Advances in Immunology Volume 70

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ADVANCES IN

Immunology VOLUME 70

This Page Intentionally Left Blank

ADVANCES IN

Immunology EDITED BY FRANK J. DlXON The Scripps Research Institute La Jolla, California ASSOCIATE EDITORS

Frederick Alt K. Frank Austen Tadamitsu Kishimoto Fritz Melchers Jonathan W. Uhr

VOLUME 70

ACADEMIC PRESS San Diego London Boston New York Sydney Tokyo Toronto

This book is printed on acid-free paper.

@

Copyright 0 1998 by ACADEMIC PRESS All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (222 Rosewood Drive, Danvers, Massachusetts 01923), for copying beyond that permitted by Sections 107 or 108 of the US. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-1998 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0065-2776/98 $25.00

Academic Press u division of Hurcourt Brace & Compuny

525 B Street, Suite 1900, San Diego, California 92101-4495, USA http:l/www.apnet.com Academic Press 24-28 Oval Road, London NW 1 7DX, UK http://www.hbuk.co.uk/ap/ International Standard Book Number: 0-12-022470-4 PRINTED IN THE UNITED STATES OF AMERICA 98 99 0 0 0 1 02 0 3 E B 9 8 7 6

5

4

3 2 1

CONTENTS

ix

CONTRIBUTORS

Biology of the Interleukin-2 Receptor

BRADH. NELSONAN11 DENNIS M. WILLERFORI) I. Introdiiction 11. The IL-2 Receptor Complex 111. IL-2 Receptor Expression IV. Cellular Responses to IL-2 Receptor Signals V. Mechani9in of IL-2 Receptor Activation VI. Intracellular Signaling by the IL-2 Receptor VII. In Viuo Studies of IL-2 Receptor Function in Lymphocyte Development VIII. In Viuo Studies of IL-2 Receptor Function in Peripheral Lymphocytes IX. Summary and Conclusions References

1

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3 1

10 19 21

42 53 64 66

Interleukin-12: A Cytokine at the Interface of Inflammation and Immunity

GIORC:IO TRINCHIEHI I. 11. 111. IV. V. VI. VII. VIII. IX. X.

Introduction IL-12 Molecule and Its Genes IL-12 Receptor and Signal Transduction Production of IL-12 Molecular Control of IL-12 Gene Expression IL-12 Effects on Heinatopoietic Stem Cells Induction of IFN--y and Other Cytokines by IL-12 Mitogenic Activity of IL-12 Activation of Cytotoxic Lyinphocytes by IL-12 Effect of IL-12 on the Differentiation of T Helper Cells v

83 86 95 101 114 119 122 127 129 134

vi

CONTENTS

XI. Effects of IL-12 on B-Cell Responses and Vaccination XII. IL-12 in Delayed-Type Hypersensitivity, & w a y Hyperresponsiveness, and Graft Rejection XIII. IL-12 in Organ-Specific Autoimmune Diseases XIV. IL-12 in the Inflammatory Response XV. IL-12 in Infectious Diseases XVI. Antitumor Effects of IL-12 XVII. Concluding Remarks References

148

152 157 166 168 187 193 194

Recent Progress on the Regulation of Apoptosis by Bcl-2 Family Members

ANDYJ. MINN,RACHEL E. SWAIN,AVERILMA, AND CRAIGB. THOMPSON

I. Significance of Programmed Cell Death 11. The Genetics of Programmed Cell Death 111. The Bcl-2 Family IV. Mitochondria Can Control A optosis V. Structure/Function Studies o Bcl-xL VI. How Do Bcl-2 Family Members Regulate Cell Sunivd? VII. Conclusion References

T!

245 247 250 257 261 266 269 271

Interleukin-18:A Novel Cytokine That Augments Both Innate and Acquired Immunity

HARUKI OKAMURA, HIROKO TSUTSUI, SHIN-ICHIRO KASHIWAMURA, TOMOHIRO YOSHIMOTO, A N D KENJINAKANISHI I. 11. 111. IV. V. VI. VII. VIII.

Introduction Molecular Structure of IL-18 and Its Gene Producing Cells Re uirement of Caspase-1 for Processing of IL-18 Bio ogical Function Receptors for IL-18 Role of IL-18 in Host Defenses PatholOgiCdl Roles O f IL-18 IX. Perspective References

7

281 282 285 286 287 294 298 301 304 305

CD4' T-cell Induction and Effector Functions: A Comparison of Immunity against Soluble Antigens and Viral Infections

ANNETTEOXENIUS, ROLFM. ZINKERNAGEL, A N D HANSHENGARTNER

I. Introduction 11. Activation of CD4' T Cells 111. CD4+ T-cell Effector Functions

313 314 330

vii

CONTENTS

IV. Conclusions References

351 352

Current Views in lntracellular Transport: Insights from Studies in Immunology

VICTOR W. Hsri

AND

PETERJ. PETERS

I. Introduction 11. A General Mechanism of Intracellular Transport 111. Complexities of Transport in V i m IV. Secretory Pathways V. Endocytic Pathways VI. Transport in Polarized Cells VII. Perspective References

369 373 383 385 392 398 402 402

Phylogenetic Emergence and Molecular Evolution of the Immunoglobulin Family

J. MARCHALONIS, SAMUEL F. SCHLUTEH, RALPIIM . BEKNSTEIN, SIIANXIANG SHEN.AND ALLENB. EDMUNDWN

JOHN

I. Introduction Evolutionary Emergence of the Combinatorial Immune System Ancient Foundations of the Coinbinatorial Irninune System Emergence of Bonn Fide Iininunoglobulins Iininunoglobulins and T-cell Receptors of Jawed Vertebrates Framework 4 of the Variable Doniain Encoded by the Joining Segment VII. Evolutioniry Comparisons of T-cell Receptors VIII. Evolution of Li lit Chains IX. Origin and Evo ution of Iininunoglobulin Heavy Chains X. Segmental Gene Organization in Evolution XI. Molecular Events Underlying the Ex h i v e Emergence of Immunoglobulins and Their Initial P ases of Evolution XI. Conclusions References 11. 111. IV. V. VI.

K

1

417 418 418 425 430 439 44 1 45 1 460 475

485 491 492

Current Insights into the “Antiphospholipid” Syndrome: Clinical, Immunological, and Molecular Aspects

DAVIDA. KAXDIAH,ANDREJSALI,YONGHIJA SHENG,EDWARD J. VICTORIA, DAVID M . MAKQLIIS, STEPHENM. COU’ITS,A N D STEVENA. KRI1.IS

I. Introduction 11. “Antiphospholipid” Antibodies 111. Clinical Features of the “Antiphospholipid’ Syndrome IV. P2-Glycoprotein I V. Iinmunogenicity and Animal Models

507 508 512 520 531

viii

CONTENTS

VI. Prothroinbin

VII. Lupus Anticoagulant Antibodies and Protein C Activation VIII. IX. X. XI. XII. XIII.

Lupus Anticoagulant Antibodies and Phos hatic~ylethanolamine Antiphospholipid Antibodies and Endothe ial Cells Pathogenesis of the Aiitiphos holipid Syndrome Laboratory Investigations of t ie Antiphos holipid Syndrome Antiphospholipid Syndrome and Future T ierapies Suminary and Conclusions References

INDEX CONTENTS OF RECENTVOLUMES

P

P

I;

533 535 536 536 537 543 545 8547 548

565 573

CONTRIBUTORS

Ralph M. Bernstein (417 ) , FDA/clm-/H FM-Fj4 I, Betliesda, Maryland 20892 Stephen M. Coutts (507, L,a Jolla Pharniaceutical Coinpiny, San Diego, California 92121 Allen B. Edmunson (417), Oklahoma Medical Reseracli Foundation, Oklahorna City, Oklalionia 6 3 104-.5046 Hans Hengartner (313), Departnient of Pathology, Institute of Experimental Immunology, Unicxmity of Ziirich, 8091 Zurich, Switzerland Victor W. Hsu (3691,Division of Rheumatology, Immuno1ogy,and Allergy, Brighani and Women’s Hospital, Haivarcl Medical School, Boston, Massachusetts 21005 David A. Kandiah (507),Department of Immiinology, Allergy, and Infectious Disease, University of Nccv South Wales School of Medicine, Saint George Hospital, Kogarah 22 17, Australia Shin-ichiro Kashiwamura (281), Laboratoiy of Host Defenses, Institute for Advances Medical Sciences, Hyogo College of Medicine, Hyogo 663, Japan Steven A. Krilis (507), Department of Immunology, University of New South Wales School of Medicine, Saint George Hospital, Kogarah 2217, Australia Averil Ma (245). Coininittee on Imiiiunology and the Department of Medicine, Universitv of Chicago. Chicago, Illinois 60637-5420 John J. Marchalonis (417), Department of Microbiology and Immu~~, nology, College of Medicine, University of Arizona, T L K S OArizona 85724 David M. Marquis (507), La J o h Pharmaceutical Company, San Diego, California 92 121 Andy J. Minn (245), Gwen Kiiapp Center for Lupus and Iniinuriology Research and the Committee on Inimiuiology, University of Chicago, Chicago, Illinois 60637-5420 I\

X

CONTRIBUTORS

Kenji Nakanishi (281),Laboratory of Host Defenses, Institute for Advances Medical Sciences, and Department of Immunology and Medical Zoology, Hyogo College of Medicine, Hyogo 663,Japan Brad H.Nelson (l),The Virginia Mason Research Center, Seattle, Washington 98101;and Department of Immunology, University of Washington School of Medicine, Seattle, Washington 98195 Haruki Okamura (281),Laboratory of Host Defenses, Institute for Advanced Medical Sciences, Hyogo College of Medicine, Hyogo 663, Japan Annette Oxenius (313),Department of Pathology, Institute of Experimental Immunology, University of Zurich, 8091 Zurich, Switzerland Peter J. Peters (369),Department of Cell Biology, Faculty of Medicine and Institute of Biomembranes, Utrecht University, 3584 CX Utrecht, The Netherlands Andrej Sali (507),Rockefeller University, New York, New York 10021 Samuel F.Schluter (417),Department of Microbiology and Immunology, College of Medicine, University of Arizona Health Sciences Center, Tucson, Arizona 85724-5049 Shanxiang Shen (417),National Institutes of Health, Bethesda, Maryland 20892 Yonghua Sheng (507),Department of Immunology, University of New South Wales School of Medicine, Saint George Hospital, Kogarah 2217,Australia Rachel E. Swain (245),Committee on Cancer Biology, University of Chicago, Chicago, Illinois 60637-5420 Craig B.Thompson (245),Gwen Knapp Center for Lupus and Immunology Research and Committees on Immunology and Cancer Biology, Department of Medicine, University of Chicago, Chicago, Illinois 60637-5420 Giorgio Trinchieri (83),Immunology Program, Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104-4268 Hiroko Tsutsui (281),Department of Immunology and Medical Zoology, Hyogo College of Medicine, Hyogo 663,Japan Edward J. Victoria (507),La Jolla Pharmaceutical Company, San Diego, California 92121 Dennis M. Willerford (11, Departments of Immunology and Medicine, University of Washington School of Medicine, Seattle, Washington 98195;and the Puget Sound Blood Center, Seattle, Washington 98104 Tomohiro Yoshimoto (281),Laboratory of Host Defenses, Institute for Advances Medical Sciences, and Department of Immunology and Medical Zoology, Hyogo College of Medicine, Hyogo 663,Japan Rolf M. Zinkernagel(313),Department of Pathology, Institute of Experimental Immunology, University of Zurich, 8091 Zurich, Switzerland

ADV4NCE5 IN 1MMUNOI.OCX VOL 70

Biology of the Interleukin-2 Receptor BRAD H. NELSON'st AND DENNIS M. WILLERFORDtr~r§ 'The Virginia Mason Research Center, Seattle, Washington, 98 101; Departments of flmmunology and #Medicine, University of Washington School of Medicine, Seattle, Washington, 98 195; and §The Puget Sound 6load Center, Seattle, Washington 98 104

I. Introduction

Homeostatic regulation of' the immune system requires extensive communication among its cellular constituents, which include lymphocytes, macrophages, dendritic cells, and stromal elements. These cell-cell interactions typically occur at close range and include activation of a wide variety of cell surface signaling molecules by direct contact, as well as signaling through secretion of an array of soluble mediators. Among the latter, interleukin-2 ( IL-2) is perhaps the most extensively studied. First identified as a growth factor for T cells in vitro (Gillis and Smith, 1977a,b; Morgan et nl., 1976), IL-2 has also been implicated in the functional differentiation of T cells, as well as in the growth and effector function of B and natural killer (NK) cells. Receptors for IL-2 are also expressed on developing T and B cells. The remarkable in vitro properties of IL-2 suggested that this lymphokine acted at the heart of the immune response -by mediating antigen-triggered T-cell expansion and promoting effector cell differentiation. This view has grown more complicated in recent years, based on a greater appreciation for the redundancy of in wivo growth and regulatory signals, as well as on unexpected observations regarding the function of IL-2. These include the studies of Lenardo (1991) demonstrating that IL-2 may promote T-cell apoptosis under some circumstances and the observation that mice lacking IL-2 have phenotypically normal lymphoid development and only moderate defects in immune responses (Kundg et al., 1993; Schorle et al., 1991). To understand the complex biologic properties of IL-2 and related cytokines, it is essential to consider the signals generated by its receptor. The IL-2R is multimeric, consisting of two obligate signaling subunits, IL-2RP (CD122) and yc (CD132), and a variably expressed IL-ZRa subunit (CD25), which regulates affinity for IL-2 (Kondo et al., 1994a; Nakainura et nE., 1994; Nelson et nl., 1994; Siege1 et al., 1987; Waldmann, 1989; Wang and Smith, 1987). As is the case with several other cytokine receptor systems, IL-2 receptor components are shared with receptors for other lymphokines, including IL-4, 7, 9, and 1Fi (reviewed in Leonard et al., 1994). This sharing of subunits explains some of the overlapping 1

Capvnght 0 I998 In Aucirmic Pirs, All ngllt, of ~ p ~ ~ d IIIt any ~ ~6 tm m rercrvrd lK)fi5-?iififlR 525 ()(I

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BRAD H NELSON AND D E N N I S M. WILLERFORD

properties of these lymphokines, and in many instances it is difficult to sort out which cytokine/receptor interaction is responsible in vivo for the generation of a given cellular signal. Like other cytokine receptors, the IL-2R contains no intrinsic enzymatic activity. Rather, the intracellular portions of the receptor chains associate with a variety of cytoplasmic proteins, including tyrosine kinases of the Jak family. Oligomerization of receptor subunits brings these regulatory enzymes into close proximity, activating the signaling complex by phosphorylation of regulatory tyrosines on the kinases themselves, as well as on the cytoplasmic domains of IL-2RP and yL.The phosphorylated cytoplasmic domain of IL-2RP plays a critical role in attracting downstream signaling molecules into the activated receptor complex, where they may serve as substrates for receptor-associated enzymes. A diversity of downstream signals are activated by the IL-BR, including those which promote cell growth and division, as well as signals that influence cell survival and differentiation. Our understanding of these downstream signals is far from complete, particularly with regard to the regulation of cellular responses other than proliferation. The biologic function of the IL-2 receptor has also been examined at a genetic level in studies of humans with immune deficiencies attributable to mutations in receptor subunits, as well as in mice generated using genetargeting techniques. The phenotypes of these mutations fall into two broad categories. Mutations in the y' gene lead to severe defects in lymphopoiesis in knockout mice and, in humans, are responsible for X-linked severe combined iinmunodeficiency (XSCID) (for review see Leonard et al., 1994). The developmental and functional defects in T and B cells associated with disruption of yc signaling reflect the participation of this subunit in multiple cytokine receptor complexes. In contrast, mutations that selectively affect signals delivered by the high-affinity IL-2R do not block T- and B-cell development, but lead to an inability to regulate the overall size of the lymphoid tissues, fimctiond defects in immune responses, and autoimmunity. This constellation of defects demonstrates that I I d - 2 ~ signals are essential for the proper regulation of immune respollses 2ind underscores their negative regulatory role in homeostasis of the peripheral lymphoid tissues. This review attempts to connect what is known regarding receptor activation from biochemical and cellular studies with the now considerable literature regarding immune system developrnent and function when various components of the IL-2R signal are disrupted. The premise is that the diverse functions that are revealed by such genetic studies can guide future investigations aimed at understanding the intracellular signals generated by the IL-2R.

HIO1,OGY OF TtlE I N T E R L E U I I N - 2 RECEPTOR

3

II. The 11-2 Receptor Complex

A. BINDIN(:OF IL-2 BY

THE

IL-2R

Depending on which of the three 1L-2R subunits are expressed by a given cell, several different binding affinities for IL-2 are observed. The a subunit alone binds IL-2 with low affinity (& 10 nM), whereas the p subunit binds IL-2 with veiy low affinity (K,, 100 nM). When coexpressed, IL-2Ra and p form a pseiido-liigli-affinity receptor complex (I& 30 pM) (Anderson et al., 1995; Arinia e f al., 1992). Unlike IL-2Ra and /3, the yc subunit has no measurable affinity for IL-2, but when coexpressed with IL-2RP forins an intermediate affinity receptor complex (& 1 nM). Finally, coexpression of the a, 0, and y( subunits results in 10 pM) that is thought to the formation of a high-affinity IL-2R (& consist of a triineric a:P:y, complex (Takeshita et al., 199213). Of all these possible receptor combinations, only two are competent to ~ ) , is expressed signal. Thus, the intermediate affinity receptor ( p : ~ which by NK cells, macrophages, and resting T cells, can signal in the presence of high concentrations of IL-2, whereas the high-affinity receptor complex (a:p:yc)is expressed on activated lymphocytes and signals even at low concentrations of IL-2 (reviewed in Smith, 1988). Although signaling by both interinediate- and high-affinity IL-2R complexes can be demonstrated readily in uitro, mice with a targeted disruption of the IL-2Ra gene show a very similar phenotype to mice lacking the IL-2 gene itself (Schorle et nl., 1991; Willerford et al., 199S), suggesting that most of the biologic effects of IL-2 in o are mediated by signals delivered through the highaffinity ( a : P : y cIL-2R ) (see Section VIII). Thus, tlie expression of the IL2Ra subunit by cells, although not required for intracellular signaling per se, is nonetheless a critical determinant of 1L-2 responsiveness. The a,0, and ycchains of the high-affinity IL-2R bind to distinct sites on IL-2, and these associations a p p e a to occur in a stepwise manner (Imler, et d., 1992; Moreau c t d . , 199.5; Sauve et al., 1991; Voss et al., 1993; Zurawski, et al., 1990).Available data suggest a model for IL-B/IL2R binding whereby IL-2 first binds to the IL-2Ra and IL-2RP chains and, subsequently, the yc chain is recruited into the complex. In support of this model, evidence shows that IL-2Ra and IL-2RP preassociate as a heterodimer in the absence of ligand. First, the affinity of the IL-2RP chain for IL-2 is very low unless IL-2RP is coexpressed with IL-2Ra. This synergistic effect does not require prior binding of ligand to IL-ZRa, as a mutant form of IL-2 (F42A) that fails to bind to the isolated IL-2Ra subunit nevertheless binds to the IL-2RdIL-2RP complex with higher affinity than to IL-2RP done (Grant et al., 1992; Roessler et nl., 1994). Second, precise ineasurenients of receptodligand interactions using tlie

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4

BRAD H. NELSON AND DENNIS M . WILLERFORD

technique of surface plasmon resonance demonstrate that the extracellular domains of IL-2Ra and IL-2RP bind IL-2 with kinetics that are indicative of simultaneous contributions from both subunits (Balasubramanian et al., 1995). Once a complex is formed between IL-2 and the IL-2Ra:IL-2RP heterodimer, the yc chain is recruited to the receptor complex, an interaction that is driven by a 10-fold increase in receptor affinity, via both an increase in the association rate constant and a decrease in the dissociation rate constant (Matsuoka et al., 1993). The recruitment of yc is required for intracellular signaling, as experiments with chimeric receptor chains have shown that signaling is initiated by ligand-induced heterodimerization of the cytoplasmic domains of IL-2RP and yc (Nakamura et al., 1994; Nelson, et aE., 1994) (see Section V,B). Thus, the association and dissociation of yewith the receptor complex serve to switch the receptor between inactive and active states. One implication of this model is that the magnitude of the IL-2R signal could be influenced by the presence of other cytokines that use yc as part of their receptors (see later), although such competitive interactions have not been demonstrated directly.

B. MOLECULAR CLONING AND STRUCTURAL FEATURES OF THE a, P, AND yc SUBUNITS The IL-2Ra chain was the first IL-2R component to be cloned, this being accomplished in 1984 by three groups (Cosman et al., 1984; Leonard et al., 1984; Nikaido et al., 1984). IL-2Ra, also known as Tac antigen or CD25, is a -55-kDa polypeptide with an extracellular domain containing 219 amino acid residues, a transmembrane domain of 19 residues, and a cytoplasmic domain containing only 13 residues. Although the cytoplasmic domain is very short, it is nonetheless highly conserved between mice and humans, suggesting that it may play an important functional role, although to date none has been identified. Unlike IL-2Rp and ye,IL-2Ra does not share the typical features of the hematopoietin receptor superfamily. The a chain of the IL-15 receptor has been cloned and found to have structural homology to IL-2Ra (Giri et nl., 1995). In particular, the two chains share an extracellular protein-binding motif known as the “sushi domain,” which is also found in complement receptor proteins. The genes encoding IL2Ra and IL-15Ra are located close to one another on chromosome 10 in humans and chromosome 2 in mice and have a similar exodintron structure, suggesting a close evolutionary relationship (Anderson et al., 1995). Based on binding studies with cloned IL-2Ra, it became clear that this protein constituted the low-affinityreceptor and that one or more additional subunits must be required to form the high-affinity IL-2R complex. A monoclonal antibody that inhibited binding of IL-2 to the putative IL2Rp chain (Tsudo et al., 1989) was utilized by Taniguchi and colleagues

BIOLOGY OF THE INTERLEUKIN-2 KECEPTOH

5

to clone the corresponding cDNA for this receptor subunit (Hatakeyaina et al., 1989b).The -75-kDa IL-BRP chain is composed of an extracellular domain of 214 residues, a transmembrane domain of 25 residues, and a long cytoplasmic domain of 286 residues. IL-2RP displays the characteristic structural features shared by members of the hematopoietin receptor superfamily, including a set of four conserved cysteine residues in the extracellular domain and a ineinbraiie-proximal WSXWS motif. The intracellular domain of IL-BRP, wliich lacks any intrinsic enzymatic function, displays the box 1 and box 2 motifs characteristic of this receptor superfanily (Hatakeyama et al., 198913).The gene encoding hunian IL-2Rp is located on chromosome 22q12-13 (Shibuya et a l , 1990) and, thus far, has not been associated with any genetic diseases in humans. Scatchard analysis of IL-2 binding to lymphoid cells expressing IL-2RP but not IL-2Ra showed the presence of an intermediate affinity receptor with & values in the nanomolar range (Hatakeyarna et al., 1989b). However, when analogous studies were performed in nonlymphoid cells, IL-2 failed to bind IL-2RP in the absence of IL-2Ra. To explain this Ascrepancy, it was proposed that the IL-2R might contain a third subunit that was expressed in lymphoid but not nonlymphoid cells (Hatakeyama et al., 1989b). This hypothesis was supported by studies showing that a -64kDa polypeptide coprecipitated with IL-2RP in the presence of IL-2 (Takeshita et al., 1990). A functional role for the 64-kDa chain in signaling by both intermediate- and high-affinity IL-2R complexes was also suggested based on correlations between expression of this protein and IL-2 responsiveness (Arima et d.,1992; Takeshita et al., 1992a; Voss et al., 1992; Zurawski et al., 1990).The 64-kDa chain was purified by coimmunoprecipitation with IL-2R0, followed by two-dimensional gel electrophoresis. Peptide sequence data obtained from the purified protein were used to isolate a full-length cDNA encoding the IL-2Ry subunit (Takeshita et al., 1992a). The newly identified IL-2Ry chain (now called yJ was found to contain 232 residues in the extracellular domain, 29 in the transmembrane domain, and 86 in the cytoplasmic domain. Furthermore, yc was a novel inember of the hematopoietic receptor superfamily by virtue of conserved cysteine residues and a WSXWS motif in the extraceIlular domain, as well as a box 1 motif in the cytoplasmic domain (Takeshita et al., 1992a). The gene encoding ycis located at Xq13 in huinans (Noguchi et al., 1993a,c; Puck et al., 1993) and region 40 of the X chromosome in mice (Cao et al., 1993; DiSanto et nl., 1994). C. SHARING OF THE IL-2RP A N D -yc CHAINS WITH OTIHEI-I CYTOKINE RECEPTORS The location of the gene encoding the ycchain proved to be extremely significant, as it corresponded to the locus for XSCID (Noguchi et al.,

6

BRAD 11. NELSON AND DENNIS M U‘ILLEKFOKD

1993c; Puck et nl., 1993). Males with this genetic disorder lack T cells and, as a result, are severely iinmunocomproinised. The developmental block in the T-cell lineage in XSCID is in marked contrast to the phenotype of IL-2 -/- mice (Schorle et nl., 1991), which produce normal numbers of functional lymphocytes and are able to mount iinrnune responses (see Section VII,E,l). This discrepancy between XSCID and IL-2 4- phenotypes suggested that ycwas a component of another receptor complex that was essential for normal T-cell development (Noguchi et al., 1993~). A number of studies soon demonstrated that in addition to the IL-2R, the yc chain was a functional component of the receptors for IL-4, IL-7, IL-9, and IL-15 (reviewed in Leonard et nl., 1994). Chemical cross-linking experiineiits demonstrated that yL associated with the IL-4, IL-7, and IL-9 receptors in the presence of ligand. Moreover, antibodies to the extracellular domain of yc were found to block proliferative responses to these cytokines (Kimura et aE., 1995; Kondo et al., 1993, 1994b; Noguchi et al., 199313; Russell et al., 1993b). Furthermore, a dominant-interfering mutant of yL,which lacked the cytoplasmic domain, was found to inhibit the proliferation of BAF3 cells in response to either IL-2 or IL-7 (Kawahara,et al., 1994). Finally, receptor reconstitution experiments showed that yL was required for intracellular signaling in response to IL-15 (Gin et al., 1994). Due to its involvement in at least five different receptor complexes, the IL-2Ry chain is now referred to as the “common y chain,” or yr (Noguchi et al., 199317). The XSCID phenotype, as well as the severe lyinphopoietic defect in mice with a targeted disruption of the yL gene (Cao et al., 1995; DiSanto et al., 1995), is now thought to result from defective signaling by multiple cytokine receptors, of which the IL-7 receptor is probably the most important (Peschon et al., 1994; von Freeden-Jeffryet nl., 1995) (see Section VII). The IL-BRP chain is also not exclusive to the IL-BR, as it has been shown to be a component of the IL-15 receptor (Bainford et al., 1994; Carson et al., 1994; Gin et nl., 1995; Grabstein et al., 1994). Indeed, the IL-2 and IL-15 receptors are very similar, in that both contain IL-2RP and yc in combination with either IL-2Ra or IL-l5Ra, respectively (Gin et al., 1994, 1995). The similarities extend further, as the IL-2Ra and IL15Ra chains are themselves structurally homologous and both are able to bind ligand independent of the IL-2RP or yr chains. Indeed, the IL-15Ra subunit alone binds IL-15 with an affinity in the 10 pM range (Gin et nl., 1995). As might be expected, IL-2 and IL-15 appear to activate the same intracellular signaling pathways in lymphocytes (Grabstein et al., 1994; Johnston et al., 1995a; Lin et al., 1995) Notably, however, a second receptor complex for IL-15 has been reported in mast cells that operates indepen-

