytokines. Similarly, CD4+ cells rather than CD8+ cells seem to control T cell hyperplasia in IL-2−/− mice (113). In light of the above findings, the mechanisms controlling the elimination of effector CD4+ and CD8+ cells may be distinctly different. For CD8+ cells, the inhibitory influence of certain cytokines, notably IFN-γ , may be sufficient to eliminate effector cells. For CD4+ cells, by contrast, it would seem that effector cell elimination, which occurs more slowly than for CD8+ cells (60, 61), reflects a tightly regulated instructional process involving multiple cell-death–inducing mechanisms acting in consort; even with inactivation of only one of these mechanisms, death is averted and effector cells survive in large numbers. The precise sequence of molecular events required to destroy CD4+ effector cells is unclear but appears to involve negative signaling by CTLA-4 and PD-1 receptors for costimulatory molecules (66, 114), activation of the Fas death pathway by dissociation of cFLIP from Fas (115–117), and onset of sensitivity to several cytokines such as IL-2 (91), IFN-γ (118), and TNF (98, 119). In addition to these active mechanisms for cell death, effector cells may undergo passive death through loss of contact with protective cytokines (120, 121). This form of death can be prevented by enforced upregulation of Bcl-2 (99). Likewise, the finding that death of effector CD4+ cells is enhanced in CD40L−/− mice (59) suggests that passive death may also be promoted through lack of CD40L contact with CD40 on APC. Based on the above data, one can obviously discard the simple notion that effector cells are intrinsically short-lived cells doomed to die by a default pathway. Instead, effector cells have a propensity to survive, and eliminating these cells involves an instructional process. As discussed below, this conclusion has important implications for the generation of memory T cells.

SELECTION OF MEMORY T CELLS In typical primary responses, the wide-scale elimination of effector T cells at the end of the response is incomplete and a small proportion of T cells survive to become long-lived memory cells. Here, a key issue is whether production of T memory cells is a stochastic process or reflects selective mechanisms. On this point, it is important to consider whether T memory cells undergo affinity maturation.

Affinity Maturation In contrast to B cells there is no firm evidence that differentiation of naive T cells into memory cells reflects somatic hypermutation. However, bearing in mind that the TCR repertoire of naive T cells is highly diverse (122), one can envisage that contact with antigen during the primary immune response leads to significant alterations in the TCR specificity of memory cells relative to naive cells. In support of this notion, the TCR repertoire of T memory cells (CD4+ cells) is narrower than

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for naive cells (122, 123). It is also likely that T memory cells have a higher average affinity for antigen than do naive T cells (124–127). Here, an important question is whether such affinity maturation occurs solely during the primary response or can also proceed after the response is completed. It is easy to envisage affinity maturation during the primary response because, especially during the later stages of the response, high-affinity T cells are likely to have a growth advantage over low-affinity T cells because of competition for antigen (127). This mechanism is the simplest explanation for immunodominance during viral infections, i.e., stronger CD8+ T cell responses to dominant (strong) antigens than to subdominant (weak) antigens (128). In considering whether affinity maturation continues after the primary response, a key question is whether the repertoires of T memory cells and typical effector cells are the same or different. For CD8+ cells responding to pathogens, the bulk of evidence suggests that the repertoire of T memory cells is imprinted solely during the primary response; differences in the TCR repertoire of primary effector cells and long-lived memory cells are minimal (129, 130). This issue is less clear for CD4+ cells, although there are reports that affinity maturation of CD4+ cells can proceed for several weeks (123, 131). Whether these latter findings signify a fundamental difference between CD4+ and CD8+ cells is unclear. One possibility is that prolonged affinity maturation of CD4+ cells simply reflects a protracted primary response because of persistence of antigen, especially in germinal centers (123). The above data refer to memory cells tested directly ex vivo or after brief restimulation in vitro. When memory cells are restimulated in vivo, i.e., in secondary (recall) responses, clonal expansion of these cells can be skewed to high-affinity cells (129). However, this is not an invariable finding (130) and, when observed, this phenomenon may simply reflect enhanced competition for antigen in secondary responses because of rapid clearance of antigen by CTL or specific antibody.

Selection of Th1 and Th2 Memory Cells As discussed earlier, differentiation of T cells into polarized vs nonpolarized cells during the clonal expansion phase of the primary response is highly complex and reflects many different factors, including the density of antigen, TCR affinity, local contact with particular cytokines, and the nature of the APC. In general, polarized cells develop as the result of prolonged exposure to antigen, whereas nonpolarized cells arise by default when antigen is limiting (50, 87, 132). For memory cell generation, measuring cytokine profiles of memory T cells derived from precursors stimulated under polarizing conditions in vitro has shown that Th1 and Th2 effector populations retain their polarity for prolonged periods in vivo (3). Likewise, the particular cytokine profiles of primary effector T cells and long-term memory T cells are quite similar (3, 132). Such findings favor the view that selection of Th1 and Th2 effector cells for death vs. survival (as memory cells) is largely a stochastic process. It is worth noting, however, that the cytokine polarity of T cells is usually assessed after brief restimulation of T cells in vitro. Here, it is interesting that clonal analysis of cytokine production by CD4+ cells

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tested directly ex vivo revealed distinct differences in the polarity of cells tested at day 9 of the primary response versus day 3 of the secondary response (132); these differences were largely masked when the cells were restimulated in vitro. In light of this finding, in terms of cytokine polarity the differentiation of effector cells into memory cells may to some extent involve selective mechanisms, at least for CD4+ cells. Despite the subtle differences observed between primary and secondary T cells in the above study (132), most of the T cells showed little evidence of cytokine polarity. This finding, which has also been reported by others (50, 87), is in line with the recent discovery that cytokine production and polarity of memory CD4+ T cells is largely restricted to a subset of activated cells marked by downregulation of CCR7 (50, 133). In contrast to these CCR7− T cells, the major population of “central” (resting) CCR7+ memory cells does not display cytokine polarity. As discussed below, this finding has important implications for memory cell selection.

Relationship of T Effectors and T Memory Cells Until recently, it was often argued that memory T cells could represent a subset of cells that failed to differentiate into effector cells during the primary response. This possibility now seems unlikely in view of evidence that the precursors of memory cells undergo extensive proliferation during the primary response (134–136) and, at least transiently, express effector functions such as perforin (136) and Granzyme B (137) synthesis and cytokine (IL-2) production (138). One has to conclude therefore that memory cells are derived from typical effector cells. What then is the difference between an effector cell that dies and an effector that differentiates into a memory cell? The simplest possibility is that there are no essential differences between typical T effectors and T memory precursors, generation of memory being largely a stochastic process (130). As suggested elsewhere (139, 140), an alternative possibility is that memory T cells are not drawn randomly from typical effector cells but are derived from a subset of precursors that arrives in the later stages of the immune response, e.g., because of slow recruitment to the draining lymph nodes from elsewhere in the body. These straggler cells may proliferate extensively and pass through an effector stage but, because of less-protracted contact with antigen, induction of the death pathways in these cells is incomplete or reversible; unlike terminally differentiated effectors, the straggler cells thus avoid death and survive to become memory cells. Though still hypothetical, this idea could explain the complete elimination of effector cells that occurs when T cells confront a very high dose of virus or other antigens (141, 142); here, continuous exposure to antigen causes exhaustive differentiation (death) of virtually all of the responding cells. For CD8+ cells, such death is partly avoided in perforin−/− mice (97). This finding may reflect that onset of CD8+ cell death is perforin-mediated (97). Alternatively, prolonged responses of normal CD8+ cells could lead to wide-scale perforin-dependent destruction of APC, T cell death being a consequence of abrupt loss of contact with antigen on APC (96, 143).

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The notion that T memory cells arise preferentially from straggler cells arriving late in the primary response has been invoked to explain the generation of nonpolarized central memory cells in humans (50). As discussed earlier, the suggestion here is that, during the terminal stages of the immune response, most APC express only low concentrations of antigen and are exhausted in terms of their capacity to produce polarizing cytokines, notably IL-12 for Th1 cells. Under these conditions, T cells recruited late in the response may avoid cytokine polarization and retain CCR7 expression; some cells die but others differentiate directly into CCR7+ longlived nonpolarized central memory cells. For cells recruited early in the primary response, by contrast, contact with high concentrations of antigen presented by activated APC plus cytokines generates CCR7− polarized effector cells, only a small proportion of which survive and differentiate into CCR7− polarized effector memory cells. Currently, the evidence that central memory cells are drawn from straggler precursors is largely indirect. In mice, useful information on this topic could be obtained by studying the efficiency of memory cell generation after exposing naive T cells to antigen-pulsed APC for various periods in vitro followed by adoptive transfer in vivo (63, 64). With this system, transfer of CD8+ cells after only brief (1 day) exposure to APC in vitro led to strong proliferation in vivo followed by differentiation into memory cells. Whether the extent of memory cell generation in this system is affected by the duration of in vitro stimulation and/or by antigen concentration and the activation status of the APC is still unclear. However, it is interesting that a recent study showed a highly efficient generation of memory cells when naive CD4+ cells were stimulated with antigen for 3 days in vitro, rested, and then adoptively transferred (144). Under these conditions of brief stimulation, the effector CD4+ cells evaded death and survived en masse to become memory cells. Viewed as a whole, the existing data indicate that memory cells arise from effector precursors, although it is not entirely clear whether these precursors represent fully differentiated or partly differentiated cells or a mixture of the two. The point to emphasize, however, is that memory cell generation appears to be an essentially passive process: Effector cells are coerced to die en masse by tightly regulated instructional mechanisms, and the few cells that escape death survive as memory cells, survival being a default pathway. This scheme applies to the initial generation of memory cells. As discussed later, the long-term survival of memory cells appears to involve instructional mechanisms.

