Killer Lymphocytes GIDEON BERKE WILLIAM R. CLARK. Department of Immunology, Weizmann Institute of Science, Rehovot, Israel. and

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2 KILLER LYMPHOCYTES

3 Killer Lymphocytes by GIDEON BERKE Department of Immunology, Weizmann Institute of Science, Rehovot, Israel and WILLIAM R. CLARK University of California, Los Angeles, California, U.S.A.

4 A C.I.P. Catalogue record for this book is available from the Library of Congress. ISBN (PB) ISBN (HB) ISBN (e-book) Published by Springer, P.O. Box 17, 3300 AA Dordrecht, The Netherlands. Printed on acid-free paper All Rights Reserved 2007 Springer No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work.

5 TABLE OF CONTENTS Preface vii Chapter 1 Basic Immunobiology: A Primer 1 Chapter 2 Cytotoxic T Lymphocytes: Generation and Cellular Properties 29 Chapter 3 Cytotoxic T Lymphocytes: Target Cell Recognition and Binding 53 Chapter 4 Cytotoxic T Lymphocytes: Target Cell Killing: Cellular Parameters 71 Chapter 5 Cytotoxic T Lymphocytes: Target Cell Killing: Molecular mechanisms 95 Chapter 6 Innate Cell-mediated Immunity 125 Chapter 7 The Role of Cytotoxicity in Allograft Rejection In Vivo 159 Chapter 8 Cytotoxicity in Immune Defenses Against Intracellular Parasites 179

6 vi Chapter 9 Killer Cells and Cancer 201 Chapter 10 Autoimmunity 217 Chapter 11 Homeostasis, Memory and CTL Vaccines 231 References 245 Index 353

7 PREFACE The existence of a unique kind of immune cell the killer lymphocyte - which destroys other cells in a highly specific manner, has fascinated immunologists for almost half a century. How do these cells, whose precursors have lived in communal harmony with their host, decide that some of their cohabitants must die? And how do they kill them? The definition of killer lymphocytes came from discovery of their roles in a wide range of in vivo phenomena such as transplant rejection, virus infection and its related immunopathologies, and anti-tumor responses. Yet for the most part almost everything we know about these cells has come from studying them in vitro. They have yielded their secrets slowly and reluctantly. To understand fully how they work, geneticists and immunologists had to unravel the major histocompatibility systems of vertebrates, a long and torturous road that provided some of the darkest hours of immunology. The search for antigen-sensing receptors on both T cells and NK cells was scarcely less frustrating. And the holy grail of cellmediated cytotoxicity defining the mechanism by which killer cells take down their adversaries sorely tested the ingenuity, patience and mutual good will of laboratories around the world. These questions have now largely been answered. But do we really understand these cells? We can tame them to a large degree in transplant rejection. It may yet turn out that we can harness their immunotherapeutic potential in treating viral and malignant disease. The pivotal role of CTL induction has become part and parcel of many vaccination schemes. But it has become less clear with time that the dramatic destruction of cells wrought by killer cells in vitro represents their true function in vivo. In writing this book we had several goals in mind. First, we wanted to provide a definitive resource for the subject of cell-mediated cytotoxicity killer lymphocytes. We felt it would be useful to have a single volume that

8 viii Preface traces the history of this field, telling its story in terms of key experiments and ideas that have shaped research into the function and biological meaning of cells that kill other cells. At the same time, we wanted to integrate, where possible, the major themes coursing through this subject in its fifty or so year history. And finally, we felt it is time to assess the evidence for and against a role of killer lymphocytes in vivo. Having worked actively in the field for over thirty years, we were still surprised by how extensive it has become. We estimate admittedly somewhat loosely that well over 100,000 papers have been published on various aspects of cell-mediated cytotoxicity (CMC) since it began. Our goal could not possibly be to cover all of this information. We have tried to identify those papers that were key to the origin of each of the many themes in CMC, and then to identify key recent reviews that allow anyone interested in a given sub-topic to work their way back through the existing literature as suits their needs. We have done our best to keep the number of papers cited to a minimum, consistent with that goal. We apologize in advance to our many friends and colleagues whose many excellent and important papers have not been cited here. All scientific fields are works in progress, and even as we bring this project to a conclusion the field of CMC is morphing in new directions. It may be worth coming back in five years or so to update both new developments and our interpretation of the history of this fascinating field. We hope readers of this book will find it useful, and we especially hope they will feel free to communicate to us their own thoughts on what we have presented, and what we have not. Finally, we would like to acknowledge the valuable assistance of Drs. Dalia Rosen, Judith Gan and Orit Gal-Garber, and Mr. Steven Manch, all of the Weizmann Institute, for invaluable assistance in preparation of this book. Gideon Berke Rehovot gideon.berke@weizmann.ac.il Bill Clark Los Angeles wclark222@cs.rr.com January, 2005

