Graft vs. Host Disease, Third Edition

Third Edition

GRAFT-VS.-HOST DISEASE Third Edition

GRAFT-VS.-HOST DISEASE Edited by

James L.M.Ferrara University of Michigan Cancer Center Ann Arbor, Michigan, U.S.A. Kenneth R.Cooke University of Michigan Cancer Center Ann Arbor, Michigan, U.S.A. H.Joachim Deeg Fred Hutchinson Cancer Research Center Seattle, Washington, U.S.A.

MARCEL DEKKER NEW YORK

This edition published in the Taylor & Francis e-Library, 2005.

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Foreword More than 100,000 individuals in the United States owe their life to the successful treatment of potentially fatal diseases by hematopoietic cell transplantation. The technique of transplantation has evolved from a relatively crude procedure into an increasingly refined treatment strategy not only for lymphohematopoietic malignancies and genetic defects but also for certain solid tumors and autoimmune disorders. The most severe remaining problem is the development of graft-versus-host disease (GVHD), i.e., the manifestation of the alloreactivity of donor cells against the patient’s tissues and organs. GVHD sets the stage for prolonged immunodeficiency, infections, chronic pulmonary disease, impaired growth and development and secondary malignancies. The increasing use of mobilized peripheral blood cells (instead of marrow) results in an increase in chronic GVHD. The use of non-myeloablative or reduced intensity conditioning regimens has changed the kinetics of the development of GVHD. Acute symptoms may occur later than previously observed. Some of the older criteria for GVHD classification are now obsolete, and staging schemes that also consider the dynamics of GVHD must be developed. These changes appear to be related in part to activation of the transplanted cells by exposure of the donor to G-CSF for mobilization, with associated alterations in the cytokine profiles in monocytes and T-cells. Reduced intensity conditioning regimens also may have different effects on the host environment regarding antigen presentation, in particular by dendritic cells, and on the release of cytokines and chemokines. The regulation of apoptosis and the apparent differences in regards to proinflammatory and apoptotic pathways in different organs are also being studied. There is evidence in murine models that manifestations of GVHD in target organs may be independent of the expression of MHC antigens. The role of cytokines such as interleukin-10, -15, -18 and others is only beginning to be understood, and a better understanding of these interactions may lead to more effective prophylactic and therapeutic strategies for GVHD. In clinical transplantation, the role of polymorphisms in the promoter regions of cytokine genes such as TNFα and IL-10 is coming into clearer focus, and should soon be able to define more precisely the risk of an individual patient for significant GVHD. Since the second edition of this book, major advances have occurred in the area of histocompatibility. HLA typing has been refined to permit selection of unrelated individuals as donors on the basis of HLA identity at the DNA level for class I and class II antigens. As a result, outcome with unrelated donor transplants has approached or is identical to those achieved with HLA-identical siblings. Furthermore, HLA-C antigens have been identified as important components in the interaction between donor and host, in particular as targets for NK cells. While still controversial, there is evidence that differences between donor and host in regard to their killer inhibitory receptors (KIR) can be exploited for more effective eradication of a patient’s leukemia without increasing the risk of GVHD. Similarly, considerable work has also been carried out on so-called minor

antigens and their tissue specific expression, permitting donor cells to be directed at antigens present on hematopoietic (and leukemic) cells but not on other tissues. Each refinement in histocompatibility typing makes it less likely that a perfect match can be found. Through the efforts of the National Marrow Donor Program, however, there are now more than 5 million HLA typed donors in the US registry and another 3 million in cooperative registries worldwide, making it increasingly possible to find a match. Finally, several new pharmacological agents with anti-inflammatory, immunosuppressive and mucosal protectant properties are being investigated in clinical trials. Results obtained with mycophenolate mofetil and with sirolimus, especially when used in combination regimens, are encouraging. In the approximately 50 years since the work of Medawar and colleagues, we have known that it is possible to achieve a state of non-reactivity between incompatible donor and recipient cells. This state of tolerance, easily achieved during fetal life, has proved to be extremely difficult to achieve in later life. We continue to struggle with the consequences of this incompatibility between donor and host, graft rejection and GvHD. Numerous investigators have contributed their expertise to this third edition of the book to provide a comprehensive overview and update on recent developments in our understanding and the management of GVHD. It is reasonable to hope that we are nearing solutions to these problems and that the material presented here will hasten that process. E.Donnall Thomas, M.D.

Preface to the Third Edition Seven years have passed since the second edition of this textbook. The editors are gratified by the enthusiastic response of our readers who have often commented that the clear and clean writing in this volume makes it accessible to many who are new to the field. We believe this book should be in the hands of every clinician caring for patients undergoing an allogeneic bone marrow transplant, as well as every scientist interested in the complex and fascinating immunologic phenomenon of GVHD. Because this text is used in over thirty countries throughout the world, we have made every effort in the third edition to maintain and enhance its accessibility. In order to keep this volume affordable to individuals, and particularly to young individuals at the early stages of their careers, we reduced its overall length. Because several new areas of investigation and expansion required inclusion, however, we decided to eliminate the introductory section of basic immunologic sciences. We have also emphasized the translational aspects of individual topics by including basic science and clinical perspectives in the same chapter wherever possible. We believe this fusion and streamlining will significantly enhance the utility of the book. Readers will note the rapid advance of certain topics. For example, in the second edition, there was not a single mention of regulatory or suppressive phenomena. Today, not only have regulatory cells become a hot topic in general immunology, they have been used very effectively to modulate GVHD in animal models. Our increased ability to identify these cells and isolate their molecular pathways leads us to hope that we will be able to manipulate these and other cells in ways that will benefit our patients in the years ahead. Readers will also note that the editorship has changed. We are grateful to Steven Burakoff for his many contributions to the immunology of GVHD and for his help in earlier editions of this text. We are delighted to be joined by Kenneth Cooke, whose enthusiasm, energy and passion for excellence will help to make this third edition the best yet. Finally we would like to dedicate this volume to our best teachers: our patients. It is our hope that by the time of the fourth edition, many of the insights illuminated in the following pages will have been translated into new therapies that will make allogeneic bone marrow transplantation a safer and more effective therapy for everyone who needs it. James L.M.Ferrara Kenneth R.Cooke H.Joachim Deeg

Preface to the Second Edition We begin this preface to the second edition of Graft-vs.-Host Disease with gratitude to our readership for the overwhelmingly positive response to the first edition, which required a second printing even while the revised volume was in progress. We appreciate the many thoughtful reviews and constructive suggestions, several of which have been incorporated into this new volume. Many of our readers have specialty training in the fields of hematology and oncology, but relatively limited backgrounds in basic immunology, an area of investigation that has exploded in the past decade. Yet nowhere is transplantation immunology more complex than in a graft-vs.-host reaction, where the immune system is both effector and target. As a result, detailed discussions of graft-vs,-host disease (GVHD) are often dense and sometimes impenetrable to the very readers for whom this book was intended. We have therefore included, as introduction, a series of chapters that review important aspects of immunobiology necessary to the understanding of GVHD pathophysiology as well as the current efforts used to control this disease. The first chapter in Part I reviews thymic ontogeny and summarizes the complex process of T-cell maturation and differentiation. The appreciation of the thymus as a target organ of GVHD helps to explain how the disease can have such devastating effects on the reconstitution of the immune system. Chapters on MHC Class I and Class II antigens review the structure and function of these molecules, which are responsible for activating CD8+ and CD4+ T cells, respectively. Overviews are presented on T-cell costimulation and anergy, molecular mechanisms of immunosuppression, and adhesion molecules critical to T-cell migration and traffic. One entire chapter is devoted to a detailed discussion of cytokine networks, an area of intense investigation that is central to multiple aspects of GVHD. Finally, reviews of target cell death by both cytolytic T lymphocytes and natural killer cells provide a context for mechanisms of specific target organ pathology. The middle part of the book has been reorganized to bring together research from experimental models and clinical protocols into single chapters. The classic triad of target organs—skin, liver, and intestinal tract—is reviewed first, followed by other, less “classic” target organs such as the immune system, the hematopoietic system, and the lung. Mechanisms of graft-vs.-leukemia effects are explored in extensive detail, with a thorough discussion of the different cellular populations that participate in this process. This part concludes with two chapters that summarize recent contributions to GVHD pathophysiology: the role of endotoxin and infections and the functional interactions between lymphocytes from the perspective of their cytokine profiles. The third and final part concentrates on clinical GVHD, its prevention and treatment. The first three chapters review the clinical spectra of GVHD after both allogeneic and autologous bone marrow transplantation (BMT), as well as transfusion-associated GVHD. Current strategies for preventing GVHD are summarized next: the matching of donor and host at HLA loci, the depletion of T cells from the donor marrow, the

nonspecific immunosuppression of the host, and the use of cytokine antagonists. The following chapter reviews the emerging role of IL-10 in the induction and maintenance of donor-host tolerance after BMT. Alternative approaches to reducing GVHD are summarized next, including the selection of stem cells, adoptive immunotherapy, and the increasing efforts at genetic modification of hematopoietic cells and T cells. The last chapter serves as a sort of coda to the entire book, with contributions from 21 investigators reviewing the most recent advances in GVHD research, many of them still in press at this time. An endeavor of this size and complexity requires the generous efforts of many contributors, whom we gratefully acknowledge. We would like to dedicate this volume to two groups of individuals who remain the bedrock of both experimental and clinical GVHD research: first, the outstanding animal care technicians and veterinarians without whose care and diligence the exploration of animal models would be impossible, and second, the nurses, residents, and fellows whose superb skills and compassion continue to offer hope to our patients for a brighter tomorrow. If this book can contribute in some small way to that hope, our efforts in bringing it forth will be more than amply rewarded. James L.M.Ferrara H.Joachim Deeg Steven J.Burakoff