Self-Specific Memory Cells Until recently, it was tacitly assumed that memory T cells are generated solely through contact with foreign antigens. In this respect, the sizeable proportion of T cells with a memory-phenotype (see below) found in normal animals is thought to reflect lifetime exposure to various environmental antigens. However, the assumption that memory cells are all descended from precursors responding to foreign antigens has been called into question by the recent finding that naive T cells begin to proliferate and differentiate into memory-phenotype

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T cells when total numbers of naive T cells are reduced below a certain threshold (4, 145). This homeostatic response of T cells is directed largely to self antigens, especially to the particular self peptides that led to initial positive selection of naive T cells in the thymus. In view of this finding, the pool of memory T cells could be drawn in part from cells responding to self components. However, it should be emphasized that, except in the neonatal period and extreme old age, the pool size of naive T cells remains high, thus precluding homeostatic proliferation and differentiation. For this reason, the proportion of self-ligand–selected pseudo-memory cells is likely to be very low in normal adult animals.

FEATURES OF MEMORY CELLS Although T memory cells seem to survive almost indefinitely at a population level, these cells display considerable heterogeneity in terms of their surface markers, tissue distribution, and activation status.

Surface Markers The expression of surface markers on T memory cells has been extensively reviewed elsewhere (1–6). In general, memory cells show distinct phenotypic differences from naive T cells. Nevertheless, a number of the markers commonly used to define memory cells, e.g., low levels of CD62L and CD45RA/B/C, seem to be partly reversible on late memory cells (134, 146–149). However, it is unclear whether this reversion is real, or reflects preferential survival of cells that failed to lose these markers during the primary responses (150), although there is strong evidence for CD45RC− → CD45RC+ reversion in rats (151). This uncertainty also applies to CCR7 expression in humans (133). Thus, it has yet to be proved whether CCR7 expression on central memory cells reflects retention of CCR7 from the time of their differentiation from naive precursors or a CCR7+ → CCR7− → CCR7+ transition; in favor of this transition, day 8 effector cells generated in LCMV infection showed total absence of CCR7, though memory cells derived from the effectors were not tested (12). Currently, high expression of CD44 seems to be the most reliable marker for memory cells in mice, both for CD4+ and CD8+ cells; in addition, memory CD8+ cells are characterized by high expression of Ly6C and CD122 (150, 152). At a population level, memory cells show marked heterogeneity in their pattern of surface markers, with some cells resembling effector cells and others displaying many (but not all) of the characteristics of naive cells. Here, it is important to consider the activation status of memory cells.

Resting Versus Activated Cells Based on their rate of turnover (proliferation) in vivo and the expression of activation markers, memory T cells comprise two broad subsets of cells. Some memory cells are in an overtly activated state and closely resemble effector cells. These effector memory cells have a rapid turnover, show direct CTL

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activity (for CD8+ cells), and tend to express activation markers such as CD69 and CD25 (148, 149, 153–155). Like typical effector cells, activated memory cells express high levels of β1 and β2 integrins and specialized chemokine receptors that allow the cells to enter nonlymphoid tissues (133, 156). Activated memory cells are also found in the blood and spleen but are largely excluded from lymph nodes because of the loss of their lymph nodes homing receptors, CD62L and CCR7 (133); however, these cells do enter lymph nodes in small numbers via afferent lymphatic vessels (153). Resting (central) memory cells have a relatively slow turnover (148), lack activation markers, and closely resemble naive T cells in terms of their distribution in the lymphoid tissues (133). Central memory cells typically express CD62L and CCR7 receptors, which allows the cells to enter lymph nodes via high endothelial venules. Despite their quiescent state, resting memory cells are clearly less inert than naive T cells. Thus, for CD8+ cells, resting memory cells divide only intermittently but express quite high levels of RNA, which suggests that many of the cells are in the G1 phase of cell cycle (157); resting memory CD8+ cells also constitutively express perforin (152, 157, 158). The implication therefore is that memory cells are not totally quiescent but are maintained in a state of low-level activation. This topic is discussed later. The relationship between central and effector memory cells is unclear. The simplest idea is that these subsets represent two distinct populations derived from different precursors during the primary immune response. However, a more likely possibility is that activated memory cells arise after the primary response through stimulation of central memory cells (50, 133, 155), e.g., by cytokines, cross-reactive environmental antigens, or trace amounts of specific antigen. This issue could perhaps be resolved by studying the features of memory cells parked in MHC−/− hosts (see below).

Functions Despite the extensive culling of T cells at the end of the primary response, the precursor frequency of antigen-specific memory cells is far higher than for naive T cells (1–6, 59). This increase in precursor frequency is the main reason why secondary immune responses are generally much more intense than primary responses. In addition, it has long been argued that, cell-for-cell, memory T cells are more responsive to antigen than naive T cells. Because of the very low frequency of antigen-specific T cells in populations of normal naive T cells, a direct comparison of naive and memory cells is possible only with transgenic mice. Using this approach, it has been shown that populations enriched in resting memory T cells may (134) or may not (158, 159) show an increase in sensitivity to limiting concentrations of antigen (peptides) relative to naive T cells. However, when stimulated by antigen, memory cells do show a shorter lag time for entering cell cycle, synthesizing cytokines, differentiating into CTL, and migrating to nonlymphoid tissues (87, 134, 142, 149, 152, 157–159); memory cells are also somewhat less dependent on costimulation than naive T cells. These data are

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consistent with the finding that memory cells are less metabolically inert than naive T cells. The above data apply to central memory cells. By contrast, effector memory cells, being in an overtly activated state, display constitutive CTL function and cytokine synthesis in vivo (50, 152, 158, 159). Residing in (or migrating through) nonlymphoid tissues such as the lung, effector memory cells are positionally poised to provide immediate immunity following secondary contact with the pathogen concerned (50, 154, 155). Secondary responses by central memory cells, by contrast, presumably hinge on initial reactivation by antigen in the draining lymph nodes before migration to nonlymphoid tissues. Because of this lag time, some workers argue that central memory cells are much less functionally relevant than effector memory cells (2). This point remains contentious.

MAINTENANCE The finding that memory cells are more metabolically active than naive T cells suggests that memory cells may be continuously signaled by extrinsic factors. Such signaling could be vital for keeping the cells alive. Here, two types of ligands could be involved.

Antigen In the past, it was argued that memory cells could be maintained through contact with trace amounts of specific antigen left over from the primary response (2). This idea now seems unlikely because memory T cells can survive in the apparent complete absence of specific antigen, both for CD8+ and CD4+ cells (21, 142, 157, 160–163). An alternative possibility is that memory cells receive survival signals through contact with cross-reactive environmental antigens (164). The obvious problem with this scenario is that it predicts that memory cells lacking crossreactivity for environmental antigens would rapidly disappear, for which there is little if any evidence. The only direct approach for testing whether memory cells are kept alive by contact with antigen is to follow the survival of these cells after transfer to MHC−/− hosts. Here, the clear-cut finding is that memory cells do survive for prolonged periods in MHC−/− hosts, both for CD4+ and CD8+ cells (144, 165, 166). However, it remains to be determined whether long-term survival in MHC−/− hosts applies to both effector and central memory cells or only to the latter. Nevertheless, at a population level, it would appear that the longevity of memory cells is independent of TCR ligation.

Cytokines As mentioned above, the turnover of memory cells, though slow relative to T cells proliferating during an immune response, is clearly faster than for naive T cells. Thus, whereas naive T cells rarely divide, memory cells divide intermittently, the period of interphase between divisions being in the order of 1–3 weeks (167). At

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least for CD8+ cells, this relatively rapid rate of cell division (compared to naive T cells) persists following transfer to MHC I−/− hosts (165). Evidence that cytokines affect the background turnover of CD8+ cells stemmed from experiments with agents that stimulate the innate immune system, e.g., Poly I:C and lipopolysaccharide (LPS) (168–171). Injecting these compounds into mice caused a brief burst of TCR-independent (bystander) proliferation, which was largely restricted to a subset of CD8+ cells with a memory (CD44hi) phenotype. Based on studies with IFN-γ −/− mice and mice lacking receptors for IFN-I, it was concluded that Poly I:C and LPS stimulate T cells via production of IFNs, both IFNI and IFN-γ . Since IFNs failed to stimulate purified T cells in vitro, it was reasoned that T cell proliferation in vivo reflects IFN-induced synthesis of another cytokine, an effector cytokine that acts directly on CD44hi CD8+ cells. IL-15 seemed a likely candidate because IFNs and IFN-inducing agents induced strong IL-15 mRNA synthesis by APC in vitro (172). In addition, CD122 (IL-2Rβ), a component of the receptor for IL-15 (and IL-2), was found to be expressed selectively on CD44hi CD8+ cells. The key finding was that IL-15 caused proliferation of purified CD44hi CD8+ cells (but not CD44hi CD4+ cells) in vitro and mimicked the capacity of IFNs to stimulate these cells in vivo. Recently, bystander proliferation of CD44hi CD8+ cells was found to be low or undetectable after transfer to IL-15−/− mice, which strongly implicates IL-15 as the effector cytokine (173). Bystander proliferation of CD44hi CD8+ cells is of brief duration and probably reflects augmentation of the natural turnover of these cells. This notion rests on the assumption that the relatively high turnover of CD44hi CD8+ cells is mediated through contact with background levels of IL-15. Strong support for this idea is provided by the finding that the normal turnover of CD44hi CD8+ cells in vivo is considerably reduced following injection of anti-CD122 mAb (174), presumably because this antibody blocks T cell contact with IL-15. It is also notable that total numbers of CD44hi CD8+ cells are selectively reduced in IL-15−/− mice (175) and increased in IL-15 transgenic mice (176, 177). Likewise, injection of IL-15 can boost numbers of antigen-specific memory CD8+ cells (178). In addition to providing a stimulus for cell division, IL-15 could also be important for keeping CD44hi CD8+ cells alive. The observation that CD44hi CD8+ cells are rare in IL-15−/− mice (175), and also in IL-15Rα −/− mice (179), is in favor of this possibility. In addition, CD44hi CD8+ cells rapidly disappear following transfer to IL-15−/− hosts (173). Collectively, the above data indicate that CD44hi CD8+ cells are strongly dependent on IL-15, both for their survival and turnover. It should be noted that dependency on IL-15 applies only to typical CD44hi CD8+ cells expressing a high density of CD122. A small proportion (about 30%) of CD44hi CD8+ cells are CD122lo, and these cells account for nearly all of the residual CD44hi CD8+ cells found in IL-15−/− mice (173). The implication therefore is that, unlike CD122hi cells, CD122lo CD44hi CD8+ cells are IL-15 independent. One recent study suggests that these latter cells could be short-lived. Thus, experiments in which CD8+ T cells were primed to specific antigen in vitro and then adoptively transferred in vivo showed that the fate of the transferred cells correlated with the level of CD122

