CYTOTOXIC CELLS

CYTOTOXIC CELLS:

Recognition, Effector Function, Generation, and Methods

M. V. Sitkovsky P. A. Henkart

Editors

Birkhauser Boston . Basel . Berlin

Michail V. Sitkovsky Laboratory of Immunology Biochemistry and Immunopharmacology Unit NIH-NIAID Bethesda, Maryland 20892 USA

Pierre A. Henkart Experimental Immunology Branch National Cancer Institute National Institutes of Health Bethesda, Maryland 20892 USA

Library of Congress Cataloging-in-Publication Data Cytotoxic cells: recognition, effector function, generation, and

methods I M.V. Sitkovsky, P.A. Henkart, editors. p. cm.

Includes bibliographical references and index. ISBN-13: 978-1-4684-6816-8 e-ISBN-13: 978-1-4684-6814-4 001: 10.1007/978-1-4684-6814-4

1. T cells. 2. Killer cells. 3. Cell-mediated cytotoxicity. I. Sitkovsky, M.V. II. Henkart, Pierre.

[DNLM: 1. T-Lymphocytes, Cytotoxic--physiology. 2. Cytotoxicity, Immunologic. QW 568 C9972 1993] QRI85.8.T2C99 1993 616.0T9--dc20 DNLM/DLC for Library of Congress

Printed on acid-free paper.

© 1993 Birkhiiuser Boston Birkhiiuser Softcover reprint of the hardcover 1st edition 1993

93-6929 CIP

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ISBN-13: 978-1-4684-6816-8

Typeset by Alden Multimedia, United Kingdom

9 8 7 6 5 4 3 2 I

CONTENTS

Preface Contributors

SECTION I: INTRODUCTION AND OVERVIEW

Introductory Remarks Herman N. Eisen

2 T-Cell-Mediated Cytotoxicity: A Historical Note

IX

X

3

Jean-Charles Cerottini 5 3 Overview of CTL-Target Adhesion and Other Critical Events in the Cytotoxic Mechanism

Eric Mart:: 9

SECTION II: TARGET CELL RECOGNITION

4 'Y.f3 T-Cell Receptor Repertoires Among Cytotoxic and Helper T Lymphocytes David M. Kran:: and Benjamin Tjoa 49

5 Class I MHC/Peptidc/ f3rMicroglobulin Interactions: The Basis of Cytotoxic T-Cell Recognition David H. Margulies, Lisa F. Boyd, Maripat Corr, Rosemarie D. Hunziker, Sergei Khilko, Steven Kozlowski, Michael Mage, and Randall K. Ribaudo 58

6 The Role of CD8-Class I Interactions in CTL Function Anne M. O'Rourke and Matthew F. Mescher 65

7 Interactions Between CD2 and T-Cell Receptor Isoforms in CTL Function Shigeo Koyasu and Ellis L. Reinher:: 72

8 Triggering Structures on NK Cells Lewis L. Lanier and Joseph H. Phillips 84

9 Targeted Cellular Cytotoxicity David M. Segal, Carolina R. Jost, and Andrew J. T. George 96

SECTION III: GENERATION OF CYTOTOXIC CELLS

10 Immunobiology and Molecular Characteristics of Peritoneal Exudate Cytotoxic T Lymphocytes (PEL), Their In Vivo IL-2 Dependent Blasts and IL-2 Independent Cytolytic Hybridomas Gideon Berke, Dalia Rosen, Denise Ronen, and Barbara Schick

II Regulatory Effects of Cytokines on the Generation of CTL and LAK Cells Mark R. Alderson and Michael B. Widmer

12 IL-2-Independent Activation of LAK Cells by a Heterodimeric Cytokine, Interleukin-12

113

128

Maurice K. Gately, Aimee G. Wolitzky, Phyllis M. Quinn, and Richard Chizzonite 138 13 Immunobiology of f3rMicroglobulin-Deficient Mice