BIOLOGY OF THE INTERLEUKIN-2 RECEPTOR

7

dent of IL-2RP and ycand generates a distinct intracellular signal involving the activation of Jak2, rather than Jakl and Jak3 (Tagaya et al., 1996a,b). 111. 11-2 Receptor Expression

A. EXPRESSION OF THE IL-2Ra CHAIN Of tlie three IL-2R subunits, the IL-2Ra chain demonstrates tlie most tightly regulated expression. In the thymus, IL-2Ra is expressed on primitive CD4-CD8-CD3- triple-negative (TN) cells, a stage that marks the first steps in T-cell receptor (TCR) rearrangement and irreversible conirnitrnent to the T-cell lineage (reviewed in Godfrey and Zlotnick, 1993). Expression of IL-2Ra is lost abruptly following successful TCRP rearranginent and expression and remains off for the remainder of thyrnocyte development (Godfrey and Zlotnick, 1993; Rothenberg, 1992). Mature, resting T cells in the periphery fail to express the IL-2Ra chain and therefore are unresponsive to IL-2. However, following TCR stimulation, IL-2Ra transcription is induced, leading to cell surface expression (reviewed in Crabtree, 1989; Rothenberg, 1992). Expression of IL-2Ra may also be induced in T cells by non-TCR stimuli, including IL-1 or tumor necrosis factor (TNF)-a (Plaetinck et nl., 1990). IL-2Ra is also expressed in tlie B-cell lineage. In mice, bone marrow pre-B cells express IL-2Ra, although the functional significance of this is uncertain, as IL-2 does not effectively support proliferation of such cells, and a similar expression pattern has not been described in humans (Chen al., 1994; Rolink et al., 1994). On mature B cells, IL-2Ra is induced following activation through the B-cell receptor (BCR), resulting in the expression of a functional, highaffinity IL-2R (Jung et al., 1984; Tsudo et al., 1984; Waldmann et al., 1984; Zubler et al., 1984). The molecular rnechanisni underlying the exquisite regulation of IL2Ra gene expression by tlie TCR has been under investigation for over a decade. A potent enhancer has been identified between positions -299 and -228 relative to the major transcription start site and is now termed PRRI (for positive regulatory region I ) (Lin et nl., 1990; Plaetinck et nl., 1990). This region contains binding sites for both NF-KB and serum response factor (SRF) and is responsive to diverse activation signals, including those resulting from TCR ligation or stimulation with phorbol esters, IL-1, and TNF-a. More recently, a second regulatory region termed PRRII has been identified between nucleotides -137 and -64 of the IL-2Ra gene (John et al., 1995). This region can function as a phorbol ester-inducible enhancer element and contains binding sites for both the Ets family protein Elf-1 and the cliromatin-associated protein HMG-I(Y). As Elf-1 has been found to associate with NF-KB p50 in vitro, it has been proposed that physical

8

BRAD 1-1, NELSON AND DENNIS M. WILLERFORD

interactions between these proteins may allow functional cooperativity between PRRI and PRRII (John et nl., 1995). IL-2 itself can also upregulate expression of the IL-2Ra chain and does so through a region of the IL-2Ra promoter/enhancer termed PRRIII, which lies distal to PRRI and PRRII (John et al., 1996; L'ecine et al., 1996; Plaetinck et al., 1990; Serdobova et al., 1997; Soldaini et al., 1995; Sperisen et al., 1995). PRRIII resembles PRRII in that it contains binding sites for Elf-1 and HMG-I(Y), but differs by also having a binding site for the transcription factor Stat5. As described in detail in Section VI,B,2, Stat5 activity is induced rapidly in lymphocytes in response to IL-2 stimulation and plays an essential role in IL-2-mediated expression of the IL-2Ra chain (John et al., 1996; L'ecine et al., 1996).

B. EXPRESSION OF THE IL-2RP CHAIN In contrast to IL-2Ra, the IL-2RP chain is constitutively expressed at low to moderate levels on resting T cells, B cells, NK cells, monocytes/ macrophages, and neutrophils (Begley et al., 1990; Djeu et al., 1993; Dukovich et al., 1987; Espinoza-Delgado et al., 1990; Sharon et al., 1990; Siegel et al., 1987; Wei et al., 1993 ;Zola et al., 1991). Expression of this subunit is modulated during T-cell development, as it is expressed at low levels on a small fraction of TN thymocytes, at very low levels on CD4+8+ double-positive (DP) thymocytes, and at moderate levels on the CD4-8' single-positive (SP) subset (Kondo et al., 1994a). Although IL-2RP differs from IL-2Ra in being constitutively expressed on cells, like IL-2Ra it is upregulated on mature T cells by stimulation with antigen or a variety of other agents (Casey et al., 1992; Cerdan et al., 1995; Hatakeyama et al., 1989b; Siegel et al., 1987). Similarly, IL-BRP expression is upregulated on B cells activated by BCR stimulation, which is enhanced by IL-4 and IL5 (Loughnan and Nossal, 1989). Studies of the 5' flanking portion of the human IL-2RP gene have led to the identification of three regulatory elements within the first 363 bp upstream of the major transcription start site (Lin et al., 1993; Lin and Leonard, 1997). One of these regions (-56 to -34) contains a binding site for Ets family proteins (specifically, Ets1- and GA-binding protein) and is involved in both basal and inducible promoter/enhancer activity. This site may also contribute to the tissuespecific expression of IL-2RP, as it was found to be active in lymphoid but not nonlymphoid cell lines. A second region (- 170 to - 139) binds the factors Spl and Sp3 as well as the immediate-early factor Egr-1, the latter being expressed in T cells on treatment with phorbol esters (Lin and Leonard, 1997). As with Ets-binding sites, mutation of the Spl- and Egr-l-binding sites severely disrupts the basal and inducible activity of the IL-2RP promoter/enhancer. Therefore, consistent with its complex pattern


9

of expression in cells, transcription of the IL-2RP gene appears to be regulated through interactions between both constitutive and inducible DNA-binding proteins.

C. EXPRESSION OF THE yLCHAIN Consistent with it being a component of at least five hematopoietic cytokine receptors, the yc chain is expressed constitutively in multiple hematopoietic lineages, including CD4+ and CD8+ T cells, B cells, N K cells, monocytes/macrophages, granulocytes, and neutrophils (Bosco et al., 1994; Epling-Burnette et al., 1995; He et al., 1995; Kondo et al., 1994a; Liu et al., 1994; Sugainura et al., 1996).The ycchain is expressed throughout thymocyte development, from the TN to SP stages. In the spleen, y,is expressed by both mature T cells and B220' B cells (Kondo et nl., 1994a). In contrast to the IL-2Ra and /3 chains, surface expression of y,decreases in response to T-cell activation rather than increasing (Kondo et a1 , 19944. The decline in expression is rapid, beginning within 4 hr of stimulation, and transient, being reversed by 24 hr poststimulation. This appears to be a T-cell-specific phenomenon, as activation of B220' B cells with LPS enhances, rather than suppresses, expression of yc(Kondo et al., 1994a). One explanation for the decreased expression of yc in activated T cells may come from work demonstrating that ycis a target of the calciumactivated neutral protease calpain, due to a so-called PEST sequence in the cytoplasmic domain of yL that serves as a recognition site for the enzyme (Noguchi et al., 1997). Activation of thymocytes by treatment with anti-CD3 was shown to induce the proteolytic degradation of the ycsubunit, presumably due to a rise in intracellular calcium and the consequent activation of calpain. The addition of A h M , an inhibitor of m-calpain, not only inhibited the anti-CD3-triggered degradation of yLin thymocytes, but also enhanced the proliferative response of CD4' thymocytes to anti-CD3 stimulation (Noguchi et al., 1997). The 5' flanhng region of the human gene encoding yc includes cis elements that appear to be involved in the transcriptional regulation of yc (Noguchi et al., 1993a; Ohbo et al., 1995). A 633-bp region upstream of the transcription start site is sufficient to drive expression of a reporter gene in hematopoietic cell lines. Within this region lie sequences characteristic for the binding of several transcription factors, including PU.l and PEA-3. Another binding site characteristic of ETS proteins is found within a short segment harboring basal promoter activity. Reporter gene expression was enhanced by treatment of cells with either phorbol esters or phytohemagglutinin (PHA), despite the fact that surface expression of the ycchain decreases on activation of T cells with similar stimuli. Furthermore, IL-2 was found to decrease expression of this reporter gene as well as endogenous yc

10

BRAD H . NELSON AND DEKNIS M . WILLEKFORD

inRNA. Therefore, it appears that the regulation of yr expression in resting and activated lymphocytes may be quite complex, involving at a minimum both transcriptional and posttranslational mechanisms. IV. Cellular Responses to 11-2 Receptor Signals

A. IL-2 AS A GHOWTII FACTOR 1. T-cell Prol$eration. IL-2 was initially identified as T-cell growth factor, a substance present in supernatants of PHA-stimulated lymphoblasts that promoted the growth of T cells in vitro (Gillis et al.,1978; Gillis and Smith, 197713; Morgan et al.,1976). This discovery led to the development of T-cell lines that could be maintained indefinitely in uitro, reagents that have contributed iinmensely to our current understanding of T-cell biology (reviewed in Smith, 1988).T cells require two sequential signals to engage in active proliferation (reviewed in Crabtree, 1989; Schwartz, 1990; Weiss and Imboden, 1987). The first is provided by TCR engagement, which induces expression of a high-affinity IL-2R complex (Cantrell and Smith, 1983; Leonard et al., 1982; Meuer et al., 1984; Robb et al., 1981; Waldmann, 1989), a process that involves de novo expression of IL-2Ra, as well as enhanced expression of the IL-2RP chain (Kondo et ul., 1994a; Leonard et al., 1982; Siege1 et al., 1987; Waldmann, 1989, 1991). TCR engagement also increases the expression of Jak3, a critical component of proliferative signaling (Kawamura et al., 1994). Once T cells are sensitized by TCR signals, IL-2 acts as a cell cycle progression factor, thus supporting proliferation. T-cell proliferation in vitro is suppressed when the IL-2/IL-2R interaction is blocked by antibodies to IL-2 or IL-2Ra (Leonard et al., 1982; Smith et nl., 1983). Thus, when standard in uitro culture conditions are used, IL2 appears to be the major factor promoting the growth of T cells. In the context of appropriate costimulatory conditions, TCR signaling also induces synthesis of IL-2; hence, T-cell proliferation is actuated by an autocrine and/or paracrine hormonal circuit (Meuer et al., 1984; Smith, 1988). One consequence for iinmunoregulation is that activation of both IL-2 and its receptor by the TCR restricts the local IL-2 response to cells with appropriate antigen specificity, thereby minimizing bystander effects. However, the intracellular signaling pathways activating IL-2 and IL-2Ra expression diverge downstream of the TCWCDS complex (Crabtree, 1989;Rothenberg, 1992), and each pathway may therefore be influenced by separate sets of conditions within the cell. For example, TCR stimulation is sufficient to induce IL-2Ra in the absence of costimulation provided by accessory cells, whereas IL-2 secretion requires an additional signal,

13IOI.OGY 01.' T l I E IKTERLEUKIN-2 RECEPTOR

11

such as that supplied by CD28 ligation (Crabtree, 1989; Schwartz, 1990). In addition, IL-2Ra expression may be induced independent of TCR signals, e.g., by IL-1 or TNF (Plaetinck et a1 , 1990). The distinct requirements for IL-2 and IL-2Ra induction are also reflected in distinct patterns of expression in T-cell sublineages: although IL-2Ra expression is common to both CD4' and CD8+T cells, IL-2 production is largely restricted to the CD4' subset, particularly, naive cells and those with a T h l profile (Schwartz, 1990). This partial dissociation of IL-2 and IL-2Ra expression underscores the paracrine nature of IL-%mediated signals and reflects the specialized functions of lymphocyte subsets. 2. Cell Cycle Regulation hi) IL-2 TCR signaling in resting, GO T cells activates metabolic pathways and generates characteristic changes in cell size and RNA content indicative of progression to the G1 phase of the cell cycle However, in the absence of IL-2, S-phase entry does not occur. Moreover, T cells deprived of IL2 undergo cell cycle arrest in G1 (Cantrell and Smith, 1984).IL-2 promotes the characteristic niorpliologic changes and RNA accumulation associated wit11 progression through G 1, induces the expression of growth-associated protooncogenes, including c-myc and c-myh (Sliibuyaet nl., 1992; Stern and Smith, 1986),and promotes entiy into S phase. The molecular mechanisms responsible for the effect of IL-2 on cell cycle progression have been investigated. In eukaiyotes, the cell cycle is coordinated by the sequential activation of cyclin-dependent hnases (CDK), which are regulated by interaction with cyclin proteins and by inhibitors of cyclin/CDK function (for reviews see Hartwell and Kastan, 1994; Hunter and Pines, 1994; Sherr, 1994; Sherr and Roberts, 199.5). TCR signals stimulate the synthesis of CDKs and G1 cyclin proteins, but this is not sufficient to generate the active CDK complexes required for progression to S phase (Ajchenbaum et nl., 1993; Firpo et nl., 1994; Karnitz and Abraham, 1996). In addition to further promoting the expression of cyclin and CDK proteins, IL-2R signals give rise to active cycliidCDK complexes (Firpo et nl., 1994). A specific mechanism for these properties of IL-2 on cell cycle progression effect has emerged based on the identification of G1 cyclidCDK inhibitors (Sherr and Roberts, 1995), the most relevant in this regard being p27"'1". Resting T cells express high levels of p27"1", which are unaffected by TCR ligation (Firpo et nl., 1994);hence the G1 cylins and CDKs that are induced by TCR signals may associate, but reinain functionally inactive. In addition to promoting the synthesis of additional cyclin and CDK proteins, IL-2R signals lead to a rapid decline in p27K'I"levels, and these changes are accompanied by the appearance of active cycliii E/CDK2 complexes and cell cycle progression (Fiipo et nl., 1994; Nourse et nl., 1994). The effects

12

BRAD H. NELSON AND DENNIS M . WILLERFOHD

of IL-2R signals on cell cycle progression, particularly the decline in ~ 2 7 ~ ' p ' levels, are inhibited by rapamycin, implying that these effects are mediated by activation of the mammalian target of rapamycin (mTOR) protein by the IL-2R (see Section VI,C,3). These studies provide a molecular explanation for the requirement for two sequential signals for T-cell proliferation and identi? a very specific role played by IL-2R signals in regulating the cell cycle in T cells. The effects of IL-2 in the cell cycle have been interpreted to indicate an in vivo role for IL-2 in postantigenic clonal expansion of T cells. This conclusion assumes that the outcome of transiting the cell cycle is the reproduction of T cells. However, it is important to consider that several alternative cell fates are also determined by processes linked to traverse of the cell cycle, notably cell death and differentiation into memory and effector cells. Importantly, these fates tend to limit or even negate the effects of cell reproduction. Thus, the IL-2R, through its effects on cell cycle progression, may promote several different fates in T cells. The specific fate adopted by a given cell is therefore likely to depend on the context provided by other signals.

3. T-Cell ProZ~erat~~n in the Absence uf IL-2R Function Proliferation of T cells in response to mitogens in vitro is largely dependent on the production of IL-2 and subsequent IL-2R signaling (see earlier discussion). These experiments have been revisited more recently utilizing T cells from mice with targeted intactivation of the genes for IL-2 (Schorle et al., 1991) or for IL-2Ra (Willerford et al., 1995). In both these strains, T-cell proliferation in vitro to lectins, antibody cross-linking of CD3, or antigen is markedly suppressed in homozygous-deficient mice (Kramer et al., 1994; Schirnpl et al., 1992, 1994; Schorle et al., 1991; Van Parijs et al., 1997; Willerford et al., 1995). However, it is important to note that in most experiments that have interrupted IL-2/IL-2R signaling, a degree of T-cell proliferation is still observed, indicating the presence of other Tcell growth factors. Indeed, IL-4, IL-7, and IL-15 all promote T-cell proliferation in a manner similar to IL-2, a property that reflects the shared utilization of yc in the receptors for these lymphokines as well as common signals generated by their specific subunits (Bamford et al., 1994; Chazen et a!., 1989; Gin et al., 1995; Grabstein et al., 1994; Keegan et al., 1994; Morrisey et al., 1989; Paul, 1991). Given that peripheral T cells from ycdeficient mice also display low-level proliferation in response to TCR signals, there must be additional, ?,-independent signals that contribute to T-cell cycle progression (Cao et al., 1995).Thus, while IL-2 is the major growth factor used by T cells stimulated through the TCR under typical cell culture conditions, this role can clearly be subserved by other factors.


13

In uiuo studies that address the role of IL-2R signals in T-cell clonal expansion are discussed fiirther in Section VIII. B. ROLEOF IL-2R SIGNALS I N LYMPHOCYTE EFFECTOR FUNCTION In addition to its role in the expansion of activated lymphocytes, IL2R signals support the functional differentiation of mature lymphocytes, including cytotoxic T lymplioyctes (CTL),natural killer cells, and B cells. As with other cytokine receptors that promote cellular differentiation, it has been difficult to separate general effects of IL-2 on the growth and survival of cells that are developing along a given pathway from receptormediated induction of lineage-specificdifferentiation events. Nevertheless, the complexity of intracellular signaIing pathways that are activated by the IL-2R leaves much room for the latter possibility. In historical terms, the concept of IL-2 as a T-cell growth factor has perhaps channeled investigative energy toward understanding proliferative responses at the expense of effects on differentiation; at a practical level, the latter are also more difficult to assay. Either way, one of the main challenges at present in understanding IL-2R signaling is to understand the connection between receptor activation and lymphocyte differentiation events. As early experiments utilized IL-2 to grow CD8' T cells in uitro, it was also determined that these IL-2-dependent cell lines retained CTL activity. Moreover, IL-2 enhances CTL activity in activated primary T cells, and this effect is inhibited by antibodies that block IL-WIL-2R interaction (Depper et al., 1983; Gillis and Watson, 1981; Maraskovsky et al., 1989). That this activity represents more than trophic effects of IL-2 is suggested by the fact that IL-2R signals induce or upregulate mRNA for FasL, perforin, and granzyme B, all of which are involved in the mechanism of CTL-mediated killing (Liu et nl., 1990; Makrigiannis and Hoskin, 1997; Smytli et al., 1990; Suda et al., 1995). NK cells also proliferate and upregulate their cytolytic activity in response to IL-2 (reviewed in Trinchieri, 1989). However, such cells do not generally express IL-2Ra! (Tsudo et al., 1987) and, as a result, these effects require relatively high doses of IL-2. In contrast, IL-15 delivers similar signals to NK cells and is effective at picomolar concentrations (Carson et al., 1997; DiSanto, 1997), suggesting that the physiologic signals promoting N K effector function are likely delivered by IL-2RP and ycin the context of the IL-15R. Host defenses medated by the huinoral arm of the immune response involve the activation of B cells by antigen-mediated BCR stimulation, as well as interactions with activated T cells. T-cell heIp for B-cell effector function includes promoting proliferation as well as differentiation into antibody-secreting cells. T-cell signals are delivered by dxect interactions, as well as by soluble mediators, including IL-2 and IL-4 (Howard, et nl.,

14

BRAD II. NELSON A N D DENNIS M. WILLERFORD

1984). Following activation by BCR signals, induction of IL-2Ra (as well as upregulation of IL-2RP) results in expression of the high-affinity IL2R (Jung et nl., 1984; Loughnan and Nossal, 1989; Tsudo et al., 1984; Waldmann et nl., 1984; Zubler et al., 1984). Analogous to its effects on T cells, IL-2 promotes the proliferation of activated B cells (Tsudo et al., 1984; Zubler et al., 1984). In addition, IL-2 promotes the secretion of IgM in primary B-cell cultures (Nakanishi et al., 1984a,b; Waldmann et al., 1984). This property reflects specific effects of IL-2R signals on gene expression required for B-cell differentiation, notably on chromatin reinodeling at the J chain locus, followed by transcriptional activation of this gene (Blackman et al., 1986). Thus, the effects of IL-2R signaling in B cells include both events in common with those in T cells, such as promoting cell cycle progression, as well as distinct patterns of downstream gene activation. OF CELLSURVIVAL AND CELLDEATII BY THE IL-2R C. REGULATION

Physiologic cell death is now recognized as a fundamental mechanism for development and tissue homeostasis in multicellular organisms (for reviews see Raff, 1992; Vaux, 1993; White, 1996; Yang and Korsmeyer, 1996). Cell death is regulated by many factors, including a long list of developmental and hormonal signals, as well as by toxic and infectious insults. Ultimately, these regulatory signals converge on a central cascade of cysteine proteases, known as caspases, which, on activation, affect the characteristic membrane and nuclear changes that are collectively recognized as apoptosis (reviewed in Martin and Green, 199s; Takahashi and Earnshaw, 1996). Cell death is a particularly important regulatory process in the lymphoid lineage. During T- and B-cell development, a large number of cells must be eliminated because the random nature of V(D)J recombination frequently results in the generation of cells bearing antigen receptors, which are either reactive with self proteins or, in the case of T cells, may lack the proper determinants to interact with MHC molecules on antigenpresenting cells. In the peripheral lymphoid compartment, the overall number of lymphocytes remains constant over time, despite a continuous input of cells from the primary lymphoid organs and the expansion of reactive clones during immune responses. Here, cell death is required both to regulate the overall size of the secondary lymphoid tissues and to adjust their composition so as to maintain a balance between naive cells with a diverse repertoire of antigen specificities and activated or memory lymphocytes with repertoires biased toward recognition of previously encountered pathogens (Tanchot and Rocha, 1997). In considering the regulatory role of cell death in the immune system, it is useful to make a distinction between death that occurs in resting

BIOLOGY OF THE INTEKLEUKIN-2 RECEPTOR

15

lyinphoctyes and that which occurs as a consequence of cellular activation, the latter being termed activation-induced cell death (AICD). Death in resting lymphocytes is generally viewed as a default pathway, which is opposed by positive survival signals. Such signals may be provided by extracellular stimuli, and many examples of trophic factors regulating cell survival have been described (Raff, 1992; White, 1996). Susceptibility of resting cells to apoptosis correlates inversely with expression of cytoprotective members of the bcl-2 family (for review see Yang and Korsmeyer, 1996); in the lymphoid system, bcl-2 and bcl-x have been characterized most extensively . Bcl-2 is overexpressed in the context of chroinosome translocations involving the immunoglobulin loci, and these appear to underlie the majority of follicular lymphomas in humans (Yang and Korsmeyer, 1996). When hcl-2 is overexpressed in peripheral lymphocytes in mice, such cells are relatively resistant to resting cell death in vitro and appear to have an extended lifespan in v i m . In particular, the accumulation of ineinory B cells is enhanced (Sentman et nl., 1991; Strasser et nl., 1990; Strasser et nl., 1991a,b). Peripheral lymphocytes require expression of bcl-2 for optimal viability, as targeted inactivation of the bcl-2 gene results in resting, mature T and B cells with heightened sensitivity to apoptosis, and involution of the peripheral lymphoid tissues in adult inice (Veis et nl., 1993). Bcl-x appears to share inany functional properties with bcl-2 and is inducible in T cells following stiinulation through TCR and CD28 (Boise et nl., 1993,1995b;Chao et nl., 1995).Bcl-x is required for thyinocyte survival at the immature DN stage (Ma et nl., 1995b). Taken together, these studies indicate a critical role of bcl-2 Family members in mediating resting cell survival in the immune system. The tendency of resting lymphocytes to undergo cell death appears to be present throughout development. At the earliest thymic stage, cells that have initiated V( D)J recombination will die unless a productive antigen receptor rearrangement occurs, leading to the assembly of a signalingcompetent pre-TCR (Rothenberg, 1992;Willerford et nl., 1996). Most DN tliyinocytes die by a default pathway in the absence of positive selection, which amounts to a survival (and differentiation?) signal delivered following interaction between TCR and MHC molecules expressed on thymic epithelial cells (Kisielow and von Boehmer, 1995) (see Section VI1,A). It has been determined that the default pathway of cell death also operates in the peripheral lymphoid compartment, as survival of peripheral T cells is dependent on the expression of MHC molecules (Kirberg et nl., 1997; Takeda et nl., 199613; Tanchot et nl., 1997). This requirement is most notable for naive T cells and occurs in the absence of cognate high-affinity antigen. MHC-dependent, antigen-independent peripheral T-cell survival is reminiscent of positive selection in the thymus and could potentially