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expression: CD122hi cells survived long term, whereas CD122lo cells disappeared within a few weeks (180). Despite the conspicuous influence of IL-15 on CD8+ memory cells, CD4+ memory cells seem to be largely independent of IL-15. This finding is not surprising because CD122 expression on memory CD4+ cells is quite low (172). By analogy with CD8+ cells, it would seem quite likely that the survival of memory CD4+ cells is cytokine-dependent. However, it is striking that long-lived CD4+ memory cells can be generated from γ c−/− precursors (181), which implies that γ c-controlled cytokines (IL-2, -4, -7, -9, -15) are not important. Other cytokines have yet to be studied.

Homeostasis of Memory Cells Despite the fact that T memory cells have a high rate of turnover, total numbers of T memory cells remain relatively constant throughout life, though there are reports that numbers of CD4+ (60) and CD8+ (178) memory cells may eventually decline in old age. The implication therefore is that memory cells are subject to strict homeostatic control, background expansion of memory cells through intermittent cell division being countered by an equivalent level of cell death (182, 183). At a population level, this equilibrium presumably reflects a balance between lifesustaining signals and proapoptotic signals. As discussed above, the longevity of memory cells appears to depend on protective signals delivered by cytokines, notably by IL-15 for CD8+ cells. How cytokines promote memory cell survival is unclear, but upregulation of antiapoptotic molecules such as Bcl-2 and Bcl-XL is a likely possibility. Here, it is of interest that Bcl-2 upregulation in T cells in vitro is controlled by γ c-controlled cytokines (184), especially by IL-15 in CD8+ cells (185), whereas Bcl-XL upregulation is elicited by other cytokines, including IFN-I (186). Here, it is notable that when tested directly ex vivo, CD8+ memory cells show high levels of Bcl-2 and, to a lesser extent, Bcl-XL relative to naive T cells (185, 187). By contrast, CD4+ memory cells show upregulation of Bcl-XL but not Bcl-2 (142, 185). These findings add to the evidence that the mechanisms governing the survival of memory CD4+ and CD8+ cells are distinctly different (see above). Thus, memory CD8+ cells may be kept alive in vivo via Bcl-2 upregulation mediated by contact with IL-15 and other γ c cytokines, whereas memory CD4+ cells may be protected through Bcl-XL upregulation, e.g., by IFN-I. However, direct evidence on this issue is still limited, and upregulation of other pro-life molecules, e.g., LKLF (188, 189), could be equally important for maintaining memory T cell survival. Under steady-state conditions, memory T cell contact with life-sustaining cytokines is presumably limiting, thus curtailing continuous expansion of these cells. If so, a protracted increase in the level of these cytokines would be expected to cause numbers of memory T cells to increase above normal. The selective expansion of CD44hi CD8+ cells in IL-15 transgenic mice (176, 177) is consistent with this idea. These cells can also expand in response to IL-7. Thus, the marked

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increase in total T cell numbers seen in IL-7 transgenic mice (190) is strongly skewed to CD44hi CD8+ cells (W.C. Kieper, R. Ceredig, C.D. Surh, unpublished data). It is unclear whether other cytokine-transgenic mice, e.g., mice expressing high levels of IL-2 or IFN-I, display a similar phenotype. However, this approach is complicated by the fact that certain cytokines can stimulate the production of other cytokines, some of which could have either positive or negative effects on T cells. Here, it is notable that two γ c cytokines, IL-15 and IL-2, have opposing effects on CD44hi CD8+ cells. Thus, the background turnover of these cells is enhanced by IL-15 (see above) but inhibited by IL-2 (135, 174). Why IL-2 is inhibitory for memory CD8+ cells is unclear, but stimulation of a suppressive population of IL2–dependent regulatory CD4+ T cells is a possibility (191, 192). Nevertheless, a balance in the relative concentrations of IL-15 and IL-2 could be a key mechanism for controlling memory CD8+ cell homeostasis. In addition to IL-2, a number of other cytokines could have an inhibitory effect on memory cells. In this respect, it is striking that bystander activation of CD44hi CD8+ cells during viral infections can promote death of these cells and thus lead to attrition of memory (193); death is IFN-γ – and Fas-dependent and appears to reflect FasL+ antigen-specific T cells interacting with IFN-γ –conditioned bystander CD44hi CD8+ cells. Hence, in this situation, a direct inhibitory influence of IFN-γ on bystander cells counters the capacity of IFNs to protect these cells through production of IL-15. Such inhibition may contribute to the normal homeostasis of memory cells, but direct data on this issue are lacking. As discussed above, the survival of memory cells is regulated by complex homeostatic mechanisms. Precise information on how these mechanisms influence the life/death fate of memory cells, however, is still minimal. Clearly, much remains to be discovered in this important area. ACKNOWLEDGMENTS We thank Ms. Barbara Marchand for typing the manuscript. This work was supported by NIH grants CA38355, AI21487, AI46710, AG01743, AI41079, and AI45809. CDS is a Scholar of The Leukemia & Lymphoma Society. Publication no. 14307-IMM from the Scripps Research Institute. Visit the Annual Reviews home page at www.annualreviews.org

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GENETIC DISSECTION OF IMMUNITY TO MYCOBACTERIA: The Human Model Annu. Rev. Immunol. 2002.20:581-620. Downloaded from arjournals.annualreviews.org by HINARI on 09/01/07. For personal use only.

Jean-Laurent Casanova and Laurent Abel Laboratory of Human Genetics of Infectious Diseases, Université René Descartes-INSERM U550, Necker Medical School, 156 rue de Vaugirard, 75015 Paris, France, European Union; e-mail: [email protected], [email protected]

Key Words Bacille Calmette-Guérin, environmental mycobacteria, leprosy, tuberculosis, Mendelian inheritance, complex inheritance ■ Abstract Humans are exposed to a variety of environmental mycobacteria (EM), and most children are inoculated with live Bacille Calmette-Guérin (BCG) vaccine. In addition, most of the world’s population is occasionally exposed to human-borne mycobacterial species, which are less abundant but more virulent. Although rarely pathogenic, mildly virulent mycobacteria, including BCG and most EM, may cause a variety of clinical diseases. Mycobacterium tuberculosis, M. leprae, and EM M. ulcerans are more virulent, causing tuberculosis, leprosy, and Buruli ulcer, respectively. Remarkably, only a minority of individuals develop clinical disease, even if infected with virulent mycobacteria. The interindividual variability of clinical outcome is thought to result in part from variability in the human genes that control host defense. In this well-defined microbiological and clinical context, the principles of mouse immunology and the methods of human genetics can be combined to facilitate the genetic dissection of immunity to mycobacteria. The natural infections are unique to the human model, not being found in any of the animal models of experimental infection. We review current genetic knowledge concerning the simple and complex inheritance of predisposition to mycobacterial diseases in humans. Rare patients with Mendelian disorders have been found to be vulnerable to BCG, a few EM, and M. tuberculosis. Most cases of presumed Mendelian susceptibility to these and other mycobacterial species remain unexplained. In the general population leprosy and tuberculosis have been shown to be associated with certain human genetic polymorphisms and linked to certain chromosomal regions. The causal vulnerability genes themselves have yet to be identified and their pathogenic alleles immunologically validated. The studies carried out to date have been fruitful, initiating the genetic dissection of protective immunity against a variety of mycobacterial species in natural conditions of infection. The human model has potential uses beyond the study of mycobacterial infections and may well become a model of choice for the investigation of immunity to infectious agents.