J. A. Frelinger, D. G. Quinn, and D. Muller 145

SECTION IV: MOLECULAR MECHANISMS OF CELLULAR CYTOTOXICITY

14 The Granule Exocytosis Model for Lymphocyte Cytotoxicity and Its Relevance to Target Cell DNA Breakdown Pierre A. Henkart, Mark P. Hayes, and John W. Shiver

15 Subpopulations of Cytotoxic T Lymphocytes with Different Cytotoxic Mechanisms David W. Lancki, Maureen McKisic, and Frank W. Fitch

16 MUltiple Lytic Pathways in Cytototoxic T Lymphocytes William R. Clark and Anna Ratner

153

166

178

VI CONTENTS

17 Properties of Cytotoxicity Mediated by CD4 + , Perforin-Negative T-Lymphocyte Clones Hajime Takayama 190

18 Direct Contact of Cytotoxic T Lymphocyte Receptors with Target Cell Membrane Determinants Induces a Prelytic Rise of [Ca2 + 11 in the Target That Triggers Disintegration Gideon Berke 196

19 Target Cell Events Initiated by T-Cell Attack John H. Russell and Scott I. Abrams 202

20 Apoptosis and Cytotoxic T Lymphocytes Richard C. Duke 213

21 Molecular Mechanisms of Lymphocyte Cytotoxicity Mark J. Smyth and John R. Ortaldo

SECTION V: GRANULE PROTEASES

22 Subtractive and Differential Molecular Biology Approaches to Molecules Preferentially Expressed in Cytotoxic and Other T Cells

223

Eric Rouvier and Pierre Golstein 237 23 Structure and Possible Functions of Lymphocyte Granzymes

Patrick Haddad, Dieter E. Jenne, Olivier Krahenbuhl, and Jurg Tschopp 251 24 The Role of Granzyme A in Cytotoxic Lymphocyte-Mediated Lysis

Mark S. Pasternack 263 25 The Granzyme A Gene: A Marker for Cytolytic Lymphocytes In Vivo

Gillian M. Griffiths, Susan Alpert, R. Jane Hershberger, Lishan Su, and Irving L. Weissman 273 26 Molecular Analysis and Possible Pleiotropic Function(s) of the T Cell-Specific Serine

Proteinase-I (TSP-I) Markus M. Simon, Klaus Ebnet, and Michael D. Kramer 278

27 Serine Protease Control of Lymphocyte-Mediated Cytolysis Dorothy Hudig, N. Janine Allison, Gerald R. Ewoldt, Ruther Gault, Dale Netski, Timothy M. Pickett. Doug Redelman, Ming T. Wang, Ulrike Winkler, Susan J. Zunino, Chih-Min Kam, Shinjiro Odake, and James C. Powers 295

SECTION VI: ALTERNATIVE MECHANISMS OF CYTOLYSIS

28 Possible Role of Extracellular ATP in Cell-Cell Interactions Leading to CTL-Mediated Cytotoxicity Frank Redegeld, Antonio Filippini, Guido Trenn, and Michail V. Sitkovsky 307

29 Cell-Permeabilizing Properties of Extracellular ATP in Relation to Lymphocyte-Mediated Cytotoxicity Francesco Di Virgilio, Paola Zanovello, and Dino Collavo 314

30 The Role of Free Fatty Acids in CTL-Target Cell Interactions Alan M. Kleinfeld 321

SECTION VII: BIOCHEMICAL AND IMMUNOPHARMACOLOGICAL MANIPULATIONS OF CYTOTOXIC CELLS

31 Identification of Protein Kinases and Protein Phosphatases Involved in CTL Effector Functions. "ON" and "OFF" Signaling and Immunopharmacological Implications Hirotaka Sugiyama, Sergei Apasov, Guido Trenn, Frank Redegeld, and Michail Sitkovsky 331