16

BRAD H. NELSON AND DENNIS M. WILLERFORD

involve the recognition of MHC/self-peptide complexes by the TCR. B cells also require an intact antigen receptor in order to survive in the periphery, based on the observation that ablation of surface Ig expression on mature cells using conditional gene knockout technology results in disappearance of the peripheral B-cell population (Lam et al., 1997).These stuhes suggest that activity of the antigen receptor signaling complex in peripheral lymphocytes is required for survival. Thus, lymphocytes have an inherent propensity to undergo apoptosis in the absence of survival signals,which appear to be delivered primarily through the antigen receptor signaling complex. This propensity to undergo cell death reflects the need for ongoing cellular turnover in the immune system and is presumably an important mechanism by which homeostasis is achieved. It has long been noted that IL-2 is required for the sui-vival of T cells in culture (Smith, 1988). This effect can even be seen on resting T cells, where apoptosis is slowed via signals delivered through the intennediateaffinity (IL-2RP:yJ IL-2R (Boise et al., 1995a; Gonzalez-Garcia et al., 1997).It is not clear how viability is enhanced in such cells, as the cytoprotective effect of IL-2 in resting cells is not accompanied by the upregulation of bcl-2, whereas bct-XL was induced in one report (Gonzalez-Garcia et al., 1997) but not in another (Boise et al., 1995a). The in vim relevance of this pathway is not known. In contrast to apoptosis in resting cells, AICD occurs in the circumstance of cellular activation induced by antigen receptor stimulation. Rather that regulating survival against a default death pathway, AICD delivers a positive death stimulus to the cell. This distinction presents difficulties, as antigen receptors may deliver survival signals under some Circumstances and death signals under others. The difference between these outcomes is commonly understood to reflect a “weak’ signal in the case of survival and a “strong” signal in the case of AICD. A firm understanding of the parameters that govern antigen receptor signal strength is elusive at present; however, examples at either end of this spectrum are fairly clear. During T- and B-cell development, AICD is termed negative selection and serves to eliminate T and B cells that express antigen receptors recognizing self-antigens with high affinity, a process that is fundamental to self-tolerance (for reviews see Goodnow et al., 1995; Green and Scott, 1994; Kisielow and von Boehmer, 1995; Rothenberg, 1992).Activation of mature T cells by antigen or bacterial superantigens also induces substantial cell death under some circumstances (Jones et al., 1990; Kawabe and Ochi, 1991; Liu and Janeway, 1990 ;MacDonald et al., 1991; Rocha and von Boehmer, 1991; Russell et nl., 1991; Shi et at., 1989; Webb et al., 1990).AICD is also one potential outcome following ligation of the B-cell receptor on mature B cells (Goodnow et al., 1995; Green and Scott, 1994).The pattern of AICD in response

HIOL0C;Y OF THE INTERLEUKIN-2 RECEPTOR

17

to superantigen immunization in vivo correlates with cell cycle progression. T-cell subsets bearing V/3 segments that react with particular superantigen expand over a period of several days; typically, deletion then occcurs such that after 10 days the reactive V/3 subsets are reduced to half their original levels. Cells undergoing apoptosis under these condition are those that have undergone DNA replication in vivo (MacDonald et al., 1991; Renno et al., 1995). Other data indicate that AICD in vitro is closely tied to cell cycle progression (Boehnie and Lenardo, 1993; Zhu and Anasetti, 1995). AICD may act as a check on clonal expansion, as well as participating in the termination of immune responses in vivo. Moreover, exuberant activation of the immune system, either in response to a genuine pathogen or by autoantigen, can damage host tissues. AICD likely plays an important role in preventing such host injury (Abbas, 1996; Critchfield et al., 1994; Green and Scott, 1994). The mechanism for the induction of AICD in T cells involves death signals delivered by Fas and other members of the TNF receptor family. Fas (CD95) contains a characteristic intracelluar death domain, a protein interaction motif that is shared by a number of proteins that regulate apoptosis. Upon receptor ligation, the death domain mediates clustering of the death adapter protein FADD/MORT-1, which in turn recruits and activates caspase 8. This leads to the characteristic cascade of caspase activation that serves as a common pathway for apoptosis induced by several pathways (reviewed in Chinnaiyan and Dixit. 1997; Nagata, 1994). Fas is expressed constitutively on T cells and is further upregulated folllowing activation. The membrane-bound ligand for Fas (FasL) is induced on T cells following TCR stimulation and can activate Fas, either by interaction on the same cell or on neighboring cells, to induce cell death in an autocrine or paracrine manner (Alderson et n l , 1995; Brunner et aZ., 1995; Dhein et nt., 1995; Ju et nl., 1995). AICD in T cells can be blocked by inhibition of the Fas/FasL interaction. In addition, T cells derived from either Fasdeficient Ipr mice (Russell et nl., 1993a) or FasL-deficient gld mice (Russell and Wang, 1993) also exhibit deficient AICD responses to TCR signals, both in vitro and in vivo (Singer and Abbas, 1994). The phenotype of Zpr and gld mice underscores the physiologic importance of Fas-mediated AICD. These mice are susceptible to lymphoid expansion and autoimmunity, indicating that peripheral AICD is an important mechanism for maintaining self-tolerance (Singer et al., 1994). In additon, mutations in Fas have been identified in humans with inherited autoimmune disorders accompanied by lymphadenopathy (Drappa et al., 1996; Fisher et al., 1995; Rieux-Laucat et al., 1995). It has been suggested that the role of Fas in lymphoid cell death may overlap with other apoptosis-iiiducing members of the TNF receptor family. In this regard, TNF-a! also medates AICD

18


in T cells (Speiser et nl., 1996; Sytwu et al., 1996; Zheng et al., 1995), whereas a potential role for the newly identifed receptors for TRAIL require further definition (Schneider et nl., 1997).Thus, physiologic AICD may involve a complex set of interactions among receptors and ligands, each with potentially distinct patterns of regulation. There appear to be at least two control points for Fas-mediated cell death signals. Because Fas is expressed consitutively, AICD is regulated in part at the level of FasL expression (Suda et al., 1995). However, induction of FasL is not itself sufficient to induce apoptosis efficiently, as upregulation of FasL occurs rapidly following TCR stimulation, whereas maximal sensitivity to AICD requires repetitive stimulation (Boehme and Lenardo, 1993;Wonget nl., 1997). Moreover, resting T cells are resistant to Fas stimulation when coculturedwith cells expressing FasL and undergoing AICD (Hornung et nl., 1997). Thus, a second control point for AICD is in the signaling machinery downstream of the Fas receptor. In T cells, an inhibitor of caspase 8 activation, which goes by many names, including cFLIP, is expressed contstitutively in the resting state and may contribute to the resistance of these cells to Fas-mediated apoptosis (Irmler et al., 1997). Regulation of cFLIP and other proteins interacting with the death signaling machinery may consitute this second control mechanism. One connection that has emerged is with the protooncogene c-myc, expression of which is associated with both cellular proliferation and apoptosis. Cell death induced by c-myc appears to depend on Fas/FasL interaction and correlates with increased cellular sensitivity to Fas signaling (Huber et al., 1997). The straightforward view of IL-2 as a T-cell growth factor was complicated by the observations of Lenardo (1991j, who found that T-cell clones cultured with exogenous IL-2 exhibited a high degree of apoptosis following restiinulation through the antigen receptor. IL-2 also promotes AICD in primary T cells that are first activated by concanavalin A in order to induce high-affinity IL-2 receptors, then exposed to IL-2 for 2 days prior to restimulation by CD3 cross-linkmg (Lenardo, 1991).The major correlate of IL-2-induced susceptibility to AICD is movement through the S phase of the cell cycle. Thus, cell death after restiinulation of T-cell clones correlates with the degree of T-cell proliferation, as measured by the incorporation of [3H]thymidine,and is inhibited when cells are blocked at the G1 phase of the cell cycle, but not when blocked in S phase (Boehme and Lenardo, 1993). The importance of cell cycle effects in explaining the IL-2-mediated susceptibility to AICD is underscored by the fact that other lymphokines that promote T-cell proliferation, including IL-4, IL-7, and IL-15, also promote AICD (Boehme and Lenardo, 1993; Van Parijs et nl., 1997). However, data of Russell and colleagues (Wang et al., 1996) suggest

BIOLOGY OF TIHE INTEHLKUKIN-2 RECEPTOR

19

that IL-2 may be more effective than other cytokines in this regard. Thus, a major consequence of the cell cycle progression signals delivered by the IL-BR is that T cells are primed for apoptosis following repeated or continuous TCR stimulation. Therefore, an important question regarding IL-2R signaling is whether susceptibility to AICD is mediated entirely by the signals that promote cell cycle progression or also includes more direct influences on cell death pathways. The requirement of IL-2R signals for the negative regulation of the immune system suggests that sensitizing cells to AICD may be an important function for these signals in uiuo (see Section VIII). V. Mechanism of 11-2 Receptor Activation

A. A GENERAL MODELFOR GIiownr FACTOH RECEPTOR ACTIVATION Receptors for soluble growth factors typically consist of an extracellular ligand binding domain connected via a short, hydrophobic membranespanning segment to a cytoplasmic domain that mediates the activation of intracellular signaling pathways. In the case of receptor tyrosine kinases (RTK, e.g., EGFR, PDGFR, insulin receptor), which have intrinsic tyrosine kinase domains within their cytoplasmic regions, signaling is initiated by ligand-induced oligomerization of the extracellular domains of receptor subunits (reviewed in Ullrich and Schlessinger, 1990). Because receptor diffusion is essentially limited to two dimensions by residence in the membrane, this clustering brings the cytoplasmic domains of the receptor components into close proximity. In the case of RTKs, low-level constitutive kinase activity permits cross-phosphoiylation of the clustered cytoplasmic domains at the site of regulatory tyrosines, resulting in the upregulation of kinase activity (Ullrich and Schlessinger, 1990). Aggregation of the extracellular domains may occur by the interaction of two monomeric receptor subunits with dimeric ligand or by the binding of a single divalent ligand to two receptor subunits, which are the either the same (homooligomerization) or different (heterooligomerization). Alternatively, the ligandreceptor interaction may be monomeric but induce a conformational change in the extracelluar domain that favors receptor oligomerization. Examples of each of these mechanisms have been described (Ullrich and Schlessinger, 1990). Although differing from RTKs by a lack of intrinsic kinase domains, members of the heinatopoietin receptor superfamily are similarly activated by ligand-induced oligomerization, as illustrated by the crystal structure of the growth hormone extracelluar domain complexed with ligand (Cunningliam et al., 1991; deVos et al., 1992). One growth hormone molecule binds to two identical receptor chains, with two dstinct facets of the ligand

20


contacting the same binding pocket on the receptor molecules. Stabilization of the receptor dimer by interaction with ligand is supplemented by contacts between the two extracellular domains C-terminal to the ligand binding site. The concept of ligand-induced dimerization as a mechanism for cytokine receptor activation extends to include heteromeric receptor complexes. For example, the IL-6 receptor consists of two chains, IL-6Ra and gp130. The a chain is required for binding of IL-6, but not for receptor signaling (Hibi et al., 1990). The binding of IL-6 to IL-6Ra leads to formation of a complex with gp130. These IL-6:IL-GRa:gp130 complexes dimerize, which brings two gp130 intracytoplasmic domains into close proximity, leading to receptor activation (Murakami et al., 1993).Therefore, the IL-6 receptor resembles the growth hormone receptor in that it is activated through ligand-induced homodimerization, but differs in its 1igand:receptor stoichiometry and by its requirement for an additional receptor subunit for ligand binding.

B. ACTIVATION OF THE IL-2 RECEPTOR Prior to the cloning of the yechain, the known components of the IL2R superficially resembled the IL-6 receptor, consisting of a ligand-binding subunit (IL-2Ra) and an essential signaling subunit (IL-2RP) with sequence homology to gp130. To test whether the IL-2R might signal through homodimerization of IL-BRP, a chimeric receptor chain was constructed to contain the transinembrane and cytoplasmic domains of IL-ZRP fused to the extracellular domain of c-kit, a receptor tyrosine kinase that homodimerizes on binding the divalent ligand stem cell factor. Ligation of this chimeric htIIL-2RP chain was sufficient for proliferative signaling in the pro-B-cell line BAF3 (Nelson et al., 1994). However, this result did not reflect a complete IL-2R signal, as induction of the IL-2Ra gene did not occur. Furthermore, when introduced into T cells, this chimeric receptor was not sufficient for proliferative signaling (Nelson et al., 1994).With the cloning of ye(Takeshita et al., 1992a),it seemed likely that the cytoplasmic domain of this receptor chain might also be required for signaling. Two groups subsequently used a chimeric receptor strategy to investigate the role of ycin IL-2R activation (Nakamura et al., 1994; Nelson et al., 1994). In both studies, dimerization of the IL-2RP and yecytoplasmic domains was accomplished by attaching these regions to the extracellular domains of heterologous receptors. In one case, the extracellular domains were derived from either c-kit or the granulocyte/macrophage colony-stimulating factor (GM-CSF) receptor, and dimerization of the chimeric chains was induced by the addition of stem cell factor or GM-CSF, respectively (Nelson et al., 1994). In the other case, the chimeric receptor chains were constructed using the extracellular domain of IL-2Ra, and dimerization

BIOLOGY OF THE INTERLEUKIK-2 RECEPTOR

21

was induced by the addition of anti-IL-2Ra antibodies (Nakaniura et al., 1994). In both studies, the dinierizing agents (i.e., stern cell factor, GMCSF, or anti-IL-2Ra antibody) were able to induce proliferative signals, provided both IL-2RP- and y,-derived chimeric chains were expressed. Therefore, it was concluded that ligand-induced heterodimerization of the cytoplasmic domains of IL-2RP and -yc is both necessary and sufficient for IL-2R-mediated proliferative signaling. Thus, the growth hormone receptor, the IL-6 receptor, and the IL-2 receptor provide three distinct examples of how ligand-induced oligomerizationcan lead to receptor activation within the hematopoietic receptor superfamily. Moreover, these studies underscore the mechanistic parallels between signaling by these receptors and members of the RTK family. VI. lntracellular Signaling by the 11-2 Receptor

One of the most intriguing aspects of hematopoietic cytokine receptor signaling is that the simple act of receptor chain dimerization is sufficient to initiate the multitude of intracellular events underlying the proliferative and differentiative responses of blood cells (Heldin, 1995). In the case of the interleukin-2 receptor, lieterodiinerization of the cytoplasmic domains of the P and ycchains can trigger such diverse physiological responses in T cells as proliferation, activation of effector function, and sensitization to death signals. Like other members of the heinatopoietin receptor superfamily, the IL-2R has no intrinsic catalytic function but instead signals through receptor-associated kinases, in particular, the Janus tyrosine kinases ( Jaks). Receptor ligation induces the catalytic activation of Jaks and other kinases, which initiate a cascade of intracellular phosphorylation events and the formation of a inultiineric signaling complex at the inner surface of the cell membrane. A number of downstream effector molecules are recruited to the IL-2R signaling complex, including the transcription factors Stat3 and Stat5, the adaptor protein Shc, and the lipid kinase PI3 kinase, each of which transmits a unique signal to target genes in the nucleus. Although much progress has been made in defining the biochemical components of several IL-2R signaling pathways, a current challenge is to link these biochemical events to the regulation of specific genes and cellular responses in lymphocytes. This section summarizes current knowledge of IL-2R signaling by first describing the signaling domains of the receptor chains themselves, followed by a brief account of some of the key molecules and pathways that are activated by the receptor. Finally, the molecular pathways that regulate cell proliferation and viability in response to IL-2 are discussed. This is not intended as an exhaustive review of IL-2R signaling mechanisms, which were covered extensively by a review article in this

22

BRAD 13. NELSON A N D D E N N I S M . WILLEKFOKU

series (Karnitz and Abraham, 1996), but instead is an attempt to highlight recent discoveries in the field particularily as they pertain to in vivo functions of the IL-2R. A. CYTOPLASMIC SUBDOMAINS OF THE P

AND

ycSUBUNITS

Following oligornerization by IL-2 binding, a multirneric signaling coinplex forms rapidly around the cytoplasmic domains of IL-2RP and yc. Formation of this complex is dependent on the catalytic activity of intracelMar kinases, particularly the Jaks, which phosphorylate tyrosine residues on both IL-2RP and yc(Asao et al., 1990; Sugamura et al., 1990) (Fig. 1). These phosphotyrosine motifs form docking sites for cytoplasmic-signaling proteins that have SH2 or phosphotyrosine-binding (PTB) domains (Kavanaugh and Williams, 1994; Pawson, 1995). Once recruited to the activated receptor, these proteins become substrates for kinases, resulting in their activation and further propagation of the intracellular signal. Therefore, to understand how the diverse cellular responses to IL-2R signaling are

FIG.1. Signaling domains of IL8R. (A) The three chains of IL-2R, with emphasis on the cytoplasmic subdomains of IL-2Rfi and y c . Box 1 and box 2 motifs on IL-2RP and yc are shown as black boxes in the membrane-proximal region. Tyrosine residues are designated “Y” and are numbered according to the human receptor sequences (Hatakeyama et nZ., 198913; Takeshita et nZ., 1992a). Also shown are the A and H regions of IL-2RP and the PROX region of (B) Tyrosine kinases associated with IL-2RP and yc chains. Jakl and Jak3 bind to box 1 and box 2 motifs in the membrane-proximal regions of IL-2RP and ye, respectively. In addition, Syk associates with the membrane-proximal region of IL-2RP, whereas the Src family kinases Lck, F p , and Lyn associate with the A region.


23

activated, a detailed account is required of the interactions between the cytoplasmic domains of IL-2RP and yoand the signaling proteins that are brought into the receptor complex. The cytoplasmic domain of the IL-BRP subunit was initially divided into three regions by Taniguchi and colleagues on the basis of amino acid sequence and restriction enzyme sites (Hatakeyama et al., 1989a). Despite their somewhat arbitrary origm, these regions have since proven to perform discrete signaling functions and therefore remain convenient designations for describing the signaling properties of IL-BRP (Fig. 1).The membraneproximal region of IL-2RP contains box 1 and box 2 motifs common to most hematopoietic cytokine receptors. These motifs generally constitute the binding sites for Janus tyrosine kinases, specifically Jakl in the case of IL-2RP (Boussiotis et al., 1994; Miyazaki et al., 1994; Russell et al., 1994; Tanaka et al., 1994). In addition, the tyrosine kinase Syk has been reported to associate with the membrane-proximal region of IL-2RP (Minami et al., 1995; Qin et al., 1994). The region encompassing box 2 was initially dubbed the S region due to the presence of a large number of serine residues; deletion of this region abrogates the activation of Jakl and Jak3 and all known signaling events downstream of the IL-2R (Hatakeyama et al., 1989a; Merida et al., 1993; Minami et al., 1995; Miyazaki et ul., 1995; Satoh et al., 1992; Shibuya et al., 1992; Witthuhn et al., 1994).The middle portion of IL-2RP, extending from amino acids 313 to 382, is rich in acidic residues, hence the name “A region.” This region of human IL2RP contains four tyrosine residues (Y338, Y355, Y358, and Y361) and contains binding sites for src-family tyrosine kinases (Lck, Fyn, and Lyn) (Hatakeyama et nl., 1991; Kobayashi et al., 1993; Minami et al., 1993), the adaptor molecule Shc (Evans et al., 1995; Friedmann et al., 1996; Ravichandran and Burakoff, 1994; Ravichandran et al., 1995, 1996), and, in some cells, Stat transcription factors (Gaffen et al., 1996) ( Lord et al., 1998).This region can be deleted from IL-2RP without compromising Jak activation or proliferative signaling in lymphoid cell lines, provided that the H region is intact (Hatakeyama et al., 1989a; Witthuhn et al., 1994). The distal portion of IL-2RP was named the H region because it constitutes one-“half” of the cytoplasmic domain (T. Miyazaki, personal communication). It contains two tyrosine residues (Y392 and Y510) that are involved in the activation of Stat transcription factors (Friedmann et al., 1996; Fujii et al., 1995; Gaffen et al., 1995, 1996). Like the A region, the H region can be deleted from IL-2RP without compromising Jak activation or proliferative signaling (Hatakeyama et al., 1989a; Lord et a[., 1998). However, as described in detail in Section C, deletion of both the A and the H regions abrogates the proliferative response, suggesting that these cytoplasmic

24

BRAD H. NELSON A N D DENNIS M . WILLEHFORD

domains are involved in the activation of parallel and partially redundant signaling pathways that regulate cell growth and division. As with IL-ZRP, the cytoplasmic domain of the ycchain has been divided into three regions based on amino acid sequence (Fig. 1).When yc was first cloned, Sugamura and colleagues noted sequence homology between the membrane-proximal region of yc and the SH2 motif found in many signaling proteins (Takeshita et al., 1992a). However, the SH2-like region of yc is short and lacks most of the residues required for binding to phosphotyrosine (Koch et al., 1991). Thus, this region of yc is unlikely to function as an SH2 domain and will be referred to simply as the membraneproximal or “PROX” region (extending from residues 284 to 321). This region contains a box 1 motif that is essential for association with, and activation of, the tyrosine kinase Jak3, as well as for IL-2R-induced cell proliferation (Asao et al., 1993, 1994; Boussiotis et al., 1994; Goldsmith et al., 1995; Miyazaki et al., 1994; Nelson et al., 1996, 1997; Russell et al., 1994).The PROX region may also play a Jak3-independent role in signaling, as it shares homology with the membrane-proximal regions of the (Y subunits of the IL-3, IL-5, and GM-CSF receptors, none of which appear to bind Jak molecules, yet are nevertheless required for receptor activation (Cornelis et al., 1995; Poloskaya et al., 1994; Quelle et al., 1994; Takaki et al., 1994). Just C-terminal to the PROX region lies 14 residues with weak homology to the box 2 motif found in many hematopoietic cytokine receptors (residues 322 to 335). This region performs a function common to other box 2 motifs, as it is essential for the association and activation of Jak3 (Asao et al., 1994; Miyazaki et al., 1994; Nelson et al., 1996; Russell et al., 1994). In fact, the PROX and box 2 regions of yc are necessary and sufficient for Jak3 activation and proliferative signaling (Goldsmith et al., 1995; Nelson, et al., 1996).The remaining C-terminal region of yc(residues 336 to 369) is highly conserved across species (Cao et al., 1993), but is not homologous to other hematopoietic cytokine receptors. The function of this region remains obscure, as it can be deleted without any apparent effect on Jak3 activation or proliferative signaling (Asao et al., 1994; Goldsmith et al., 1995; Nelson, et al., 1996). B. SIGNALING PATHWAYS DOWNSTREAM OF THE IL-2R 1. The Janus Tyrosine Kinases Jakl and Jak3 It has long been recognized that stimulation of hematopoietic cytokine receptors induces the rapid tyrosine phosphorylation of multiple cytoplasmic substrates, despite the fact that these receptors lack intrinsic kinase activity. The general mechanism by which this occurs was resolved with the discovery that members of the Janus tyrosine kinase (or Jak) family


25

associate with the cytoplasmic domains of hematopoietic cytokine receptors and are activated rapidly in response to receptor oligomerization. There are four known Janus kinases in mammals: Jakl, Jak2, Jak3, and Tyk2 (reviewed in Ihle, 1996a). In the case of the IL-2R, the catalyhc activation of two Jailus kinases, Jakl and Jak3, is induced within minutes of IL-2 binding (Asao et al., 1994; Brunn et al., 1995; Johnston et al., 1994, 1995a; Musso et al., 1995; Tanaka et al., 1994; Tortolani et al., 1995; Wakao et al., 1995; Witthuhn et al., 1994). By coprecipitation experiments, Jakl has been shown to associate constitutively with the IL-2RP chain and Jak3 with the yc chain (Boussiotis et al., 1994; Miyazaki et al., 1994; Russell et al., 1994; Tanaka et al., 1994) (Fig. 1).When the IL-2R is ligated, JakS appears to also associate with IL-gRP, perhaps by transfer from 7‘. In both cases, the Jak molecule interacts with the membrane-proximal cytoplasmic region of the receptor chain, specifically the box 1 and box 2 motifs (Asao et al., 1994; Higuchi et al., 1996; Miyazaki et al., 1994; Nelson, et al., 1996; Russell et al., 1994; Witthuhn et al., 1994). Conversely, the N-terminal region of Jak3 appears to mediate association with yc (Chen et al., 1997a; Kawahara et al., 1995), and a similar model may apply to the association of Jakl with IL-2RP. Catalytic activation of Jakl and Jak3 upon ligation of the IL-2R is associated with the phosphorylation of multiple tyrosine residues that are thought to perform a regulatory function. The conventional model for this process is that receptor dimerization brings the associated Jak molecules in close proximity, which promotes cross-phosphorylation and transactivation of the kinases (Darnell, 1997; Ihle, 1995; Ullrich and Schlessinger, 1990). This model is supported by the finding that Jakl fails to become activated in the absence of Jak3 catalytic activity (Kawahara et al., 1995; Nelson, et al., 1996; Oakes et al., 1996; B. H. Nelson, unpublished observations). However, the converse does not appear to be true, as Sugmura and colleagues have introduced point mutations into the box 1 motif of IL-2RP (P257S and P259S) that abrogate the ability of the receptor chain to bind or activate Jakl; surprisingly, these mutant IL-2RP chains can still interact with ycto promote normal activation of Jak3 in response to ligand (Higuchi et al., 1996). The mechanism of Jak activation by the IL-2R was also addressed in a study in which the cytoplasmic domain of yc was replaced by a covalently attached Jak3polypeptide on the assumption that this would allow ligand-induced interaction of Jak3 with Jakl and other components on the IL-2RP chain (Nelson et al., 1997). Contrary to the result expected under the “proximity” model, the attached Jak3 molecule failed to become activated in response to ligand unless the PROX regon of yc was retained as part of the receptor chain. This requirement for the PROX region might reflect a structural role in orienting Jak3 with respect to the IL-BRP