Preface to the First Edition There are two major reasons for producing a broad overview of graft-vs.-host disease (GVHD). From a clinical standpoint, GVHD can be a devastating complication of bone marrow transplantation. Significant progress has been made; however, several obstacles remain in overcoming this problem, particularly in patients given histoincompatible grafts. Multiple approaches, discussed in the clinical sections of this book, have been taken, each with advantages and disadvantages. Frequently, those approaches were based on experimental models. Thus, it is only logical that the clinical sections are preceded by a description of preclinical studies. It is these animal models that have been critical in advancing our understanding of the physiology of GVHD. Control of genetic variables as well as the ability to produce GVHD in a variety of models have permitted insights not possible in a strictly clinical context. In addition, these systems have also provided models for understanding lymphocyte development and function in vivo irrespective of GVHD. The increasing sophistication and precision of cellular and molecular probes have initiated an analysis of cellular mechanisms in physiological environments rather than in a petri dish or test tube. The abnormalities of GVHD thus provide a perspective from which to understand the complexities of normal lymphocyte differentiation and activation. The insights from these studies should lead to improved understanding of fundamental lymphocyte biology and thus to new therapeutic strategies, not only in bone marrow transplantation, but in other areas, such as autoimmune diseases. It is our hope that future editions of this book will reflect this increased understanding and will witness further movement from experimental models to clinical realms. The production of a book this size is a long and complex undertaking, and we are grateful to the many people who have given generously of their time and effort. We would like to thank particularly Joshua Hauser and Michele Fox for their labors in the preparation of the index. We also wish to acknowledge the continued support of our colleagues and teachers who have encouraged us over many years, especially David G.Nathan, Fred S. Rosen, Baruj Benacerraf, Rainer Storb, and E.Donnall Thomas. Steven J.Burakoff H.Joachim Deeg James L.M.Ferrara Kerry Atkinson

Contents Foreword E.Donnall Thomas Preface to the Third Edition

iv

Preface to the Second Edition

vii

Preface to the First Edition

ix

Contributors

vi

xiii

I. GRAFT-vs.-HOST DISEASE PATHOPHYSIOLOGY

Immune Activation and Dysregulation 1. The Pathophysiology of Graft-vs.-Host Disease James L.M.Ferrara, Kenneth R.Cooke, and Takanori Teshima 2. Animal Models of Graft-vs.-Host Disease Robert Korngold, Thea M.Friedman, and Stephen C.Jones 3. Graft-vs.-Host Disease as a Th1-Type Process: Regulation by Th2-Type Cells Daniel H.Fowler and Ronald E.Gress 4. Role of T-Cell Costimulation and Regulatory Cells in Allogeneic Hematopoietic Cell Transplantation Bruce R.Blazar and Patricia A.Taylor 5. Chemokines and Graft-vs.-Host Disease Kenneth R.Cooke, Jon S.Serody, Lisbeth A.Welniak, and William J.Murphy 6. Graft-vs.-Leukemia Effects Jeffrey J.Molldrem and Warren D.Shlomchik

1 36 62

89

131 165

Pathophysiology of Target Organ Damage 7. The Immune System in Graft-vs.-Host Disease: Target and Effector Organ Crystal L.Mackall, Frances T.Hakim, and Andrea Velardi 8. Pathology and Pathogenesis of Cutaneous Graft-vs.-Host Disease John L.Wagner and George F.Murphy

207 243

9. Graft-vs.-Host Disease of the Liver Chen Liu and James M.Crawford 10. Intestinal Graft-vs.-Host Disease Allan Mowat and Gerard Socié 11. The Pathophysiology of Lung Injury After Hematopoietic Stem Cell Transplantation Kenneth R.Cooke and James L.M.Ferrara II. CLINICAL GRAFT-vs.-HOST DISEASE: PREVENTION AND TREATMENT 12. Clinical Spectrum of Acute Graft-vs.-Host Disease Joseph H.Antin and H.Joachim Deeg 13. Role of Minor Histocompatibility Antigens in the Development of Graft-vs.Host Disease and Graft-vs.-Leukemia Reactivity Anna Barbui, Lisette van de Corput, and Frederik J.H.Falkenberg 14. Advances in Tissue Typing and Their Potential Impact on GVHD Incidence and Severity Effie W.Petersdorf and Takehiko Sasazuki 15. Immunosuppression and Immunophilin Ligands: Cyclosporin, Tacrolimus, and Sirolimus Barbara E.Bierer 16. Prophylaxis and Treatment of Acute Graft-vs.-Host Disease Nelson J.Chao, Ernst Holler, and H.Joachim Deeg 17. Graft Manipulation to Prevent Graft-vs.-Host Disease Robert J.Soiffer 18. Graft-vs.-Host Disease in Nonmyeloablative Transplant Daniel R.Couriel and Sergio Giralt 19. Adoptive Immunotherapy in Stem Cell Transplantation David L.Porter, John E.Levine, and Hans-Jochem Kolb 20. Clinical Spectrum and Therapeutic Approaches to Chronic Graft-vs.-Host Disease Görgün Akpek, Charles S.Via, and Georgia B.Vogelsang 21. Recent Advances and Future Directions Pavan Reddy, Kenneth R.Cooke, James L.M.Ferrara, and H.Joachim Deeg Index

276 299 352

398 413

432

459

495 518 549 563 595

651

674

Contributors Görgün Akpek University of Maryland Medical System, Baltimore, Maryland, U.S.A. Joseph H.Antin Harvard Medical School and Dana-Farber Cancer Institute, Boston, Massachusetts, U.S.A. Anna Barbui Ospedali Riuniti de Bergamo, Bergamo, Italy Barbara E.Bierer Harvard Medical School and Brigham and Women’s Hospital, Boston, Massachusetts, U.S.A. Bruce R.Blazar University of Minnesota, Minneapolis, Minnesota, U.S.A. Nelson J.Chao Duke University, Durham, North Carolina, U.S.A. Kenneth R.Cooke University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan, U.S.A. Lisette van de Corput University Medical Center Utrecht and Wilhelmina Children’s Hospital, Utrecht, The Netherlands Daniel R.Couriel The University of Texas M.D. Anderson Cancer Center, Houston, Texas, U.S.A. James M.Crawford University of Florida College of Medicine, Gainesville, Florida, U.S.A. H.Joachim Deeg Fred Hutchinson Cancer Research Center and University of Washington, Seattle, Washington, U.S.A. Frederik J.H.Falkenburg Leiden University Medical Center, Leiden, The Netherlands James L.M.Ferrara University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan, U.S.A. Daniel H.Fowler National Institutes of Health, Bethesda, Maryland, U.S.A. Thea M.Friedman Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania, U.S.A. Sergio Giralt The University of Texas M.D. Anderson Cancer Center, Houston, Texas, U.S.A. Ronald E.Gress National Institutes of Health, Bethesda, Maryland, U.S.A. Frances T.Hakim Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, U.S.A. Ernst Holler University of Regensburg, Regensburg, Germany Stephen C.Jones Trudeau Institute, Inc., Saranac Lake, New York, U.S.A. Hans-Jochem Kolb University of Munich, Munich, Germany Robert Korngold Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania, U.S.A. John E.Levine University of Michigan, Ann Arbor, Michigan, U.S.A. Chen Liu University of Florida College of Medicine, Gainesville, Florida, U.S.A. Crystal L.Mackall Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, U.S.A. Gerald F.Marshall Consultant, Niles, Michigan, U.S.A.

Jeffrey J.Molldrem The University of Texas, M.D.Anderson Cancer Center, Houston, Texas, U.S.A. Allan Mowat University of Glasgow, Glasgow, Scotland George F.Murphy Thomas Jefferson University, Philadelphia, Pennsylvania, and Harvard Medical School and Brigham and Women’s Hospital, Boston, Massachusetts, U.S.A. William J.Murphy Department of Microbiology and Immunology, University of Nevada, Reno, Nevada, U.S.A. Effie W.Petersdorf University of Washington and Fred Hutchinson Cancer Research Center, Seattle, Washington, U.S.A. David L.Porter University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A. Pavan Reddy University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan, U.S.A. Takehiko Sasazuki Research Institute, International Medical Center of Japan, Tokyo, Japan Jon S.Serody Departments of Medicine, Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, U.S.A. Warren D.Shlomchik School of Medicine, Yale University, New Haven, Connecticut, U.S.A. Gerard Socié Hospital St. Louis, Paris, France Robert J.Soiffer Dana-Farber Cancer Institute, Boston, Massachusetts, U.S.A. Patricia A.Taylor University of Minnesota, Minneapolis, Minnesota, U.S.A. Takanori Teshima Department of Medicine II, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan Andrea Velardi Department of Clinical and Experimental Medicine, Division of Hematology and Clinical Immunology, University of Perugia School of Medicine, Perugia, Italy Charles S.Via University of Maryland Medical System, Baltimore, Maryland, U.S.A. Georgia B.Vogelsang Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A. John L.Wagner Thomas Jefferson University, Philadelphia, Pennsylvania, and Harvard Medical School, Boston, Massachusetts, U.S.A. Lisbeth A.Welniak Department of Microbiology and Immunology, University of Nevada, Reno, Nevada, U.S.A.

1 The Pathophysiology of Graft-vs.-Host Disease JAMES L.M.FERRARA and KENNETH R.COOKE University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan, U.S.A. TAKANORI TESHIMA Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan The graft-vs.-host (GVH) reaction was first noted more than a half century ago when irradiated mice were infused with allogeneic marrow and spleen cells. Although mice recovered from radiation injury and marrow aplasia, they subsequently died with “secondary disease,” a syndrome consisting of diarrhea, weight loss, skin changes, and liver abnormalities (1). This phenomenon was subsequently recognized as GVH disease (GVHD). In 1966, Billingham formulated the three requirements necessary for GVHD. First, the transplanted graft must contain immunologically competent cells; second, the recipient must be incapable of rejecting the transplanted cells; third, the recipient must express tissue antigens that are not present in the transplant donor that could thus be recognized as foreign (2). It is now known that mature T lymphocytes are the immunocompetent cells in the graft that fulfill the first precondition for GVHD (3). After allogeneic hematopoietic stem cell transplantation (SCT), the severity of GVHD correlates with the number of donor T cells transfused (4). Because experimental models have shown that the ability of marrow T cells to induce GVHD is much less potent than mature circulating T cells (5), the contamination of bone marrow with peripheral blood at the time of marrow harvest may contribute significantly to the development of GVHD. The seond precondition for GVHD stipulates that the recipient must be immunocompromised. A normal immune system will usually reject T cells from a foreign donor. This requirement is found most commonly during allogeneic SCT, where patients have traditionally received immunoablative doses of chemotherapy and/or radiation before stem cell infusion. Conditions are also met in other situations, such as during solid organ allografts and blood transfusion, where recipients are often immunosuppressed. There are, however, exceptions to this requirement. GVHD can occur in an immunocompetent recipient of tissues from a donor who is homozygous for one haplotype of the recipient (e.g., transfusion of blood from an HLA homozygous parent to a heterozygous child). This genetic situation prevents the recipient from rejecting the donor cells even though he or she is not immunocompromised (6).