9 Chapter 1 BASIC IMMUNOBIOLOGY: A PRIMER This chapter is intended for those readers whose primary field of interest is not immunology, and for whom some degree of orientation in the basic structure and function of the immune system would be useful. A comprehensive discussion of the immune system is beyond the scope of this book; for that purpose the reader is referred to any of a number of current excellent textbooks and reviews in immunology, such as Paul s Fundamental Immunology 1 or various volumes of Annual Reviews of Immunology. However, for purposes of orientation to those aspects of immunity most relevant to understanding the immunobiology of killer lymphocytes, we offer the following greatly simplified summary. Many of the topics that follow will be dealt with in more detail throughout this book. 1 THE IMMUNE SYSTEM The immune system is composed of a number of interacting organs, tissues and cell types, whose principal function is to protect the host organism from uncontrolled growth of potentially pathogenic microorganisms. The immune system, including killer lymphocytes, may also be involved in suppressing the development of malignancy, but from an evolutionary viewpoint this is likely to be a secondary function. The immune system can also cause harm, through such hypersensitivity reactions as allergy and asthma, through overshoot reactions to intracellular parasites (whether the parasites are harmful or not), and through autoimmune disease. These are presumed to be evolutionarily acceptable costs of an otherwise efficiently protective microbial defense system. And the immune system in 1 Fundamental Immunology, 5th ed.. Lippincott, Williams & Wilkins, 2003.

10 2 Chapter 1 particular killer lymphocytes constitutes the major barrier to potentially life-saving organ transplants. From a functional point of view, it is useful to think of immune reactions as falling into two major categories: innate immune responses, and adaptive immune responses. Innate immune reactions are evolutionarily older, occurring in one form or another in virtually all animals and most if not all plant species. Innate responses are part of the germline inheritance of each organism, and require no further modification to be maximally effective. In general, innate immune reactions in vertebrates rely on recognition of relatively invariant molecular motifs that are, in turn, part of the genetic heritage of potentially harmful microorganisms (Janeway and Medzhitov, 2002). In vertebrate immune systems, various cells have this capacity, in particular dendritic cells (DC), but also macrophages and granulocytes. Among killer lymphocytes, natural killer (NK) cells are considered part of the innate system of defense (Chapter 6). Innate immune responses are highly effective and absolutely essential for survival. Many diseases based on an absence of innate immunity are lethal. The possibility of self-harm from innate immune responses is for the most part neutralized phylogenetically through natural selection. The major shortcoming of innate immune responses is that they cannot respond quickly to a changing pathogenic environment. One wonders how many established plant or animal species, dependent entirely on innate immune defenses (all living organisms other than vertebrates), have disappeared from the planet because of the sudden emergence of a new and lethal (for the affected species) pathogen. The ability to change quickly in response to a changing antigenic environment is the hallmark of adaptive immunity. Adaptive immune responses are based on the principle of ontogenetic generation of essentially unlimited numbers of different receptors with which to probe the antigenic universe. This highly polymorphic receptor repertoire is generated completely randomly, by recombination of a limited number of germline gene fragments. The survival of any particular genetic recombination is dependent on selection by antigen, and a system for negative selection of self-reactive receptors to avoid autoimmunity. Although defects in the adaptive immune system can be lethal, often they are not. 1.1 Anatomy of the immune system The mediators of adaptive immunity in vertebrates consist of the B lymphocytes, which produce antibodies, and T lymphocytes, which include both T helper cells and killer T cell - the cytotoxic T lymphocytes, or CTL. Antibodies are produced in response to the presence, in the extracellular