26


chain. Alternatively, the PROX region could facilitate activation of Jak3

by generating an additional activation signal. Indeed, it was found that the PROX region alone, although insufficient to activate Jakl or Jak3, nonetheless induced low-level tyrosine phosphorylation of IL-2RP and the associated tyrosine phosphatase SHP-2 in response to ligand. Furthermore, upon stimulation of the wild-type IL-2R in T cells, tyrosine phosphorylation of IL-2RP was shown to precede the activation of Jak3. Taken together, these data suggest an alternate model for early activation of IL-2R signaling, in which the PROX region of yc interacts with another as yet unidentified tyrosine kinase. Upon receptor dimerization, this second kinase would induce the phosphorylation of Jak3 as well as the IL-2RP chain. Once activated, Jak3 may then induce phosphorylation and activation of Jakl (Nelson et al., 1997). It appears that Jakl is dispensible for the mitogenic response to IL-2R signaling, as mutations in IL-2RD that abrogate Jakl binding can still mediate robust proliferation in serum-starved MOLT-4 cells. These mutant IL-BRP chains appear to interact normally with ycto induce the activation of Jak3 and Stat5 (Higuchi et al., 1996). One note of caution, however, is that these experiments were performed in a transformed T-cell line that can grow independent of IL-2 unless deprived of serum. Therefore, it remains possible that Jakl is required for proliferative signaling in normal, nontransformed lymphocytes. In contrast, it is clear that the catalytx activation of Jak3 is a critical event in IL-2R signaling. Taniguchi and colleagues overexpressed a kinase-deficient form of Jak3 in the IL-3-dependent proB cell line BAF3 and evaluated the effect on signaling through a reconstituted IL-2R complex (Kawaharaet al., 1995).This dominant-interfering form of Jak3 abrogated the activation of Jakl and the induction of c-fos in these cells and markedly inhibited cellular proliferation and the induction of c-myc. Intriguingly, induction of the antiapoptotic gene bcl-2 occurred normally in response to IL-2. This latter event was blocked by overexpression of a truncated yc chain lacking the cytoplasmic domain, suggesting that yc induces expression of bcl-2 by a Jak3-independent pathway. In addition to these experiments, studies of IL-2R signaling in a human B-cell line lacking Jak3 have shown that Jak3 is required for tyrosine phosphorylation of IL-2RP, Jakl, and Stat5 (Oakes et al., 1996). Other studies using site-directed mutants of the cytoplasmic domain of yc have demonstrated a strict correlation between the ability of mutants to activate Jak3 and their ability to induce multiple downstream events, including activation of Jakl, expression of c-myc and c-fos, and cell proliferation (Nelson, et al., 1996). Conversely, mutants of yc that retained the ability to activate Jak3 remained competent to induce these events. Regarding the induction of the c-myc and c-fos genes, there appear to be differences


27

between the results obtained in nontransformed T-cell lines and the results of other studies using the fibroblast cell line L929 or the transformed Tcell line ED40515(-). In the latter studies, a truncation mutant of ye (EDE30-4) that was competent to induce catalytic activation of Jak3 and expression of c-myc failed to induce the c-fos gene (Asao et al., 1993, 1994), suggesting that the C terminus of yc provides a signal for c-fos induction that is required in addition to the activation of Jak3. The discrepancy between these and the former studies (Nelson et al., 1996) has not been resolved. The in vivo substrates of Jakl and Jak3 have not been definitively identified; however, several proteins demonstrate diminished tyrosine phosphorylation in cells in which the catalytic activity of Jak3 has been disrupted. In the case of IL-2R signaling, these include Jakl, the IL-BRP chain, the adaptor molecule Shc, and the transcription factor Stat5 (Kawahara et al., 1995) (Oakes et al., 1996) (B. H. Nelson, unpublished results). The only enzymes reported to undergo normal tyrosine phosphorylation or activation in the absence of Jak3 catalytic activity are the tyrosine kinase Lck (Gonzdez-Garcia et al., 1997) and the tyrosine phosphatase SHP-2 (Adachi et al., 1997). Thus, Jak3 appears to play a major role in the proximal activation of the IL-2R-signaling complex. Other cytokine receptors that utilize the yc chain, including the IL-4, IL-7, IL-9, and IL-15 receptors, also activate both Jakl and Jak3, which raises the issue of how the specificity of intracellular signals is achieved. One potential mechanism is at the level of the substrates of Jakl and Jak3 that are recruited to the receptor complex through binding interactions with either the receptor chains themselves or with various adaptor molecules in the complex. Because the individual receptor chains differ in the bindmg sites contained within their cytoplasmic domains, each may present a distinct set of substrates to Jakl and Jak3, thereby imparting unique biochemical characteristics to cytokine signals. It should also be noted that cytokine receptors that share yc mediate a number of overlapping functions in lymphocytes, particularly with respect to cell proliferation. Accordingly, Jakl and Jak3 appear to also mediate a common set of biochemical events for this group of receptors. 2. Stat Transcription Factors Jak kinase activity is generally associated with the tyrosine phosphorylation and activation of one or more Stat proteins, a family of molecules that was first discovered through studies of interferon signaling (reviewed in Darnell, 1997). Stat molecules are latent transcription factors that, in unstimulated cells, are localized to the cytoplasm. Cytohne stimulation induces tyrosine phosphorylation of receptor chains, thereby creating docking sites for Stats, which have an SH2 domain near the C terminus. The

28


recruited Stats undergo rapid tyrosine phosphorylation, which induces them to form homo- or heterotypic dimers that subsequently translocate to the nucleus, where they bind to characteristic DNA sequence motifs within specific target genes. The IL-2R activates both the A and the B isoforms of Stat5 and, in several cell types, including primary T cells, can also activate Stat3 (Brunn et al., 1995; Frank et al., 1995; Fujii et nl., 1995; Gaffen et al., 1995; Gilmour et al., 1995; Hou et al., 1995; Johnston et al., 1995a; Lin et al., 1995; Pernis et al., 1995; Wakao et al., 1995). Stat activation by the IL-2R requires the tyrosine hnase activity of Jak3 (Fujii et al., 1995; Oakes et al., 1996; B. H. Nelson, unpublished results) but not Jakl (Higuchiet a!.,1996).In addition, Stat activation requires the presence of either Y392 or Y510 on the IL-SRP chain (Friedmann et al., 1996; Fujii et al., 1995), although in the T-cell lines CTLL2 and HT-2 Stats can also be activated via Y338 of IL-2RP (Gaffen et al., 1996; Lord et al., 1998) Site-directed mutants of IL-2RP that lack all cytoplasmic tyrosine residues fail to activate Stat factors, despite normal activation of Jakl and Jak3 (Friedmann et al., 1996; Gaffen et al., 1996; Lord et al., 1998). Thus, as is the case with other cytokine receptors, the IL-2R appears to induce nuclear signals through Stat proteins by the following series of events: (1) Jak-dependent phosphorylation of one or more tyrosine residues on the receptor chain, (2) recruitment of Stats through their SH2 domains to phosphorylated sites on the receptor, (3) tyrosine phosphorylation of the recruited Stat molecules, either by Jak3 or another receptor-associated kinase, (4) homo- or heterodimerization of Stat molecules via reciprocal interactions between their SH2 domains, (5) translocation of Stat dimers to the nucleus, and (6) binding to specific regulatory sequences within target genes. In addition to tyrosine phosphorylation, Stat factors also require serine phosphorylation for optimal transcriptional activation (reviewed in Darnell, 1997). With the IL-2R, this is carried out by a serine/ threonine kinase that has not been identified but is distinct from p42/44 MAP kinase, mTOR (the mammalian target of rapamycin), or p70 S6 kinase (Beadling et al., 1996; Kirken et al., 1997). Among the target genes of IL-2 that are Stat5 dependent are (1)cis (Matsumoto et al., 1997), a member of a novel family of proteins that are negative regulators of signaling by other cytokine receptors (Endo et al., 1997; Naka et al., 1997; Starr et al., 1997; Yoshimura et al., 1995);although neither cis nor other members of this protein family have yet been shown to associate with the IL-2R complex or modulate IL-2R signaling, data from other receptors suggest that such interactions are likely to be described in the near future; (2) osm, which encodes the cytokine oncostatin M (Yoshimura et al., 1996); and ( 3 ) the IL-2Ra gene (see later). So far, no role


29

for Stat3 or Statrj has been defined in the regulation of growth-related genes such as c-myc, c-fos, bcl-2, or bcl-x in the context of the IL-2R. Of the just-mentioned Stat5-dependent genes, IL-2Ra has been the most extensively characterized in terms of its mechanism of regulation. As described in Section III,A, a transient wave of IL-2Ra expression is induced in T cells by TCR signals, whereas maximal and sustained IL-2Ra transcription requires additional stimulation with IL-2 (reviewed in Nabholz et al., 1995). Experiments with transgenic mice harboring a reporter gene containing the 5’ flanking region of the IL-2Ra gene, together with DNase hypersensitivityanalyses, led to the identification of a 78 nucleotide element that is responsive to the IL-2 signal and is located 1.3 kb upstream of the major transcription start site (Soldaini et nl., 1995). This segment, which has since been named PRRIII (for positive regulatory region 111), was found to contain two potential binding sites for Stat proteins in addition to potential binding sites for Ets and GATA proteins (Sperisen et al., 1995). A homologous segment was subsequently identified in the human ZL-2Ra gene -4 kb upstream of the transcription start site (John et al., 1996; L’ecine et al., 1996). PRRIII of the human gene has been shown to bind both Stat5A and Stat5B, apparently via the more distal of the two potential Stat-binding sites (Lecine et al., 1996). Additional transcription factors, including Elf-1, GATA-1, and HMG-I(Y), also bind to sites in PRRIII but, in contrast to Stat5A and B, may not require IL-2 stimulation for their activation or expression (John et nl., 1996; L‘ecine et al., 1996; Serdobova et al., 1997). Experiments with reporter genes have shown that none of the DNA-binding sites in PRRIII is sufficient for IL-2-inducible transcription, whereas all three sites are necessary for optimal activity (John et al., 1996; L’ecine et al., 1996; Serdobova et al., 1997). Thus, it appears that the three known DNA-binding sites within the PRRIII element interact cooperatively to induce transcription of the IL-2Ra gene in response to IL-2 and that Stat5 is a critical inducible factor that switches the element to an active state. The essential role of Stat5 in regulation of the IL-2Ra gene is highlighted by the fact that splenocytes from mice with a targeted disruption of the Stat5A gene demonstrate defective induction of IL-2Ra expression in response to IL-2 and consequently require high doses of IL-2 for proliferation (Nakajima et al., 1997a). Notably, however, there appears to be at least one other inducible activity required for induction of the IL-2Ra gene in response to IL-2, as the IL-3 receptor can also activate Stat5A and StatB yet fails to induce expression of IL-2Ra (Ascherman et al., 1997).It is not known whether this additional activity represents a posttranslational modification of one or more proteins already known to bind PRRIII or the involvement of additional regulatory factors.

30


Although the physiological role of Stat factors in IL-2R signaling is not fully defined, the just-mentioned list of target genes suggests that Statmediated signals may be involved in both positive and negative regulation of IL-2R activity via induction of the ZL-2Ra and cis genes, respectively. The requirement for Stat5 for expression of the cytokine oncostatin M raises the additional possibility that Stats may be involved in immunomodulatory aspects of IL-2R signaling. Finally, the essential role of Stat4 and Stat6 in the differentiation of CD4' T cells to the T h l and Th2 subclasses, respectively (Kaplan et al., 1996a,b; Shimoda et al., 1996; Takeda et al., 1996a; Thierfelder et al., 1996),suggests that Stat5 may also play a role in lymphoid differentiation. In this regard, Stat5 might be expected to function in the development of NK cells and T C R y 8 T cells, as these lymphoid classes are severely reduced in IL-2RP-l-mice (Suzuki et al., 1997a) (see Section VII). 3. MAP Kinase Pathways The IL-2R activates Ras and downstream components of the MAP kinase pathway, including Raf, MEK, and p42/44 MAP kinase (Duronio et al., 1992; Graves et al., 1992; Izquirdo et al., 1992; Karnitz et al., 1995; Perkins and Collins, 1993; Satoh et al., 1991; Turner et al., 1991; Welham et al., 1994a,b),which are associated with mitogenic signaling by a wide variety of growth factor receptors (reviewed in Marais and Marshall, 1996; Marshall, 1994).Activation of this pathway involves recruitment of the adaptor molecules Shc and Grb2 and the guanine nucleotide exchange factor mSOS to the IL-2R complex (Burns et al., 1993; Cutler et al., 1993; Karnitz et al., 1995; Liu et al., 1994; Ravichandran and Burakoff, 1994; Zhu et aE., 1994). Downstream of this pathway lies the protooncogene cfos, which is induced through the semm-response element in the promoter-proximal region, as well as fra-1, c-jun, and junB (Hatakeyama et al., 1992; Shibuya et al., 1992). Much has been learned about the components of the IL-ZR complex required for activation of the Ras/MAP kinase pathway in lymphocytes. First, the catalytic activity of Jak3 is essential, as tyrosine phosphorylation of Shc and induction of cfos fail to occur with a kinase-deficient form of Jak3 (Kawahara et al., 1995) (B. H. Nelson, unpublished results). Second, the A region of IL-2RP is required, as deletion of this region has been shown to abrogate the phosphorylation or activation of Shc, Ras, and p42/44 MAP kinase and the induction of cfos, c-jun, and other related protooncogenes (Evans et al., 1995; Hatakeyama et al., 1992; Ravichandran et al., 1996; Satoh et al., 1992; Shibuya et al., 1992). Third, within the A region, Y338 is required, as point mutation of this residue to phenylalanine abrogates the phosphorylation of Shc and p42/44 MAP kinase and the induction of c-fos (Evans et al., 1995; Friedmann et al., 1996; Gaffen et al., 1996) (Lord et al., 1998). Fourth, recruitment and tyrosine phosphoryla-


31

tion of Shc by the IL-2R appear sufficient to initiate the activation of this pathway. This was shown by replacing the entire A and H regions of IL2RP with a covalently linked Shc molecule (Lord et nl., 1998). Specifically, the N terminus of Shc was attached just C-terminal to the box 1 and box 2 regions of IL-2RP. As expected, the resulting IL-2RPIShc fusion protein mediated normal activation of Jakl and Jak3 and underwent tyrosine phosphorylation in response to ligand. More importantly, the fusion protein mediated normal phosphorylation of ~ 4 9 4 4MAP kinase and induction of c-fos in CTLLB and BAF3 cells, despite lacking Y338 and all other residues in the A region. Collectively, these and other results suggest a linear sequence of events by which the IL-2R induces activation of the Ras/MAP kinase pathway: (1)receptor dirnerization activates Jakl and Jak3, (2)Y338 of IL-2RP is phosphorylated, ( 3 ) Shc binds to this site through its PTB domain (Friedmann et aE., 1996; Ravichandran et al., 1996), (4) Shc undergoes tyrosine phosphorylation, ( 5 ) Grb2 binds to phosphorylated tyrosine residues in the CH domain of Shc (Harmer and DeFranco, 1997; Ravichandran and Burakoff, 1994; Zhu et al., 1994), and (6) mSOS is recruited to the receptor complex (Ravichandran and Burakoff, 1994; Ravichandran et al., 1995) and induces the activation of Ras. Subsequent events downstream of Ras leading to activation of p42/44 MAP kinase and the induction of c-fos are thought to occur by the standard Ras/Raf/MEK/MAP kinase pathway defined for other growth factor receptors. PI3 kinase also appears to be involved in IL-2R-mediated MAP kinase activation, as wortmannin (a pharmacologic inhibitor of PI3 kinase) significantly lminishes the catalpc activity of both MEK and MAP kinase in IL-2-stimulated T cells without affecting Raf activity (Karnitz et al., 1995). Although much is known about the mechanism by which IL-2R activates the Ras/MAP kinase pathway, the physiological function of this pathway in the cellular response to IL-2 remains undefined. Importantly, the A region of IL-2RP, although absolutely required for activation of the Ras/ MAP kinase pathway and induction of c-fos, is redundant with the H region for IL-2R-induced proliferation and viability of lymphoid cell lines (see Section V1,C). It remains possible, however, that the dispensibility of the A region merely reflects the permissive growth properties of cultured cell lines and that the proliferative response of primary lymphocytes in vivo requires activation of the Ras/MAP hnase pathway by the IL-2R. In neuronal cells, activation of the p42/44 MAP kinase pathway has been associated with the delivery of cell survival signals (Xia et al., 1995), raising the additional possibility that the role of this pathway in the biologc response to IL-2 may include the regulation of lymphocyte viability. Furthermore, evidence shows that this pathway may affect lymphocyte differentiation, as overexpression of a dominant p42/44 MAP kinase mutation in the thymus

32


leads to preferential differentiation of the CD4+ T-cell lineage at the expense of the CD8' lineage (Sharp et al., 1997). In addition to the conventional p42/44 MAP kinases, alternative MAP kinase pathways have been described that involve proteins of the c-jun amino-terminal kinase (JNK) and p38 kinase pathways. Generally asssociated with inflammatory cytokines or stress responses, JNK and p38 pathways are also activated by IL-2R signals (Crawley et al., 1997). It was reported that the activity of p38 MAP kinase is essential for IL-2R-mediated proliferation, as a pharmacological inhibitor of p38 was found to suppress T-cell proliferation in a dose-dependent manner (Crawley et al., 1997). However, this group has since found that the activation of p38 MAP kinase in BAF3 cells appears to require the A region of IL-BRP, which itself is dispensible for proliferation, therefore an essential role for p38 in the proliferative response seems unlikely (B. M. J. Foxwell, personal communication). In other receptor systems, these kinases have been asssociated with a number of cellular responses, including cellular activation, differentiation, and apoptosis (Fanger et nl., 1997; Kyriakis and Avruch, 1996; Verheij et at., 1996). Further investigation into the functional consequences of JNK and p38 activation by the IL-2R will thus be of great interest. 4. Other Components of the IL-2R Signaling Complex a. Src Family Kinases. The first tyrosine kinase shown to be activated by the IL-2R was the Src family member Lck. Lck was shown to physically associate with the acidic domain of IL-2RP through an unconventional interaction involving the kinase domain of Lck (Hatakeyama et al., 1991) (Fig. 1).Moreover, IL-2 was found to induce the catalyhc activity of Lck within minutes of stimulation, an event that is dependent on the presence of the A region (Hatakeyama et al., 1991; Horak et al., 1991; Kim, et al., 1993; Minami et al., 1993). In cells lacking Lck, the related kinases Lyn and Fyn have been shown to undergo catalytic activation in response to IL-2 (Kobayashi et al., 1993; Torigoe et al., 1992), therefore the IL-2R appears to be somewhat permissive in its interactions with Src family kinases. Activation of Src family kinases by the IL-2R is associated with the catalytic activation of PI3 kinase (Taichman et al., 1993). In T-cell lysates, PI3 kinase activity coprecipitates with Fyn, and the amount of coprecipitating activity is enhanced severalfold by stimulation with IL-2 (Augustine et al., 1991). The interaction between these two molecules is mediated by the SH3 domain of Fyn and two proline-rich regions in the p85 subunit of PI3 kinase (Karnitz and Abraham, 1996; Karnitz, et al., 1994;Pleiman et al., 1994; Prasadet al., 1993).It should be noted, however, that Src family kinases are likely to represent only one of several means by which the IL-2R activates PI3 kinase. Given the fact that the acidic


33

region of IL-2RP is redundant for proliferative signaling in lymphocytes, the physiological role of Lck and Fyn in IL-2R signaling remains unclear. A correlation between IL-2R-mediated activation of Lck and the prolongation of T-cell viability has been described, which is discussed in Section D.

b. ZRS-1 and IRS-2. In addition to Shc and Grb2, two other adaptor proteins have been reported to participate in IL-2R signaling. Insulin receptor substrate 1 (IRS-1) and its homolog IRS-2 are large cytoplasmic proteins that were initially identified as downstream components of the insulin and IL-4 receptors, respectively (Sun et al., 1995; Wang et al., 1992; White and Jahn, 1994). These molecules contain multiple tyrosine residues that are phosphorylated in response to receptor stimulation and serve as docking sites for several cytoplasmic signal-transducing molecules containing SH2 domains, including Crk, Grb-2, Nck, PI3 kinase, and SHPTP2 (Gustafson et al., 1995; Myers et al., 1996; Skolnik et al., 1993; Sun et al., 1993). Consistent with their central role in recruiting substrates to activated receptor complexes, IRS-1 and IRS-2 are required for optimal cell proliferation in response to insulin and IL-4. IL-2 has been shown to induce the tyrosine phosphorylation of IRS-1 and IRS-2 in PHA-activated human peripheral blood T cells, as well as human NK cells and B cells (Johnston et al., 1995b). Moreover, IRS-1 was shown to physically associate with Jakl and Jak3, and IRS-2 with Jakl, in IL-%stimulated T cells. One functional consequence of IRS-1 phosphorylation in response to IL-2 may be activation of PI3 kinase, as the p85 regulatory subunit of PI3 kinase coprecipitates with IRS-1 after IL-2 stimulation (Johnston et al., 1995b). c. SHP-2. Two SH2 domain-containing tyrosine phosphatases, SHP-1 and SHP-2, have been implicated in signaling by hematopoietic cytokine receptors (reviewed in Ihle, 1996b). Tyrosine phosphorylation of SHP2, but not SHP-1, occurs in response to IL-2R activation; however, no corresponding change in the catalytic activity of this enzyme has been reported (Adachi et al., 1997; Nelson et aZ., 1997). Both the membraneproximal and A regions of IL-2RP are required for phosphorylation of SHP-2, whereas the catalytic activity of Jak3 is not (Adachi et al., 1997). Indeed, a truncation mutant of 'ye that fails to bind or activate Jak3 can nevertheless interact with IL-BRP to induce modest tyrosine phosphorylation of SHP-2 (Nelson et al., 1997). One clue to the physiological role of SHP-2 in IL-2R signaling is suggested by studies of receptor tyrosine kinases, where it may contribute to the activation of Ras by serving as an adaptor molecule (Bennett et al., 1994; Li et al., 1994; Noguchi et al., 1994).A second clue may come from studies of the IL-6 receptor subunit gp130, which is thought to promote cell proliferation in part through the

34


recruitment of SHP-2 to a cytoplasmic tyrosine residue (Fukada et al., 1996). Notably, however, similar roles for SHP-2 in IL-2R signaling have yet to be established.

C. MITOGENICSIGNALING BY THE IL-2R Of all the cellular responses regulated by the IL-2R, the role of the receptor in controlling lymphocyte proliferation is by far the best characterized at the molecular level. Therefore, the following section summarizes current knowledge and hypotheses concerning the molecular mechanism of proliferative signaling by the IL-BR, and many of the key factors are illustrated in Fig. 2. Although some of the following signaling events have been mentioned in previous sections, they are included here as well to provide a comprehensive overview of the process of mitogenic signaling. 1. Role of Jak Molecules and Other Tyrosine Kinases Signals that promote cell cycle progression are initiated by IL-2-mediated heterodimerization of the IL-BRP and ycchains, which results in the rapid activation of several tyrosine kinases, including Jakl, Jak3, Syk, and Lck

FIG.2. A model for mitogenic signaling by IL-2R. Depicted are the components of the IL-2R complex known or hypothesized to be involved in transduction of the proliferative signal, including the cytoplasmic domains of IL-2RP and ye, the tyrosine kinase Jak3, the STAM protein, three tyrosine residues on IL-BRP, the adaptor molecule Shc, and the transcription factor Stat5 Also shown are examples of target genes involved in the regulation of cell proliferation and viability. Other factors involved in mitogenic signaling, such as PI3 kinase and mTOR, are not shown, as they have not yet been localized to specific pathways or sites in the receptor complex.


35

or Fyn (Fig. 1).Of these, Jak3 has been shown to be essential for mitogenic signaling. Evidence supporting this conclusion includes: ( 1) fibroblasts expressing a reconstituted IL-2R complex will not proliferate in response to IL-2 unless Jak3 is coexpressed (Miyazah et al., 1994); (2)overexpression of a catalytically inactive version of Jak3 markedly inhibits IL-2R-mediated proliferation of the pro-B cell line BAF3, presumably by a dominantnegative mechanism (Kawahara et al., 1995); (3) deletion of either the S region of IL-2RP or the PROX or box 2 regions of yc abrogates the activation of Jak3 by IL-2 and similarily ablates the proliferative response (Asao et al., 1993, 1994; Goldsmith et al., 1995; Hatakeyama et al., 1989a; Mori et al., 1991; Nelson et al., 1996, 1997; Witthuhn et al., 1994); (4) T cells from Jak3-null mice fail to proliferate in response to IL-2 (Nosaka et al., 1995; Park et al., 1995; Thomis et al., 1995); and (5) signaling can be restored to nonfunctional truncation mutants of yr by covalent attachment of the Jak3 molecule (Nelson et at., 1997), but this requires that the Jak3 molecule have a functional catalytic domain (B. H. Nelson, unpublished results). In contrast to the critical role of Jak3, Jakl is apparently dispensible for proliferative signaling, as point mutants of IL-BRP that eliminate the binding and activation of Jakl do not affect proliferative response to IL-2R activation (Higuchi et al., 1996). Importantly, however, this result has yet to be confirmed in a nontransformed lymphocyte. As described earlier in Section VI,B,4, the Src family kinases Lck, Fyn and Lyn are also activated by the IL-2R, however, deletion of the A region of IL-SRP abrogates the binding and activation of these kinases without disrupting proliferative signaling (Hatakeyama et al., 1989a, 1991; Minami et at., 1993), suggesting that these kinases are either not involved in the mitogenic signal or are redundant with other factors. Sykis activated rapidly by the IL-2R and associates with the S region of IL-BRP, which itself is essential for mitogenic signaling (Minami et al., 1995; Qin et al., 1994). Moreover, when artificially clustered at the cell surface in the form of a chimeric CD16/Syk receptor, Syk can mediate induction of the c-myc gene in BAF3 cells (Minami et al., 1995).Nevertheless, Syk has not been directly demonstrated to be essential for IL-2-induced proliferation of lymphocytes, and in fact may not be, as it is expressed at very low levels in IL-2responsive peripheral T cells (Chan et al., 1994). Thus, of the tyrosine kinases known to be activated by the IL-2R, Jak3 appears to play the predominant role in mitogenic signaling (Fig. 2). This raises the critical issue of which factors downstream of Jak3 transmit the proliferative signal to the nucleus.