Graft-vs.-host disease

2

The third precondition for GVHD, the expression of recipient tissue antigens not present in the donor, became the focus of intensive research with the discovery of the major histocompatibility complex (MHC). Human leukocyte antigens (HLA) are the gene products of the MHC. HLA are expressed on the cell surfaces of all nucleated cells in the human body and are essential to the activation of allogeneic T cells (7). During lymphocyte development, T cells are selected in the thymus to recognize self-MHC molecules, but when confronted with allogeneic (nonself) MHC molecules, the activated T cells mount a formidable attack that culminates in the destruction of the allogeneic tissues. In fact, MHC differences between donor and recipient are the most important risk factor for the induction of GVHD. In addition, there are minor histocompatibility antigens (mHAg) derived from the expression of polymorphic genes. Surprisingly, GVH reactions can occur between genetically identical strains and individuals (8,9). These rare observations have prompted a revision of the third postulate to include the inappropriate recognition of host self antigens. GVHD can be considered both acute and chronic, given the timing of its occurrence. This chapter will focus on acute GVHD; chronic GVHD will be reviewed in Chapter 20. I. ACUTE GVHD PATHIOPHYSIOLOGY: A THREE-STEP MODEL The pathophysiology of acute GVHD can be considered as a three-step process where the innate and adaptive immune systems interact (Fig. 1). The three steps are 1) tissue damage to the recipient by the radiation/chemotherapy pretransplant conditioning regimen, 2) donor T-cell activation and clonal expansion, and 3) cellular and inflammatory factors. This schema underscores the importance of mononuclear phagocytes and other accessory cells to the development of GVHD after complex interactions with cytokines secreted by activated donor T cells. In step 1, the conditioning regimen (irradiation and/or chemotherapy) leads to damage and activation of host tissues throughout the body and the secretion of inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-1. These cytokines may enhance donor T-cell recognition of host alloantigens by increasing expression of MHC antigens and other molecules on host antigen-presenting cells (APCs). Inflammatory cytokines may also stimulate chemokine release, recuiting donor T cells into host target organs. In step 2, host APCs present alloantigen (an HLA-peptide complex) to the donor T cells. Costimulatory signals are required for T-cell activation, and these signals further activate APCs, which in turn enhance T-cell stimulation, characterized by cellular proliferation and the secretion of cytokines. IL-2 expands the T-cell clones and induces cytotoxic T cell (CTL) responses, whereas interferon (IFN)-γ has multiple effects, including the priming of mononuclear phagocytes to produce TNF-α and IL-1. In step 3, effector functions of mononuclear phagocytes and neutrophils are triggered through a secondary signal provided by mediators such as lipopolysaccaride (LPS) that leak through

The pathophysiology of graft-vs.-host disease

3

Figure 1 The pathophysiology of acute GVHD. GVHD pathophysiology can be summarized in a three-step process. In phase 1, the conditioning regimen (irradiation, chemotherapy, or both) leads to the damage and activation of host tissues, especially the intestinal mucosa. This allows the translocation of lipopolysaccharide (LPS) from the intestinal lumen to the circulation, stimulating the secretion of the inflammatory cytokines TNF-α and IL1 from host tissues, particularly macrophages. These cytokines increase the expression of MHC antigens and adhesion molecules on host tissues, enhancing the recognition of MHC and minor histocompatibility antigens by mature donor T cells. Donor T-cell activation in phase 2 is characterized by the predominance of Th1 cells and

Graft-vs.-host disease

4

the secretion of IFN-γ, which activates mononuclear phagocytes. In phase 3, effector functions of activated mononuclear phagocytes are triggered by the secondary signal provided by LPS and other stimulatory molecules that leak through the intestinal mucosa damaged during phases 1 and 2. Activated macrophages, along with CTL, secrete inflammatory cytokines that cause target cell apoptosis. CD8+ CTL also lyse target cells directly. Damage to the GI tract in this phase, principally by inflammatory cytokines, amplifies LPS release and leads to the “cytokine storm” characteristic of severe acute GVHD. This damage results in the amplification of local tissue injury, and it further promotes an inflammatory response. the intestinal mucosa damaged during step 1. This inflammation, along with direct lysis of target cells by CTL, causes pathological changes in target organs. It should be noted from the outset that not all these steps carry equal weight in the pathogenesis of acute GVHD. The pivotal interaction occurs in step 2, where host APCs activate allogeneic donor T cells. Although there is substantial evidence for inflammatory cytokines as major effectors of target organ damage, blockade of individual cytokines may not reverse established GVHD when other cellular effectors such as CTL are present. GVHD can also occur when no conditioning of the host has occurred, e.g., transfusion-associated GVHD. This schema is not exhaustive and is intended to provide perspective on how the innate and adaptive immune systems interact in this complicated, inflammatory disease. A. Step 1: Effects of SCT Conditioning The first step of acute GVHD occurs before donor cells are infused. Prior to SCT, a patient’s tissues have been damaged, sometimes profoundly, by several factors such as the underlying disease, its treatment, infection, and transplant conditioning. Highintensity chemoradiotherapy characteristic of SCT conditioning regimens can activate host APCs and help to recruit donor T cells infused in the stem cell inoculum. This inflammatory environment may help to explain a number of unique and seemingly unrelated aspects of GVHD. For example, a number of reports have noted increased risks

The pathophysiology of graft-vs.-host disease

5

of clinical GVHD associated with advanced-stage leukemia, certain intensive conditioning regimens, and histories of viral infections (10–12). Total body irradiation (TBI) is particularly important in this process because it activates host tissues to secrete inflammatory cytokines, such as TNF-α and IL-1 (13), and it induces endothelial apoptosis that leads to epithelial cell damage in gastrointestinal tract (14). After autologous SCT, injury to the gut is transient and self-limited. After allogeneic SCT, however, GVHD amplifies gastrointestinal (GI) damage by allowing the translocation of microbial products such as LPS into systemic circulation, increasing secretion of inflammatory cytokines. This scenario helps to explain the increased risk of GVHD associated with intensive conditioning regimens as observed both in animal models (15) and in clinical SCT (10,11). B. Step 2: Donor T-Cell Activation and Cytokine Secretion 1. Donor T-Cell Activation Donor T-cell activation occurs during the second step of acute GVHD. Murine studies have demonstrated that host APCs alone are both necessary and sufficient to stimulate donor T cells (16,17). After allogeneic SCT, APCs derived from both the host and the donor are present in secondary lymphoid organs. T-cell receptors (TCRs) of donor T cells can recognize alloantigens either on host APCs (direct presentation) or donor APCs (indirect presentation). During direct presentation, donor T cells recognize either the peptide bound to allogeneic MHC molecules or the foreign MHC molecules themselves (18). During indirect presentation, T cells respond to the peptides generated by degradation of the allogeneic MHC molecules that are presented on self MHC (19). In GVHD to minor H antigens (mHAg), direct presentation is dominant because APCs derived from the host rather than from the donor are critical (16). CD4 and CD8 proteins are coreceptors for constant portions of MHC class II and MHC class I molecules, respectively. MHC class I (HLA-A, B, C) differences stimulate CD8+ T cells, and MHC class II (HLA-DR, DP, DQ) differences stimulate CD4+ T cells (20). After HLA-identical SCT, GVHD is usually induced by mHAg, which are peptides derived from polymorphic cellular proteins presented on the cell surface by MHC molecules (21). Because the genes for these proteins are located outside of the MHC, two siblings will often have many different peptides in the same MHC that can be recognized by donor T cells and lead to GVHD. It remains unclear how many of these peptides behave as mHAg, although over 50 different minor histocompability genetic loci have been defined among inbred strains of mice (22). The actual number of so-called “major” minor antigens that can potentially induce GVHD is likely to be limited. In murine models, the protein H60 has been shown to be one such major minor (23). In humans, of five previously characterized mHAg (HA-1, 2, 3, 4, 5) recognized by T cells in association with HLA-A1 and A2, only mismatching of HA-1 significantly correlated with acute grade II–IV GVHD (24). Several minor histocompatibility antigens are encoded on the male-specific Y chromosome, and there is an increased risk of GVHD when male recipients are transplanted from female donors (25,26). mHAg with tissue expression limited to the hematopoietic system are potential target antigens of GVL

Graft-vs.-host disease

6

reactivity (27), and separation of GVHD and GVL by using CTLs specific for such antigens is an area of intense research (28). Signaling through the TCR after antigen recognition induces a conformational change in adhesion molecules, resulting in higher-affinity binding to the APC (29). Full T-cell activation also requires costimulatory signals provided by APCs in addition to TCR signals (Table 1). Two primary costimulatoty pathways signal through either CD28 or TNF receptors. Currently, there are four known CD28 superfamily members expressed on T cells: CD28, cytotoxic T-lymphocyte antigen 4 (CTLA-4), inducible costimulator (ICOS), and programmed death (PD)-1. In addition, there are four TNF receptor family members: CD40 ligand (CD154), 4-1BB (CD137), OX40, and HSV glycoprotein D for herpesvirus entry mediator (HVEM). The best-characterized costimulatory molecules, CD80 and CD86, deliver positive signals through CD28 that lower the threshold for Tcell activation and promote T-cell differentiation and survival, while signaling through CTLA-4 is inhibitory (30,31). This topic is reviewed in detail in Chapter 15.

Table 1 T Cell-APC Interactions T Cell

APC

Adhesion ICAMs

LFA-1

LFA-1

ICAMs

CD2 (LFA-2)

LFA-3

TCR/CD4

MHC II

TCR/CD8

MHC I

CD28

CD80/86

CD152 (CTLA-4)

CD80/86

ICOS

B7H/B7RP-1

PD-1

PD-L1, PD-L2

unknown

B7-H3

CD154 (CD40L)

CD40

CD134 (OX40)

CD134L (OX40L)

CD137 (4-1BB)

CD137L (4-1BBL)

HVEM

LIGHT

Recognition

Costimulation

HVEM, HSV glycoprotein D for herpesvirus entry mediator; LIGHT, homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D for herpesvirus entry mediator (HVEM), a receptor expressed by T lymphocytes.