32 Cytolytic Granules as Targets for Immunosuppressive Therapy: Selective Ablation of CTL by Leucyl-Leucine Methyl Ester Dwain L. Thiele and Peter E. Lipsky 340

SECTION VIII: FUNCTIONS OF CYTOTOXIC CELLS IN VIVO

33 Role of CD8+ rxf3 T Cells in Respiratory Infections Caused by Sendai Virus and Influenza Virus Peter C. Doherty, William Allan, Maryna Eichelberger, Sam Hou, Jacqueline M. Kat::, Rudolf Jaenisch, and Maarten Zijlstra 351

CONTENTS VII

34 CD4 + and CD8 + Cytolytic T Lymphocyte Recognition of Viral Antigens Vivian Lam Braciale 358

35 Can CTL Control Virus Infections Without Cytolysis? The Prelytic Halt Hypothesis Eric Martz 366

36 Immunologic Control of Toxoplasma Gondii Infection by CD8+ Lymphocytes: A Model for Class I MHC- Restricted Recognition of Intracellular Parasites Ricardo T. Gazzinelli, Eric Denkers, Frances Hakim, and Alan Sher 370

37 Antigen-Specific Suppression of Antibody Responses by Class II MHC-Restricted CTL Nobukata Shinohara 378

38 The Immunosenescence of Cytolytic T Lymphocytes (CTL): Reduction of Pore-Forming Protein and Granzyme Levels Eda T. Bloom and Judith A. Horvath 384

39 Bone Marrow Graft Rejection as a Function of T NK Cells Gunther Dennert 394

40 Class I MHC Antigens and the Control of Virus Infections by NK Cells Raymond M. Welsh, Paul R. Rogers, and Randy R. Brutkiewicz 400

41 Clinical Trials of Immunotherapy of Cancer Utilizing Cytotoxic Cells Stephen E. Ettinghausen and Steven A. Rosenberg 407

SECTION IX: MACROPHAGE-MEDIATED CYTOTOXICITY

42 Macrophage-Mediated Cytotoxicity Penelope J. Duerksen-Hughes and Linda R. Gooding 439

SECTION X: METHODS

43 The 5lCr-Release Assay for CTL-Mediated Target Cell Lysis Eric Mart:: 457

44 DNA Fragmentation and Cytolysis Assayed by lH-Thymidine Eric Martz 468

45 The JAM Test: An Assay of Cell Death Polly Matzinger 472

46 Target Cell Detachment Assay Scott I. Abrams and John H. Russell 478

47 Protocol for Assaying CTL Activity Against Toxoplasma gondii Ricardo T. Ga::zinelli, Eric Denkers, Frances Hakim, and Alan Sher 481

48 Granule Exocytosis Assay of CTL Activation Michail V. Sitkovsky 482

49 Measurement of Cytolysin Hemolytic Activity Pierre Henkart 484

50 SPDP Crosslinking of Antibodies to Form Heteroconjugates Mediating Redirected Cytotoxicity David M. Segal 485

51 Derivatization of Cells with Antibody Anna Ratner and William R. Clark 487

52 Mixed Lymphocyte Culture for the Generation of Allospecific CTL Eda T. Bloom 488

53 Generation of CD4 + and CD8 + Antiinf'luenza CTL and Assay of In Vitro Cytotoxicity Vivian Lam Braciale 490

54 Generation of Antigen-Specific Murine CTL Under Weakly Immunogenic Conditions Sergey G. Apasov 492

55 Commercial Liposomes and Electroporation Can Deliver Soluble Antigen for Class I Presentation in CTL Generation Weisan Chen and James McCluskey 494

56 Stimulation of CTLs on Antibody-Coated Plates Anna Ratner and William R. Clark 497

V111 CONTENTS

57 CTL Recognition of Purified MHC Antigens and Other Cell Surface Ligands Matthew F. Mescher, Paul Champoux, and Kevin P. Kane 498