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INTRODUCTION The genus Mycobacterium, together with Corynebacterium and Nocardia, forms a monophyletic taxon within the family of Actinomycetes (1). These gram-positive bacteria have very waxy cell walls owing to the presence of mycolic acids. This renders them acid- and alcohol-fast, a feature that specifically distinguishes them from other bacteria. Unique biochemical pathways differentiate Mycobacteria from the related Corynebacterium and Nocardia genera. Extensive biochemical and genetic studies have made mycobacteria one of the groups of microorganisms about which we know the most (2, 3). The genus Mycobacterium is highly diverse, with 85 different species identified since the identification of M. leprae in 1873 (4). In addition there are almost certainly many more species that remain to be discovered. Based on sequencing of the 16S RNA (5), RNA polymerase (RpoB) (6), or hsp 65 (7) genes, species can be rapidly identified and phylogenetic trees established, illustrating the diversity of the mycobacterial world. This genotypic classification is consistent with the phenotypic classification of cultivable strains into rapidly growing and slowly growing species in vitro (2). There are also a number of Bacille Calmette-Guérin (BCG) vaccine substrains, derived from an attenuated M. bovis strain obtained in 1921 (8). Finally, the individual Mycobacterium species, such as M. tuberculosis, display significant diversity (9). The vast majority of mycobacterial species are environmental free-living saprophytes (10). Mycobacteria have adapted to various environmental conditions and grow in the soil (11) and water (12) of various regions of the world. They can live in a variety of natural waters, including fresh- and saltwater, and treated water, including swimming pools and drinking water, from which they are readily spread via aerosols. However, little is known about the metabolic requirements of these bacteria in their natural niches. For example, in drinking water distribution systems, M. avium is preferentially recovered from water samples, whereas the closely related M. intracellulare is recovered from biofilms (13). A few mycobacterial species, such as M. tuberculosis and M. bovis, first identified in infected humans and cattle, respectively, but both capable of infecting other animal species, have never been identified in the environment (14–16). This suggests that these organisms are obligate parasites of humans or animals. However, definitive conclusions cannot be drawn, as there may be many unexplored environmental niches. For example, M. ulcerans was only recently identified in the environment (17, 18). Along these lines, M. leprae was thought to be strictly human-tropic until natural infection of a rare animal, the nine-banded armadillo, was documented (19, 20). Caution is therefore required in observations concerning the natural reservoirs and hosts of mycobacteria and their resulting classification. All humans are exposed to water- and air-borne environmental mycobacteria (EM), which frequently come into contact with the skin and mucous membranes (principally the digestive and respiratory epithelia). In addition, the vast majority (85%) of children worldwide are inoculated intradermally or subcutaneously with live BCG vaccine. A large proportion of humans, mostly in the poorest countries

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or regions of the world, are also exposed to primarily air-borne human-tropic mycobacteria such as M. tuberculosis and M. leprae. Although some EM can infect animals (21) and certain mycobacterial species appear to be animal-tropic (e.g., M. bovis) (16), improvements in hygiene have reduced the level of human exposure to these species via contact with infected animals. Mycobacteria do not seem to engage in commensalism, as they have never been cultured from the skin or mucous membranes of healthy individuals (whereas they can be cultured from the tissues of infected patients). It is unclear whether humans and mycobacteria derive indirect benefit from each other via the environment. There seems to be little if any direct mutual benefit between Homo sapiens, a recently evolved vertebrate species (22, 23), and any species of the ancient Mycobacterium genus (24, 25). Rather, mycobacteria cause a broad epidemiological, clinical, and pathological spectrum of diseases in humans. M. leprae is the causal agent of leprosy, 700,000 new cases of which are reported annually worldwide (26). M. leprae resides within Schwann cells and macrophages. Although rarely life-threatening, leprosy is a chronic granulomatous disease of the skin and peripheral nerves and presents either as polar leprosy (lepromatous/multibacillary or tuberculoid/paucibacillary) or as one of several intermediate forms (19, 20). M. tuberculosis and related species (e.g., M. bovis) cause tuberculosis (14–16). About 8 million new cases of tuberculosis are reported annually worldwide, resulting in almost 1.9 million deaths (27). M. tuberculosis survives within macrophages, often despite the formation of surrounding granulomas. Tuberculosis is primarily a pulmonary disease, but organs other than the lungs may be affected. Buruli ulcer, caused by environmental M. ulcerans, is the third most common mycobacterial disease (18). The global incidence of this disease is not known, but the number of cases reported has increased in recent years (28). Unlike other mycobacteria, M. ulcerans grows extracellularly, and its pathogenicity seems to result largely from the secretion of a toxin. This chronic disease causes painless, expanding skin ulcers. Many other EM (e.g., M. avium) may cause occasional cases of localized or disseminated clinical disease, with each species being potentially pleiotropic yet generally showing a particular pattern of tissue-tropism (10). Similarly, BCG vaccines may cause local or disseminated disease (29). It is difficult to distinguish between exposure to and infection with mycobacteria other than BCG (which is inoculated) because individual host exposure cannot be strictly ascertained (30) (Figure 1). Exposure to most mycobacteria probably results only rarely in infection. The frequency of infection is itself probably underestimated because it is often based on the detection of immunological phenotypes, which reflect an adaptive memory immune response. Innate immunity may suffice to control the infection, and possible poor memory responses may make it difficult to identify the phenotype of interest. It is clear that only a minority of the individuals infected go on to develop clinical disease. This holds true not only for mildly virulent EM and BCG but also for the more virulent species M. tuberculosis (31) and M. leprae (20), which cause clinical disease in less than 10% of infected individuals. It may also be true for M. ulcerans (17). Thus, vulnerability to mycobacteria is the

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Figure 1 The various steps in the interaction between humans and mycobacteria. Exposure to mycobacteria does not always result in infection. Whether or not an established infection further develops depends on innate immunity, alone or in conjunction with adaptive immunity. Immunological and clinical phenotypes may be detectable once mycobacterial infection is established and adaptive immunity to mycobacteria is involved. Each of the three steps in this process is under host and environmental control. Host factors may be genetic (e.g., mutation in a gene involved in immunity to mycobacteria) or nongenetic (e.g., skin lesion) and may have an impact at each stage of the interaction. Environmental factors may be mycobacterial (e.g., virulence factors) or related to the mode of exposure (e.g., direct inoculation) and may have an impact at each stage of the interaction.

exception rather than the rule in humans. A combination of environmental microbial and nonmicrobial factors and host genetic and nongenetic factors determines the outcome of exposure and infection. Whatever the relative contributions of these factors, the occurrence of clinical disease implies that host defense to mycobacteria has failed. Immunity to mycobacteria has been extensively investigated in several animal models (32). The greatest strides forward have been made in the mouse model, particularly since the infection of a series of knockout mice with mycobacteria (mostly BCG and M. tuberculosis and more recently M. avium). These remarkable studies (33–35, 35a) established the relative contributions of various cell subsets, such as α/β and γ /δ T cells and CD4 and CD8 α/β T cells, to the destruction of mycobacteria by macrophages. They also identified a number of key molecules involved in recognition (e.g., Tlr2), regulation (e.g., IL-12, IFNγ , TNFα), and effector (e.g., NO) processes. An inherent limitation of these genotype-to-phenotype

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studies is that they require the definition of candidate genes. It has been shown, in a study using a phenotype-to-genotype approach, that the natural vulnerability of inbred mouse strains to mycobacteria is controlled by a recessive allele of the autosomal Bcg locus (36). A positional cloning approach has been used to identify a natural mutation in Nramp1/Bcg, which encodes a phagosomal membrane protein (37). This remarkable series of investigations has opened up new avenues of research into mycobacterial immunity (38, 39). Novel Mendelian mycobacterial vulnerability genes are unlikely to be identified in inbred strains of mice, and further progress will probably be achieved by investigations of complex vulnerability to mycobacteria (40). Despite its considerable impact in mycobacterial research, the mouse model suffers from the inherent limitation of being an experimental model of infection. The mycobacterial species with which the mice are inoculated are not natural mouse pathogens. M. microti is the natural agent of rodent tuberculosis, but mice are generally infected with M. tuberculosis, M. bovis BCG, or M. avium in this experimental model. In addition, the modes of infection used (e.g., the intravenous inoculation of mice kept in an artificial environment with large amounts of a laboratory mycobacterial strain) differ markedly from natural modes of infection (e.g., exposure of wild animals to low densities of natural, air-borne mycobacteria). Finally, laboratory mice form a small and poorly diverse group of inbred strains, which are less resistant and healthy than outbred wild mice. In contrast, humans form an expanding, outbred population in which most mycobacterial infections are natural, with BCG infection paradoxically serving as an “experimental” control. In addition, as human medicine is more developed than veterinary medicine, a comprehensive and up-to-date description of the phenotypic traits associated with each human mycobacterial disease is available. Finally, the technical difficulties that previously hampered biological research in humans have been largely overcome by recent major advances in human genetics (Figure 2). Considerable efforts are now focused on the genetic dissection of human protective immunity to mycobacteria in natural conditions of infection. We review the current state of knowledge in this novel and rapidly expanding field.

MILDLY VIRULENT MYCOBACTERIA: BCG AND MOST ENVIRONMENTAL MYCOBACTERIA Epidemiological Studies Environmental M. ulcerans is not discussed in this section because it is clearly more virulent than other known EM. Although mildly virulent, BCG and EM other than M. ulcerans may cause clinical diseases in humans. BCG vaccination may cause a variety of infectious adverse effects, from local adenitis (BCG-itis) to disseminated disease (BCG-osis) (29). The lung is the organ most frequently damaged, but clinical EM disease displays considerable diversity, reflecting the diversity of EM species and predisposing factors (10, 41, 42). For example,

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Figure 2 Methods and strategies for identifying human mycobacterial susceptibility genes. The molecular basis of rare Mendelian predisposition to mycobacterial disease may be investigated using several strategies. Linkage analysis is usually the first step in the positional cloning approach, although the identification of visible cytogenetic abnormalities may be helpful. The candidate gene approach (“by hypothesis”) involves the prior selection of genes (generally based on studies of animal models in vivo or human cells in vitro, or comparison with other human inherited disorders with a related clinical phenotype), which are then tested by functional assays and/or mutation detection. Another potentially fruitful strategy is based on studying the differential expression of genes in tissues from affected and healthy individuals. To determine the molecular basis of complex predisposition to common mycobacterial diseases, linkage studies (which may be model-based or model-free) search for a chromosomal region that segregates nonrandomly with the infectious disease-related phenotype of interest, within a number of families. The role of polymorphisms within candidate genes identified “by experiment” (i.e., located within this candidate region) is tested in association studies (which may be population-based or family-based). Candidate genes may also be selected by hypothesis (as in Mendelian investigations) and tested by association studies. Statistical evidence for an association should be validated by functional studies aimed at determining the impact of the polymorphism studied on gene function and, potentially, on the mycobacterial infection-related phenotype of interest.