The pathophysiology of graft-vs.-host disease

7

The most potent APCs are dendritic cells (DCs); however, the relative contribution of DCs and other semiprofessional APCs such as monocytes/macrophages and B cells to the development of GVHD remains to be elucidated. DCs can be matured and activated during SCT by (1) inflammatory cytokines, (2) microbial products such as LPS and CpG entering systemic circulation from damaged intestinal mucosa, and (3) necrotic cells that are damaged by recipient conditioning. All of these stimuli may be considered “danger signals” (32) and may make the difference between an immune response and tolerance (33). When T cells are exposed to antigens in the presence of adjuvant such as LPS, the migration and survival of T cells are dramatically enhanced in vivo (34). Recent studies have shown that activation of DCs through Toll-like receptors that bind molecules such as LPS and CpG can induce an immune response and prevent the generation of tolerogenic T-regulatory cells (35). The role of host APCs in acute GVHD is an area of intense current research. Enhanced allostimulatory activity of APCs in older hosts may also help explain the increased incidence of acute GVHD in older recipients (36). The distribution of APCs may help explain the unusual target organ distribution of GVHD. For example, selective removal of APCs from a specific organ may reduce GVHD in that target organ but not in others (37). The general elimination of host APCs by activated natural killer (NK) cells can prevent GVHD (38). This suppressive effect of NK cells on GVHD has been confirmed in humans: HLA class I differences driving donor NK-mediated alloreactions in the GVH direction mediate potent GVL effects and produce higher engraftment rates without causing severe acute GVHD (38,39). 2. Cytokine Secretion by Donor T Cells T-cell activation involves multiple, rapidly occurring intracellular biochemical pathways that in turn activate transcription of genes for cytokines, such as IL-2, IFN-γ, and their receptors. Cytokines secreted by activated T cells are generally classified as Th1 (secreting IL-2 and IFN-γ) or Th2 (secreting IL-4, IL-5, IL-10, and IL-13) (40). Several factors influence the ability of DCs to instruct naive CD4+ T cells to secrete Th1 or Th2 cytokines. These factors include the type and duration of DC activation along with the DC:T-cell ratio and the proportions of DC subsets present during T-cell interactions (41,42). Differential activation of Th1 or Th2 cells has been evoked in the immunopathogenesis of GVHD and the development of infectious and autoimmune diseases. In each setting, activated Th1 cells: (1) amplify T-cell proliferation by secreting IL-2, (2) lyse target cells by Fas/FasL interactions, (3) induce macrophage differentiation in the bone marrow by secreting IL-3 and GM-CSF, (4) activate monocytes and macrophages by secreting IFN-γ and by their CD40-CD40L interactions, (5) activate endothelium to induce macrophage binding and extravasation, and (6) recruit macrophages by secreting monocyte chemoattractant protein-1 (MCP-1) (43,44). During step 2 of acute GVHD pathophysiology, IL-2 has a pivotal role in amplifying the immune response against alloantigens. IL-2 is secreted by donor CD4+ T cells in the first several days after GVHD induction (45). In some studies, the addition of low doses of IL-2 during the first week after allogeneic BMT enhanced the severity and mortality of GVHD (46,47). The precursor frequency of host-specific IL-2-producing cells (pHTL) predicts the occurrence of clinical acute GVHD, often preceding the onset of acute

Graft-vs.-host disease

8

GVHD by approximately 2 weeks (48–50). Monoclonal antibodies (mAbs) against IL-2 or its receptor can prevent GVHD when administered shortly after the infusion of T cells (45,51,52), but this strategy was not particularly successful in reducing the incidence of severe GVHD (53,54). IL-15 is another critical cytokine in initiating allogeneic T-cell division in vivo (55), and elevated serum levels of IL-15 are associated with acute GVHD in humans (56). IL-15 may therefore also be important in the clonal expansion of donor T cells in step 2. IFN-γ is a cytokine that can both amplify and suppress acute GVHD. In terms of amplification, increased levels of IFN-γ are associated with acute GVHD (15,57–59), and a large proportion of T-cell clones isolated from GVHD patients produce IFN-γ (60). In many experimental models of GVHD, IFN-γ levels peak between days 4 and 7 after transplant before clinical manifestations are apparent. CTLs that are specific for mHAgs and produce IFN-γ also correlate with the severity of GVH reaction in a skin-explant assay of human disease (28), but serum levels of IFN-γ were not significantly increased in a small series of patients with GVHD (61). IFN-γ modulates several aspects of the immune response of acute GVHD. First, IFN-γ increases the expression of numerous molecules, including adhesion molecules, chemokines, MHC antigens, and Fas, resulting in enhanced antigen presentation and the recruitment of effector cells into target organs (62–64). Second, IFN-γ alters target cells in the GI tract and in the skin, making them more vulnerable to damage; the administration of anti-IFN-γ mAbs prevents gastrointestinal GVHD (65), and high levels of both IFN-γ and TNF-α correlate with the most intense cellular damage in skin (66). Third, IFN-γ mediates the immunosuppression seen in several experimental GVHD systems in part by the induction of nitric oxide (NO) (67–72). Fourth, IFN-γ primes macrophages to produce pro-inflammatory cytokines and NO in response to LPS (73,74). GVHD effector mechanisms can sometimes be inhibited if donor T cells produce fewer Th1 cytokines. Transplantation of Th2 cells into nonirradiated recipients results in reduced secretion of TNF-α and protection of recipient mice from LPS-induced, TNF-αmediated lethality (75). Furthermore, cell mixtures of Th2 donor cells with otherwise lethal numbers of naïve T cells also protect recipient mice from LPS-induced lethality, demonstrating the ability of Th2 cells to modulate Th1 responses after aliogeneic transplantation (76). Similarly, the injection of donor Th2 cells that have been polarized in an MLR with host stimulators in the presence of IL-4 fails to induce acute GVHD to MHC class I or class II antigens (77). Polarization of donor T cells toward a Th2 phenotype by pretreatment donors with granulocyte colony-stimulating factor (G-CSF) also results in less severe GVHD (78). Interestingly, GVHD still occurs if donor mice lack the signal transducer and activator of transcription (STAT) 4, which is crucial to a Th1 response, although GVHD is less severe than if the donor cells lacked STAT6, a molecule critical for Th2 polarization (79). These experiments support the concepts that Th1 cells induce GVHD more efficiently than Th2 cells and that the balance of Th1 and Th2 cytokines is critical in the development of a GVH reaction. It should be noted, however, that systemic administration of Th2 cytokines IL-4 or IL-10 as experimental prophylaxis of GVHD was either ineffective or toxic (80–82). In this light it is surprising that administration of Th1 cytokines can also reduce GVHD. High doses of exogenous IL-2 early after bone marrow transplant (BMT) protect animals from GVHD mortality (83). IL-2 may mediate its protective effect via inhibition

The pathophysiology of graft-vs.-host disease

9

of IFN-γ (57). Furthermore, the injection of IFN-γ itself can prevent experimental GVHD (84), and neutralization of IFN-γ accelerates GVHD in lethally irradiated recipients (70). Interestingly, the use of TFN-γ-deficient donor cells can also accelerate GVHD in lethally irradiated recipients (85,86) but reduces GVHD in sublethally irradiated or unirradiated recipients (87,88). These paradoxes may be explained by the complex dynamics of donor T-cell activation, expansion, and contraction. Activation-induced cell death (AICD) is a major mechanism of clonal deletion and is largely responsible for the rapid contraction of activated T cells following an initial massive expansion (89). IFN-γ contracts the pool of activated CD4+ T cells by inducing AICD, and the complete absence of IFN-γ may result in an unrestrained expansion of activated donor T cells, leading to accelerated GVHD. This phenomenon may be of particular importance in recipients of intensified conditioning, which causes greater T-cell activation of host APCs (15). Similarly, administration of IFN-γ-inducing cytokines such as IL-12 or IL-18 early after BMT protects lethally irradiated recipients from GVHD in a Fas-dependent fashion (86,90,91). Thus, moderate amounts of Th1 cytokine production after donor T-cell expansion may amplify GVHD; extremes in production (either low or high), particularly early during Tcell expansion, may hasten the death of activated donor T cells, aborting T-cell expansion and reducing GVHD. Further consideration of Th1 and Th2 cytokines in this process is presented in Chapter 3. Subpopulations of regulatory donor T cells can prevent GVHD. Repeated in vitro stimulation of donor CD4+ T cells with alloantigens results in the emergence of a population of regulatory T-cell clones (Treg cells) that secretes high amounts of IL-10 and TGF-β (92). The immunosuppressive properties of these cytokines are explained by their ability to inhibit APC function and to suppress proliferation of responding T cells directly. The addition of IL-10 or TGF-β to MLR cultures induces tolerance (93), with alterations in biochemical signaling similar to costimulatory blockade (94). So-called “Th3” cells that produce large amounts of TGF-β can also act as regulatory T cells. CD8+ suppressor cells have also been identified in both mice and humans (95–98). Natural suppressor (NS) cells reduce GVHD in a variety of experimental BMT models (99). NK1.1+ T cells (NKT) may possess such NS cell function (100), and both peripheral blood and marrow NKT cells can prevent GVHD by their IL-4 secretion (5,100,101). Recent research has also focused on CD4+CD25+ regulatory T cells that prevent lethal GVHD in several animal models (102). These topics are explored in detail in Chapter 4. C. Step 3: Cellular and Inflammatory Effectors The pathophysiology of acute GVHD culminates in the generation of multiple cytotoxic effectors that contribute to target tissue injury. At a minimum, step 3 includes (1) several inflammatory cytokines, (2) specific antihost CTL activity using Fas and perforin pathways, (3) large granular lymphocytes (LGL) or NK cells, and (4) nitric oxide. Significant experimental and clinical data suggest that soluble inflammatory mediators act in conjunction with direct cell-mediated cytolysis by CTL and NK cells to cause the full spectrum of deleterious effects seen during acute GVHD. As such, the effector phase of GVHD involves aspects of both the innate and adaptive immune response and the synergistic interactions of cells and cytokines generated during step 1 and step 2.