11 1. Basic Immunobiology: A Primer 3 fluids of the body, of non-self or altered-self biological materials, and are released into the bloodstream, where they trigger a series of reactions culminating in inactivation and/or removal of the non-self material (antigen) from the system. CTL participate in the destruction of self cells compromised by intracellular infection or oncological transformation. Both B-cell and CTL responses are aided by a subset of T lymphocytes called helper T cells, which in addition to their help function are also potent mediators of inflammation. We explore the basic parameters of these reactions in the remainder of this chapter. The major organs of the vertebrate immune system are the bone marrow, the thymus, the spleen and various lymph nodes scattered throughout the body, which comprise both adaptive and innate immune elements. The bone marrow is the locus of hematopoietic stem cells characterized by, among other things, self renewal, and the presence of a surface marker called CD34. (A list of CD designations used to define immune system cells is given in Table 1.1). These stem cells give rise to the cells - called generically white cells or leukocytes - involved in immune responses (Figure 1.1), as well as red blood cells. As such, CD34 cells are critical in bone marrow transplantation, and are used for various strategies in gene therapy for diseases such as AIDS and SCID (severe combined immune deficiency). CD34 cells may also serve as pluripotent adult stem cells for generation of non-lymphoid cells and tissues. Table 1-1. CD designation CD antigen Previous Designation(s) Function(s) CD1 T6; Leu 6 Presentation of non-peptide (lipid) antigens to T cells (, /, NKT) CD2 LFA-2 Intercellular adhesion (with CD58/LFA-3) on NK cells, T cells; activation CD3 OKT3; T3; Leu 4 T-cell receptor signal transduction CD4 OKT4; T4; Leu 3a; L3T4 T-cell coreceptor for MHC class II CD8 OKT8; T8; Lyt2,3; Leu 2 T-cell coreceptor for MHC class I CD11a LFA-1 Intercellular adhesion (with CD54/ICAM- 1) CD16 Leu 11a; Fcγ RIIIA Low affinity Fc receptor on NK cells CD28 T44 T-cell receptor for B7 (CD80, 86) CD30 Ber-H2; Ki-1 TNF-family receptor CD34 gp 105 Adhesion molecule; hematopoietic stem cell marker CD40 Bp50 Growth factor receptor CD54 ICAM-1 Intercellular adhesion (with CD11a) CD56 Intercellular adhesion (immune system ligand unknown CD57 NKH-1; Leu 7 Intercellular adhesion (?) CD58 LFA-3 Intercellular adhesion (with CD2) CD64 Fc RI Mediates ADCC

12 4 Chapter 1 CD antigen Previous Designation(s) Function(s) CD80 B7.1 Ligand for CD28, CTLA-4 CD86 B7.2 Ligand for CD 28, CD95 Fas; APO-1 Mediation of apoptosis CD152 CTLA-4 Negative regulation of T-cell function CD159a NKG2A NK cell signaling (inhibition) CD161 NKR-P1A Regulation of human NK function CD178 Fas ligand CD95L Mediation of apoptosis There are now over two hundred cell-surface markers used to define cell populations within the immune system. These markers were defined by antibodies developed in laboratories around the world, and the initial names for these markers were those chosen by the producing laboratory. It was not unusual for the same marker to have more than one name. In an attempt to bring uniformity to the nomenclature for such markers, an ongoing series of international workshops and conferences on Human Leukocyte Differentiation Antigens are periodically held, in which candidate antiens are tested by multiple laboratories and assigned a CD number ( While these workshops focus on human CD antigens, where identity is clear the same designation is used for mouse antigens as well. A current list of CD designations can also be obtained at Figure 1-1. The origin of B and T lymphocytes. B-cells complete their maturation in bone marrow and seed directly into B cell compartments of lymph nodes and spleen. T-cells complete their maturation in the thymus. Bone marrow is also the site of B-cell maturation. After maturation, the B cells seed into specific regions of the lymph nodes and spleen, where they encounter antigen. Once activated to become plasma cells (the activated,