2. Role of Tyrosine Residues on the IL-2RP Chain There is strong evidence from mutational studies that tyrosine residues within the cytoplasmic region of the IL-2RP chain play an essential role in

36


proliferative signaling downstream of Jak3. By mutating the six cytoplasmic tyrosine residues of the human IL-2RP chain to phenylalanine, Leonard and colleagues completely abrogated IL-2R-mediated proliferation of the myeloid cell line 32D (Friedmann et al., 1996). Add-back experiments in which one or more of these residues were retained as tyrosine revealed that at least one of three specific tyrosine residues (Y338, Y392, or Y510) is required for the mitogenic signal (Fig. 2). Intriguingly, any one of these three residues was sufficient for a proliferative response, whereas simultaneous elimination of all three tyrosines completely abrogated mitogenesis. Similar conclusions were drawn from studies in the T-cell lines HT-2 (Gaffen et al., 1996) and CTLL-2 (Lord et al., 1998). In the experiments with CTLL-2 cells, it was established that tyrosine residues on IL2RP are required for proliferation even in the face of normal Jak activity. The requirement for specific tyrosines on IL-2RP is in contrast to analogous experiments with the erythropoietin receptor, where mitogenesis can occur in the absence of receptor tyrosine residues, apparently through the activation of Jak2 alone (Joneja and Wojchowski, 1997; Nakamura et al., 1996). It also differs from studies of the yc chain, where cytoplasmic tyrosine residues have been found to be dispensible for proliferative signaling (Gaffen et al., 1996; Nelson et al., 1996). The apparent functional redundancy of Y338, Y392, and Y510 with respect to proliferative signaling is entirely consistent with studies involving large deletions of the IL-2RP chain. Taniguchi and colleagues showed that mitogenesis is largely unaffected by deletion of the acidic region of IL2RP (which contains Y338) or of the H region (which contains Y392 and Y510) (Hatakeyama et al., 1989a). However, if both the A and the H regions are deleted simultaneously, the proliferative response is abrogated, despite the retention of normal Jak activity (Lord et al., 1998). Thus, Jak3 catalytic activity is not sufficient for proliferative signaling by the IL-2R. Rather, mitogenic signaling proceeds from the catalytic activation of Jak3 to phosphorylation of one or more tyrosine residues in the A or H regions of IL-2RP. Studies in CTLL-2 and BAF3 cells have shown that the shared ability of the A and H regions of IL-2RP to promote cell proliferation also applies to the induction of certain protooncogenes. Thus, induction of the c-myc, bcl-2, and bcl-x genes proceeds normally, provided either the A or the H region of IL-2RP is present (Miyazaki et al., 1995; Shibuya et al., 1992) (J. D. Lord and B. H. Nelson, unpublished results). Further paralleling the proliferative response, induction of these genes is dependent on the presence of one or more tyrosine residues in these two regions of IL2RP ( J . D. Lord and B. H. Nelson, unpublished results). In contrast to these major protooncogenes, other gene induction events are fully or


37

partially dependent on either the A region (e.g., c-fos) or the H region as described in previous sections. (e.g.,Stat targets such as cis and IL-~RcY), These studies are consistent with a model in which the cytoplasmic region of IL-BRP, after being phosphorylated on specific tyrosine residues by Jak3, serves as a protein interaction domain to recruit cytoplasmic signaling molecules to the activated receptor complex, where, in turn, they are activated by receptor-associated kinases. Experimental observations suggest that different sections of the IL-2RP chain mediate interaction with distinct, but partially overlapping, sets of cytoplasmic signaling proteins, thereby giving rise to discernible patterns of downstream biochemical events.

3. Role of Factors Downstream of IL-2Rp Phosphorylated tyrosine residues on growth factor receptors generally serve as binding sites for cytoplasmic signaling molecules containing SH2 or PTB domains (Pawson, 1995), serving to recruit these proteins into the receptor complex where they may be activated, principally by phosphorylation. As described in the preceding section, three tyrosine residues on IL2RP (Y338, Y392, and Y510) have been implicated in transduction of the proliferative signal. Therefore, there is currently great interest in identifying the cytoplasmic factors that bind to these tyrosine-containing motifs and transmit the mitogenic signal. As illustrated in Fig. 2, two candidates for such factors are the adaptor molecule Shc, which binds to phosphorylated Y338 and is involved in activation of the RasIMAPK pathway (Burns et al., 1993; Cutler et al., 1993; Friedmann et al., 1996; Karnitz et al., 1995; Liu et al., 1994; Ravichandran and Burakoff, 1994; Zhu et al., 1994), and the transcription factor Stat5, which can be activated through Y392, Y510 (Friedmann et al., 1996; Fujii et al., 1995), or, in some cells, Y338 (Gaffen et nl., 1996) (Lord et al., 1998). The role of Shc in mitogenic signaling was investigated by linking the Shc protein covalently to the IL2RP chain in place of the A and H regions (Lord et al., 1998). In this experiment, the signals induced by Shc could be studied in isolation from other A or H region-dependent events. As expected, the IL-BRPI Shc fusion protein induced Jak activation in response to ligand and also demonstrated normal aspects of Shc function, including phosphorylation of p42/44 MAP kinase and induction of c-fos. Moreover, the the IL-2RPI Shc fusion protein was able to promote a robust proliferative response in both the T-cell line CTLL2 and the pro-B cell line BAF3, which corresponded with normal induction of the c-myc, bcl-2, and bcl-x genes. Thus, the proliferative function contributed by the A and H regions of the IL2RP can be replaced by a covalently associated Shc protein. Intriguingly, the IL-2RPIShc fusion protein was unable to support the long-term

38


viability of transfected cells, despite the fact that induction of bcl-2 and bcl-x was normal. Thus, it appears that Shc is able to mediate some, but not all, of the signaling events normally induced through Y338, suggesting that Y338 may mediate cell expansion through both Shc and a second downstream molecule that has yet to be identified. If this is the case, it would imply that individual phosphotyrosine motifs on the cytoplasmic domain of the IL-2RP can interact with multiple downstream factors. The evidence implicating Stat5 in proliferative signaling is so far strictly correlative, in that Y392 and Y510 in the H region of IL-2RP can promote both Stat5 activation and cell proliferation (Friedmann et al., 1996; Fujii et al., 1995; Gaffen et al., 1996); thus, the possibility that a second, unidentified factor binds to Y392 and Y510 and is responsible for mitogenic signaling is not precluded. Indeed, if such a factor existed and were able to also interact with Y338, it would provide a simple explanation for the apparent functional redundancy of the A and H regions (and their respective tyrosine residues) with respect to proliferative signaling. To detect the activity of such a factor, it will be necessary to simultaneously abrogate the activity of Shc and Stat5 in cells while retaining one or more phosphorylated tyrosine residues on the IL-2RB chain. Although such an experiment has not been reported for the IL-2R, a mutant form of the EPO receptor has been described that can induce the proliferation of BAF3 cells independent of the Shc and Stat5 pathways (Klingmuller et al., 1997), indicating that alternative proliferative pathways do in fact exist in lymphocytes. At this time, all that can be concluded for the IL-2R is that the factors responsible for transmitting the mitogenic signal downstream of Y338, Y392, and Y510 of IL-2RP have yet to be definitively characterized; however, the available evidence suggests Shc and Stat5 as the best candidates. 4. Role of PI3 Kinase and Akt

Ligation of the IL-2R induces the catalytic activation of PI3 kinase, a lipid and serinelthreonine kinase that has been implicated in mitogenic signaling by a number of growth factor receptors (Merida, et al., 1991, 1993; Monfar et al., 1995; Reif et al., 1997; Remillard et al., 1991) (for reviews see Karnitz and Abraham, 1996; Vanhaesebroeck et al., 1997). Several biochemical events have been reported to lie downstream of PI3 kinase in the context of IL-2R signaling, including activation of the serine/ threonine kinases Akt and p70 S6 kinase (Ahmed et al., 1997; Karnitz et al., 1995; Karnitz and Abraham, 1996; Monfar et al., 1995; Reif et al., 1997) and the threoninehyrosine kinase MEK (Karnitz et al., 1995).Wortmannin,


39

which is a potent inhibitor of PI3 kinase [and, at high concentrations, of mTOR as well (Brunn et al., 1996)], has been shown to cause a modest reduction in IL-2-induced proliferation of the T-cell line CTLL2 (Karnitz et al., 1995). Gain-of-function experiments involving the PI3 kinase pathway have yet to provide a consistent picture of its role in lymphocyte proliferation. Overexpression of constitutively active forms of Akt in the pro-B-cell line BAF3 resulted in constitutive expression of the protooncogenes c-myc and bct-2 and inhibited cell cycle arrest and apoptosis on cytokine withdraw1 (Ahmed et al., 1997; Songyang et al., 1997). In contrast, expression of a constitutively active form of PI3 kinase in the T-cell line Kit225 was not sufficient to induce DNA synthesis or GUS phase transition (Brennan et al., 1997). Thus, PI3 kinase may contribute to lymphocyte proliferation, however, it appears to be neither necessary nor sufficient for this response. Cantrell and colleagues reported that IL-2 increases the activity of the proinitogenic transcription factor E2F in T cells through a pathway involving PI3 kinase and Akt (Brennan et al., 1997). This was shown using a transient transfection assay in which expression of a reporter gene linked to two tandem E2F-binding sites was induced by IL-2R activation. This inducible E2F activity was blocked by LY294002, a pharinacologic inhibitor of PI3 kinase, or by a dominant-negative version of PI3 kinase. Conversely, constitutively active forms of PI3 kinase or Akt were sufficient to induce reporter gene activity. LY294002 was shown to also inhibit IL-2-mediated induction of the cyclin D3 gene and downregulation of p27kipl expression. These two events are thought to promote phosphorylation of Rb, which results in the release of free and active E2F (Brennan et al., 1997). Given the general involvement of E2F in cell cycle regulation, these results suggest a mechanism by which PI3 kinase and Akt could contribute to proliferative signaling by the IL-2R. The PI3K pathway has also been implicated in the maintenance of cell viability, as described in Section D. 5. Role of mTOR

An important clue to how the IL-SR transduces signals affecting cell cycle progression is provided by the antibiotic rapamycin, a lipophilic inacrolide derived from the bacterium Streptonigces hygroscopicus (for reviews see Abraham and Wiederrecht, 1996; Karnitz and Abraham, 1996). Rapainycin inhibits T-cell proliferation in late G1 in response to IL-2 (Bierer et al., 1990; Duinont et al., 1990; Morice et al., 1993a) and indeed blocks cell cycle progression in response to a number of growth factors, both in inainmals and in yeast. This conserved activity suggests that rapamycin acts at a key control point for cell growth regulation, and hence, there has been great interest in defining the biochemical pathways that

40


are affected by the agent. In mammalian cells, the effects of rapamycin appear to be mediated through mTOR (the mammalian target of rapamycin) (reviewed in Karnitz and Abraham, 1996). The structure of mTOR initially suggested that it was a lipid kinase, although evidence shows that it may in fact function as a serinehhreonine kinase. Indeed, Abraham and colleagues reported that the PHAS-1 protein, which is involved in the regulation of protein translation, is directly phosphorylated on serine and threonine residues by mTOR (Brunn et al., 1997). Another molecule that is activated through mTOR, although not thought to be a direct substrate, is p70 S6 kinase (Brown et al., 1995; Calvo et al., 1992; Chung et al., 1992; Ferrari et al., 1993; Kuo et al., 1992; Price et al., 1992; reviewed in Chou and Blenis, 1995). This enzyme, which is essential for G1 progression in fibroblasts (Lane et al., 1993), phosphorylates the ribosomal protein S6 and thereby regulates the translation of a distinct subset of cellular mRNAs (Terada et al., 1994). These findings suggest that rapamycin may inhibit cell cycle progression by suppressing the translation of critical proteins involved in DNA replication or cell growth. Rapamycin has been shown to block the ability of IL-2R to downregulate expression of the cyclindependent kinase inhibitor ~ 2 7 ~ Iin ' ' T cells (Nourse et al., 1994), with a concomitant block in cyclin-dependent kinase activation (Firpo et al., 1994; Morice et al., 1993a,b),thus demonstrating one clear mechanism by which this agent inhibits cell growth. However, this is not the only pathway through which rapamycin antogonizes IL-2R signaling: in mice lacking p27, IL-2R-mediated cellular proliferation is intact and is still sensitive to inhibition by rapamycin (Nakayama et al., 1996). The precise mechanism by which mTOR is activated by the IL-2R has yet to be defined. There is evidence, however, in IL-3-dependent cell lines that one pathway to mTOR activation may operate through PI3 kinase (R. Abraham, personal cominunication). 6. Role of STAM

An addition to the list of factors that participate in mitogenic signaling by the IL-2R is the STAM protein (Takeshita et al., 1996, 1997). STAM may function as an adaptor protein, as it contains both an SH3 domain and an immunoreceptor tyrosine-based activation motif (ITAM) region, the latter being characteristic of signaling proteins associated with TCR and BCR. Overexpression of a putative dominant-interfering mutant of STAM, consisting of a deletion of the SH3 domain, was shown to inhibit both IL-2- and GM-CSF-induced proliferation of the pro-B-cell line BAF3 in a transient assay (Takeshita et al., 1997). It is unclear at present how STAM functions in the IL-2R mitogenic pathway. The ITAM region of STAM is reported to bind directly to Jak3 (or Jak2 in the case of the GMCSF receptor); however, it is not known whether STAM influences the


41

catalytic activity of these kinases (Takeshita et al., 1997). Similarly, it is not known whether STAM has an effect on known signaling pathways for IL-2R-mediated cell cycle progression, such as those involving Shc, Stat5, mTOR, or PI3 kinase.

7. Target Genes of tlze IL-2R In addition to signal transducing proteins, the IL-2R activates a number of target genes involved in cell growth and division. These include protooncogenes such as c-myc, cfos, and c-jun, as well as G1 cyclins (Shibuya et al., 1992). Use of a differential hybridization technique has led to the identification of several new genes that are induced in an iminediate-early manner by IL-2 (Beadling et al., 1993), and this number is likely to grow with the application of newer techniques, such as high-density DNA array hybridization, to the study of cytokine signaling. Of the target genes induced by IL-2, the pathways leading to cfos and c-jun are relatively well defined (see Section VI,3). In contrast, little is known about the pathway(s) that regulates expression of the c-myc and cyclin genes, despite the ubiquitous and central role they play in the control of cell growth. The importance of c-myc expression to mitogenic signaling is underscored by the fact that, in all the mutational analyses that have been performed to date with IL-2R and other cytokine receptors, a perfect correlation has been observed between the expression of c-myc and the induction of cell proliferation. Regulation of the c-myc gene has been studied for over a decade in a wide variety of cells under many different stimulatory conditions, and three general control mechanisms have been identified, including modulation of transcriptional initiation, transcriptional elongation, and inRNA stability (reviewed in Spencer and Groudine, 1991). For most growth factors, including IL-2, it is not known which of these mechanisms is primarily responsible for regulating expression of c-nzyc. However, one clue may come from the observation that IL-2 can induce expression of a reporter gene containing a 2.3-kb fragment froin the 5' region of the c-myc gene, an effect that is enhanced by overexpression of STAM (Takeshita et al., 1997). This suggests that the IL-2R induces cmyc expression through cis regulatory elements located in the upstream 2.3-kb region of the gene. Studies with the IL-3 receptor further indicate that cytokine regulation of c-nzyc may involve the transcription factor E2F (Watanabe et al., 1995). Although these results are intriguing, more work is clearly required to establish whether such a mechanism is relevant to regulation of the endogenous c-rnyc gene by the IL-2R.

D. SIGNALINGPATHWAYS FOR CELLVIABILITY An important element of responses to signals generated by cytokine receptors, including the IL-2R, is the promotion of cell viability. In prolifer-

42

BRAD 11. NELSON AND DENNIS M . WILLERFORD

ating cells, this effect is somewhat difficult to separate from cellular reproduction. However, withdraw1 of cytokines, e.g., from proliferating T-cell lines, leads to apoptosis, indicating that positive survival signals are one component of the cytokine response. As discussed in Section V, IL-2R signals can promote the survival of T cells in the absence of proliferation (Akbar et al., 1996; Boise et al., 1995a; Gonzalez-Garcia et al., 1997), although the high concentrations of IL-2 required for this effect raise the issue of physiological relevance. The biochemical signal for T-cell viability appears to differ from that for proliferation in that it can occur without prior antigen stimulation and with only trace expression of Jak3 or phosphorylation of Jakl. The effects of IL-2 on the viability of resting T cells correlates with the catalytic activation of the tyrosine kinase Lck and is reduced markedly in the presence of LY294002, an inhibitor of PI3 kinase. Cell survival was also associated with induction of bcl-x expression in one study (Gonzalez-Garcia et nl., 1997), but not another (Boise et al., 1995b). The importance of the PI3 kinase pathway in signaling viability is supported by studies of Akt (also known as protein kinase B), a serinehhreonine kinase that is activated by IL-2R signals in a PI3 kinase-dependent manner (Ahmed et al., 1997; Brennan et al., 1997; Franke et al., 1995; Reif et al., 1997). Expression of a constitutively active Akt kinase is sufficient to induce bcl-2 expression and prolong the viability of BAF3 and 32D cells after cytokine withdrawal (Ahmed et nl., 1997; Songyang et al., 1997). Taken together, these studies suggest that the IL-2R may promote T-cell viability through a Jak-independent pathway involving PI3 kinase and Akt and the target genes bcl-x and/or bcl-2. This inodel is consistent with results from Taniguchi’s group demonstrating that IL-2R-mediated induction of the bcl-2 gene in the pro-B-cell line BAF3 does not require the cataytic activity of Jak3 (Kawahara et al., 1995). Although signals for cell viability and expression of bcl-2 family genes may be generated by a Jak-independent mechanism, they resemble the proliferative signal in requiring the presence of one or more tyrosine residues on the IL-2RP chain (Lord et al., 1998); perhaps these sites on IL-2RP are phosphorylated by Lck and serve as docking sites for the p85 regulatory subunit of PI3 kinase. VII. In Vivo Siudies of IL-2 Receptor Function in Lymphocyte Development

IL-2R components are expressed by iininature T and B cells and may therefore influence lymphocyte development at one or more stages. As IL-2RP and yc are shared with other cytokine receptors, investigations into the specific role of IL-2 and the high-affinity IL-2R in lymphocyte development must be considered together with experiments that address the contribution of signals generated by individual receptor components.

43

BIOLOGY OF THE INTERLEUKIN-2 KECEPTOH

Studies to date show that neither IL-2 nor its high-affinity receptor is required for the development of T, B, or NK cells; nevertheless, the immune deficiencies and inflammatory disorders associated with mutations of IL-2 or IL-2R components raise the possibility that the lymphocytes that develop in these animals have undergone abnormal selection processes. Because of its shared use in multiple lymphokine receptors, the yc chain is required for effective lymphopoiesis, principally due to the activities of the IL-7R in promoting the expansion and/or survival of early T- and Bcell progentiors. Mutations in the gene encoding yc are responsible for XSCID in humans and engender a severe lyinphophoietic defect in knockout mice. Moreover, the IL-2RP chain, which is shared by the IL-2 and IL-15 receptors, is required for the development of NK cells. The effects on lymphoid development of induced mutations in mice involving IL-2R components and related genes are summarized in Table I, and are discussed in detail below.

A. OVERVIEW OF T- AND B-CELLDEVELOPMENT The development of the imtnune system has two major components: the generation of lymphocytes with a large repertoire of antigen receptor specificities and the shaping of this repertoire by selection processes that exlude autoreactive cells and ensure optimal recognition under conditions of antigen presentation by accessory cells. The capacity of the immune system to respond to a nearly boundless array of different antigens is based TABLE I T- A Y D &CELL.DEVELOPMENT I N M l C E WITI-I MUT.4TIONS AFFECTINC: I L 9 R SI(:NALINC: Gene IL-2

T-cell Development

TCR Transgene Effects

B-Cell Development

Normal

Normal antigen-iuduced deletion in thymus Normal antigen-induced deletion in thymus Norinal positive and negative selection

Normal

I L - ~ R c Y Normal IL-2RP

x Jak3 IL-7 IL-7Ra IL-4

Normal; small thynus due to systemic disease Leaky block at DN stage Leaky block at DN stage Leaky block at DN stage Severe block at DN stage Normal

Incomplete rescue of T-cell development

Incoinplete rescue of T-cell development

Normal Nonnal

Leaky block at Stage Leaky block at stage Leaky block at stage Leaky block at stage Normal

pro-B pro-B pro-B pro-B

44


on the ability of developing T and B cells to generate antigen receptors with a vast diversity of binding specificities. This is accomplished by the unique process of somatic gene rearrangement, involving the variable (V), diversity (D), and joining ( J ) gene segments of T-cell receptor and immunoglobulin genes (reviewed in Bogue and Roth, 1996; Gellert, 1997). Enormous combinatorid possibilities result from the large number of recombining segments, particularly V segments. Imprecision in joining these segments, which is an inherent property of the recombinase apparatus, further contributes to diversity of the receptor repertoire. Antigen receptor rearrangement is lineage specific and follows an ordered developmental pattern. In B cells, Ig heavy chains are rearranged prior to light chains, whereas in the major T-cell lineage, the TCRP locus rearranges before TCRa. Antigen receptor rearrangement is not merely a consequence of lymphocyte development, it also drives it: the stepwise expression of antigen receptor proteins triggers the major developmental events in both T- and B-cell lineages (reviewed in Kisielow and von Boehmer, 1995; Willerford et al., 1996).In the thymus, early TN cells have germline antigen receptor genes or have begun to rearrange TCRP. In-frame rearrangement of TCRP results in the expression of a functional pre-T-cell receptor, which includes the nascent TCRP chain, the pre-Ta chain, and components of the CD3 signaling apparatus, This complex signals a major developmental transition in the thymus, termed “beta selection,” involving a burst of cell division and expression of CD4 and CD8 to populate the DP thymocyte compartment. This transition also results in the activation of V(D)J recombination at the TCRa locus, and expression of the TCRa chain creates a complete ap T-cell receptor. The TCR drives the next set of developmental choices, based on binding to self-peptides presented by MHC molecules on thymic epithelial cells. The characteristics of this interaction determine negative and positive selection events, which result in the maturation of a population of exportable CD4’ or CD8’ single-positive (SP) T cells. In the B-cell compartment, in-frame rearrangement of IgH results in the expression of p chain, which associates with V-preB and h5 to form the pre-B-cell receptor. This receptor triggers cellular expansion and loss of CD43 expression. As in the DP thymocyte compartment, pre-B cells rearrange their light chains (IgK or Igh) and become immature IgM’AgD’ B cells. Thus, the major steps of T- and B-cell development are driven by successive signals generated following the stepwise rearrangement and expression of antigen receptor proteins.

B.

INTERLEUKIN-8 RECEPTOR FUNCTION DURING T- A N D B-CELLDEVELOPMENT Primitive thymocytes, comprising the CD4- CDB-CD3- TN population, contain a heterogeneous mix of cells, which span the developmental spec-

BIOLOGY OF T H E INTERLEUKIN-2 RECEPTOR

45

trum from multipotent progenitor cells to committed T cells having undergone productive rearrangement of one antigen receptor gene (for reviews see Godfrey and Zlotnick, 1993; Rothenberg, 1992; Shortnian and Wu, 1996; Willerford et al., 1996).The expression of the IL-2Ra chain (CD25) correlates with the major developmental events at the TN stage. Thus, cells within the CD25' subpopulation express Rag-1, Rag-2, and germline transcripts from the TCRP locus. Divergence of cells constituting the y6 and crp T-cell lineages occurs during this period, and rearrangement of TCRP is accomplished. Productive rearrangement and expression of the TCRP chain completes the pre-TCR, resulting in downregulation of CD25, and initiates a burst of proliferation and differentiation, which results in the filling of the CD4TD8' DP thymocyte compartment. Appropriate expansion and/or survival of the TN thymocyte population is critically dependent on yL,a property that is largely due to signaling in the context of the IL-7R. JL-7 stimulates the proliferation of this cellular subset (Conlon et al., 1989; Okazaki et nl., 1989; Watson et al., 1989). In additon, TCRP rearrangement in TN thymocytes in vitro is largely dependent on IL-7 (Muegge et al., 1993), an effect that likely reflects a trophic requirement for IL-7 at this stage, but could also indicate a more direct influence on the efficiency of V(D)J recombination (Cand'eias et al., 1997; Corcoran et al., 1996). Does CD25 expression in TN thymocytes indicate that high-affinity IL-2 receptors are functional at this stage? Experiments addressing this question have provided contradictory results. Human TN thymocyte subpopulations expressing either intermediate (i.e., CD25-) or high-affinity (i.e., CD25+) IL-2 receptors proliferate in response to IL-2 (Toribio et al., 1989). In mice, conflicting results have been obtained, with the clearest evidence for proliferative responses to IL-2 being found in fetal thymocytes (Raulet, 1985; Zuniga-Pflucker et al., 1990), with less convincing responses in cells derived from adult animals, which internalize the ligand-bound receptor inefficiently (Lowenthal et al., 1986; Raulet, 1985).In these studies the concentrations of IL-2 required for proliferation were often higher than expected for high affinity IL-2R interactions; however, high-affinity IL-2R binding has been demonstrated in the case of fetal thymocytes (Zuniga-Pfluckeret al., 1990). Nevertheless, it is possible that proliferation is not the only relevant response to IL-2R signals in TN thymocytes. Indeed, there is a suggestion that IL-2 also promotes differentiation of human immature thymocytes (Toribio et al., 1988) and antibodies to CD25, which block IL-2 signaling and inhibit thymocyte development in 14-day mouse fetal thymic organ culture (Jenkinson et al., 1987). The role of IL-2R signals in thymic development in vivo has been similarly examined using antibody inhibition studies. Administration of antibodies to CD25 in pregnant mice resulted in a profound block in

46


T-cell development in neonates (Tentori et al., 1988). Following sublethal irradiation of mice, reconstitution of the thymus was slowed in animals receiving anti-CD25, but not an irrelevant antibody (Zuniga-Pflucker and Kruisbeek, 1990; Zuniga-Pflucker et al., 1990). These studies suggest that IL-2R signals could potentially play a role either in the early expansion of T-cell progenitors or in promoting efficient differentiation. These effects may be most evident under special conditions of kinetic stress, which were present in these experiments. As such, these results could be reconciled with data from IL-2 or IL-2Ra knockout mice that show no obvious defect in T-cell development under steady-state conditions. Alternatively, the observations made using antibody inhibition could reflect toxic effects of the reagent, rather than a specific block in IL-R signaling. As is the case for TN thymocytes, B-cell progenitors are importantly influenced by ye signals delivered by the IL-7R. IL-7 is secreted by bone marrow stromal cells and supports the proliferation of pro- and pre-B cells (Namen et al., 1988). IL-7R signals also promote B-cell antigen receptor rearrangement. Using a retroviral gene transfer system, cultured B-cell progenitors from IL-7Ra-deficient mice were reconstituted with wild-type or mutant IL-7Ra chains. Distinct mutations affected the proliferation of B-cell progenitors and the induction of antigen receptor rearrangement, suggesting either that IL-7R signals directly stimulate V( D)J recombination in B cells or that the efficiency of this process is enhanced by IL-7Rmediated survival signals (Corcoran et al., 1996). In developing mouse B cells, CD25 is expressed at the CD45RtIgM-CD43- pre-B-cell stage (Chen et al., 1994; Rolink et al., 1994), but is apparently not expressed in normal human B-cell precursors. The functional importance of CD25 expression in mouse pre-B cells is not clear, as such cells are apparently not responsive to IL-2. NK cells perform a cytolytic function in vivo that is thought to enhance host defenses against viruses, and possibly transformed cells as well. Unlike CTL, which recognize antigen via TCR interaction with MHC class I/ peptide complexes, NK activity is greatest against cells with low or absent class I expression, due to expression by NK cells of inhibitory receptors recognizing class I molecules (for reviews see Gumperz and Parham, 1995; Raulet, 1996; Spits et al., 1995). NK cells share some common features with T cells and appear to develop from a common progenitor (Carlyle et al., 1997; Rodewald et al., 1992; Sanchez et al., 1994). NK cells can develop in the thymus; however, extrathymic venues, particularly the bone marrow, may be the major sites of NK cell production. Although IL-2 stimulates the proliferation and activation of NK cells, this generally requires much higher doses than are required for T-cell proliferation, as IL-2Ra is not expressed on this subset (Trinchieri, 1989). Recent interest has focused


47

on IL-15, which promotes the development of NK cells from bone marrow progenitors in vitro, and supports their survival at concentrations consistent with signaling through the high-affinity IL-15R (Mr'ozek et al., 1996; Williams et d., 1997). Thus, IL-15 may represent the physiologic cytokine for NK cell development in vivo, whereas IL-2 may simply mimic this activity in vitro.