Graft-vs.-host disease

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1. Cellular Effectors The Fas/Fas ligand (FasL) and the perforin/granzyme (or granule exocytosis) pathways are the principal effector mechanisms used by CTLs and NK cells to lyse their target cells (103,104). Perforin is stored together with granzymes and other proteins in cytotoxic granules of CTLs and NK cells (105). Following recognition of a target cell through TCR-MHC interaction, perforin is secreted and inserts itself into the target cell membrane, forming “perforin pores” that allow granzymes to enter the cell and induce apoptosis through various downstream effector pathways (105). Ligation of Fas results in the formation of the death-inducing signaling complex (DISC) and the subsequent activation of caspases (106). A number of ligands on T cells also possess the capability to trimerize TNF receptor—like death receptors (DR) on their targets, such as TNF-related apoptosis-inducing ligand (TRAIL: DR4,5 ligand) and TNF-like weak inducer of apoptosis (TWEAK: DR3 ligand) (107–109). The involvement of these pathways in GVHD has been tested by utilizing donor cells that are genetically deficient in each molecule. Transplantation of perforin-deficient T cells results in a marked delay in the onset of acute GVHD in transplants across mHAg (110), across both MHC and mHAg (111), and across isolated MHC I or II disparities (112,113). However, lethal GVHD occurs even in the absence of perforin-dependent killing, demonstrating that the perforin/granzyme pathway plays a significant, but not exclusive, role in target organ damage. CD4+ CTLs preferentially use the Fas/FasL pathway during acute GVHD, while CD8+ CTLs primarily use the perforin/granzyme pathway, consistent with other pathological onditions that involve cell-mediated cytolysis. Fas is a TNF receptor (TNFR) family member that is expressed by many tissues, including GVHD target organs. Inflammatory cytokines such as IFN-γ and TNF-α can increase the expression of Fas during GVHD (114). FasL expression on donor T cells is increased during experimental GVHD (115–117), and elevated serum levels of soluble FasL and Fas have been observed in some patients with acute GVHD (118–121). FasLdefective donor T cells cause markedly reduced GVHD in the liver, skin, and lymphoid organs (110,122,123). The Fas-FasL pathway is particularly important in hepatic GVHD, consistent with the marked sensitivity of hepatocytes to Fas-mediated cytotoxicity in models of murine hepatitis (124). Fas-deficient recipients are protected from hepatic GVHD, but not from GVHD in other target organs (125). Administration of anti-FasL (but not anti-TNF) mAbs significantly blocked hepatic GVHD damage in one model (126), whereas the use of FasL-deficient donor T cells or the administration of neutralizing FasL mAbs had no effect on the development of intestinal GVHD in several studies (110,126,127). Cytotoxic double-defficient (cdd) (absence of both perforin/granzyme and FasL pathways) donor T cells do not induce lethal GVHD across MHC class I and class II disparities after sublethal irradiation (111). However, the lack of acute GVHD was probably caused by the rejection of the donor cdd T cells because they lacked the effector mechanisms to eliminate host resistance to GVHD (128,129). When recipients were conditioned with a lethal dose of irradiation, cdd CD4+ T cells produced similar mortality to wild-type CD4+ T cells (129).

The pathophysiology of graft-vs.-host disease

11

2. Inflammatory Effectors In the effector phase of acute GVHD, inflammatory cytokines synergize with CTLs, resulting in the amplification of local tissue injury and the development of target organ dysfunction in the transplant recipient. The cytokines TNF-α and IL-1 are produced by an abundance of cell types involved in innate and adoptive immune responses, and they have synergistic and redundant roles during several phases of acute GVHD. A central role for inflammatory cytokines in acute GVHD was confirmed by a recent murine study using bone marrow chimeras in which alloantigens were not expressed on target epithelium but on APCs alone (17). GVHD target organ injury was induced in these chimeras even in the absence of epithelial alloantigens, and mortality and target organ injury were prevented by the neutralization of TNF-α and IL-1. These observations were particularly true for CD4-mediated acute GVHD; in CD8-mediated disease, donor CTL effectors contributed to GVHD mortality. A critical role for TNF-α in the pathophysiology of acute GVHD was first suggested almost 15 years ago when high levels of TNF-α mRNA were noted in GVHD tissues (130,131). Target organ damage could be inhibited by infusion of anti-TNF-α mAbs, and mortality could be reduced from 100% to 50% by the administration of the soluble form of the TNF-α receptor (sTNFR) (13). TNF-α plays a central role in intestinal GVHD (126,130,132) as well as in skin and lymphoid tissue (126,130,133,134) and to a lesser extent in the liver (17,135). TNF-α may be involved at many points within GVHD pathophysiology because TNF-α can (1) cause cachexia, a characteristic feature of GVHD, (2) induce maturation of DCs, thus enhancing alloantigen presentation, (3) recruit effector T cells, neutrophils, and monocytes into target organs through the induction of inflammatory chemokines, and (4) cause direct tissue damage by inducing apoptosis and necrosis (136). TNF-α is also involved in donor T-cell activation directly through its signaling via TNFR1 and TNFR2 on T cells; TNF-TNFR1 interactions promote alloreactive T-cell responses (137), whereas TNF-TNFR2 interactions are critical for intestinal GVHD (138). An important role for TNF-α in clinical acute GVHD has also been suggested by multiple studies. Elevations of TNF-α protein (serum) and mRNA (peripheral blood mononuclear cells) have been measured in patients with acute GVHD and other endothelial complications, such as hepatic veno-occlusive disease (VOD) (139–142). Furthermore, a phase I/II trial using TNF-α receptor mAbs during the conditioning regimen as prophylaxis in patients at high risk for severe acute GVHD showed reduction in lesions of the intestine, skin, and liver, although GVHD flared after discontinuation of treatment (132). Collectively, these data might suggest that approaches to limit TNF-α secretion will be a very important avenue of investigation in allogeneic SCT. IL-1 is the second major pro-inflammatory cytokine that contributes to acute GVHD toxicity. Secretion of IL-1 appears to occur predominantly during the effector phase of GVHD in the spleen and skin, two major GVHD target organs (143). A similar increase in mononuclear cell IL-1 mRNA has been shown during clinical acute GVHD (141). Although the use of an IL-1 receptor antagonist (IL-1RA) appeared promising in murine studies (144,145), a recent randomized trial of IL-1RA to prevent acute GVHD was not successful (146). Finally, macrophages can produce significant amounts of NO as a result of activation during GVHD. Several studies have shown that NO contributes to the deleterious effects

Graft-vs.-host disease

12

GVHD on target tissues and specifically to GVHD-induced immunosuppression (72,147). NO also inhibits repair mechanisms of target tissue by inhibiting proliferation of epithelial stem cells in the gut and skin (148). In humans and rats, development of GVHD is preceded by an increase in NO products in the serum (149,150). 3. Lipopolysaccharide and Innate Immunity During GVHD, cytokines (including IFN-γ) prime mononuclear cells. The secretion of inflammatory cytokines by these primed monocytes/macrophages occurs after stimulation by a second signal. This signal may be provided by LPS and other microbial products that have leaked though an intestinal mucosa damaged initially by SCT conditioning regimens during step 1. Since the GI tract is known to be particularly sensitive to the injurious effects of cytokines (130,151), damage to the GI tract incurred during the effector phase can lead to a positive feedback loop wherein increased translocation of LPS results in further cytokine production and progressive intestinal injury. Thus, the GI tract may be critical to propagating the “cytokine storm” characteristic of acute GVHD; increasing experimental and clinical evidence suggests that damage to the GI tract during acute GVHD plays a major role in the amplification of systemic disease (152). This conceptual framework underscores the role of LPS in the development of acute GVHD, is suggested by several groups (74,151,153). LPS is a major structural component of gram-negative bacteria and is a potent stimulator of cellular activation and cytokine release (154,155). LPS shed from bacteria that comprise normal bowel flora can elicit a broad range of inflammatory responses from macrophages, monocytes, and neutrophils. In particular, LPS may stimulate gut-associated lymphocytes and macrophages (74). Following allogeneic SCT, LPS accumulates in both the liver and spleen of animals with GVHD prior to its appearance in the systemic circulation (153). Elevated serum levels of LPS have been shown to correlate directly with the degree of intestinal histopathology occurring after allogeneic SCT (151,156,157). The severity of GVHD also appears to be directly related to the level of macrophage priming (74). Injection of small, normally nonlethal amounts of LPS caused elevated TNF-α serum levels and death in animals with GVHD, which could be prevented with anti-TNF-α antiserum. Mice known to be sensitive or resistant to LPS have been used as stem cell donors in order to determine the effect of donor responsiveness to LPS on the development of acute GVHD. C3Heb/Fej animals exhibit normal sensitivity to LPS challenge (LPS-s), whereas the receptor responsible for LPS signaling, a genetic mutation in the Toll-like receptor 4 of C3H/Hej mice, has made this strain resistant to LPS (LPS-r) (158–161). These defects are specific to LPS, and, importantly, T cells from LPS resistant mice respond normally to mitogen and alloantigen (162,163). In an irradiated parent (P)→F1 hybrid murine model of GVHD, SCT with LPS-r donor cells resulted in a significant reduction of TNFα levels. GI tract histopathology, and systemic GVHD compared to animals receiving LPS-s SCT (151). Systemic neutralization of TNF-α further decreased the severity of intestinal and systemic GVHD in LPS-r SCT recipients (151). Recent experiments using animals deficient in CD14 as SCT donors extend these observations. CD14, a cell surface protein on mononuclear cells and macrophages, is the principal receptor for the complex of LPS and its soluble LPS-binding protein (LBP)