M. marinum is associated with swimming pool skin granulomas, whereas M. malmoense is associated with pneumonitis in Northern Europe. The clinical spectrum of local disease associated with a single species, such as M. avium, may extend from benign cervical adenitis in childhood to more severe pulmonary disease in adulthood. In addition, not only well-known M. avium, but also much less-virulent

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species such as M. smegmatis and M. peregrinum may cause disseminated disease (43, 44). BCG and EM infections are generally considered to be rare but may be frequent in certain epidemiological contexts. Skin lesions and nosocomial inoculation favor the development of these infections (45). They are also favored by various acquired immunodeficiencies, including AIDS, hairy cell leukemia, and bone marrow and organ transplantation (10, 41, 42, 46). Human inherited disorders may favor the development of BCG/EM disease as well. Cystic fibrosis, the most frequent Mendelian disorder in Caucasian populations, results in a paucity of water in mucus secretions. Despite an apparently normal immune function, these patients suffer from chronic respiratory EM infections (47). In other patients disseminated BCG/EM disease almost invariably attests to an underlying Mendelian immune disorder, which may fall into one of two groups: classical primary immunodeficiency (PID) and Mendelian susceptibility to mycobacterial disease (MSMD) (Table 1) (46). Purely local disease (e.g., EM pneumonitis, BCG-itis) may also be favored by genetic factors, which may turn out to be simple or complex, but no epidemiological or molecular evidence is yet available either way (42).

Primary Immunodeficiencies SEVERE COMBINED IMMUNODEFICIENCY PIDs constitute a group of more than 100 Mendelian disorders (48–50). Patients with these disorders are generally susceptible to infection with a variety of viruses, bacteria, fungi, and protozoans. The genetic defect responsible predisposes the patient to severe BCG or EM disease in only a few of these disorders (46) (Table 1). Children with severe combined immunodeficiency (SCID) lack autologous T cells and are highly vulnerable to BCG (51–58), irrespective of the presence of B and/or NK cells and the underlying genetic defect. Only two SCID patients with EM disease have been reported, one with M. avium (59) and the other with M. marinum (60) disease. The small number of cases reported may be due to the low level of exposure to EM and of EM infection in children with SCID, most of whom die before their first birthday if they do not receive a bone marrow transplant. Alternatively, innate immunity may be sufficient to control EM, at least partially. Two thirds of SCID children inoculated (and hence infected) with BCG do not develop disseminated disease (58). Nevertheless, these cases demonstrate that human T cells are crucial for protective immunity against poorly virulent mycobacterial species such as BCG and some species of EM. HYPER-IgE SYNDROME Hyper-IgE syndrome (HIES) is a rare systemic autosomaldominant disorder combining a susceptibility to bacterial and fungal infections, eczema, and high serum IgE levels (48–50). The cellular defect responsible for the disease and its molecular basis have remained elusive. Disseminated BCG disease has been described in one child (61), and pneumonia caused by M. intracellulare has been reported in one adult patient (62). In some patients with HIES low levels of IL-12 and IFNγ production by blood cells may account for the occurrence

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TABLE 1 Mendelian immune disorders predisposing the patient to mycobacterial diseasea Condition Clinical form and molecular basis Mycobacterium species

References

SCID

HIES

T(−)B(−)NK(−):

BCG

(51–57)

Reticular dysgenesis, ADA deficiency T(−)B(−)NK(+): RAG-1/RAG-2, Artemis defects T(−)B(+)NK(−): γ -chain, JAK-3 defects T(−)B(+)NK(+): IL-7Rα, CD45 defects

M. avium, M. marinum

(59, 60)

M. tuberculosis

(131)

BCG

(61)

M. intracellulare

(62)

XR-CGD:

BCG

(56, 64–69)

gp91-NADPH oxidase AR-CGD: p22, p47, p67-NADPH oxidase

M. avium, M. flavescens, (70–73, 133) M. fortuitum, Mycobacterium spp. M. tuberculosis (133, 134)

XR-EDA-ID: NEMO defect XR-OL-EDA-ID: NEMO defect Cleft lip/palate-EDA-ID: Not identified

M. avium, M. kansasii, Mycobacterium spp. M. chelonae M. tuberculosis

(74–79)

HIGM

XR-HIGM: CD154

M. tuberculosis, M. bovis

(132)

MSMD

Response to IFNγ abolished:

BCG

(44, 79, 90, 95, 97, 99a, 99b, 101)

CGD

EDA-ID

Not identified

c-AR-IFN-γ R1 deficiency c-AR-IFN-γ R2 deficiency

Impaired response to IFNγ : p-AR-IFN-γ R1 deficiency p-AD-IFN-γ R1 deficiency p-AR-IFN-γ R2 deficiency p-AD-STAT-1 deficiency Impaired IFNγ production: c-AR-IL-12Rβ1 deficiency c-AR-IL-12p40 deficiency a

(76, 135)

M. avium, M. kansasii, (43, 44, 90, 94, 96–102, 99b) M. szulgai, M. chelonae, M. fortuitum, M. abscessus, M. smegmatis, M. peregrinum BCG

(86, 87, 106, 107, 109a, 109b)

M. avium, M. kansasii, M.chelonae, M. abscessus, M. gorvonac, M. asiaticum M. tuberculosis

(86, 87, 90, 107, 109, 109a, 109c)

BCG

(110–113, 115)

M. avium, M. chelonae M. tuberculosis

(112–114, 116) (115)

(106)

The conditions are indicated, followed by their clinical forms and molecular defect. The Mycobacterium species isolated from patients suffering from the various conditions are indicated in three groups [BCG, EM (second line), and M. tuberculosis and related species (third line)]. References corresponding to each of the three groups of mycobacteria, for each condition or clinical form considered, are also indicated. Abbrevations: SCID, severe combined immunodeficiency; CGD, chronic granulomatous disease; HIES, hyper-IgE syndrome; EDA-ID, anhidrotic ectodermal dysplasia with immunodeficiency; OL-EDA-ID, anhidrotic ectodermal dysplasia with immunodeficiency, lymphedema, and osteopetrosis; HIGM: hyper-IgM syndrome; MSMD, Mendelian susceptibility to mycobacterial disease; AR, autosomal recessive; XR, X-linked recessive; c-AR, complete autosomal recessive; p-AR, partial autosomal recessive; p-AD: partial autosomal dominant; BCG: Bacille Calmette-Guérin.

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of mycobacterial disease (63). However, HIES patients seem to be only mildly susceptible to BCG/EM, as many of the HIES patients reported were not only inoculated with BCG in childhood, but survived into adulthood, ruling out the possibility that they were not exposed to a variety of EM. CHRONIC GRANULOMATOUS DISEASE Patients with chronic granulomatous disease (CGD) have mutations in the genes encoding the NADPH oxidase complex, rendering phagocytes incapable of killing certain ingested microorganisms via oxygen-dependent pathways (48–50). They suffer from severe recurrent bacterial and fungal infections. Disseminated BCG-osis may occur in CGD patients (64–67), although local disease (BCG-itis) is more frequent (56, 68, 69). Disease caused by EM is less frequently reported, despite the large number of affected children and adults worldwide. Disseminated M. flavescens infection (70), M. fortuitum pneumonitis (71) and osteomyelitis (72), and M. avium pneumonitis (73) have been reported in CGD patients. These studies demonstrate that the phagocytic respiratory burst contributes to the control of weakly virulent mycobacterial species, such as BCG and a few EM. ANHIDROTIC ECTODERMAL DYSPLASIA WITH IMMUNODEFICIENCY Children with a rare multi-systemic disorder known as anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) (48–50) are vulnerable to a variety of bacterial infections. The genetic basis for X-linked EDA-ID was recently determined. Hypomorphic mutations in the NEMO gene, which encodes an essential component of the NF-κB activation machinery, were identified in affected males. Six patients with EDA-ID presented with disseminated M. avium disease in the first three years of life (74–76). Other patients with more severe NEMO mutations and the related syndrome, EDA-ID with osteopetrosis and lymphedema (OL-EDA-ID) (75), presented with M. kansasii (77) or an unidentified EM (78) infection in the first year of life. Disseminated M. chelonae infection was diagnosed in a girl with a related syndrome consisting of cleft lip/palate and EDA-ID (79). Mycobacterial disease is thought to result from the impairment of innate and adaptive immunity, as phagocytic (TNFα-R, Tlrs) and T-cell (IL-1α-R, IL-18-R) receptors signal through NF-κB. The high frequency and severity of early-onset EM infection in EDA-ID and related syndromes, particularly in OL-EDA-ID, indicates that NEMOdependent NF-κB activation is crucial for protective immunity against EM. PRIMARY IMMUNODEFICIENCIES, BCG, AND ENVIRONMENTAL MYCOBACTERIA Unexpectedly, mycobacterial disease is only rarely associated with PIDs. However, anticipated advances in the identification of EM in clinical samples and the diagnosis of more patients with PIDs may somewhat qualify this conclusion. Only four PIDs are associated with a predisposition to BCG and EM infection, and mycobacterial disease occurs only in a minority of affected individuals (from a small percentage of patients with CGD and HIES to approximately one third of patients with SCID and EDA-ID). Remarkably, most of the known PIDs do not

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predispose patients to mycobacterial infection (48–50). Complement and antibody deficiencies and more surprisingly most antigen-presenting cell and T-cell defects do not predispose patients to mycobacterial disease. Notably, these PIDs include HLA class I and HLA class II deficiencies (caused by molecular defects in trans) (46). There is conclusive evidence that NF-κB activation and T-cell development are required for immunity to BCG and EM. In contrast, antigen presentation by HLA class I or HLA class II molecules is apparently not required to ensure protective immunity to poorly virulent mycobacterial species. A number of children with severe mycobacterial disease and an ill-defined PID remain to be investigated at a molecular level, and the identification of novel mechanisms that contribute to mycobacterial immunity is therefore expected.