58 Use of Protease Inhibitors as Probes for Biological Functions: Conditions, Controls, and Caveats Dorothy Hudig and James C. Powers 502

59 The Murine T Cell-Specific Serine Proteinase-I: Cleavage Activity on Synthetic and Natural Substrates M. D. Kramer, U. Vettel, K. Ebnet, and M. M. Simon 516

60 Detection of Specific mRNAs by In Situ Hybridization Gillian M. Griffiths, Susan Alpert, R. Jane Hershberger, Lishan Su, and Irving L. Weissman 521

Index 525

PREFACE

Our motivation for putting together this book was the need for a single source reference that could be used as an introduction to cell-mediated cytotoxicity for newcomers to this field, such as students and fellows beginning work in our laboratories. At present no such book is available, and we felt that it would be useful as a teaching tool and as a way of conveying our enthusiasm about recent progress in the cytotoxicity field to our colleagues in allied areas. It was with some hesitation that we approached our colleagues with the proposal for this book, and we were pleased to find them very supportive of the idea and willing to participate. We thought it important to broaden the scope of the book to include historical, molecular, cell biological, and clinical aspects of cell-mediated cytotoxicity. To our knowledge this is the first book on cell-mediated cytotoxicity with such a broad scope.

Historically, studies on cellular cytotoxicity were part of cellular immunology from its origin. One development of tremendous import was the advent of the 51 Cr assay, which allowed this arm of the immune response to be measured easily and quantitatively. Thus, a readout of this effector pathway is available within a few hours; other immune effector functions can take days or even longer to assay, and the assays are often less quantitative. For example, using the 51 Cr-release assay, studies of cytotoxic T cells played a crucial role in discovering the rules of antigen recognition and in establishing the concept of major histocompatibility complex (MHC) restriction. In turn, the major breakthroughs in the understanding of the structure and functions of target recognition receptors on cytotoxic cells led to the development of such powerful approaches as retargeted cytotoxicity, which is further extending the utility of cytotoxic cells.

Natural killer (NK) cells and cytotoxic T cells are to be considered as two different but related arms of host defense, e.g., against virus infection. Indeed, some interesting interrelations between the class I MHC expression and sensitivity of virus-infection cells to destruction by cytotoxic T lymphocytes (CTL) or NK cells are found. Both positive and negative feedback mechanisms could be at play here, since virus-induced interferon makes cells resistant to NK cells as these infected cells become increasingly sensitive to CTL. The least studied, but most clinically used, cytotoxic cells [lymphokine-activated killer (LAK) cells, tumor-infiltrating lymphocytes (TIL)] may provide the efficient means of antitumor treatments.

The studies of the mechanisms of effector functions are more controversial than other areas (e.g., CTL recognition), since they are linked to broader issues of cell biology and physiology. The diversity of opinion is reflected in the various contributions to this book and provides a flavor of the excitement of this fast-moving area.

We have been immensely pleased with the quality of the chapters we have received for this book. We are confident that it will fulfil the need we have set out to address. We thank the various contributors for their efforts and hope that the readers will get as much out of these chapters as we have.

Michail V. Sitkovsky Pierre A. Henkart Editors

CONTRIBUTORS

Scott I. Abrams, Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110, USA. Current Affiliation: Laboratory of Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA

Mark R. Alderson, Department of Immunology, Immunex Research and Development Corporation, Seattle, Washington 98101, USA

William Allan, Department of Immunology, St. Jude Children's Research Hosptial, Memphis, Tennessee 38105, USA

N. Janine Allison, Cell and Molecular Biology Program, School of Medicine and College of Agriculture, University of Nevada, Reno, Nevada 89557-0046, USA

Susan Alpert, Department of Pathology and Howard Hughes Medical Institute, Stanford, California 94305, USA

Sergey G. Apasov, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA

Gideon Berke, Department of Cell Biology, The Weizmann Institute of Science, 76100 Rehovot, Israel