The pathophysiology of graft-vs.-host disease

13

(164). The engagement of CD14 by LPS/LPB results in the association of the CD14 molecules with Toll-like receptors and subsequent intracellular signaling and cytokine synthesis during innate immune responses (49). Mononuclear cells from CD14-deficient (CD14−/−) animals are insensitive to LPS stimulation in vitro, and such mice are resistant to the lethal effects of LPS challenge in vivo (165). Transplantation of CD14−/− donor cells into irradiated allogeneic recipients reduces serum TNF-α levels and decreases gut histopathology compared to controls, resulting in less GVHD and improved long-term survival (166). Data supporting a role for LPS in the GVH response are also provided by both experimental and clinical studies examining the effects of decontamination of the GI tract on the incidence of GVHD. Early animal studies showed that death from GVHD could be prevented if transplanted mice were given antibiotics to decontaminate the gut; normalization of the gut flora at or before day 20 abrogated this effect (167). After clinical SCT, endotoxemia is associated with biochemical parameters of intestinal injury, hepatic toxicity, acute GVHD, and fever in the absence of bacteremia (156,157). Further support is derived from studies where gram-negative gut decontamination during SCT reduces GVHD (168,169), and the degree of the decontamination predicts GVHD severity (170,171). Naturally occurring antibody titers to a rough-mutant strain of Escherichia coli J5 are also associated with a decreased incidence of acute GVHD after allogeneic SCT (172). Use of a polyclonal antiserum against E. coli J5 as prophylaxis for acute GVHD in a prospective, placebo-controlled trial reduced overall GVHD from 63% to 42%. The antiserum was particularly efficacious in a subset of patients with severe GVHD (173). Blockade of cellular activation by LPS after allogeneic SCT has been studied using B975, a synthetic analog of E. coli lipid A that belongs to a family of potent antagonists of LPS-induced cellular activation (174). These molecules competitively inhibit LPS binding to its receptors and block NFκB activation and nuclear translocation. They are active both in vitro and in vivo and are devoid of agonistic activity even at high doses (174). Treatment of allogeneic BMT recipients with B975 for the first week after transplant blocked mononuclear cell responses to LPS and resulted in decreased inflammatory cytokine release and reduced GVHD severity. Importantly, LPS antagonism had no effect on donor T-cell responses to host antigens, and treatment with B975 did not diminish GVL effects and improved leukemia-free survival. Collectively, the data outlined above demonstrate a critical role for LPS to the early inflammatory events responsible for GI toxicity and subsequent mortality from GVHD; LPS acts via a positive feedback mechanism to amplify intestinal injury by triggering cytokine release. These data provide the rationale for testing strategies that strengthen the mucosal lining of the GI tract in order to reduce or prevent GVHD. Hypothetically, such an approach would block immune dysregulation before the inflammatory cytokine cascade is initiated, maintain the integrity of the GI mucosal barrier, and limit translocation of immunostimulatory molecules (including endogenous LPS) into the circulation. Because direct shielding of the GI tract is not feasible, this effort would utilize pharmacological agents that provide a “cytokine shield” to reduce gut mucosal injury incurred initially by the effects of SCT conditioning regimens and ultimately by the direct effects of GVHD.

Graft-vs.-host disease

14

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family (FGF-7) that stimulates growth of cells that express its receptors such as gut epithelial cells, hepatocytes, keratinocytes, and alveolar type II cells (175–177). KGF has been studied extensively for its cytoprotective properties against chemotherapeutic and radiation toxicity to a variety of tissues including the bowel, lung, and bladder (178–181). KGF administration prior to autologous SCT in mice protected the GI tract from damage resulting from lethal conditioning (178) and may do so by promoting potent trophic effects on intestinal epithelium and enhancing crypt stem cell survival (175,182). The mechanism of action of KGF is believed to involve regulation of genes that reduce oxidative damage and enhance DNA repair (175,183). Several recent studies have examined the effects of human recombinant KGF alter experimental allogeneic SCT (184–188). Administration of KGF prior to lethal SCT conditioning significant reduced GVHD mortality and the severity of target organ damage in surviving animals (184,185). Extension of the dosing schedule from day −3 to day +7 abrogated GVHD of the GI tract early after SCT and resulted in further improvement in long-term survival (184). Consistent with the hypothesis that protection of the GI mucosa could block the inflammatory cascade of GVHD, KGF prevented the translocation of LPS into the circulation and resulted in a significant reduction in systemic TNF-α. Thus the protection afforded by KGF was attributed directly to its trophic effects on the intestinal epithelium, aborting the destructive and atrophic phase of GI GVHD, and indirectly to a significant reduction in systemic cytokine dysregulation. KGF has also been shown to attenuate glutathione depletion and organ injury from reactive oxygen species and upregulate the secretion of anti-inflammatory cytokines after experimental SCT (186–188). Moreover, KGF has also been shown to facilitate alloengraftment and ameliorate GVHD in mice by a mechanism that is independent of a reparative response to conditioning-induced tissue damage (186). Importantly, KGF administration does not protect malignant cell lines from chemoradiotherapy and preserved donor cytolytic T-cell responses and allospecific GVL effects after SCT (184). Based upon these preclinical data, two phase I/II clinical trials are underway to determine the safety and efficacy of KGF when administered along with methotrexate and tacrolimus or cyclosporine prior to either matched related or mismatched related/ unrelated donor SCT. In the mismatched related/unrelated donor setting, KGF is administered 3 consecutive days prior to the conditioning regimen and then for 3 consecutive days each week starting at day 0, with a dose escalation planned at 2-week intervals to a total of 36 possible injections of KGF (9 weeks of therapy). Preliminary data from this schedule escalation trial suggest that KGF can be administered safely and that its potential efficacy in preventing GVHD may be schedule dependent; of 9 evaluable patients, all 4 receiving 6 doses of KGF developed grade II–IV acute GVHD compared to 1 of 5 patients receiving 12–18 doses of KGF (189). These early results at higher KGF doses compare favorably to historical controls in whom the incidence of acute GVHD is 50–75% in this high-risk group of patients. Taken together, experimental and preliminary clinical results suggest that KGF may hold promise as a non-crossreactive adjunct to standard GVHD prophylaxis and as a novel strategy to effectively separate the toxicity of GVHD from the desirable GVL effects.

The pathophysiology of graft-vs.-host disease

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4. Inflammatory Chemokines Regulation of effector cell migration into target tissues during the development of GVHD occurs in a complex milieu of chemotactic signals where several chemokine receptors may be triggered simultaneously or successively. Inflammatory chemokines expressed in inflamed tissues are specialized to recruit effector cells such as T cells, neutrophils, and monocytes (190). Chemokine receptors are differentially expressed on subsets of activated/effector T cells. Upon stimulation, T cells can rapidly switch chemokine receptor expression, acquiring new migratory capacity (34,191). The involvement of inflammatory chemokines and their receptors in GVHD has been recently investigated in mouse models of GVHD. MIP-1α recruits CCR5+ CD8+ T cells into the liver, lung, and spleen during GVHD (192,193), and levels of several chemokines are elevated in GVHDassociated lung injury (194). Chemokine gradients may play a role in the unusual target organ distribution of GVHD; a recent paper showed that CCR5 appears essential for donor T-cell homing to Peyer’s patches and the subsequent development of lethal GVHD (195). Further studies will help to determine whether expression of specific chemokines and their receptors control the homing of effector cells to GVHD target organs (skin, gut, and liver), and whether these molecules will prove to be potential targets for modulation of GVHD. This topic is reviewed in detail in Chapter 5. II. GVHD PREVENTION AND TREATMENT: TRANSLATION TO CLINICAL SCT In general, strategies to prevent and treat GVHD have been based on an “oncological” model of the disease, i.e., the notion that since GVHD is dangerous and life-threatening, management requires the utilization of high-dose immunosuppressant regimens. High doses of multiple immunosuppressive agents have been used much like we use combination chemotherapy to treat malignancies. While there is often reasonable control of the clinical manifestations of GVHD, this broadly aggressive approach is often associated with opportunistic infections, lymphoproliferative disorders, or relapse of the underlying malignancy. Indeed, infection often results in mortality while GVHD is in remission. The three-phase model of GVHD presented here suggests that the disease is an exaggerated and dysregulated response of a normal immune sy stem to tissue damage that is intrinsic to transplantation. The donor’ s immune system reacts as if there is a massive and uncontrolled infection, and its attempts to deal with this injury result in the clinical manifestations of GVHD. The tissue injury intrinsic to the administration of high-dose chemoradiotherapy initiates the breakdown of mucosal barriers, allowing endotoxin into the tissues. Toll-like receptors on dendritic cells bind to endotoxin and activate signal transduction pathways that lead to dendritic cell maturation and induce inflammation (196). The upregulation of co-stimulatory molecules, MHC molecules, adhesion molecules, cytokines, chemokines, prostanoids, and other inflammatory mediators prime and trigger attack on target tissues, fueling the fire. It is likely that components of a

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cytokine storm (197), the danger hypothesis (32), and more traditional notions of adaptive and innate immunity (198) all apply to the disease process. A. Step 1: Reduced Intensity Conditioning Regimens The three cardinal organs affected by GVHD are skin, intestinal tract, and liver. One common thread linking the three would seem to be exposure to the environment. Skin and gut have very obvious barrier functions and a well-developed reticuloendothelial system. Similarly, the liver is the first line of defense downstream of the gut. One would expect the lung to be a similar target, and we argue elsewhere in this volume (Chapter 11) that it should be considered as such. It may be that less intense exposure to organisms, particularly gram-negative rods, reduces the manifestations of acute GVHD in the lung. All of these organs are rich in dendritic cells, a probable prerequisite for antigen presentation and the generation of injurious cytokines. They are all subject to injury from conditioning, and breaches of the barrier would allow organisms or endotoxin into the circulation. One prediction of this model is that less intense conditioning regimens would be associated with less GVHD. For example, when donor lymphocyte infusions were administered without pharmacological GVHD prophylaxis, the resultant GVHD has been less frequent and severe than would be expected in the absence of immunosuppressive drugs (reviewed in Chapter 19). Similarly, the GVHD expected after reduced-intensity conditioning regimens might be less severe both through less cellular injury per se and through a reduction in endotoxin exposure by virtue of less mucosal injury (see also Chapter 18). Thus, while data on reducing both colonization with bacteria and reducing tissue injury with less intense regimens are suggestive and support the basic concepts described above, the effect is insufficient to control GVHD adequately in human transplantation. B. Step 2: Modulation of Donor T Cells 1. Reduced T-Cell Numbers Elimination of T cells with monoclonal antibodies, immunotoxins, lectins, CD34 columns, or physical techniques is effective at reducing GVHD. T-cell depletion of marrow is relatively straightforward because the number of T cells in the marrow is low compared with the total nucleated cell count, making it relatively easy to manipulate in the laboratory. As the use of peripheral blood stem cells has become more prevalent, the 10-fold increase in number of T cells is a technical challenge. A typical unmanipulated marrow transplant entails the infusion of ~107 T cells per kg of recipient weight. A T-cell dose of ≤105 per kg has been associated with complete control of GVHD (4). More recently, the combination of very high stem cell numbers and CD3 T cell numbers Tc2 cells; less GVHD with Tc2>Tc1