Inherited Disorders of the IL-12-IFNγ Axis MENDELIAN SUSCEPTIBILITY TO MYCOBACTERIAL DISEASE BCG and EM may also cause disseminated disease in otherwise healthy individuals with no classical primary immunodeficiency (80–83). These patients do not generally have associated infections, apart from salmonellosis, which affects less than half of the cases. Parental consanguinity and familial forms are frequently observed, and this syndrome was therefore named Mendelian susceptibility to mycobacterial disease (Table 1) (MIM 209950) (84). The syndrome is heterogeneous, although its clinical features seem to be restricted to a predisposition to mycobacterial infection. First, the genetic basis of the syndrome is not the same in all affected families. In most familial cases, inheritance is autosomal and recessive, but X-linked recessive inheritance seems to be involved in one family (83, 85), and autosomal dominant inheritance has been reported in several other families (86, 87, 109b,c). Second, clinical outcome differs between patients and has been found to correlate with the type of BCG granulomatous lesion present (88) and osteopontin expression in situ (88a). Children with granulomas of the lepromatous type (poorly delimited, multibacillary, with no epithelioid or giant cells) generally die of overwhelming infection, whereas patients with tuberculoid granulomas (well delimited, paucibacillary, with epithelioid and giant cells) have a favorable outcome. Positional cloning and a candidate gene approach have led to the identification of five Mendelian mycobacterial susceptibility genes that are mutated in children and adults with this syndrome: IFNGR1 and IFNGR2, encoding the two chains of the receptor for IFNγ , a pleiotropic cytokine secreted by NK and T cells; STAT1, encoding an essential transducer of IFNγ -mediated signals; IL12B, encoding the p40 subunit of IL-12, a potent IFNγ -inducing cytokine secreted by macrophages and dendritic cells; and IL12RB1, encoding the β1 chain of the receptor for IL12, expressed on NK and T cells. The type of mutation (recessive/dominant, hypomorphic/loss-of-function) also accounts for clinical heterogeneity, as the various mutations define nine disorders. All defects result in impaired IFNγ -mediated immunity. IFNγ secretion is impaired in patients with IL-12p40 and IL-12Rβ1 deficiency, whereas the response to IFNγ is impaired or abrogated in patients

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with partial or complete IFNγ R1, IFNγ R2, and STAT-1 deficiencies, respectively. We review these disorders and discuss their possible immunological implications. Clinical aspects of these conditions have been recently reviewed elsewhere (89–93). COMPLETE IFNγ R1 AND IFNγ R2 DEFICIENCY Complete interferon γ receptor ligand-binding chain (IFNγ R1) deficiency was the first genetic etiology of susceptibility to mycobacteria to be identified and was initially identified in two kindreds (94, 95). Seven more families have since been reported (43, 44, 96–99b). Two kindreds with complete IFNγ receptor signaling chain (IFNγ R2) deficiency have also been reported (90, 100). The recessive IFNGR1 and IFNGR2 mutations identified are null, as they preclude cell surface expression of the receptor. A lack of cellular responses to IFNγ in vitro has been demonstrated. Four other unrelated families have recently been identified in which children with complete IFNγ R1 deficiency were found to have normal expression of IFNγ R1 molecules on the cell surface (101, 102). The mutations in IFNGR1 in these children were loss-of-function mutations because they prevented the binding of the encoded surface receptors to their natural ligand, IFNγ . In all forms of complete IFNγ R deficiency, levels of circulating IFNγ in the blood are highly elevated (103). Disseminated disease owing to BCG and/or EM, including slow-growing (M. avium, M. kansasii, and M. szulgai) and fast-growing (M. chelonae, M. abscessus, M. peregrinum, M. smegmatis, and M. fortuitum) species, were diagnosed in all patients with IFNγ R deficiency. Remarkably, M. smegmatis (44) and M. peregrinum (43) are among the least virulent mycobacteria, and infection with these bacteria had never before been reported to cause disseminated disease in humans. All BCG-vaccinated patients developed BCG disseminated disease. In all cases, EM infections occurred before the age of 3 years. No mature mycobacterial granulomas were seen. No other opportunistic infections were observed, and the course of infections owing to common childhood pathogens was unremarkable, with the exception of Salmonella enterica (94), Listeria monocytogenes (97), and a few viruses (99a, 104, 105), each reported in one patient only with the exception of cytomegalovirus in two children. Mycobacterial (BCG and EM) infections resulted in the death of about half the patients and required continuous antimycobacterial treatment in the survivors. One child recently died of tuberculosis (see More Virulent Mycobacterial Species). Complete IFNγ R deficiency thus results in susceptibility to early-onset, disseminated, and diverse BCG/EM infection, implying that IFNγ is essential for protective immunity to mycobacteria. That IFNγ R-deficient children’s vulnerability to mycobacteria is more pronounced than that of children with SCID and EDA-ID suggests that residual IFNγ -mediated immunity in the latter two conditions probably accounts for the milder course of mycobacterial disease. PARTIAL RECESSIVE IFNγ R1 AND IFNγ R2 DEFICIENCY Two siblings with partial, rather than complete, IFNγ R1 deficiency have also been reported (106). A homozygous recessive missense mutation causing an amino-acid substitution in the

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extracellular domain of the receptor was identified. The receptor is expressed on the cell surface. Cells from children with partial IFNγ R1 deficiency and transfected cells carrying the mutant allele respond to IFNγ , but only at high concentrations. The missense IFNGR1 mutation probably reduces the affinity of the encoded receptor for its ligand, IFNγ . A patient with partial, as opposed to complete, IFNγ R2 deficiency was also reported (107). A homozygous nucleotide substitution was found in IFNGR2, causing a single amino-acid substitution in the extracellular region of the encoded receptor. Membrane-bound IFNγ R2 molecules were detected on the patient’s cells. The response of the patient’s cells to stimulation with IFNγ was impaired but not abolished. Transfection with the wild-type IFNGR2 gene restored full responsiveness to IFNγ . Thus, there is a causal relationship between the IFNGR2 missense mutation and weak cellular responses to IFNγ . The molecular mechanism underlying this condition remains to be determined. One IFNγ R1-deficient child had disseminated BCG and Salmonella enteritidis infections with a favorable outcome. His sibling, who had not been vaccinated with BCG, had curable symptomatic primary tuberculosis (see More Virulent Mycobacterial Species). Both are currently well at 17 and 20 years of age, with no treatment. The IFNγ R2-deficient patient with a history of BCG and M. abscessus infection is now well at 22 years of age. The clinical phenotype of the three patients with partial recessive IFNγ R deficiency is milder than that of children with complete IFNγ R deficiency. They had well-circumscribed and differentiated tuberculoid BCG granulomas. Thus, there is a correlation between the IFNGR1 and IFNGR2 genotype (loss-of-function or hypomorphic mutation), the cellular phenotype (complete or partial defect of response to IFNγ ), the histological phenotype (lepromatous or tuberculoid granulomas), and the clinical phenotype (poor or favorable outcome). The level of human IFNγ -mediated immunity seems to be the crucial factor determining the pathological lesions associated with, and the clinical outcome of, mycobacterial infections (108). PARTIAL DOMINANT IFNγ R1 DEFICIENCY Patients from 12 unrelated kindreds were found to have a dominant form of partial IFNγ R1 deficiency (86). These patients have a heterozygous small frameshift deletion in IFNGR1, downstream from the segment encoding the transmembrane domain. An interesting genetic feature of this disorder is that position 818 of IFNGR1 is the first small deletion hotspot to be identified in the human genome. Four families, each with a different dominant mutation, and five other families with a mutation at position 818, were subsequently identified (90, 105, 109, 109a,b,c). All the mutant alleles encode truncated receptors that reach the cell surface, bind IFNγ with normal affinity, dimerize and form a tetramer with two IFNγ R2 molecules, but do not transduce IFNγ -triggered signals owing to the lack of intracellular binding sites for the cytosolic molecules involved in the signaling cascade. The receptors also accumulate at the cell surface owing to the lack of an intracellular recycling site, thereby exerting a dominantnegative effect. Most IFNγ R1 dimers in heterozygous cells are nonfunctional, as they contain at least one defective molecule. The few wild-type IFNγ R1 dimers