Eda T. Bloom. Division of Cytokine Biology (HFB-826), Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, USA

Lisa F. Boyd, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA

Vivian Lam Braciale, University of Virginia, Beirne Carter Center for Immunology Research and Department of Microbiology, Health Sciences Center, Charlottesville, Virginia 22908, USA

Randy R. Brutkiewicz, Department of Pathology, University of Massachusetts Medical Center, Worcester, Massachusetts 01655, USA

Jean-Charles Cerottini, Ludwig Institute for Cancer Research, Lausanne Branch, 1066 Epalinges, Switzerland

Paul Champoux, Division of Membrane Biology, Medical Biology Institute, La Jolla, California 92037, USA

Weisan Chen, Department of Clinical Immunology, Flinders Medical Center, Bedford Park, 5042, South Australia, Australia

Richard Chizzonite, Molecular Genetics Department, Hoffmann-La Roche, Inc., Nutley, New Jersey 07110-1199, USA

William R. Clark, Department of Biology, The Molecular Biology Institute, University of California, Los Angeles, California 90024-1606, USA

Dino Collavo, Chair of Immunology, University of Padova, 1-35100 Pad ova, Italy

Maripat Corr, Laboratory ofImmunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA

Eric Denkers, LPD, NIAID, National Institutes of Health, Bethesda, Maryland 20892, USA

Gunther Dennert, Department of Microbiology, University of Southern California, Los Angeles, California 90033, USA

Penelope J. Duerksen-Hughes, Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia 30322, USA

CONTRIBUTORS xi

Francesco Di Virgilio, Institute of General Pathology, University of Ferrara, 1-44100 Ferrara, Italy

Peter C. Doherty, Department ofImmunology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA

Richard C. Duke, Immunology Program, University of Colorado Cancer Center, University of Colorado School of Medicine, Denver, Colorado 80262, USA

Klaus Ebnet, Max Planck-Institute fur Immunbiologie, 0-7800 Freiburg/Br., Germany

Maryna Eichelberger, Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA

Herman N. Eisen, Center for Cancer Research, EI7-128, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

Stephen E. Ettinghausen, Surgery Branch, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA

Gerald R. Ewoldt, Cell and Molecular Biology Program, School of Medicine and College of Agriculture, University of Nevada, Reno, Nevada 89557-0046, USA

Antonio Filippini, Institute of Histology and Embriology, University of Rome "La Sapienza", 00161 Rome, Italy

Frank W. Fitch, Department of Pathology, The University of Chicago, Chicago, Illinois 60637, USA

Jeffrey A. Frelinger, Department of Microbilogy and Immunology, University of North Carolina, Chapel Hill, North Carolina 27599-7290, USA

Maurice K. Gately, Immunopharmacology Department, Hoffmann-La Roche, Inc., Nutley, New Jersey 07110-1199, USA

Ruth A. Gault, Cell and Molecular Biology Program, School of Medicine and College of Agriculture, University of Nevada, Reno, Nevada 89557-0046, USA

Ricardo T. Gazzinelli, LPD, NIAID, National Institutes of Health, Bethesda, Maryland 20892, USA

Andrew J. T. George, Immune Targeting Section, Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA

Pierre Golstein, Centre d'lmmunologie, INSERM-CNRS de Marseille-Luminy, Case 906, 13288 Marseille Cedex 9, France

Linda R. Gooding, Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia 30322, USA

Gillian M. Griffiths, Basel Institute of Immunology, Postfach CH-4005 Basel, Switzerland

P~trick Haddad, Institute of Biochemistry, University of Lausanne, 1066 Epalinges, Switzerland

Frances Hakim, EIB, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA

Mark P. Hayes, Division of Cytokine Biology, CBER, FDA, Bethesda, Maryland 20892, USA