49

AKR T cell 3×107 leukemia/ lymphoma 5×104/ mouse

0% with DLI on D21

A protection assay; DLI given on day 21, AKR leukemia on day 28

33– 35

5×107 spleen 0.5×105 B6dom1

0%

B6dom1 specific T cells alone do not cause GVHD; GVL more effective in recipients not expressing B6dom1

47

107 spleen

varied

Mice immunized against

48

AKR

>50% by D7

1−3×106 3×106 spleen cells 90% D10 1×106 0% D100

Graft-vs.-host disease

172

or LN leukemia 105/animal

donors with MHC disparate 3rd party cells

3rd party cells had increased GVL without GVHD; mechanism not known,

FasLdeficient T cells

B6→B6D2F1 12 Gy

wt 50% GVL does not require p815 105/ 2×107 mouse D-2 spleen cells D28 gld FasL; GVHD attenuated L1210 104/ 0% with FasL−/− T cells mouse D-2 PKO 0%

FasLdeficient T cells

B6→B6D2F1 B6→B6C3F1 13 Gy

32D/p210 1− 5×103/ mouse p815

1−2×106

wt 50% D30 PKO 50% D35 gld 70& D90

Regulatory DCs (rDC)

B6→DBA/2 10 Gy

p815, 2×105 D-2

1.5×107

no rDC, rDC suppress alloimmune 74 100% responses via generation D8 of regulatory T cells; sufficient alloreactivity to mediate GVL

Regulatory

CD4+CD25+ cells

B6→BAL B/c

BCL1luciferase A20luciferase

GVL does not require FasL; GVHD attenuated with FasL−/− T cells; GVHD CD4 dominated with FasL mechanism; GVL CD8 dominated with perforin-mediated killing

40

43

Overall 84 suppression of alloreactivit y; intact CD8 CTL activity

peptides must be endogenously expressed in host APCs, and one would expect that for leukemia cells to be sensitive to spontaneous GVL reactions, they must also express the genes encoding immunodominant epitopes expressed in APC. 2. Mechanisms of T-Cell Killing Is GVL mediated by CD4+ T cells or CD8+ T cells alone, or are both required? In part, the answer depends upon the model, but effective GVL by either CD8 cells or CD4 cells alone has been demonstrated, while in some models maximal GVL depends on a mix of CD4 and CD8 cells. The roles of FasL and perforin in killing have been evaluated using FasL−/− or perforin−/− T cells (39–44). Perforin appears to be more critical than FasL-

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mediated killing for CD8 CTL responses. The role of TNF-α has also been shown to be important in GVL responses via experiments with anti-TNF-α antibodies, TNF-α−/−, and TNFR1−/− T cells (37,40,46). It has been difficult to directly implicate TNF-α as a direct cytolytic mediator since TNF-α also plays a role in the initiation of alloimmune responses (61). For example, in one model, anti-TNF-α antibodies decreased GVL while the target cells themselves were resistant to TNF-α in vitro (40). Another TNF-α family member expressed on T cells, TRAIL (TNF-related apoptosis-inducing ligand), has also been implicated as being important for GVL (46). The role of TRAIL was examined in MHCdisparate and MHC-identical allo HCT models against p815 and 32D-p210 cell lines. Overall, the absence of TRAIL had little effect on GVHD but significantly reduced GVL in the three model systems tested. The authors suggested that augmentation of TRAILmediated killing might induce more effective GVL without increasing GVHD. We have studied GVL in an MHC-identical, multiple mHA-disparate system using a murine model of chronic phase CML (mCP-CML) induced by retrovirus expressing the human p210 cDNA (28,62–65). Because mCP-CML is induced by a retrovirus-encoding gene, we have been able to transfer it to mice that are deficient in genes that might be important mediators of GVL killing. An advantage of this approach over using genedeficient T cells is that the T-cell response is left intact and only the posited effector mechanisms are tested. For CD8 CTL-mediated GVL in this model, mCP-CML targets deficient in Fas or doubly deficient in TNFR1 and TNFR2 were as sensitive to GVL as wild-type targets (W.Shlomchik, unpublished data). For CD4 T cell-mediated GVL, we have found that GVL is impaired, although not absent, against MHC II- mCP-CML (W. Shlomchik, unpublished data), suggesting that direct cognate interactions are required for maximal CD4-mediated killing. 3. Antigen Presentation in GVL In alloimmune responses, naïve T-cell responses are primed by so-called professional APC, including DC, B cells, and macrophages. These APC provide requisite costimulatory signals to T cells critical for their activation, proliferation, and maturation into effector and memory T cells. Antigen presentation has been the focus of numerous GVHD studies, but has been less well examined in GVL responses. Costimulatory molecule blockade has been proposed as a method to prevent GVHD, and obviously the impact of these interventions on GVL responses would be of particular interest. A priori, one would expect that interventions that block costimulation would weaken GVL, whereas those that promote antigen-driven T-cell expansion will promote it. To study this question, Blazar and colleagues (66) have examined antibody-mediated blockade of B7 engagement with both CD28 and CTLA-4 and blockade of B7 engagement only with CTLA-4 in an MHC-disparate DLI model. Blocking antibodies to B7-1 and B7-2 inhibited GVL against C1498 cells as completely as depleting antibodies to both CD4 and CD8. The same study suggested that B7-1 overexpression on leukemic cell lines might promote GVL responses. This left unanswered whether the infusion of a small number of B7-modified leukemia cells could enhance protection against unmodified leukemic cells, which could be a useful clinical strategy. In contrast to B7 blockade, blocking antibodies to CTLA-4 augmented DLI-mediated GVL against C1498

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cells (36). Although GVHD was not noted to be increased in recipients of anti-CTLA-4 as compared to controls, overall survival was not improved due to the number of deaths from GVHD. The same study showed that anti-CTLA-4, when given on day 0, augmented GVHD, suggesting that its use in the DLI setting would likely still carry an increased risk for GVHD. Nevertheless, these murine experiments provide important preclinical data supporting the use of CTLA-4 blockade to augment GVL, especially against relatively GVL-resistant neoplasms or in patients who do not develop GVHD after DLI given for relapsed or persistent disease. These studies suggest that blocking B7-mediated activation of CD28 will impair alloimmunity in general, as would be expected. This effect could be T-cell dose dependent, as CD28−/− T cells have been shown to cause GVHD in MHC I- and MHC II-disparate models, although their capacity to do so at lower T-cell doses is impaired (36,67,68). These studies also highlight that the net effect of B7 blockade reflects the activity of both the CD28 and CTLA-4 pathways. It remains untested whether B7 blockade begun on day 0 or in MHC identical models would enhance GVL over GVHD. Blazar et al. used the same MHC-disparate DLI model to examine augmentation of signaling via 4-1BB (69). 4-1BB is a TNF-receptor family member expressed on activated cytolytic and helper T cells (70–72). The 4-1BB ligand is expressed on B cells, macrophages, and DC. Infusion of an agonist antibody to 4-1BB augmented GVL mediated by only a modest dose of donor spleen cells. However, at higher DLI doses, survival was decreased due to more severe GVHD, supporting the conclusion that a dose effect of T cells might separate GVL and GVHD in the setting of modified T-cell costimulation. As with anti-CTLA4, this could be a promising approach in some highrisk clinical settings, albeit with a significant increased risk for probability of GVHD. In the mCP-CML GVL model system described above using MHC-dentical, multiple mHA-mismatched pairings, CD8-mediated GVL was intact if donor bone marrow was MHC-I- and, thus, unable to interact with donor CD8 T cells. We examined the role of antigen presentation by leukemia cells via generation of mCP-CML in B7.1/ B7.2−/− cells and found that GVL mediated by a mix of CD4 and CD8 cells was intact (W.Shlomchik, unpublished data). These studies are ongoing and may help shed more light on the role of costimulation in GVL. 4. Separation of GVL and GVHD The holy grail of allo SCT has been the ability to deliver a potent antitumor effect without GVHD. Table 2 summarizes some different approaches. Separation of GVHD and GVL could be achieved in several general ways. Overall T-cell alloreactivity can be reduced such that it is sufficient for GVL but insufficient for GVHD. For example, T-cell doses can be decreased to numbers that can mediate GVL without GVHD, and this has already been demonstrated clinically (73). A novel approach for globally suppressing alloimmune responses is the infusion of regulatory DC (rDC) at the time of transplant (74). In a Pto F1 model, this approach achieved GVHD suppression with sufficient remaining alloreactivity to kill p815 mastocytoma cells. The data from this innovative study suggest that GVHD is suppressed via the generation of donor-derived CD4+/CD25+ regulatory T cells, a subject discussed elsewhere in this book.