C. GENETIC STUDIESOF IL-2 AND IL-2Ra FUNCTION IN LYMPHOCYTE DEVELOPMENT 1. IL-2 and IL-2Ra Transgenic Mice Overexpression studies in mice have utilized transgenes encoding human IL-2 and IL-2Ra in order to distinguish transgenic from endogenous proteins. In one transgenic line in which human IL-2 was driven by the H-2K' promoter, expression of human IL-2 was detected in the thymus, but no resulting developmental abnormalities were reported (Ishida et aZ., 1989). A second study reported a human IL-2 transgene expressed under the control of the metallothionin promoter, which caused upregulation of IL-2Ra on thymocytes, but no other developmental consequences (Kromer et nl., 1991). Overexpression of human IL-2Ra using the H-2Kd promoter resulted in the acquisition of high-affinity receptors on the majority of thymocytes and conferred the ability to proliferate in response to IL-2, but again no developmental abnormalities were reported (Nishi et nl., 1988). However, a second report of mice bearing a human IL-2Ra transgene expressed under the SV40 promoter and enhancer indicated a modest decrease in CD4+CD8+thymocytes, with a concomitant increase in the DN subpopulation (Gutierrez-Ramos et al., 1989), suggesting a partial inhibition of T-cell development. Mice expressing a combination of IL-2 and IL-2Ra transgenes under the control of the H-2Kdpromoter developed a greatly expanded thymic population of large granular lymphocytes with NK activity and unrearranged T-cell receptor genes. The mice were small and died by 4 weeks of age, exhibiting infiltration of the cerebellum by NK cells and neuronal loss. This dramatic phenotype likely reflects the abnormally broad expression of high-affinity IL-2R and does not necessarily reflect a physiologic function of IL-WIL-2R interactions in the thymus. Given the probable role of IL-15R signals in NK cell development, this transgenic sytem probably generates an overly active IL-2RP and signal, and perhaps can be interpreted as equivalent to an overly active IL15R signal. 2. Interleukin-2- and IL-2Ra-Deficient Mice The functional role of IL-2 and the high-affinity IL-2R in lymphocyte development have been examined in mice with targeted disruption of

48


either IL-2 or IL-2Ra genes (Schorle et al., 1991; Willerford et nl., 1995). No apparent defects in T- or B-cell development were identified in young adults of either strain, indicating that steady-state production of phenotypically normal lymphocytes does not require IL-2 or high-affinity IL-2R. Detailed analysis of CD4- CD8- CD3- TN thymocyte subsets in either IL-2Ra-deficient or IL-2Ra-/- X Rag-24- mice did not reveal any differences in either the phenotype or the distribution of cells in this subpopulation other than absence of CD25 expression (D. M. Willerford, unpublished observation). The development of NK cells also appears normal in mice lacking IL-2 (Schimpl and Hunig, 1994). In IL-2-deficient mice expressing a class I MHC-restricted TCR transgene, antigen-induced deletion of DP thymocytes was normal, both in vitro and in vivo (Kramer et al., 1994). Similar results were observed in IL-2Ra-deficient mice bearing a class I1 MHC-restricted TCR transgene (D. T. M. Leung and D. M. Willerford, manuscript in preparation). Thus, these gene targeting experiments do not support an essential role for IL-2R signaling in T-or B-cell development. However, a more subtle or redundant role for such signals, e.g., in promoting efficient progression through the TN thymocyte stage, is not ruled out by these studies. 3. Human IL-2 and IL-2Ra Deficiencies

Cases of human patients with severe combined immune deficiency have been reported with deficiencies in IL-2 secretion (Chatila et al., 1990; Pahwa et al., 1989; Weinberg and Parkman, 1990) or IL-2Ra expression (Roifman, 1997; Sharfe et al., 1997). T and B cells were present in these patients, consistent with the finding of phenotypically normal development of mature lymphocytes in mice deficient in IL-2 or IL-2Ra. In the report by Roifman and colleagues, a frameshift mutation was identified near the 5' end of the IL-2Ra gene, which abolished expression of the IL-2Ra protein (Sharfe et al., 1997). Detailed examination of the thymus revealed normal size and immunohistochemical staining patterns for CD4, CD8, and class I and I1 MHC. A striking absence of staining for CDla on cortical thymocytes was observed, whereas CDla expression could be normally upregulated on activated monocytes, suggesting that the defect was specific to thymic development. CD1 molecules are structurally related to class I MHC and include five genes in humans, designated CDla-e (Calabi and Milstein, 1986; Martin et al., 1986), and two in mice (CD1.l and CD1.2) (Bradbury et al., 1988), both of which are homologous to human CDld. Human CDlb appears to be involved in antigen presentation for glycolipids (Beckman et al., 1994), and the mouse CD1.l gene is required for efficient thymic selection of the NK1.l-bearing T-cell subset (Chen et al., 199%; Mendiratta et al., 1997). The finding that CDla expression is absent in


49

IL-2Ra-deficient human thymocytes cannot be investigated adequately in IL-2Ra-deficient mice, as no clear homolog to CDla has been identified. Nevertheless, abnormal thymus composition in this patient with IL-2Ra deficiency raises that possibility that abnormalities in T-cell development might as yet be unrecognized in knockout mice and may contribute to the immune and homeostatic abnormalities associated with this mutation. I N IL-2RP CHAIN-DEFICIENT MICE D. LYMPHOCYTE DEVELOPMENT

The role of IL-2RP-dependent signals (including, at a minimum, those induced by IL-2 and IL-15) in lymphocyte development has been characterized in mice with a targeted null mutation of the IL-2RP gene (Suzuki et al., 1995). As in mice deficient in IL-2 and IL-2Ra, the development of T and B lymphocytes is phenotypically normal in young animals laclang IL-2RP, although the thymus is small. This observation is thought to be a secondary consequence of the inflammatory disease that affects these animals, resulting in corticosteriod-mediated thymic involution, as T-cell development in fetal thymus organ cultures is normal. In contrast, the development of several specialized T-cell subsets appears to be impaired, including NKl' T cells and the the population of gut intraepithelial lymphoctyes utilizing yFfCR (Ohteki et al., 1997; Suzuki et al., 1997a). NK cell development is also abrogated in IL-ZRP-deficient mice. IL-2RP-deficient mice are susceptible to a severe and complex inflammatory disorder, beginning at 4weeks of age, that is dependent on T cells (see Section VIII,A,2). One potential explanation for this disorder is abnormal thymic repertoire selection leading to the maturation of autoreactive T cells. The role of IL-2RP in positive and negative selection processes in the thymus was investigated by introducing a TCR transgene specific for the male antigen, HY, into the IL-2RP-deficient background (Suzuki et al., 199713). Positive and negative selection were found to be normal in thymi from, respectively, female and male IL-2RP-deficient mice. However, given that the HY transgene is selected on class I MHC, and several aspects of the autoimmune disease in IL-2RP-deficient mice are dependent on CD4+ cells, a defect in class I1 MHC-dependent thymic selection remains a formal possibility.

E. ROLEOF yc IN LYMPHOCYTE DEVELOPMENT 1. Mutations ($yc in XSCZD XSCID represents the most common form of severe combined immunodeficiency in children, accounting for approximately half of patients in several series (Buckley et al., 1997; Fischer et al., 1997; Stephan et al., 1993). The disease gene (termed SCIDX1) was mapped to the vicinity of Xq12-13.1 (de Saint-Bade et al., 1987; Puck et al., 1989). ShortIy after

50


the molecular cloning of yo it was determined that the ye gene localized to Xq13 and mapped near markers associated with SCIDX1. Moreover, mutations in the the ye coding sequence were identified from XSCID patients (Noguchi et al., 1993c; Puck et al., 1993). Subsequent analysis has identified yc mutations in most XSCID patients (Buckley et al., 1997; Fischer et al., 1997). Boys with XSCID have severe thymic hypoplasia and lack peripheral T cells, demonstrating a critical role for ye in the early stages of human T-cell development (reviewed in Conley, 1992; Fischer et al., 1997). B-cell numbers are generally preserved, indicating that, in contrast to the situation in mice (described later), yc is not required for human B-cell development. However, XSCID B-cells have intrinsic functional abnormalities, including defective mitogen-induced proliferative responses (Gougeon et al., 1990). These observations are supported by X chromosome inactivation studies in female carriers of XSCID mutations, which demonstrate a nonrandom pattern of X inactivation in T cells (i.e., only the wild-type X chromosome is active). A random X inactivation pattern is seen in immature B cells, suggesting that no competitive disadvantage is engendered by the mutant yc during development, whereas mature B cells are biased toward the wild-type X chromosome, indicating that optimal survival and/or expansion of peripheral B cells requires yc (Conley et al., 1988). XSCID is a lethal condition, for which the only longterm treatment is bone marrow transplantation

2. Mice Lacking ye Detailed studies of the role of ycin lymphocyte development and function have been facilitated by the generation of mice with null mutations in the ye gene (Cao et al., 1995; DiSanto et al., 1995; Ohbo et al., 1996). The development of T cells in these mice differs somewhat compared to human XSCID patients in that T cells, while markedly reduced in number, are present in the periphery and accumulate with age. Unlike the phenotype in humans, B-cell development is markedly impaired in ye-deficient mice. The developmental block in T- and B-cell lineages occurs at the transition from the TN to DP thymocyte stage and the pro- to pre-B-cell stage, respectively. This suggests that early lymphocytes do not efficiently achieve complete formation of the pre-TCR and pre-BCR in ye-deficient mice. At this point in development, yc could potentially mediate signals for the expansion or survival of early lymphoid progenitors. In addition, such signals could facilitate efficient V( D )J recombination, although the fact that the impairment in lymphoid development is leaky indicates that this process does not absolutely require ye. Furthermore, developmental defects due to disrupton of V(D)J recombination in T cells are rescued by expression of an appropriately selecting T-cell receptor ap transgene


51

(Shinkai et al., 1993). However, expression of either MHC class I- or class II-restricted TCR transgenes in the 7,-deficient background produces only partial rescue of thymic cellularity (DiSanto et al., 1996; Nakajima et al., 1997b), indicating that 7, has a role in early thymic development that is independent of the antigen receptor rearrangement process. A subtle role of 'ye in thymic negative selection has been suggested based on the partial imparrment in 7,-deficient mice of the deletion of Vflll' T cells, which normally occurs in the presence of the Mtv-9 provirus (Nakajima and Leonard, 1997), and a similar result was reported in inice lacking Jak3, which resemble 7,-deficient mice (Saijo et al., 1997, see later). However, negative seletion of T cells bearing VPS was normal in ?,-deficient mice, as was deletion of VP6' T cells in the presence of the self-superantigen Mls-ld (Nakajima and Leonard, 1997). Negative selection of transgenic T cells recognizing the H-Y antigen was also normal in male 7,-deficient mice (DiSanto et al., 1996). Development of the 76 T-cell sublineage is markedy impaired in mice lacking yc. Furthermore, NK cells are also markedly diminished, in parallel with the N K defect observed in mice lacking IL-2RP (Suzuki et al., 1995). Taken together, these data indicate that the production of NK cells is critically dependent on signals delivered by the IL-2RP and 7, chains, but not IL-2Ra, a description that is consistent with the IL-15 receptor. The generation of mice deficient in IL-15 or the IL-15Ra chain will assist in resolving this issue. Of the cytokine receptors that utilize yc,the IL-7R appears to have the most profound effects on early lymphocyte proliferation and differentiation (see Section B). These observations suggest that the developmental phenotype of 7,-deficient mice is in large part due to deficient IL-7R signaling at early stages. This view is supported by studies of mice rendered IL-7 deficient by antibody injection (Grabstein et al., 1993) or by gene targeting (von Freeden-Jeffry et al., 1995) or of mice lacking the IL-7Ra chain (Peschon et al., 1994). These animals resemble 7,-deficient mice in that they all display a marked reduction in both T- and B-cell production. For T cells , expression of a transgenic TCR in mice lacking IL-7Ra results in only partial rescue of thymic cellularity, similar to what is observed in 7,-deficient mice, suggesting that the defects in this T-cell sublineage are primarily mediated by IL-7R in the early stages of thymic development (Crompton et al., 1997). However, careful comparison of thymic development in mice lacking y,, IL-7, or IL-7Ra reveals subtle differences. In mice lacking IL-7Ra, the generation of mature T cells appears more severely limited than in mice lacking either IL-7 or ye,suggesting that IL7Ra may generate developmental signals in addition to those delivered in the context of the IL-7Rdyc heterodirner. Indeed, the novel lyinphokine TSLP, which is derived from thymic stroma, also supports the growth of

52


early T- and B-cell progenitors in a manner similar to IL-7 (Friend et al., 1994). The receptor for TSLP includes IL-7Ra (Peschon et al., 1994), as well as a novel TSLP receptor, but is independent of yc. In studies that utilized a pre-B-cell line responsive to both IL-7 and TSLP, the TSLP response was inhibited either by antibodies to IL-7Ra or TSLP receptor, but not by antibodies to 7, (S. Levin and A. Farr, personal communication). Thus, the more severe defect in T-cell development in IL-7Ra-deficient mice likely reflects impairment of signals delivered by both IL-7 and TSLP. Although disrupted IL-7R signaling explains many of the defects seen in 7,-deficient mice, there are also additional IL-7R-independent signals generated by yc during lymphocyte development. First among these are signals required for the development of NK cells, which are absent in mice lacking ye,but intact in IL-7Ra-deficient mice (He and Malek, 1996). As discussed earlier, this difference likely reflects the participation of yc in a cytokine receptor distinct from IL-7 and IL-2, most likely the IL15R. There may also be IL-7R-independent, 7,-dependent signals for ap T-cell development. In bone marrow reconstitution studies in which recipients were treated with antibodies to y,, defective T- and B-cell development similar to that seen in 7,-deficient mice was observed. With donor bone marrow derived from IL-7Ra-deficient mice, an additional inhibition of T-cell development was observed with antibody treatment, suggesting that yc delivers additional, IL-7Ra-independent signals to early thymic progentors (He et al., 1997). One possibility is that this IL-7independent signal is provided by the high-affinity IL-2R, which is redundant at the TN thymocyte stage in the presence of intact IL-7R signaling. Alternatively, these observations could suggest a role for another cytokine that utilizes yc in its receptor. 3. Deficiency of y, Is Mimicked by Defects in Jak3 Signaling One crucial function of yc in the activation of the IL-2R is to recruit the tyrosine kinase Jak3 to the receptor complex, an event that is required for most of the identified events downstream of the IL-2R (see Section VI).The importance of Jak3 in IL-2R function in viwo is underscored by developmental defects seen in mice made deficient in Jak3 by gene targeting (Nosaka et al., 1995; Park et al., 1995; Thomis et al., 1995). These mice have a severe defect in both T- and B-cell development, as well as in T-cell activation, which parallels the defects seen in ?,-deficient mice. Similarly, a subset of patients with an XSCID-like phenotype (i.e., SCID with circulating B cells) but a normal 7, gene have been found to have mutations in Jak3 (Macchi et al., 1995; Russell et al., 1995). Jak3 activity also appears to correlate with the degree of immunodeficiency: a patient with combined T- and B-cell deficiency, but partial immune function, was

BIOLOGY OF THE INTERLEUKIN-2 RECEFTOR

53

described with a mutation in the cytoplasmicdomain of ycthat only partially interfered with Jak3 binding (Russell et al., 1994). Therefore, similar to in vitro studies of IL-2R signal transduction, the phenotypic correlation between Jak3 activity and yc function in both mice and humans indicates that Jak3 activation is a required proximal event in signaling by cytokine receptors that utilize yc. VIII. In Vivo Studies of IL-2 Receptor Function in Peripheral Lymphocytes

The concept of IL-2 as a T-cell growth factor suggested that interruption of IL-2R signals should impair the amplification of immune responses and result in immune deficiency. Thus, the initial desciiption of mice laclang IL-2, which exhibited a lymphoid system with a relatively normal appearance and function, was somewhat surprising (Schorle et al., 1991). One approach to reconciling this in vivo finding with the well-established role of IL-2 as a T-cell growth factor in vitro is to invoke redundancy in cytokmes mediating T-cell growth, a concept that has gained support with a better appreciation of the large family of cytokine receptors that share yc and have overlapping cellular effects. However, such explanations do not address the two major abnormalities in mice deficient in IL-2 or IL2R components: an inability to appropriately control the size of secondary lymphoid tissues and the emergence of inflammatory disorders, at least some of which represent autoimmunity. Thus, these studies indicate that the clearest in vivo function for IL-2R signals is to negatively regulate the size and functional activity of the peripheral lymphoid compartment. In addition, such studies should encourage further attempts to reframe questions regarding IL-2R signaling mechanisms in the context of negative regulation of iinmune functions. The peripheral consequences of mutations in mice affecting IL-2R signaling are summarized in Table 11. A. IL-2R SIGNALS REGULATETHE SIZEA N D CONTENT OF THE SECONDARY LYMPHOID TISSUES 1. Peripheral Consequences of lL-2- and IL-2Ra Mutations in Mice

Mice lacking the capacity to make either IL-2 (Schorle et al., 1991) or the IL-2Ra chain (Willerford et al., 1995) have a similar phenotype. Young adult mice develop polyclonal expansion of the peripheral lymphoid compartment, with all the major cellular subsets represented at levels 5- to 10-fold higher than in wild-type littermates, suggesting a global defect in lymphoid homeostasis. T cells in these animals are characterized by a memory cell phenotype, with high expression of CD44 and low expression of CD62L, suggestive of previous activation. Depending on the age of the animals, avarying increase in the proportion of peripheral T cells expressing

TABLE I1 PERIPHERAL PHENOTYPES OF MICEWITH MUTATIONSAFFECTING I L B R SIGNALING Gene

T Cells

IL-2

Expanded, activated, memory phenotype

IL-2Ra

Expanded, activated, memory phenotype

IL-2RP Expanded, activated blastic

x

B Cells

Cellular Immunity

Humoral Immunity

Idammatory Disorders

Hemolyhc anemia Expanded initially, decline Expansion to Near-normal with age superantigens intact antiviral antibodies Inflammatory bowel disease Partly reduced CD4 Increased Ig levels and CD8 responses to virus Hemolyt~canemia Expanded initially, decline Expansion to Inflammatory bowel with age superantigens intact disease Increased Ig levels Partial reduction of T-cell expansion in oioo to antigen Wasting, granulocytosis Decreased initially decline Expansion to Absent with organ infiltration further with age superantigens intact Death by 12 weeks Increased Ig levels Absent antiviral responses Proliferative typhlitis Decreased Expansion to described in a subset superantigens delayed

Decreased; activated, memory phenotype, accumulate with age Jak3 Decreased; activated, Decreased memory phenotype, accumulate with age IL-7 Decreased Decreased IL-7Ra Nearly absent Normal IL-4 Normal

CD4+ T-cell subset alterations

Impaired IgE production

None reported


55

the activation marker CD69 is also observed. In addition to increased numbers, B-cell activity is increased, as evidenced by marked elevations in serum immunoglobulins. Older adult mice experience a progressive loss of B cells as a consequence of impaired bone marrow production; the etiology of this process is not clear. IL-2- and IL-2Ra-deficient mice are subject to two types of pathologic autoreactivity (Sadlack et al., 1993, 1994; Willerford et al., 1995). The first is a fatal, severe anemia, accompanied by markedly increased erythropoiesis in the spleen. This is likely a hemolytic anemia due to red cell autoantibodies. Only a minority of adult animals manifest anemia, but the hemolyhc process is likely present in a compensated form in the majority, since even animals with a normal red cell mass usually have increased splenic erythropoiesis and high numbers of circulating reticulocytes. Other autoantibodies, including those specific for colon tissue or DNA, can also be demonstrated (Sadlack et al., 1993; D. T. M. Leung and D. M. Willerford, unpublished observations). Older mice uniformly develop an inflammatory bowel disease, with diarrhea and wasting, that is ultimately fatal. The inflammatory process is restricted to the colon and includes infiltration with activated lymphocytes and neutrophils, mucosal ulceration, absecces of intestinal crypts, and signs of abnormal crypt regeneration. Histologically, these features resemble human ulcerative colitis. Bowel inflammation requires intestinal flora, as it is abrogated in IL-2-deficient mice raised under gnotobiotic conditions (Sadlack et al., 1993). Most or all of the regulatory abnormalities observed in IL-2- or IL-2Radeficient mice can be traced to T cells. Thus, enlarged lymph nodes and spleen, loss of B cells, and inflammatory bowel disease were abrogated in IL-24- X nude mice (Kramer et al., 199.5). Similarly, inflammatory bowel disease did not develop in IL-24- X Rag-24- mice, which lacked T and B cells, but did develop in IL-24- X JH-/- mice which lacked B cells (Ma et al., 1995a). In the B-cell lineage, the hypersecretion of immunoglobulins includes isotypes characteristically T-cell dependent: IgG1, IgG2a, IgGzb, IgA, and IgE, but not IgM or IgG3. Moreover, this abnormality is abrogated in IL-24- X n d n u mice (Kramer et al., 1995). Regulation of the peripheral lymphoid compartment by IL-NL-2R interactions is autonomous to hematopoietic cells, as the phenotype of enlarged lymph nodes and spleen occurred in Rag-24- mice receiving IL-2-deficient bone marrow. The critical regulatory function of IL-2 appears to involve prominent paracrine effects, since the lymphoid expansion in IL-24- bone marrow recipients is prevented by admixture of a 30% fraction of IL-2+/+ bone marrow (Kramer et al., 1995). The most straightforward conclusion from the phenotypes of IL-2- and IL-2Ra-deficient mice is that the major role of IL-UIL-2R interactions in

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vivo is to negatively regulate the peripheral lymphoid compartment. These negative effects include maintaining homeostasis in terms of the overall size of the secondary lymphoid tissues, as well as controlling the emergence of autoreactive clones in the periphery. These functions may involve separate mechanisms or represent progressive steps in a unitary process, as defects in homeostasis are manifest as early as 4 weeks of age in IL-2Radeficient mice, whereas autoimmune disorders usually occur at a later age. The apparent similarity in the phenotypes of IL-2- and IL-2Ra-deficient strains suggests that, for the most part, the biologic function of IL-2 is mediated through the high-affinity form of the IL-2R.