13 1. Basic Immunobiology: A Primer 5 antibody-producing form of B cells), many of them migrate back to the bone marrow, which provides space for expanded plasma cell replication and antibody production. And finally, bone marrow can also be a site of T cell encounter with antigen (Feurer et al., 2003), although this is probably a minor venue in comparison with lymph nodes and spleen. The thymus is the site of T-cell maturation, the T in fact denoting the role of the thymus in their developmental history. Like B cells, T cells originate in the bone marrow, but early in their life history they translocate to the thymus for their final stages of maturation. In the thymus, the T cells differentiate into killer cells (CD8) and helper cells (CD4) (Germain, 2002), and undergo a series of positive and negative selection steps to fine-tune their ability to distinguish self from non-self. From an immunological point of view, self refers to biological materials derived from normal healthy tissues and cells of the host organism. Non-self may refer to biological material of extra-self origin (whether pathogenic or not); to altered self (self materials that have been mutated, damaged or in some other way altered); or self materials that appear only secondary to processes such as tumorigenesis. This ability to distinguish between self and non-self is largely a property of T lymphocytes. B cells with potential self-reactivity are often present in adult animals, but their ability to react with self components is circumvented by various strategies, such as functional deprivation of T-cell help. 1.2 Lymphocyte circulation The lymph nodes serve as filters for antigenic materials brought to them in lymph fluid, which drains virtually every location in the vertebrate body. The spleen filters antigens only from arterial blood. Cells such as macrophages, follicular dendritic cells and B lymphocytes resident in lymph nodes can entrap these materials. More importantly, particularly for killer cell responses, highly specialized antigen-entrapping cells such as dendritic cells can also capture antigen in various tissues of the body (e.g. at the site of a skin allograft), and transport the antigen via the lymphatic circulation to a regional draining lymph node (Figure 1.2). In either case, T cells then scan the entrapping cells for anything interpreted as non-self and thus a potential threat to the host organism s existence. We will discuss the mechanics of this process, and the ensuing activation of T cells, in greater detail below in Antigen processing and presentation. The spleen plays a role similar to that of the lymph nodes in the entrapment of antigen and as a site for lymphocyte activation. Once activated by antigen, T cells leave the lymph nodes or spleen via efferent lymphatic vessels and circulate throughout the body, looking for the source of the activating antigen.

14 6 Chapter 1 The circulation of lymphocytes and many of the cells with which they interact takes place in both the blood and lymphatic fluid. Lymph fluid is created when plasma leaves capillaries to deliver oxygen and nutrients to body tissues, and to gather waste products excreted by cells. This fluid collects into lymphatic sinuses, which give rise to lymphatic vessels, which in turn coalesce into larger lymphatic trunks that ultimately empty into the bloodstream at the great veins of the neck. Lymphatic vessels drain every region of the body served by blood vessels; failure of the lymphatic drainage system results in local pooling of lymph fluid in tissue spaces (lymph edema). Figure 1-2. Lymphocyte circulation. Antigenic material from a skin allograft is carried by dendritic cells into a draining lymph node, where it interacts with T lymphocytes. Lymphocytes recognizing graft antigens leave the node via the efferent lymphatic and eventually enter the bloodstream and circulate throughout the body until they encounter the graft site. At different points in their life cycle, T cells may travel in either lymph or blood. Newly generated lymphocytes arrive at lymph nodes from the thymus or marrow via arterial blood, and with the aid of surface receptors home to T- or B-cell-specific regions of the nodes. They leave the blood

15 1. Basic Immunobiology: A Primer 7 circulation by squeezing between endothelial cells lining post-capillary venules in the middle and deep zones of the lymph node cortex, which brings them into the lymphatic circulation. Upon activation by antigen, they exit the lymph nodes via the efferent lymphatic vessels, and after passing through varying numbers of downstream lymph nodes, re-enter the blood circulation at the neck. In the antigen-activated state, T cells do not express the surface markers required to interact with post-capillary venules in lymphoid tissues, and thus remain in the blood stream. However, they can interact with chemokine receptors on post-capillary venules at sites of inflammatory reactions, which allows them to leave the blood and enter tissue spaces, where they are once again in the lymphatic circulation. As will be discussed in detail later, some of the activated T cells differentiate into a memory state, and once again express receptors that allow them to take up long-term residence in lymphoid tissue until they encounter cognate antigen again, resulting in their reactivation. As mentioned above, dendritic cells scattered throughout the body are very important in bringing antigen into the lymph nodes, and converting it to a form recognized by T cells. Dendritic cells that have acquired antigen become mobile, and slip away from their tissue-resident sites into the surrounding lymphatic fluid. From there they travel to the nearest downstream lymph node, where they enter T-cell-rich areas and can be examined by T cells. Dendritic cells spend very little time in the blood circulation. 1.3 T cells and their functions T cells are divided into two main functional types: helper T cells and killer T cells (CTL). Helper T cells, distinguished by the presence of the CD4 surface marker, are mainly amplifier cells (Figure 1.3). They release chemical signals (cytokines) used by cells of both the innate and adaptive immune systems to carry out their functions (Table 1.2). B cells, for example, require certain T-helper cell cytokines to mature into antibodyproducing plasma cells. There are also receptors for many of these cytokines on non-lymphoid tissues, including even the brain, suggesting the possibility of two-way communication between the nervous system and the immune system. T-helper cells are often further divided into T h -1 and T h -2 subsets based on the pattern of cytokine production. It is likely that the division into these subsets occurs at the time of initial activation of CD4 cells by antigen.