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GVL and GVHD could also be separated on the basis of T-cell subset selection. This has been studied in human allo HCT as well, with the suggestion that CD8-depleted DLI or the selective depletion of CD8 T cells from initial grafts can still mediate GVL against chronic phase CML, but with less GVHD (75,76). We have found this to be true in the mCP-CML GVL model where CD4 cells alone can mediate GVL without GVHD when performed in a strain pairing in which CD4 cells alone do not cause GVHD (28). These data suggest that there can be CD4 T-cell-mediated reactivity against malignant hematopoietic cells without clinical GVHD. However, when we tested the efficacy of CD4 cells in a strain pairing in which CD4 cells mediate GVHD, GVL was accompanied by GVHD (W.Shlomchik, unpublished data), suggesting that this might be also be true in humans if CD4 T cells did not contribute to GVHD, which does not appear likely. In a separate clinical trial of a small group of CP-CML patients, CD4 T-cell-depleted grafts that were augmented with CD8 CTL also showed less GVHD and better engraftment, without higher than expected relapse rates (77). Furthermore, graded doses of DLI have shown that GVL can be induced with less GVHD for treatment of CML (78). Taken together, this may imply that both CD4 and CD8 subsets are important mediators of both GVL and GVHD in humans, and that any method that produces a modest degree of T-cell depletion can translate into a reduction in GVHD with retention of some GVL. This is supported by a larger study from the IBMTR that shows that “narrow specificity” antibodies are associated with better preservation of the GVL effect, as measured by leukemia-free survival (79). In some Pto F1 models, GVHD is primarily mediated by CD4 T cells, while GVL against at least some cell lines has been found to be mostly CD8 CTL-dependent. This difference has been exploited by manipulations that suppress the alloreactivity of CD4 cells greater than that of CD8 cells. For example, a number of mediator reagent-based interventions in the B6to B6D2F1 Pto F1 model are capable of suppressing CD4 T-cellmediated GVHD while leaving CD8 CTL responses relatively intact (37,39,44,80–83). Regulatory CD4+ CD25+ cells have also been tested for their ability to suppress GVHD while leaving GVL intact (84). In an MHC-disparate model with the BCL1 lymphoma and A20 leukemic cell lines, CD4+CD25+ cells suppressed GVHD while leaving adequate alloreactivity to mediate effective GVL. Specifically, perforin-mediated CD8+ CTL activity was shown to be intact. Another novel feature of this model was the use of leukemia cell lines that express luciferase, which allowed simultaneous in vivo imaging based on bioluminescence. GVL and GVHD might also be separated on the basis of unique effector mechanisms. In some models, GVHD is FasL-dependent, but GVL can be mediated by perforin, TNFα, and TRAIL, independent of FasL (40,43). Furthermore, it may also be possible to separate GVL and GVHD via the use of T cells that recognize only a single dominant mHA, as single mHA differences have been shown to be insufficient for GVHD (51,85). In an MHC-matched model, CD8 T cells only recognizing the B6 mHA B6dom1 mediated GVL without inducing GVHD (47). Finally, in another approach, T cells that recognize hematopoietically restricted antigens might also be able to mediate GVL with less GVHD (86–93). This is a very attractive approach for augmenting GVL without GVHD, although it would not solve the problem of reconstituting T cells without inducing GVHD. A novel way in which this approach might be used therapeutically would be to elicit T cells from a donor, for

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example, of MHC type A, against a hematopoietically restricted antigen presented by recipient cells of MHC type B (91–93). The T cells would need to retain their peptide specificity and also have the necessary MHC type B restriction. If used in the MHCdisparate allo HCT setting, recipient hematopoietic cells should be susceptible, but donor hematopoietic cells would not be as they are of a different MHC type. This approach would not be limited to mHA, but would apply to any hematopoietic-restricted peptide that sufficiently bound to two or more HLA alleles (see below). 5. NOD/SCID Mice for the Study of GVL Effects with Human T Cells and Natural Killer Cells As introduced above, the use of CD8+ T-cell lines or clones that recognize antigens restricted to hematopoietic cells restricted antigens has been suggested as a potential strategy for inducing GVL without GVHD. In vitro studies demonstrated that such cells could kill bulk populations of leukemia blasts. However, it has been more difficult to determine whether clonogenic leukemia blasts are susceptible to such killing. The use of NOD/SCID mice has been suggested as an in vivo model reservoir of adoptively transferred patient leukemia cells, which can then be assayed to determine the spectrum of primitive hematopoietic cells, including clonogenic AML blasts (94–96). Cells that can colonize NOD/SCID mice have been termed SCID leukemia-initiating cells, or SLIC. Using this model, human CD8+ CTL clones when cultured with AML blasts were shown to reduce the engraftment of those blasts in NOD/SCID mice (29). If T-cell clones are to be applied clinically, such a model system could be useful in predicting their in vivo efficacy in patients. Questions about toxicity or epitope spread could not be easily addressed due to potential differences in gene expression and tissue distribution of the target antigen between humans and mice and due to differences in MHC. The NOD/SCID system has also been used to demonstrate the efficacy of alloreactive natural killer cell (NK) lines and clones to kill clonogenic AML cells (30). However, unlike human T cells, human alloreactive NK cells were shown to kill AML blasts in vivo in NOD/SCID mice after engraftment. This result was important in providing evidence that alloreactive NK cells directly kill AML blasts in humans rather than mediating an indirect effect (see below). IV. PRECLINICAL AND CLINICAL STUDIES OF GVL EFFECTS IN HUMANS A. Effector Cells of GVL Because T-cell depletion of donor cells is associated with significant relapse rates after HCT, and because enrichment of T cells in either the original graft or in subsequent DLI increases the likelihood of remaining in durable remission, it is thought that αβ-T lymphocytes are the most significant mediators of GVL (Table 3). Although unconventional T-cell subsets such as γδ T cells, NKT cells, or certain other CD1restricted αβ T cells have been studied for their GVL potential, these cells may have only limited, if any, involvement. Likewise, the humoral immune response is unlikely to play

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a significant role in GVL. However, recent data have emerged that NK cells may contribute to GVL very early after HCT in AML patients that are transplanted with donor cells mismatched at specific MHC class I loci. In this section, we will examine the biology of these mediators of GVL by reviewing relevant preclinical and clinical studies.

Table 3 Clinical Evidence of LymphocyteMediated GVL Effects Relapse increased in recipients of T-cell-depleted transplants (79) Relapse decreased in recipients who develop acute/chronic GVHD (240) Remission associated with GVHD flares (7,241) Relapse higher in recipients of T-cell-depleted matched related donor allograft recipients who develop GVHD compared to recipients of matched related donor allografts who do not develop GVHD (242) Remission in up to 90% of CML patients who receive DLI administered for relapse after transplant (2,243)

B. T Lymphocytes T cells recognize peptide antigens that are presented on the cell surface in combination with major histocompatibility complex (MHC, in humans termed HLA) antigens (Fig. 1). Peptides derived from intracellular cytoplasmic proteins that are 8–11 amino acids in length bind within the groove of class I MHC molecules and are transported via the endoplasmic reticulum to the cell surface and are subsequently recognized by CD8 T cells. Larger peptides, 12–18 amino acids in length, usually derived from extracellular proteins, bind class II MHC molecules and are presented on the cell surface to CD4 T cells. Both peptide/MHC I and peptide/MHC II are recognized by the heterodimeric αβ T-cell receptor (TCR-αβ) with very rapid off rates and kD that are typically in the range of 10−4– 10−5 M (97) (Fig. 2). Points of contact between the TCR and the peptide/MHC surface include surface amino acids contributed by the two alpha helical domains of the MHC molecule that flank the peptide antigen binding pocket as well as amino acids from the peptide itself. Following an initial limited contact of surface TCR-αβ heterodimers with their cognate peptide/MHC target antigen, a complex signaling network leads to the formation of an immune synapse (98–101). Further modulation occurs via costimulatory molecules and cell adhesion molecules (99,100,102). In this way the initial signaling event is amplified, leading to further downstream events such as upregulation of IL-2 and the IL-2 receptor. These events are essential for subsequent T-cell functions (103).

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Figure 1 Antigen recognition by T lymphocytes. Peptide antigen, presented in the peptide-binding groove of an MHC molecule, is recognized and bound by the T-cell receptor (TCR), as shown for a CD8 Tlymphocyte response. The T lymphocyte also receives other costimulatory signals from antigenpresenting cells that modulate the antigen-specific signal. As an example, the CD28 receptor on CD8 Ty lymphocytes binds CD80 on the antigen-presenting cell, which decreases the activation threshold of the T lymphocyte.

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Figure 2 T-cell receptor (TCR) signaling. After binding to cognate antigen/MHC complexes on the antigen-presenting cell (APC) surface, TCRs assemble toward the center of an enlarging synapse between the T lymphocyte and the APC. Phosphorylation of the immune receptor tyrosine activation motif (ITAM) on the various CD3 subunits associated with the TCR complex then occurs mediated by tyrosine kinases such as lck/fyn. The strength of the signal that is thereby transduced is related to the degree of ITAM phosphorylation. TCE-binding affinity and avidity contribute to prolonged signaling, and the signal strength progressively increases from activation threshold to possible overstimulation with saturation of the ITAMs, which may lead to eventual cell death. Our understanding of the nature of antigen-specific T cell responses has been greatly improved by the discovery that antigen-specific TCR can be reversibly labeled with soluble peptide/MHC tetramers (104) (Fig. 3). Up to 45% of all peripheral circulating T cells may be specific for a single dominant antigen at the height of an immune response

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to EBV infection (105), and similar dominance may be seen during other viral infections (106,107). Tetramers have also been used to discover and study immune responses to tumor antigens (108,109). C. Potential GVL T-Lymphocyte Antigens Tissue-restricted mHA that are derived from proteins expressed only in hematopoietic tissue have been shown to be the targets of alloreactive T cells. These mHA may result from polymorphic differences between donor and recipient in the coding regions of peptide antigens. Heterologous T-cell clones with alloreactivity toward mHA have been

Figure 3 Peptide/MHC tetramers can be used to identify antigen-specific T lymphocytes. Recombinant MHC heavy chains are folded together with β2-microglobulin and synthetic peptide antigens, and they are then attached via a biotin-streptavidin link at the C terminus of the MHC heavy chain to a fluorochrome, such as phycoerythrin (PE). The avidity of the TCRpeptide/MHC binding is increased by increasing the number of peptide/MHC monomers attached to the streptavidin up to four, with the geometry of the resulting tetramers being optimal for TCR binding. established from patients with severe GVHD following HCT with an HLA-matched donors (110–113). Some of these mHA-specific CTL clones react only with hematopoietic-derived cells targets, suggesting tissue specificity (112), and therefore may be useful because of potentially shared antigens with leukemia cells. In one study, GVHD

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correlated closely with differences in the minor antigen HA-1 in HLA identical sibling transplants (114). Expression of two human mHAs, identified as HA-1 and HA-2, is confined to hematopoietic tissues, and HA-2 was identified as a peptide derived from the non-filament-forming class I myosin family by using mHA-reactive CTL clones to screen peptide fractions eluted from MHC class I molecules (115). This methodology is extremely labor intensive, and it is unclear whether CTL specific for any minor antigens identified thus far convey leukemia-specific immunity without concomitant GVHD. Adoptive cellular immunotherapy of leukemia using antigen-specific T cells that target mHA may promote GVL and reduce GVHD if appropriate mHA restricted to hematopoietic cells could be targeted. However, a practical limit of any immunotherapy approach targeting these mHAs is that only 10% of individuals would be expected to have the relevant HA-1 alternate allele, and