Pierre A. Henkart, Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA

R. Jane Hershberger, Department of Pathology and Howard Hughes Medical Institute, Stanford, California 94305, USA

Judith A. Horvath, Division of Cytokine Biology (HFB-826), Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, USA

Sam Hou, Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA

Xli CONTRIBUTORS

Dorothy Hudig, Cell and Molecular Biology Program, School of Medicine and College of Agriculture, University of Nevada, Reno, Nevada 89557-0046, USA

Rosemarie D. Hunziker, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA

Rudolf Jaenisch, Whitehead Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA

Dieter E. Jenne, Institute of Biochemistry, University of Lausanne, 1066 Epalinges, Switzerland

Carolina R. Jost, Immune Targeting Section, Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA

Chih-Min Kam, School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332, USA

Kevin P. Kane, Department of Immunology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

Jacqueline M. Katz, Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA

Sergei Khilko, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA

Alan M. Kleinfeld, Medical Biology Institute, La Jolla, California 92037, USA

Steven Kozlowski, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA

Shigeo Koyasu, Laboratory of Immunobiology, Dana-Farber Cancer Institute, Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA

Olivier Krahenbiihl, Institute of Biochemistry, University of Lausanne, 1066 Epalinges, Switzerland

Michael D. Kramer, Institut fur Immunologie und Serologie der Universitiit, Immunpatholgie, D-6900 Heidelberg, Germany

David M. Kranz, Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, USA

David W. Lancki, Department of Pathology, The University of Chicago, Chicago, Illinois 60637, USA

Lewis L. Lanier, DNAX Research Institute of Molecular and Cellular Biology, Department of Immunology, Palo Alto, California 94304, USA

Peter E. Lipsky, The University of Texas Southwestern Medical Center at Dallas, Rheumatic Diseases Division, Department of Internal Medicine, Dallas, Texas 75235-8887, USA

Michael Mage, Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA

David H. Margulies, Molecular Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA

Eric Martz, Department of Microbiology and Program in Molecular and Cell Biology, University of Massachusetts, Amherst, Massachusetts 01003, USA

Polly Matzinger, National Institutes of Health, Bethesda, Maryland 20892, USA

James McCluskey, Department of Clinical Immunology, Flinders Medical Center, Bedford Park, 5042, South Australia, Australia

Maureeen McKisic, Department of Pathology, The University of Chicago, Chicago, Illinois 60637, USA

Matthew F. Mescher, Division of Membrane Biology, Medical Biology Institute, La Jolla, California 92037, USA

CONTRIBUTORS Xlll

Dan Muller, Division of Rheumatology, Department of Medicine, University of Wisconsin, Madison, Wisconsin 53792, USA

Dale M. Netski, Cell and Molecular Biology Program, School of Medicine and College of Agriculture, University of Nevada, Reno, Nevada 89557-0046, USA

Shinjiro Odake, School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332, USA

Anne M. O'Rourke, Division of Membrane Biology, Medical Biology Institute, La Jolla, California 92037, USA

John R. Ortaldo, Laboratory of Experimental Immunology, National Cancer Institute, Frederick Cancer Research Facility, Frederick, Maryland 21702-1201, USA

Mark S. Pasternack, Infectious Disease Units (Children's and Medical Services), Massachusetts General Hospital, Boston, Massachusetts 02114, USA

Joseph H. Phillips, DNAX Research Institute of Molecular and Cellular Biology, Department of Immunology, Palo Alto, California 94304, USA

Timothy M. Pickett, Cell and Molecular Biology Program, School of Medicine and College of Agriculture, University of Nevada, Reno, Nevada 89557-0046, USA

James C. Powers, School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332, USA

Daniel G. Quinn, Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, North Carolina 27599-7290, USA

Phyllis M. Quinn, Immunopharmacology Department, Hoffmann-La Roche, Inc., Nutley, New Jersey 07110-1199, USA

Anna Ratner, Departments of Anatomy and Cell Biology, University of California, Los Angeles, California 90024-1606, USA