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that form in response to IFNγ account for the defect being partial rather than complete. Patients with partial dominant IFNγ deficiency are generally vulnerable to BCG; EM infections, mostly with M. avium, are frequent, generally occurring after the age of three years and occasionally in adulthood. Severe disease owing to Histoplasma capsulatum (86) and varicella zoster virus (105) were each diagnosed in one patient. One parent who died of tuberculosis was not genetically tested owing to a lack of genetic material (see More Virulent Mycobacterial Species). The prognosis of these patients is relatively good, as only three of the reported patients died, and the survivors are all well without treatment. BCG granulomas are invariably tuberculoid in these patients. The clinical features of patients with partial dominant IFNγ R1 deficiency are clearly milder than those of patients with complete deficiencies and somewhat more severe than those with partial recessive IFNγ R deficiency. This is consistent with the respective cellular phenotypes and suggests that human IFNγ -mediated immunity is a genetically controlled quantitative trait that determines the outcome of mycobacterial invasion (108). It appears that no alternative immunological pathways can partially or completely compensate for the decrease or total lack of IFNγ signaling in response to a mycobacterial challenge. PARTIAL STAT-1 DEFICIENCY Two kindreds with the same heterozygous mutation in STAT1 causing partial dominant STAT-1 deficiency have recently been described (87). STAT-1 is a critical transducer of IFN-mediated signals, either as STAT-1 homodimers, designated gamma-activating factor (GAF), or as STAT-1/STAT-2/p48 trimers, known as interferon-stimulated gamma factor 3 (ISGF3). This heterozygous STAT1 mutation decreased cellular responses to both IFNγ and IFNα in terms of the activation of GAF, but not ISGF3. The mutation results in a loss of function for both cellular phenotypes but is dominant for GAF and recessive for ISGF3 activation in the patients’ heterozygous cells stimulated with IFNs. Clinically, one patient suffered from disseminated BCG infection with tuberculoid granulomas, whereas the other had disseminated M. avium infection. They are now 36 and 10 years old and well. The clinical and cellular phenotypes of the patients were similar to those of patients with partial recessive IFN-γ R deficiency, in terms of mycobacterial disease and GAF activation. This further documents the strict genotype-phenotype correlation in the IFNγ signaling pathway, with complete lack of response to IFNγ in vitro associated with a severe clinical outcome in vivo, and partial lack of response to IFNγ associated with a good clinical outcome (108). Moreover, this observation implies that human IFNγ -mediated mycobacterial immunity is dependent on STAT-1 and GAF. The lack of severe viral illness in the patients suggests that IFN-mediated viral immunity is STAT-1–independent and/or ISGF3-dependent. COMPLETE IL-12p40 DEFICIENCY A kindred with a loss-of-function recessive mutation in the IL12B gene encoding the p40 subunit of IL-12 was reported in 1988 (110). Five other kindreds with IL-12p40 deficiency have recently been identified

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(111). Neither monocytes nor dendritic cells were capable of secreting IL-12 upon stimulation. The lymphocytes of these patients secreted less IFNγ than do those of normal individuals. Impaired IFNγ secretion was successfully complemented in a dose-dependent manner by treatment with exogenous recombinant IL-12. This suggests that IFNγ deficiency is not a primary event but a consequence of inherited IL-12 deficiency. Another kindred with impaired, but not abolished, IL-12 production has also been reported (83, 85). The genetic defect was not identified, but the familial pedigree suggests recessive X-linked inheritance rather than autosomal recessive IL-12p40 deficiency. All patients but one with complete IL-12 deficiency owing to IL12B mutations had BCG infection, associated in only one child with EM infection (M. chelonae) and in another with tuberculosis (see More Virulent Mycobacterial Species); one third had S. enterica infections (which was the only infection in one child), and one had Nocardia asteroides infection. Only five children died of infection, and the survivors are all well without treatment. Thus, IL-12–deficient children probably suffer from mycobacterial infection primarily because their IFNγ -mediated immunity is impaired. Residual, IL-12–independent secretion of IFNγ probably accounts for the clinical phenotype being milder than that of children with complete IFNγ R deficiency. The clinical outcome of IL-12– deficient patients, however, varies from case to case. Mutations in the IL12RB1 gene encoding the β1 subunit of the IL-12 receptor were initially identified in six kindreds (112, 113). Three additional families were subsequently identified (114–116). All patients were homozygous for recessive mutations precluding the surface expression of IL-12Rβ1, and IFNγ secretion in vitro by otherwise functional NK cells and T cells was impaired (112, 113, 116a). The molecular complementation of defective cells by transfection with the wild-type IL12RB1 gene has recently been reported, confirming the pathogenic role of missense mutations in two kindreds (115, 116). A heterozygous IL12RB1 missense mutation was identified that may have contributed to another patient’s predisposition to mycobacteriosis (116b). A patient with a related phenotype of vulnerability to mycobacterial and staphylococcal disease was found to respond poorly to IL-12 despite having normal IL-12Rβ1 (117). The clinical phenotype of IL-12Rβ1–deficient patients appears to be similar to that of IL12p40–deficient children, suggesting that IL-12Rβ1–independent IL-12 signaling (118) has little impact on immunity to mycobacteria. BCG infections were curable, and EM infections (M. chelonae in one child, M. avium in the other cases) occurred only after the age of three years, with three cases diagnosed in adulthood. Half the patients had associated S. enterica infections, but no other infections were reported. One patient presented with abdominal tuberculosis (see More Virulent Mycobacterial Species). Only one patient died of EM infection, and the other patients were well at the last follow-up. The histological phenotype also appears to be milder, as BCG granulomas were found to be well delimited and well differentiated. Unlike the case of defects of cellular responses to IFNγ , there seems to be significant interfamilial and intrafamilial clinical heterogeneity, as two patients with IL-12Rβ1 DEFICIENCY

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IL-12Rβ1 deficiency were resistant to BCG (114, 115), one of whom did not even develop atypical mycobacteriosis (115). She was vaccinated three times with live BCG and did not show any adverse effect, whereas her brother had disseminated BCG-osis (115). Accordingly, EM disease generally presented in childhood but occurred in adulthood in three patients (113, 116) and we recently identified asymptomatic affected siblings in several kindreds (unpublished data). The occurrence of strictly asymptomatic individuals with genetic mutations abolishing IL-12– mediated immunity is a conceptually challenging observation that should perhaps lead to the revision of certain immunological dogmas (119). Unlike with IFNγ R deficiency there is apparently no correlation between the IL12B and IL12RB1 genotypes and clinical phenotype. There may be alternative pathways that compensate for the loss of IL-12 signaling and result in different cellular phenotypes (e.g., different levels of production of IFNγ ) in the patients. This suggests that IL-12 is one of several inducer cytokines and IFNγ is the only effector cytokine involved in immunity to mycobacteria.

Immunological Issues and Genetic Implications The identification of Mendelian defects predisposing patients to BCG/EM disseminated disease has revealed that T cells, NEMO-dependent NF-κB activation, and IL-12–dependent, STAT-1–mediated activation by IFNγ are crucial for protective immunity to mycobacteria. The clinical course of mycobacterial infection in patients with anhidrotic ectodermal dysplasia with immunodeficiency, lymphedema, and osteopetrosis (OL-EDA-ID) and complete IFNγ R deficiency is more severe than that in severe combined immunodeficiency (SCID) patients, which is itself more severe than that in patients with other forms of EDA-ID and Mendelian susceptibility to mycobacterial disease (MSMD). Among patients with MSMD, the level of IFNγ -mediated immunity determines the severity of mycobacterial disease. This suggests that impaired IFNγ -mediated immunity is the principal pathogenic mechanism underlying mycobacterial disease in patients with MSMD, EDA-ID, and SCID and that quantitative variations in production of, or response to, IFNγ between the different types of defect account for the differences in clinical phenotype. The identification of novel molecular defects in patients with PIDs and mycobacterial disease should enable a genetic dissection of the immunological pathways connected with IFNγ -mediated immunity. The identification of novel molecular defects upstream of IL-12 and downstream of STAT-1 in patients with MSMD should facilitate a genetic dissection of the principal axis of human immunity to mycobacteria. A lack of development of T cells results in a broad vulnerability to viruses, bacteria, and fungi, and impaired NF-κB activation results in a vulnerability to different types of bacteria. In contrast, patients with an inherited defect of the IL-12-IFNγ axis show a vulnerability that is mostly restricted to mycobacteria (120, 121). Infections by S. enterica in less than half the patients, and by H. capsulatum, L. monocytogenes, N. asteroides, and a few viruses in only one patient

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each, somewhat qualify this conclusion and suggest that other intracellular microorganisms may occasionally threaten these patients. The observation that IL12(R)–, IFNγ R–, and STAT-1–deficient patients are vulnerable to mycobacteria was expected, based on the mouse model (35, 120), but their apparent resistance to most other micro-organisms is surprising. In natural conditions of infection, it appears that the IL-12-IFNγ axis exerts some form of specificity for protective immunity to mycobacteria. This observation may reflect in part an ascertainment bias, and the molecular and clinical investigation of patients with various infectious diseases, living in different environments, is required. The lack of T cells obviously accounts for mycobacterial disease in SCID patients. In patients with EDA-ID and MSMD the cells responsible for vulnerability to mycobacteria are unknown. As IFNγ is a major macrophage-activating cytokine, macrophages probably play a key role in the pathogenesis of mycobacterial infections in patients with IFNγ R and STAT-1 deficiency. However, lymphocytes and other immune cell subsets may also be involved, either directly, as they express IFNγ R molecules, or indirectly, because impaired macrophage activation by IFNγ may restrict their activation by monokines (80, 98). Along these lines, the cells responsible for mycobacterial disease in IL-12p40 deficiency may be dendritic cells and/or macrophages, and those responsible in IL-12Rβ1 deficiency may be NK and/or T cells. Finally, NEMO and NF-κB are ubiquitous and triggered by different stimuli, raising questions about which pathways and which cells are responsible for the phenotype. Antigen-presenting cells, expressing the Toll-like receptors and the TNFα receptor, along with lymphocytes, expressing the IL-1β and IL-18 receptors, are good candidates. The development of conditional knock-out mice will be necessary to address the question of the cellular basis of mycobacterial disease in patients with impaired NF-κB or IFNγ immunity.