2. Peripheral Phenotype of IL-2RP-Deficient Mice Mice homozygous for a null mutation of IL-2Rp exhibit a complex phenotype involving both lymphoid and nonlymphoid hematopoietic lineages (Suzuki et al., 1995, 199713). Beginning at approximately 4 weeks of age, mice display growth retardation and signs of ill health. The lymph nodes and spleen are enlarged, with an expansion of activated T cells that are blastic and express CD69. Although analogous to what is seen in IL2- and IL-2Ra-deficient mice, the T-cell activation phenotype appears to be more dramatic in mice lacking IL-2RP. B-cell numbers are decreased in young adult mice, but display evidence of increased function, as serum levels of IgGl and IgE (but not other isotypes) are markedly elevated. Autoantibodies reacting with red blood cells, nuclear antigens, and DNA are also detected. As with IL-2- and IL-2Ra-deficient mice, B-cell numbers decline further with time. A generalized increase in granulopoiesis in the bone marrow and spleen is also observed in IL-2RP-I- mice, with granulocyte infiltration of liver and lymph nodes. IL-2RP-deficient mice die by 12 weeks of age, although a specific cause of death has not been identified. Similar to the situation in IL-2- or IL-2Ra-deficient mice, many of the phenotypic abnormalities in IL-2RP-I- mice can be traced to T cells. Depletion of CD4' T cells following the injection of anti-CD4 antibodies improves the overall health of IL-2RP-I- mice and, specifically, prevents the development of autoantibodies and depletion of B cells. Transfer of IL-2RP-I- T cells into nude mice also recapitulates the B-cell abnormalities. The disorder resulting in the accumulation of granulocytes could represent an autonomous defect of myeloid cells, as IL-2RP is expressed in the myeloid lineage, or could be secondary to aberrant regulation of T cells, which can secrete myelopoietic cytokines. The granulopoietic defect was not abrogated by depletion of CD4' T cells; however, this abnormality did develop in nude mice receiving IL-2RP-I- T cells, suggesting that this disorder may be a result of deregulated T-cell function. Inflammatory bowel disease is not described in mice lacking IL-2RP; however, these


57

animals typically die at about the age that this disorder begins to manifest in IL-2- or IL-2Ra-deficient mice. Thus, taken with the results of targeted deletion of IL-2 or IL-2Ra, the phenotype of mice lacking IL-2Rp supports the general notion that one of the major physiologic functions of IL-2R signals is to negatively regulate the peripheral lymphoid compartment. The similarities between these two groups of mice, particularly the B-ceI1 abnormalities, presumably reflect the same defect in intracellular signals normally delivered by the highaffinity IL-2R. The apparently more severe abnormalities in T-cell regulation in IL-2RP-I- inice suggest that negative regulation of the peripheral lymphoid compartment also involves additional signals delivered by IL2RP independent of the IL-2R. Thus, T-cell homeostasis appears to require at least two nonredundant signals: one delivered by the high-affinity IL2R and the other by a second receptor utilizing IL-2RP, such as the IL15R. It will be of great interest to see whether mice with null mutations in IL-15 or IL-15Ra also manifest deregulation of peripheral T cells. 3. Peripherul Consequences of yc and Jak3 De$ciency in Knockout Mice

As noted earlier, mice lacking yc or Jak3 have a severe but incomplete impairment in T- and B-cell development, indicating that while yJJak3 signals are not essential for lymphocyte differentiation, they are critical for expansion of early progenitors and/or efficient transition through ratelimiting developmental steps (Cao et d.,1995; DiSanto et d.,1995; Nosaka et aE., 1995; Ohbo et al., 1996; Park et al., 1995; Thomis et al., 1995). Peripheral lymph nodes are essentially absent in 7'- and Jak3-deficient mice; only the mesenteric lymph node is readily identified (Cao et al., 1995; Ohbo et al., 1996; Park et al., 1995; Thomis et al., 1995).The absence of peripheral lymph nodes is rather striking, as lymph nodes are found readily in Rag-2-deficient mice, which have no mature lymphocytes. This apparent difference may indicate that yJJak3 signals are required for the development of the peripheral lymph node structure, a property that could be mediated by a nonantigen receptor-bearing cell. With age, lymphocytes accumulate abnormally in the spIeen and mesenteric lymph node (but apparently not peripheral lymph nodes) of yc- and Jak3-deficient mice. Expansion of T cells is accompanied by an activated phenotype in the CD4' subset, including an increased proportion of larger cells, upregulation of CD44 and CD69, and downregulation of CD62L. Bowel lesions were reported in one strain of ?,-deficient mice, but were initially described as a proliferative typhlitis seen in association with helicobacter-like organisms and apparently distinct histologically from the condition resembling ulcerative colitis that occurs in IL-2- and IL-2Ra-deficient mice (Cao et al., 1995).Peripheral T cells in ycmice exhibit increased uptake of bromodeoxy-

58


uridine, which, considering the markedly hypoplastic thymus, indicates that the increase in mature T cells is most likely due to expansion in the periphery. In Jak3-deficient mice reconstituted with a Jak3 transgene expressed in the thymus but not in peripheral T cells, T-cell development was restored; however, the peripheral phenotype of activated T cells and lymphoid expansion was preserved, indicating that these abnormalities reflect the peripheral regulatory function of Jak3 (Thomis and Berg, 1997). Aside from the developmental abnormalities that reflect the participation of yc in multiple cytokine receptors, the peripheral T-cell phenotype of y,-deficient mice can be at least partly explained by the lack of signals delivered by the high-affinity IL-2R, underscoring the role of the IL-2R in peripheral lymphoid homeostasis. Mutation of ycor Jak3 is not, however, associated with the autoreactivity characteristic of mice lacking IL-2, IL2Ra, or IL-2RP. This could reflect the developmental abnormalities present in yc- and Jak3-deficient mice, e.g., the fact that there are relatively fewer T cells than in mice deficient in other IL-2R components. However, this explanation is undermined by the the fact that restoration of T-cell development in Jak3-deficient mice by thymic expression of a Jak3 transgene does not lead to autoimmunity (Thomis and Berg, 1997). An alternative explanation is that while IL-2R function in controlling the size of the peripheral T-cell compartment consists of negative regulatory signals, the emergence of autoimmunity (or, for that matter, the generation of normal immune responses) additionally requires positive signals delivered by other cytokine receptors that utilize yc. One candidate for such a positive effect is IL-4, which is also a growth factor for T cells. However, the combined deficiency of IL-2 and IL-4 does not reverse the autoimmune manifestations characteristic of IL-2 deficiency, demonstrating that IL-4 is not required for autoimmunity to develop (Sadlack et al., 1994). Whether other cytokines that utilize yc positively influence autoimmunity remains to be tested.

B. ROLEOF IL-2R SIGNALSIN IMMUNE FUNCTION 1. Immune Responses in IL-2- and IL-2R-Dejicient Mice Immune responses have been studied in detail in IL-2-deficient mice (Kundig et al., 1993; Schimpl and Hunig, 1994; Schimpl et al., 1992, 1994) and, to a lesser extent, in IL-2Ra-deficient mice (Van Parijs et al., 1997). Secondary antibody responses to the model T-cell-dependent antigen TNPkeyhole limpet hemocyanin were normal or enhanced in IL-2-deficient mice (Schimpl et al., 1992). Antibody responses were also assessed following vesicular stomatitis virus infection of IL-24- mice (Kundig et al., 1993), where the induction of neutralizing IgM antibodies was normal, but the kinetics of the delayed IgG response were slowed, but not dampened. This


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suggests that T-cell help for Ig class switching is dependent on IL-2 for the fastest response. CTL responses to vaccinia virus infection in IL-2deficient were found to be indistinguishable from wild-type mice, although the frequency of specific CTL was reduced threefold in IL-24- mice after infection with LCMV. The functional consequences of the latter observation were minimal, however, as local swelling at the site of inoculation was normal and LCMV-primed IL-24- mice were resistant to rechallenge with virus. However, a second group found that clearance of LCMV was delayed following infection in IL-24- mice and that expansion of CD8+ CTL in the spleen was inhibited markedly (Cousens et al., 1995). Induction of NK activity on day 3 following LCMV infection was seen in IL-24mice; however, it was reduced in magnitude three- to ninefold (Kundig et al., 1993).These studies demonstrate that humoral and cellular immune responses in IL-%deficient mice are largely intact, suggesting either that the primary role of IL-2 is not in mediating the obligatory expansion of antigen-reactive T cells during immune responses or that this function is largely redundant with other cytokines. These experiments also indicate that mice lacking IL-2 are not especially immunocompromised. How, then, can these observations be reconciled with the reports of human SCID in patients with defective IL-2 production (Weinberg and Parkman, 1990) or a null mutation in IL-2Ra (Shade et aE., 1997)?One possible explanation is suggested by the observation that older IL-24- mice fail to generate CTL responses to viral infection in vivo (Kundig et al., 1993). It could be then that immune deficiency in the absence of IL-WIL-2R signals does not directly reflect the participation of these signals in immune responses, but rather is a secondary consequence of the abnormal, expanded peripheral lymphoid compartment that develops over time and may result in the inability to mount a properly organized immune response. In contrast to the situation in IL-24- mice, immune responses to viral challenge are essentially absent in IL-2RP-deficient mice, including T-celldependent and -independent phases of the humoral response to vesicular stomatitis virus, as well as both CD4+ and CD8+ T-cell-dependent phases of the response to LCMV. These results have at least two possible interpretations. One is that amplification of immune responses in vivo is absolutely dependent on cytokine receptor signals utilizing IL-BRP, but that either IL-2R or IL-15R signals can subserve this function. Alternatively, the severe illness that manifests early in life in IL-2RP-I- mice may itself contribute to the observed immune deficiency, much as appears to be the case in older IL-2-deficient mice. 2. Immunodeficiency in Patients with Defective IL-2 or IL-2R Function Severe combined immunodeficiency in humans is a clinical syndrome involving loss of function in both cellular and humoral arms of the immune

60

BRAD H. NELSON A N D DENNIS M. WILLERFORD

response, resulting in susceptibility to infection (Rosen et al., 1995). The clinical definition of SCID encompasses individuals with developmental defects in the gerierative lymphoid organs, as well as functional defects in settings where T and B cells are present. Among the latter group of patients, several have been described with impaired secretion of IL-2 by activated T cells (Chatila et al., 1990; Doi et al., 1988; Paliwa et al., 1989; Weinberg and Parkman, 1990). Although none of these cases described a defect in the IL-2 gene, in one instance (Pahwa et al., 1989) a clinically significant improvement in immune function was reported following therapy with IL2. Roifman and colleagues described a child with clinical SCID and a lack of IL-2Ra expression due to a mutation in the IL-2Ra gene (Roifman, 1997; Sharfe et al., 1997). This patient had several features reminiscent of the phenotype of IL-2Ra-deficient mice, including lymphadenopathy, splenomegaly, and autoimmune manifestations involving the skin and gut. Thymic abnormalities were also described in this patient (see Section VI,C,3). The SCID phenotype associated with yc or Jak3 deficiency in humans is readily explained by the characteristic absence of peripheral T cells. However, a mutation in the extracellular domain of yc has been described in two patients with SCID who had normal T-cell numbers (Sharfe et al., 1997).Expression of ycwas not affected, but reduced receptor affinity for IL-2 was observed, along with deficient responses to IL-2. This mutation therefore apparently differentially affects yc participation in different cytokine receptors, permitting ye to function in the context of receptors required for lymphoid development, but with reduced activity with respect to IL-2R function. These clinical observations illustrate an important point that is perhaps underappreciated in studies of IL-2- and IL-2Ra-deficient mice: that peripheral IL-2/IL-2R interactions are required for effective host defenses. C. ROLEOF IL-2R

SIGNALS IN

T CELL GROWTH A N D SURVIVAL

IN

VWO

1. T-cell Expansion in Response to Antigen

In order to test the long-standing hypothesis that IL-2R signals mediate expansion of T cells in vivo during immune responses, the behavior of T cells after activation in vivo was examined in mice with mutations in IL-WIL-2R signaling. In the first 3-5 days following immunization with bacterial superantigens, the expansion of T cells expressing the corresponding VP segments was intact in mice lacking IL-2Ra (Willerford et al., 1995), IL-2 (Kneitz et al., 1995), and IL-2RP (Suzuki et al., 199%). In y,-deficient mice, expansion of SEB-reactive T cells was less than wildtype mice after 3 days, but reached an equivalent peak at 5 days. Using a class I MHC-restricted TCR transgene bred into the IL-%deficient

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background, Schiinpl and colleagues (Kramer et al., 1994) demonstrated that antigen-stimulated blast transformation and transition through the cell cycle in vivo did not require IL-2. These studies demonstrate that T cells can progress through the cell cycle and expand in vivo in the absence of IL-2/IL-2R signals, and indeed in the absence of signals for all lymphokines using yc, raising the question as to what other T-cell growth factors are utilized in uiuo. It is possible that a role for IL-2R is masked by the singular potency of superantigen signals in these experiments. Expansion and differentiation of T cells following exposure to antigen in vivo can be followed using the system reported by Jenkins and colleagues (Kearney et al., 1994), wherein T cells bearing a transgenic TCR are adoptively transferred into a syngeneic, nontransgenic host. Under these conditions, the TCRtransgenic T cells represent only a few percent of the normal T-cell complement, but can be followed accurately in viuo using a monoclonal antibody specific for the TCR clonotype. In addition to measuring antigen responses, this system has the advantage of examining mutant T cells in the context of a normal lymph node structure. D. T. M. Leung and D. M. Willerford (manuscript in preparation) bred the DO1l.10 TCR transgene (Murphy et al., 1990),which is class I1 MHC restricted and specific for an ovalbuminderived peptide, onto the IL-2Ra-deficient background and examined Tcell expansion in response to the antigenic peptide following adoptive transfer into normal BALB/c mice. A modest (approximately 50%) reduction in the degree of T-cell expansion was observed for IL-2Ra-deficient T cells compared with wild-type mice, suggesting that T-cell expansion following encounter with antigen may be at least partially dependent on high-affinity IL-ZR signals. Similar experiments have been performed using DO1l.10 X IL-24- T cells. In these experiments, following immunization with high doses of antigen subcutaneously, T-cell expansion was equivalent in IL-24- and IL-2+ transgenic T cells (Khoruts et al., 1998). However, when lower doses of antigen are used, IL-24- T cells exhibit impaired expansion in vivo (M. Jenkins, personal communication). Hence, the longstanding view that IL-2 amplifies immune responses by promoting T-cell expansion in vivo finds some support in these experiments. Further work with this system may help further clarify the role of IL-2R signals in immune responses and perhaps contribute to understanding the defect in host defenses in humans with abnormalities in IL-2 or IL-2Ra expression.

2. Role of IL-2R Signals in Antigen-Mediated T-cell Deletion in Vivo Lymphoid homeostasis requires that the majority of cells generated during iinniune responses undergo cell death (Sprent and Tough, 1994). Given the studies of Lenardo (1991), indicating that IL-2R signals may promote death as an outcome of TCR stimulation, it has been postulated

62


that the defect in lymphoid homeostasis in IL-2R-deficient mice reflects a relative decrease in the proportion of cells undergoing AICD following antigenic encounter (Kneitz et al., 1995; Willerford et al., 1995). The role of IL-2 and IL-2R interactions in activation-induced T-cell death has been investigated in vivo by examination of superantigen (SEB)-mediated peripheral T-cell deletion in IL-2Ra-deficient mice (Willerford et al., 1995). In these experiments, deletion of VPS' T cells 10 days following immunization with SEB was partially impaired. Similar results have been obtained for the CD4+ T-cell subset in mice deficient in IL-2 (Kneitz et al., 1995) and for T cells in mice lacking yc (Nakajima and Leonard, 1997). These results would appear to support the notion that lymphoid accumulation in IL-2R-deficient mice could result from defective AICD following antigen exposure. However, one important caveat in interpreting these experiments is that the subset distribution of T cells in the mutant mice was biased toward a memory phenotype prior to SEB immunization, which could affect sensitivity to AICD in vivo. More recent work has provided data that conflict with the hypothesis that IL-2R signals are required for efficient AICD in vim. In contrast to the foregoing results, SEB-mediated T-cell deletion is intact in young mice lacking IL-2RP (Suzuki et al., 1997b). The role of IL-2R in antigenmediated AICD in vivo has also been investigated using IL-2Ra-deficient mice bearing the class I1 MHC-specific DO1l.10 TCR transgene (Murphy et al., 1990), where subcutaneous administration of an antigenic peptide resulted in the efficient deletion of peripheral T cells after 8 days, with no differences compared with wild-type littermates. Antigen-induced peripheral deletion was also normal in D011.10+, IL-2Ra-/- T cells that were transferred adoptively into normal BALB/c mice (D. T. M. Leung and D. M. Willerford, manuscript in preparation). These studies suggest that the IL-2R may not have an indispensable role in regulating AICD following the activation of T cells by high-affinity antigens in vivo. It is probably fair to say that experiments done to date have not exhausted the physiologic diversity of antigen dose, receptor affinity, and site of encounter with the immune system, so the precise in vivo role of IL-2R signals in AICD following T-cell stimulation with antigen remains incompletely defined. AICD in T cells is mediated through activation of Fas, via interaction with FasL, although data suggest that TNFa, and perhaps other TNF family members, may have overlapping roles in this process (see Section IV,C). Therefore, an important question regarding the lymphoproliferative defects in IL-2 and IL-2R-deficient mice is to what extent these are mediated by abnormalities in Fas/FasL regulation. Expression of Fas and its upregulation following TCR stimulation is normal in mice deficient in IL-

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2 or IL-2Ra (Kneitz et al., 1995; Van Parijs et al., 1997). IL-2 has also been reported to contribute to the upregulation of FasL on activated T cells (Suda et al., 1995). Moreover, because T cells are differentially sensitive to cell death mediated by Fas ligation, depending on their state of activation, IL-2R signals could also affect the Fas pathway by modifying the signaling apparatus downstream of the Fas receptor. One possible mechanism for such an effect is the upregulation of c-myc by IL-2R signals, which may increase the cellular sensitivity of Fas-mediated apoptosis (Huber et al., 1997). In support of this, activated T cells derived from IL-2 and IL-2Radeficient T cells are resistant to killing by antibody-mediated cross-linking of Fas (Kneitz et al., 1995; Van Parijs et al., 1997).Whether these observations reflect a specific effect of IL-2 receptor signals on the Fas signaling pathway or simply reflect differences in T-cell proliferation in vitro in the absence of 1L-2R signals is not yet clear (Boehme and Lenardo, 1993; Zhu and Anasetti, 1995). In contrast to the preceding results, Fas sensitivity was found to be normal in T cells derived from IL-2RP-deficient mice (Suzuk~et al., 1997b). One approach to evaluating the potential role of the Fas pathway in the disordered T-cell homeostasis in mice lacking IL-2 or IL-2Ra is to compare the phenotypes of these mutations with mice deficient in Fas or FasL. Superficially, both types of mutations are associated with enlarged lymph nodes and autoimmunity. However, the peripheral lymphoid expansion in IL-2- and IL-2Ra-deficient mice is general, involving more or less normal proportions of all lymphoid subsets, indicating a global defect in lymphoid homeostasis. In contrast, mice with genetic defects in the Fas pathway acquire large lymph nodes specifically because of the accumulation of an unusual CD4-CD8- T-cell subset (Cohen and Eisenberg, 1991),which is not increased in mice lacking IL-2 or IL-2Ra. Although exibiting strainspecific variation, the spectrum of autoimmune manifestations in Fas pathway-defective mice predominantly consists of autoantibody and immune complex &orders (Cohen and Eisenberg, 1991). While IL-2- and IL-2Ra-deficient mice develop a hemolytic anemia, which is presumably antibody mediated, other antibody or immune complex disorders seen in Fas/FalsL-deficient mice have not been described. Finally, the inflammatory bowel disease, which is a major component of the phenotype in IL2- and IL-2Ra-deficient mice, is not seen in Fas pathway-defective mice. Thus, a phenotypic cornparison indicates that the disrupted regulation of the peripheral lymphoid compartment in IL-2- and IL-2Ra-deficient mice is not due primarily to a defect in the Fas pathway, although a contribution of impaired Fas-mediated AICD to some of the abnormalites in these mice cannot be excluded.

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IX. Summary and Conclusions

Studies of the biology of the IL-2 receptor have played a major part in establishing several of the fundamental principles that govern our current understanding of immunology. Chief among these is the contribution made by lymphokines to regulation of the interactions among vast numbers of lymphocytes, comprising a number of functionally distinct lineages. These soluble mediators likely act locally, within the context of the microanatomic organization of the primary and secondary lymphoid organs, where, in combination with signals generated by direct membrane-membrane interactions, a wide spectrum of cell fate decisions is influenced. The properties of IL-2 as a T-cell growth factor spawned the view that IL-2 worked in vivo to promote clonal T-cell expansion during immmune responses. Over time, this singular view has suffered from increasing appreciation that the biologic effects of IL-2R signals are much more complex than simply mediating T-cell growth: depending on the set of conditions, IL-2R signals may also promote cell survival, effector function, and apoptosis. These sometimes contradictory effects underscore the fact that a diversity of intracellular signaling pathways are potentially activated by IL-2R. Furthermore, cell fate decisions are based on the integration of multiple signals received by a lymphocyte from the environment; IL-2R signals can thus be regarded as one input to this integration process. In part because IL-2 was first identified as a T-cell growth factor, the major focus of investigation in IL-2R signaling has been on the mechanism of mitogenic effects in cultured cell lines. Three critical events have been identified in the generation of the IL-2R signal for cell cycle progression, including heterodimerization of the cytoplasmic domains of the IL-2RP and yc chains, activation of the tyrosine kinase Jak3, and phosphorylation of tyrosine residues on the IL-2Rp chain. These proximal events led to the creation of an activated receptor complex, to which various cytoplasmic signaling molecules are recruited and become substrates for regulatory enzymes (especiallytyrosine kinases) that are associated with the receptor. One intriguing outcome of the IL-2R signaling studies performed in cell lines is the apparent functional redundancy of the A and H regions of IL2RP, and their corresponding downstream pathways, with respect to the proliferative response. Why should the receptor complex induce cell proliferation through more than one mechanism or pathway? One possibility is that this redundancy is an unusual property of cultured cell lines and that primary lymphocytes require signals from both the A and the H regions of IL-2RP for optimal proliferative responses in vivo. An alternative possibility is that the A and H regions of IL-2RP are only redundant with respect to proliferation and that each region plays a unique and essential

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role in regulating other aspects of lymphocyte physiology. As examples, the A or H region could prove to be important for regulating the sensitivity of lymphocytes to AICD or for promoting the development of NK cells. These issues may be resolved by reconstituting IL-2RP -/- mice with Aand H-deleted forms of the receptor chain and analyzing the effect on lymphocyte development and function in uivo. In addition to the redundant nature of the A and H regions, there remains a large number of biochemical activities mediated by the IL-2R for which no clear physiological role has been identified. Therefore, the circumstances are ripe for discovering new connections between molecular signaling events activated by the IL-2R and the regulation of immune physiology. Translating biochemical studies of IL-2R function into an understanding of how these signals regulate the immune system has been facilitated by the identification of natural mutations in IL-2R components in humans with immunodeficiency and by the generation of mice with targeted mutations in these genes. Efficient lymphopoiesis requires the yc chain, and mutations in yc are responsible for human XSCID, an important cause of congenital immunodeficiency. Boys with XSCID and mice with targeted disruption of the ycgene share defects in early T-cell development due to the participation of yL in multipte cytokine receptors. Signals from IL-7R are probably most important at this stage, but other 7,-containing cytokine receptors may also be involved. In the peripheral lymphoid compartment the most striking abnormality in mice lacking IL-2, IL-2Ra, or IL-BRP is an impaired ability to control the overall size of the secondary lymphoid tissues. The immune system is not encompassed by a physical capsule, as are other solid organs, and is subject to cellular fluxes from continuous production of cells by the primary lymphoid organs and the expansion of lymphocytes in the periphery during immune responses. Yet, under normal circumstances, the overall size of the peripheral lymphoid compartment is strictly controlled over time. Thus, one of the most important physiologic functions of the IL-2R in vivo appears to be the homeostatic regulation of lymphoid tissues. IL-2R signals may exert a negative regulatory influence by promoting AICD following encounter with antigen, thus limiting clonal expansion and participating in the termination of immune responses. However, data indicate that MHC (and presumably TCR-)-dependent signals may also control T-cell survival independent of encounter with antigen. It is worth considering whether such survival signals could be counterbalanced by IL-2R-dependent negative regulatory influences. IL-2R defects in mice also lead to the development of fatal inflammatory disorders, which are accompanied by autoantibodies, suggesting that IL-2R signals play a role in maintaining peripheral immune tolerance. One proposed mechanism for this effect is in preventing

66


the emergence of autoreactive clones during immune responses through IL-2R-mediated promotion of AICD, although the in vivo evidence for such an effect is conflicting. Thus, the pathogenic mechanism of inflammatory disorders in IL-2R-deficient mice is not well understood. Nevertheless, the fact that IL-2R signals are now known to be required to suppress these conditions may help illuminate the causes of similar conditions in humans. Efforts by many investigators to define the biologic role of the IL-2R now span more than 20 years and represent a substantial fraction of the scientific output of the immunology community. The identification of IL2R mutations in humans and the creation of such mutations in mice have provided some surprising and puzzling insights into the function of IL2R signals in vivo. These phenotypes provoke a necessary check on the assumptions that underlie experiments performed in cell systems and provide a more complete context in which to interpret results. More importantly, these complex phenotypes in vivo have stimulated a broader consideration of signals delivered by the IL-2R and will hopefully lead to a fuller understanding of the diversity of intracellular pathways that are utilized by this model cytokine receptor.

ACKNOWLEDGEMENTS The authors thank Phil Creenberg, Ken Kaushansky, Robert Abraham, Averil Ma, Raymond Doty, and Chaim Roifman for reading the manuscript and making many helpful suggestions. We are grateful to Drs. Roifinan and Abraham, along with Warren Leonard, Marc Jenkins, Steve Levin, and Andy Farr for sharing unpublished data. D.M.W. is supported by NIH Grants AI-01173 and AI-41051.

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Watson, J. D , Morrissey, P. J., Namen, A. E., Conlon, P. J., and Widmer, M. B. (1989). J Inmunol 143, 1215-1222. Webb, S., Morns, C., and Sprent, 1. (1990). Crdl 63, 1249-1256. l . 1979Wei, S., Blanchard, D., Liu,&J..Leo!lard, W., and Djeu, J. (1993).J I n ~ m u t ~ o150, 1987. Weinberg, K., and Parkman, R. (1990). N . Engl. J . Mecl. 322, 1718-1723. Weiss, A,, and Imboden, J. (1987). Arlo. Itntnunol. 41, 1-38. Welham, M. J., Duronio, V., Leslie, K. B., Botwell, D., and Schrader, J. W. (199421).J. Biol. Chem. 269, 21165-21176. Weham, M. J., Duronio, V., and Schrader, J. W, (1994b).J . Biol. Cheni. 269, 586555873, White, E. (1996). Genes Deo. 10, 1-15. White, M. R., and Jahn, C. R. (1994).J . B i d . C h e m 269, 1-4. Willerford, D. M., Chen, J., Ferry, J. A,, Davidson, L., Ma, A., and Aft, F. W. (1995). btiinunity 3, 521-530. Willerford, D. M., Swat, W., and Alt, F. W. (1996). Cum. Opin. Genet. Deo. 6, 603-609. Williams, N . S., Moore, T. A,, Schatzle, J. D., Puzanov, I. J., Sivakumar, P. V., Zlotnik, A,, Bennett, M., and Kumar, V. (1997).J. Exp. Med. 186, 1609-1614. Witthuhn, B. A,, Silvennoinen, O., Miura, 0..et cil. (1994). Nature 370, 153-157. Wong, B., Arron, J., and Choi, Y. (1997).J , Exp, Med. 186, 1939-1944. Xia, Z., Dickens, M., Raingeaud, J., Davis, R. J., and Creenberg, M. E. (1995).Science, 13261331. Yang, E., and Korsmeyer, S. J. (1996). Blood 88, 386-401. Yoshimura, A,, Ichihara, M., Kinjyo, I., Moriyama, M., Copeland, N. G., Gilbert, D. J., Jenkins, N . A,, Hara, T., and Miyajima, A. (1996). EMBO J. 15, 1055-1063. Yoshimura, A,, Ohkubo, T., Kiguchi. T., Jenldns, N. A,, Gilbert, D. J., Copeland, N. G., Hara, T., and Miyajima, A. (1995). EMBO J , 14, 2816-2826. Zheng, L., Fisher, G., Miller, R. G., Peschon, J., Lynch, D. H., and Lenardo, M. J. (1995). Nature 377, 348-351. Zhu, L., and Atlasetti, C. (1995).J . Zmt~unol.154, 192-200. Zhu, X., Suen, K.-L., Barbacid. M., Bolen, J. B.. and Fargnoli, J. (1994). J.Biol.Chern. 269, 5518-5522. Zoh, H., Weedon, H., Thompson, C., Fung, M., Ingley, E., and Hapel, A. (1991). Ittiniunology 1991, 167-173. Zubler, R. H., Lowenthal, J. W.. Erard, F., Hashimoto, N., Devos, R., and Macdonald, H. R. (1984).J. Exp. Med. 160, 1170-1183. Zuniga-Pflucker, J. C., and Kruisbeek, A. M . (1990)./. Ioimunol. 144, 3736-3740. Zuniga-Pflucker,J. C., Smith, K. A,, Tentori, L., Pardoll, D. M., Longo, D. L., and Kruisbeek, A. M. (1990). Dea. Zrnmtinol. 1, 59-66. Zurawski, S. M., Imler, J.-L., and Zurauaki, C. (1990). EMBO J. 9, 3899-3905. This article was accepted for publication on February 4, 1998.