Frank Redegeld, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA

Doug Redelman, Sierra Cytometry, Reno, Nevada 89509, USA

Ellis L. Reinherz, Laboratory of Immunobiology, Dana-Farber Cancer Institute, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA

Randall K. Ribaudo, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA

Paul R. Rogers, Department of Molecular Genetics and Microbiology, University of Massachusetts Medical Center, Worcester, Massachusetts 01655, USA

Denise Ronen, Department of Cell Biology, The Weizmann Institute of Science, 76100 Rehovot, Israel

Dalia Rosen, Department of Cell Biology, The Weizmann Institute of Science, 76100 Rehovot, Israel

Steven A. Rosenberg, Surgery Branch, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA

Eric Rouvier, Centre d'Immunologie, INSERM-CNRS de Marseille-Luminy, Case 906, 13288 Marseille Cedex 9, France

John H. Russell, Department of Molecular Biology & Pharmacology, Washington University Medical School, St. Louis, Missouri 63110, USA

Barbara Schick, Department of Cell Biology, The Weizmann Institute of Science, 76100 Rehovot, Israel

David M. Segal, Immune Targeting Section, Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA

XIV CONTRIBUTORS

Alan Sher, LPD, NIAID, National Institutes of Health, Bethesda, Maryland 20892, USA

Nobukata Shinohara, Laboratory of Cellular Immunology, Mitsubishi Kasei Institute of Life Sciences, Tokyo 194, Japan

John W. Shiver, Merck, Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486, USA

Markus M. Simon, Max Planck-Institut fUr Immunbiologie, D-7800 Freiburg/Br., Germany

Michail V. Sitkovsky, Chief, Biochemistry and Immunopharmacology Section, Laboratory ofImmunology, National Institutes of Health, NIAID, Bethesda, Maryland 20892, USA

Mark J. Smyth, Cellular Cytotoxicity Laboratory, Austin Research Institute, Heidelberg 3084, Victoria, Australia, and Laboratory of Experimental Immunology, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland 21702-120 I, USA

Lishan Su, Department of Pathology and Howard Hughes Medical Institute, Stanford, California 94305, USA

Hirotaka Sugiyama, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA

Hajime Takayama, Laboratory of Cellular Immunology, Mitsubishi Kasei Institute of Life Sciences, Tokyo 194, Japan

Dwain L. Thiele, The University of Texas Southwestern Medical Center at Dallas, Liver Unit, Department of Internal Medicine, Dallas, Texas 75235-8887, USA

Benjamin Tjoa, Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, USA

Guido Trenn, Department of Hematology, University of Essen, D-4300 Essen, Germany

Jiirg Tschopp, Institute of Biochemistry, University of Lausanne, 1066 Epalinges, Switzerland

U1rike Vettel, Institut fUr Immunologie und Serologie der Universitat, Immunpathologie, D-6900 Heidelberg, Germany

Ming T. Wang, Cell and Molecular Biology Program, School of Medicine and College of Agriculture, University of Nevada, Reno, Nevada 89557-0046, USA

Irving L. Weissman, Department of Pathology and Howard Hughes Medical Institute, Stanford, California 94305, USA

Raymond Welsh, Department of Pathology, University of Massachusetts Medical Center, Worcester, Massachusetts 01655, USA

Michael B. Widmer, Department of Immunology, Immunex Research and Development Corporation, Seattle, Washington 98101, USA

U1rike Winkler, Cell and Molecular Biology Program, School of Medicine and College of Agriculture, University of Nevada, Reno, Nevada 89557-0046, USA

Aimee G. Wolitzky, Immunopharmacology Department, Hoffman-La Roche, Inc., Nutley, New Jersey 07110-1199, USA

Paola Zanovello, Chair of Immunology, University of Padova, 1-35100 Padova, Italy

Maarten Zijlstra (Deceased), Department of Molecular Genetics, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands

Susan J. Zunino, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9050, USA