MORE VIRULENT MYCOBACTERIAL SPECIES (M. TUBERCULOSIS, M. LEPRAE, M. ULCERANS) Epidemiological Studies Factors known to contribute to the development of clinical tuberculosis include nongenetic host factors, such as acquired immunodeficiency (e.g., AIDS) or acquired immunity (e.g., BCG vaccination), and environmental factors, such as microbial virulence (e.g., resistance to antibiotics) and social conditions (e.g., poverty) (Figure 1) (14, 15, 122). The same factors, with the apparent exception of HIV infection and immunodeficiency, affect the development of leprosy (19, 20). Less is known about the factors that influence the development of Buruli ulcers (17). Many epidemiological studies have indicated that the host’s genetic makeup plays a role in the considerable variability of clinical response to infection with M. tuberculosis and M. leprae (123). Several studies have shown that an individual’s level of resistance to M. tuberculosis infection is correlated with the geographical origin of the family, with the most vulnerable individuals tending to have

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ancestors originating from areas once free of tuberculosis (124). The incidence of tuberculosis is particularly high during outbreaks in populations with no ancestral experience of the infection, such as native Americans (125). Black populations have also been shown to be more susceptible than Caucasians to tuberculosis (124, 126). Familial aggregation studies have also provided convincing evidence. Twin studies have shown much higher concordance rates for monozygotic than dizygotic pairs for both clinical tuberculosis (127) and leprosy (128). Finally, several segregation analyses (129) have clearly shown that susceptibility to leprosy has a significant genetic component. In particular, a segregation analysis performed on Desirade Island in the French West Indies found evidence for a recessive major gene controlling susceptibility to leprosy per se (i.e., regardless of clinical subtype) (130). To our knowledge, the role of host genetic factors in M. ulcerans infection has yet to be investigated (17). The following sections therefore deal with the principal genetic studies carried out for tuberculosis and leprosy.

Mendelian Disorders PRIMARY IMMUNODEFICIENCIES Virtually nothing is known regarding the relationships between any Mendelian disorder and infection with M. leprae or M. ulcerans. In contrast, a number of children with Mendelian disorders, including cystic fibrosis and primary immunodeficiencies (PIDs), have been found to be highly vulnerable to M. tuberculosis and related species (46). This was shown for one child with SCID (131), two patients with X-linked hyper IgM syndrome associated with CD154 mutations (132), one of 368 CGD patients from North America (133) and six of seven CGD patients from Hong Kong (134), where tuberculosis is endemic, and two EDA-ID patients including one child with a NEMO mutation (76, 135). The underlying immunodeficiency probably favored the development of tuberculosis in these patients because these types of immunodeficiency are known to predispose patients to infection with less virulent mycobacteria (except CD154 deficiency; see Mildly Virulent Mycobacteria), and the course of tuberculosis was invariably severe and often fatal. Two adults with tuberculosis were reported to suffer from common variable immunodeficiency (136) and mild T-cell deficiency (137). However, a causal relationship cannot be demonstrated in these two cases, as tuberculosis may have been coincidental. The susceptibility to mycobacterial infections observed in these conditions demonstrates that T cells, T cell–associated CD154, phagocytic NADPH oxidase, and NEMO-dependent NF-κB activation are essential components of immunity to M. tuberculosis. It is not entirely clear, however, which PIDs predispose patients to infection with virulent mycobacteria because exposure and infection are generally not detected and are relatively rare in countries in which PID diagnosis is possible, whereas constant exposure and infection are likely to occur in endemic countries where these PIDs are largely underdiagnosed. In Hong Kong, an endemic region where PIDs are well diagnosed, most patients with CGD suffer from severe tuberculosis (134). Thus, the number and type of PIDs predisposing patients to severe disease are currently underestimated, and more studies in endemic areas are

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required to document more cases. It is therefore less clear which genes and immune pathways are essential for protective immunity against M. tuberculosis than BCG and EM. MENDELIAN DISORDERS OF THE IL-12-IFNγ AXIS Patients with MSMD are vulnerable to M. tuberculosis. The classical phenotype of these patients is vulnerability to poorly virulent mycobacterial species such as Bacille Calmette-Guérin (BCG) and environmental mycobacteria (EM), but five patients developed tuberculosis, which in three cases was the sole clinical manifestation of their underlying genetic disorder. One child with IL-12p40 deficiency (111) and another with complete IFNγ R1 deficiency (unpublished data) had curable BCG-osis and died of tuberculosis. The reason only two patients with an inherited defect of the IL-12-IFNγ axis and BCG/EM clinical disease also had tuberculosis is probably that most patients were not exposed to M. tuberculosis. One parent of a child with partial dominant IFNγ R1 deficiency died of tuberculosis, but no material was available for genetic analysis (86). One child who was not vaccinated with BCG developed a symptomatic form of primary tuberculosis (106). The child was diagnosed with partial recessive IFNγ R1 deficiency because her older brother had symptomatic BCG-osis. Otherwise, she would have probably remained one in a multitude of cases of symptomatic primary tuberculosis, as she suffered no other unusual infections, including other mycobacterial infections. Finally, another patient who was vaccinated three times with BCG with no adverse effect (and can be considered as being truly resistant to BCG, as the tuberculin skin tests were positive, implying that live BCG had indeed been inoculated) and who did not develop atypical mycobacteriosis presented with full-blown abdominal tuberculosis at 18 years of age (115). This observation suggests that susceptibility to severe forms of tuberculosis, such as extra-pulmonary tuberculosis, may be caused in BCG-resistant and otherwise healthy individuals by purely Mendelian disorders of the IL-12-IFNγ axis (138). The precise frequency of such types of Mendelian predisposition to tuberculosis is unknown.

Complex Inheritance In more common situations, the strategy used to investigate the genetic component of the response to M. tuberculosis and M. leprae is based on population genetic epidemiology studies (139). The ultimate goal of these analyses is to identify the genes, and the alleles of these genes, that significantly account for the phenotype of interest (e.g., affected/unaffected) and possible interactions of these alleles with environmental risk factors. Recent developments, such as the establishment of a genetic map of the human genome based on highly polymorphic markers (140), the growing availability of intragenic single nucleotide polymorphisms (141, 142), and the sequencing of the human genome (22, 23) have led to the creation of tools essential for these genetic studies. Numerous methods have been (and are being) developed. They generally fall into two categories (143, 144): linkage analysis methods, which seek to locate a chromosomal region that segregates nonrandomly

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with the phenotype of interest within families, and association studies, which test for a significant association between a specific genetic polymorphism and a phenotype within a population (Figure 2). One major advantage of linkage studies is that they can be used to explore the whole genome (genome screen) ensuring that all major loci involved in the control of a phenotype are identified and making it possible to discover new genes (and, potentially, new immunological pathways). Association studies investigate the role of polymorphisms (or alleles) of candidate genes defined on the basis of either their location (in regions identified by linkage analysis) or function (genes involved in the immune response to mycobacteria). After presenting the main candidate genes that have been investigated by linkage and/or association studies in both diseases, we review the two genome screens recently reported in tuberculosis and leprosy (Table 2). THE MAJOR HISTOCOMPATIBILITY COMPLEX Human leukocyte antigen (HLA) molecules are highly polymorphic and present antigenic peptides to α/β T cells. This has led to extensive studies of the role of the HLA genes in tuberculosis and leprosy. Most of the studies carried out were population-based association surveys in adults, comparing HLA class I and/or class II alleles in unrelated cases and unrelated controls. No consistent findings were reported for HLA-I alleles. Several studies have reported a higher frequency of HLA-DR2 in patients with pulmonary tuberculosis (PTB) from Indonesia (145) and India (146, 147), with an associated odds ratio (OR) of 1.8–2.7. If the prevalence of the disease is lower than 10%, which is generally the case for tuberculosis, the OR is a valid estimate of the relative risk (i.e., the risk of disease with a particular genotype versus the risk of disease without this genotype). A family-based association study also provided evidence that HLA-DR2 is involved in PTB (148), by showing a skewed transmission of DR2 (around 80%) to affected offspring from DR2 heterozygous parents in a sample of 25 multiple-case Indian families. Other case-control studies failed to replicate the HLA-DR2 association in Chinese (149), Mexican (150), and Indian (151) populations. Nevertheless, the use of HLA serologic techniques has not provided an accurate resolution of HLA class II types (152). Recent studies with molecular DNA-based typing methods have reported a high frequency of DRB1∗ 1501 (a DR2 allele) in PTB patients from India (153, 154) and Mexico (155), with estimated ORs of 2.7–8. Two DQ1 alleles, DQB1∗ 0503 in Cambodia (156) and DQB1∗ 0501 in Mexico (155), were also found to be associated with PTB. HLA has been reported to be involved in tuberculoid and lepromatous leprosy, but not in leprosy per se (i.e., all clinical forms of leprosy). Case-control association studies (157) have shown that HLA-DR3 frequencies are high in patients with tuberculoid leprosy and low in patients with lepromatous leprosy. A high frequency of HLA-DR2 was also reported in both tuberculoid and lepromatous patients (158). Two family-based association studies in India (159) and Egypt (160) found a skewed distribution of the DR2 allele in siblings with tuberculoid leprosy. Using molecular HLA typing, an association was found between tuberculoid leprosy and DR2 alleles DRB1∗ 1501 and DRB1∗ 1502 in India (161, 162). Sib-pair

Genome-wide linkage analysis

10p13

224 multicase families

1 large pedigree of Aboriginal Canadians 20 leprosy families

44 families

(176) (178) (179) (177) (180) (184) (199)

4.1 (1.9–9.1) 1.8 (1.1–3) 0.5 (0.3–0.7)

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