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interleukin-12: A Cytokine at the interface of inflammation and Immunity GlORGlO TRlNCHlERl The Wshr fflstiluk of Anubmy und Biokgy, Phi/ude$hio, Pennsybuniu

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I. Introduction

Adaptive immunity in higher organisms is an extremely specialized mechanism of resistance that is characterized by fine recognition of specific antigens and immunological memory. Thus, it represents a much more effective and specialized mechanism of defense against infections or pathologicd alteration than innate and natural resistance, which are present even in lower organisms and are characterized by a lack of memory and specificity for antigen. However, because adaptive immunity is based on the clonal expansion of B or T lymphocytes carrying the receptor for a specific antigen, it becomes effective only a few days after exposure to the antigen, especially in primary infections. Thus, even in higher organisms with a developed adaptive immune system, the first line of defense against primary infections is provided by the effector cells and mechanism of innate resistance. For example, production of antiviral substances such as interferon and activation of macrophages and natural killer ( N K ) celIs is observed within 1 or 2 days of viral infection and efficiently contributes in controlling the infection, although complete eradication of the infection usually requires the production of specific antibodies and the generation of cytotoxic T cells, which occur about 1 week after initial infection. The relationship between innate resistance and adaptive immunity is not only temporal, but also profoundly interactive via a complex crosstalk between inflammatory cells [including phagocpc cells, other antigenpresenting cells (APC), and N K cells] and the antigen-specific T and B lymphocytes (1). This regulatory cross-talk underlies the phenomenon of inflammation and is mediated by both direct cellular interactions and soluble factors, including cytokines and other pharmacological mediators. The role of cytokines produced by lymphocytes, i.e., lymphokines, in inflammation and in the immune response is well characterized, and the physiologic significance of lymphokines such as interleukin-2 ( IL-2), interferon-? (IFN-.)I), and IL-4 has been analyzed in depth. Phagocytes act as APC and as accessory cells for lymphocyte responses by providing membrane-bound costimulatory molecules (e.g., B7 antigen binding to the CD28 receptor on T cells) (2) and by secreting iminunoregulatory cyto83

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kines. Although cytokines such as tumor necrosis factor (TNF-a), IL-1, IL-6, and IL-8 that are derived in part from phagocytic cells have been studied extensively with respect to their role in inflammation, shock, and tissue damage, much less is known about their ability to regulate lymphocyte functions. Thus, many of the regulatory functions of accessory phagocytic cells on the immune response mediated by T and B lymphocytes remain unexplained. However, the primary role of phagocytw cells as a first line of defense against bacterial and parasitic infections implies that the ability of these cells to direct the generation of subsequent immune response is instrumental in determining the success or failure of the organism’s mechanisms. The interaction between phagocytic cells and lymphocytes is not unidirectional, and lymphocytes produce factors that are potent regulators of phagocytic cell functions, with both enhancing and suppressing effects. These factors include IFN-?, (the most potent enhancer), granulocyte/macrophage colony-stimulating factor (GM-CSF), macrophageCSF (M-CSF), IL-4, IL-10 (the most effective inhibitor), and others (3-5). The ability of bacterial products to activate phagocyhc cells has been utilized widely in the attempt to boost the immune system, by inducing an immune or inflammatory response against tumors or by providing an adjuvant effect in vaccination. Pure antigens are notoriously poor immunogens unless they have a complex and repetitive structure, and for the generation of efficient humoral or cellular immunity, they should be mixed with adjuvants to facilitate presentation of antigen to the immune system. Although adjuvants differ chemically, they are often irritants that induce an inflammatory response, and bacterial preparations have often been used effectively for this purpose. Furthermore, there have been sporadic observations since the 1700s of certain cancer patients undergoing bacterial infections and a concomitant remission of their malignant growth (6). In 1893 Coley compiled several observations of tumor regression associated with bacterial infections, primarily with streptococcus-induced erysipelas (7). He initiated treatment of cancer patients, first unsuccessfully, with Streptococcus pyogenes and then, in a large number of patients, with S. pyogenes in association with the gram-negative bacterium Bacillus prodigi osus (Serratia marcexens) (7,s).Although his results are difficult to evaluate by modern standards for clinical trials, Coley observed a high proportion of partial and even complete remissions or cures, especially in the case of soft tissue sarcomas. Among the most plausible explanations for those therapeutic benefits are the antitumor effects of infection-induced hyperthermia and the secretion of cytokines, of which TNF is thought to play a major role (8,9). However, despite the dramatic curative effects of TNF, alone or in association with IFN-7, on sarcomas when used at extremely high concentrations in isolated limbs, it is difficult to envision that such a

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highly toxic molecule, acting alone by a systemic effect, could induce the antitumor response associated with Coley's toxin treatments or with spontaneous infection in certain cancer patients (8). The immune response to infectious agents and to nominal antigens is often characterized by a dominance of either cell-mediated or liumoraltype effector mechanisms ( l o ) ,which has been attributed to a dichotomy in the cytokine production pattern of Th CD4+cells (11).T h l cells produce IL-2, IFN-y, and lyinphotoxiii (LT) and favor cell-mediated immunity, delayed-type hypersensitivity (DTH), niacrophage activation, and production of opsonizing antibodies. Th2 cells produce IL-4, IL-5, IL-6, and IL10 and favor humoral responses, production of IgE and IgA, and activation of eosinophils and basophils. The differentiation of Th cells toward a Th1 or Th2 phenotype occurs early during an immune response and is influenced by many interrelated factors, including the nature and the concentration of the antigen, the anatomical localization of the immune response, the nature of the APC, and the inflammatory cytokine milieu at the site of the immune response (12). The dichotomy in the cytokine production pattern is not limited to CD4+ Th cells, but is also clearly demonstrated for CD8' T cells (13-16), for T cells with y6 T-cell receptor (TCR) (17), and, to a certain extent, for NK cells (18). The discovery of natural killer cell stimulatory factor (NKSF) or IL-12 (19-22) has, at least in part, provided an explanation for some of the immunoregulatory functions of phagocytic cells and APC, including their ability to direct Th cell differentiation, and may represent the missing link in the cross-talk between phagocytic cells and lymphocytes. IL-12 is produced by phagocybc cells, B cells, dendritic cells, and possibly other accessory cells and acts on T cells and NK cells by inducing proliferation and production of cytokines, especially IFN-y, and by enhancing generation and activity of cytotoxic lymphocytes. Acting directly or indirectly through generation of other cytokines or a cascade of cellular interactions, IL-12 is a key factor in the induction of T-cell-dependent and -independent activation of macrophages, generation of T helper type 1 (Thl) cells, generation of cytotoxic T lymphocytes, suppression of IgGl and IgE production, induction of organ-specificautoimmunity, and resistance to bacterial and parasitic infections as well as to tumors. Treatment of animals with recombinant IL-12 has been shown to potentiate the immune response against a large variety of infectious agents, to have a potent antitumor effect, and to act as a potent vaccine adjuvant inducing botli cellular and humoral immunity. These activities of endogenous and exogenous IL- 12 have generated much interest in its study, with rapid progress in our understanding of the immunobiology of this cytokine and in pursuing its use in clinical trials for a variety of pathological conditions.

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II. 11-12 Molecule and Its Genes

A. DISCOVERY, PURIFICATION, A N D CLONING OF IL-12

NK cell stimulatory factor, later named IL-12, was originally identified as a factor secreted by Epstein-Barr virus (EBV)-transformed human Bcell lines (BCL) that mediates several biological activities on human T and NK cells, including induction of IFN-7 production, enhancement of NK cell-mediated cytotoxicity, and cornitogenic effects on resting T cells (19). Human BCL can facilitate the growth and expansion of NK and T cells (23-25), and coculture of human thymocytes with BCL was reported to induce the production of IFN-7 (26). Because BCL produce a variety of cytokines, Kobayashi et nl. (19), while purifying lymphotoxin and other cytokines affecting the formation of hematopoietic colonies (27) from the EBV-transformed BCL supernatant fluid, investigated whether the effects of BCL on NK and T cells were mediated by a soluble factor(s) and identified a cytokine, then termed NKSF, which mediated induction of IFN-7, mitogenesis, and enhancement of cytotoxicity in T and NK cells. NKSF was purified to homogeneity from the conditioned medium of the phorbol diester-stimulated RPMI-8866 EBV-transformed BCL. Unlike any other cytokine, NKSF was shown to have a heterodimeric structure, composed of covalently linked chains designated p40 and p35 based on their apparent molecular weight (19). Purified NKSF (9200-fold enriched) exhibited its biological activities at concentrations in the range of 0.1 to 10 pM (19). The genes encoding the two polypeptide chains of NKSF were cloned on the basis of partial amino acid sequences of several peptides obtained from the purified proteins, and biologically active recombinant IL-12 was produced in eukaryotic cells transfected with the cDNA for both NKSF chains (21). The cytotoxic lymphocyte maturation factor (CLMF) was later identified in the conditioned medium of the NC37 cell line, an EBV-transformed BCL, on the basis of its ability to synergize with IL-2 in inducing the generation of lymphokine-activated killer (LAK) cells and to induce proliferation of human phytohernagglutin (PHA)-activated T-cell blasts (20). Purification and cloning of the genes encoding CLMF showed that NKSF and CLMF were the same cytokine (19-22), and the unifying term of IL-12 is now universally accepted.

B. IL-12 ~ 4 S0U B U N I T The gene encoding the IL-12 p40 subunit of the 70-kDa heterodimer has been mapped to human chromosome 5q31-q33 (28) and to a syntenic region in mouse chromosonie 11 (29-31), in a region containing genes encoding several cytokines and cytokine receptors, including IL-4. However, the mouse IL-12 p40 gene has been positioned 9.3 cM proximal to

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the IL-4 gene and thus the IL-12 gene appears to be outside the region of closely linked cytokine genes (29). The human p40 gene is composed of eight exons and seven introns (32; S. Wolf, personal communication), similar to the mouse p40 gene (30, 31). It contains a single long-open reading frame encoding a 328 amino acid polypeptide with a 22 amino acid long hydrophobic signal peptide and a characteristic cleavage site immediately preceding the N-terminal sequence of the natural p40 protein (21, 22). Tlie p40 cDNA also contains a relatively long (1.32 kb) 3’untranslated sequence that includes one copy of the Alu repetitive sequence element and multiple copies of ATTTA mRNA destabilizing sequence common to many cytokine and protooncogene inRNAs (21). Tlie mature protein (calculated M , 34,700; pZ 5.4) contains 10 cysteine residues, four consensus sequences for asparagine-linked glycosylation, and one theoretical heparin-binding site (21,22). Immunoprecipitation or Western blotting of both natural and recombinant IL-12 p40 reveals heterogeneity from -36 to more than 40 kDa, with at least two dominant bands (33; G. Carra, personal communication). Treatment with N-glycosidase or chemical deglycosylationwith trifluorometlianesulfonic acid eliminated the heterogeneity and reduced the molecular inass to yield and single band of 36 kDa. Overall, these results indicate that the p40 subunit is composed of -10% N-linked carbohydrates, with a heterogeneity in the extent of glycosylation and no evidence of O-linked oligosaccharides (33). Of the four possible N-glycosylation sites, two were analyzed in sequenced peptides from the natural human IL-12, and Asnzoobut not AsnIo3was found to be N-glycosylated (33). The intracliain disulfide pairing of cysteine residues has been determined (34) and is shown in Fig. 1. Eight cysteine residues are involved in intrachain binding, whereas Cysli7is involved in is not paired the interchain binding with Cysi, of the p35 subunit. with any other cysteine in IL-12, but it was determined to be cysteinylated (disulfide bonded with a cysteine) and to contain thioglycolate (paired with the sulfur in thioglycolic acid) (34). Cysteinylation was demonstrated previously in other proteins, but IL-12 is the first protein in which thioglycolation is known to be involved. C. IL-12 ~ 3 SUBUNIT 5 The gene encoding IL-12 p35 was mapped on human chromosome 3p123q13.2 (28) and in the mouse, to a syntenic region on murine chromosome 6 in one study utilizing fluorescence in situ hybridization (31),but on mouse chromosome 3 using backcross analysis in another study (30). The gene structure of the human IL-12 p35 gene has not been reported, but in the mouse it consists of either eight (30) or seven (31)exons. This discrepancy appears to rest in the multiple transcription initiation sites on the mouse

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FIG.1. Intrachain and interchain disulfide bonds in the human IL-12 p70 heterohmer. The chromosome mapping of the two genes encoding the p40 and p35 chains is also indicated. Cysln on the p40 subunit is involved in the interchain binding with Cys7, of the p35 subunit; Cyseseis not paired with any other cysteine in IL-12, but it is cysteinylated and contains thioglycolate (34).

p35 gene, as determined by primer extension analysis (31) and by rapid amplification of cDNA ends (30), and as further suggested by the isolation of murine p35 cDNA with alternative 5’-untranslated regions (30, 35). The nucleotide sequence of human p35 cDNA isolated from human BCL contains a single long open reading frame encoding a 253 amino acid polypeptide. This sequence contains two potential translation initiation codons (residues 1 and 35) 5’ to the N-terminal sequence determined from the natural p35 protein (21). The upstream translation initiation codon is maintained in p35 genes in nonhuman primates (36) and in pigs (GenBank accession number SSVO8317), but not in the murine p35 (22) or in several other mammals. The sequence initiated from the second methionine encodes a typical hydrophobic signal peptide (residues 35-56) with a consensus cleavage site immediately adjacent to the N-terminal sequence of the mature p35 protein. The hypothetical 34 amino acid sequence beginning with the methionine at residue 1 is less hydrophobic and includes several basic residues (21). Based on sequence data, together with information from other species lacking the upstream initiation site and transfection data using cDNA lacking the methionine at residue 1, it was concluded that the second methionine is sufficient for expression of the functional IL-12 p35 subunit (21). However, other sequences similar to the sequence 1-34 in the IL-12 p35 are found linked to signal peptides of membrane-associated proteins, raising the possibility that this sequence may be involved in generating a membrane form of p35; the possible

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expression of IL-12 as a membrane-bound forin in both human and murine macrophage cell lines has been suggested in an isolated study using cytofluoriinetric staining with anti-IL-12 antibodies (37). Immediately upstream of the first methionine of the p35 cDNA isolated from BCL, there is a possible TATA box that in the mouse gene has been suggested to be part of an ancestral IL-12 p35 promoter (31).However, the picture now emergmg is that although the major transcription initiation site in human BCL is upstream of that TATA sequence and is reflected in the published cDNA sequences (21, 22), other physiological IL-12-producing cells, e.g., IFN-.)I- and LPS-activated human nionocytes, predominantly use a transcription site downstream of the TATA box, generating a shorter mRNA that includes only the second methionine (M. Hayes, personal cominunication; X. Ma and G. Trinchieri, unpublished observation). The functional significance of these alternative transcripts in different cell types remains unknown. The mature IL-12 p35 peptide is 197 amino acids long (calculated M , 22,500; pZ 6.5) and contains seven cysteine residues and three consensus N-linked glycosylation sites. The 3’-untranslated 450-bp cDNA contains multiple copies of the ATTTA mRNA-destabilizing sequences. Like p40, p35 subunits appear on gels as a heterogeneous band, possibly due to differential glycosylation. Results from chemical and enzymatic deglycosylation suggest that the p35 subunit comprises -20% carbohydrates, 40% of which are 0 linked (33).

D. IL-12 FROM NONHUMAN SPECIES The two IL-12 genes have been cloned in several other mammalian species. The IL-12 p40 and p35 genes from nonhuman primates (rhesus macaque, pigtailed macque, and sooty mangabey) showed -96% homologywith the human genes (36). However, a Val-Ser-Leu at position 27-29 of mature human IL-12 p35 is replaced by a Gln-Pro-Pro sequence in the p35 inolecules of nonhuman primates (36). Such a change probably generated conformation and immunogenicity differences that might underlie the production of antihuman IL-12 antibodies in primates injected for 15-20 days with human recombinant IL-12 (38,39). Comparison of the sequence of the murine IL-12 subunits with their human counterparts revealed that die p40 subunits are more highly conserved than the p35 subunits (70 and 60% homology, respectively) (35).Although human IL12 binds to the mouse IL-12 receptor, it is not active on murine cells, whereas inurine IL-12 has biological activity on both human and mouse lymphocytes (35).A hybrid heterodimer consisting of murine p35 and huinan p40 was also biologically active on both human and mouse cells, whereas the combination of human p35 and mouse p40 was completely

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inactive on murine cells, indicating that the inability of human IL-12 to act on murine cells is largely determined by the ~ 3 subunit ~ 5 (35). Five residue changes in three discontinuous sites of the murine p35 molecule eliminate bioactivity on mouse but not on human cells, suggesting that these residues are important elements in determining the species specificity of IL-12 (40). Only a partial sequence is available for rat IL-12 (41). Complete sequences for both chains have been reported for cat (42, 43; GenBank accession numbers Y07762, Y07761), red deer (g1223907, g1223905),woodchuck (g1262373,g1262371),cow (44, g555795, g555917), and pig (45, g984510, g927204), and sequences for the p40 chain are available for goat (g2253433) and sheep (g2199555) IL-12.

E. IL-12 HETERODIMERS AND ~ 4 MONOMERS 0 AND HOMODIMERS Transient transfection of COS cells or stable transfection of CHO cells with either p40 or p35 cDNA induces secretion of the respective IL-12 chains; cotransfection with both cDNAs in the same cells is required for secretion of the biologically active p70 form of IL-12 (21, 22). Unlike the cells transfected with p35 cDNA, primary cells or cell lines have never demonstrated production of the free p35 chain (46), raising the possibility that p35, in the absence of p40, is not secreted spontaneously from cells and that the apparent secretion from transfected cells is due to a release subsequent to cell death and lysis. Consistent with this interpretation is the fact that the majority of p35 protein is found to be cell associated in p35 cDNA-transfected cells (S. Wolf, personal communication). However, p40 cDNA-transfected cells produce large amounts of p40 protein not associated with p35, and BCL as well as other physiological producer cells of IL-12 produce the p40 chain in excess from severalfoldto more than 100fold over the production of the biologically active p70 heterodimer (20,46). The report by Mattner et al. (47) that supernatant fluid from murine IL-12 p40-transfected cells inhibited the biological activity of the murine IL-12 p70 heterodimer raised much interest in determining whether the free p40 chain might be a physiologic antagonist of IL-12 activity. Analysis of the p40 protein produced by p40 cDNA-transfected COS cells revealed that about 70% of the subunit was secreted as monomers and the remaining 30% as 80-kDa homodimers which, under reducing conditions, dissociated into 40-kDa species, suggesting that the human p40 homodimers are covalently linked (48). Peptide analysis confirmed that the 80-kDa molecules were dimers of the 40-kDa forms (48),but because analysis was performed under reducing conditions, the site of the hypothesized covalent linkage was not determined. The p40 hoinodimers competed for the binding of '251-labeledIL-12 to KIT225/K6 human T cells expressing the IL-12 receptor (IL-l2R), with ICs0values 5- to 10-fold higher than that of the hetero-

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dimers. Unlike the homodimers, the p40 monomers inhibited '251-labeled IL-12 binding only at concentrations at least 100 times higher than that of the heterodimer and never reached complete inhibition (48).The human p40 homodimers inhibited the proliferation of human PHA-activated Tcell blasts induced by 0.2 n g h l of IL-12 with an ICjo of -50 ng/ml, i.e., a -250-fold excess of homodimer was required for 50% inhibition (48). Chinese hamster ovary (CHO) cells stably transfectecl with murine p40 cDNA also produce monomeiic and homodimeric forms of p40 protein (49). Peptide analysis demonstrated that the murine homodimer arises from formation of a single intermolecular bond at CYS,,~, the same residue used in the p70 heterodimer for the interchain disulfide bond with the p35 chain, whereas in the monomeric p40, this cysteine is capped by cysteinylation (49). As with human p40, the murine p40 homodimer was 100-fold more efficient than the monomers in inhibiting IL-12 receptor binding or biological functions; however, unlike the poor inhibition of IL-12 function observed with human p40 homodimers, the mouse homodimer was equivalent to the p70 heterodimer in competing for IL-12 binding to the mouse cellular receptor and induced a 50% reduction in biological functions (proliferation, IFN-.)Iinduction, and NK cell activation) at concentrations approximately equivalent to those of IL-12 in the assay (47). A subsequent study (50) confirmed the highly inhibitory activity of the murine p40 homodiiner, but only with an IC5{,requiring a 33-fold molar excess of the homodimer over the heterodimer. Because the mouse p40 homodimer was able to inhibit IL-12 biological function at very low concentrations without mediating any detectable biological activity, it was of much interest that approximately 20% of the circulating mouse p40 in vivo, produced either constitutivelyor induced by lipopolysaccharide (LPS) injection, was present as a homodimer, as demonstrated by gel filtration and by SDS-PAGE analysis under reducing or nonreducing conditions (50). Moreover, injection of mice with 40 pg of recombinant murine p40 homodimer significantly inhibited the endotoxin-induced 1FN-y production (50).The ability of the IL-12 p40 homodirner to block IL-12 action in vivu has been extended to suppression of Thl responses and DTH (51). Overall, data argue in favor of the role of the p40 homodimer as a physiological antagonist of IL-12 action in vivo in the mouse, perhaps being produced at later times during an inflammatory response and thus contributing to the extinction of IL-12 biological effects (52). However, this is most likely not the case in humans. The human IL-12 p40 homodiiner has only a modest ability to compete for IL-12 biological activity (48), even considering that human IL-12 p40 may be secreted in vitru and in vivo at concentrations up to 100 times higher than those of the IL-12 p70 heterodiiner. Furthermore, careful analysis of the stoichiometry of the p40

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and p35 chains in the -40- and -70-kDa peaks of IL-12 produced by IFN-y- and LPS-stimulated human monocytes revealed no significant amounts of covalently linked p40 homodimer (G. Carra, F. Gerosa, and G. Trinchieri, unpublished results). Thus, the role of p40 homodimers as a physiological antagonist of IL-12 may represent a species difference between mice and humans. F. IL-12 STRUCTURE The primary amino acid sequence of the IL-12 p35 chain indicates an a-helix bundle structure, similar to most cytokines (40, 53). Comparison of the p35 amino acid sequence with those of IL-6 and G-CSF showed that many of the amino acid positions conserved between these two cytokines are also conserved in IL-12 p35 (54). Interestingly, three leucine zipper motifs located near the N terminus of the human p35 molecule are conserved in other species (42); whether these motifs are involved in binding of p35 to molecules other than p40 remains to be determined. The p40 sequence is not homologous with any other known cytokine, but rather belongs to the hematopoietic cytokine receptor family, which is characterized by four cysteines and one tryptophan in conserved positions in the extracellular portions and by a WSXWS motif (55).The p40 sequence has significant sequence homology with the extracellular portions of the IL-6 receptor and the ciliary neurotrophic factor (CNTF) receptor (35, 55). IL-6R, CNTF-R, and IL-12 p40 have an N-terminal immunoglobulinlike domain followed by the sequence characteristics of the receptor family; the WSXWS motif (which in the p40 sequence is modified by the insertion of an alanine) is near the C terminus in the p40 molecule. The intrachain disulfide pairing of the p40 molecule confirms the homology with the cytokine receptor family (34, 49). The second fibronectin-like domain of human IL-12 p40 contains an RGD sequence and in the mouse, the sequence QEDV, both halImarks of adhesion molecules binding to the integrin receptor family, and also the sequence VTCG similar to adhesion sequences in thrombospondin and properdin (53).Thus, it appears that the second domain of p40 resembles the active sites of adhesion molecules. Furthermore, the third domain of IL-12 p40 and other members of the cytokine receptor family has similarities with members of the gastrointestinal peptide family of hormones, particularly secretin and glucagon prohormones (53).These findings suggest that the different regions of the IL12 molecule may compose a functional mosaic relevant for the action of IL-12 in various tissue, e.g., at the mucosal surface. Most cytokine receptors can be released by cells in soluble forms, which usually have a C terminus immediately following the WSXWS motif and are produced either by proteolytic digestion of the transmembrane form or

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by alternative splicing of the message with elimination of the exons encoding the transmembrane and cytoplasmic portions (56).The binding of IL6 to the IL-6R is a low-affinity interaction, but a high-affinity hexameric complex forms on association of a diiner of gp130 (a nonligand-binding, signal-transducing transmembrane protein), and signa1 transduction through gp130 is triggered (57). The soluble form of the IL-6R, unlike most other soluble receptors, does not compete for binding of IL-6 to the cellular receptors; rather, it binds in solution with IL-6 and this complex can bind to gp130 on the cell surface, mediating signal transduction and IL-6 biological activities (56,57).The CNTF-R is composed of three chains: gp130, sharedwith the IL-6R, the leukemia inhibitory factor (LIF)receptor p chain, and a CNTF-Ra chain. Like the IL-GR, the CNTF-Ra chain is released as a soluble protein that binds to CNTF, and the complex mediates signal transduction on cell types expressing gp130 and LIF-RP chain (58). IL-11, another member of the IL-6 family of cytokines which share the use of the gp130 chain as part of their receptor (59), also associates in solution to the soluble form of the a chain of its receptor and binds in this coinplexed form to the transmembrane gp130 monomer, inducing signal transduction and biological responses (60). Thus, it is possible that a primordial cytokine (the p35 equivalent), which, like IL-6, CNTF, and IL-11, had a multichain receptor, gave rise to the heterodimeric IL-12 during evolution. The transmembrane form of one chain of the receptor (the p40 equivalent) was lost, but an efficient association of the primitive cytokine and the primitive soluble receptor was maintained by the presence of a covalent linkage between the two chains. The heterodimeric complex, like the soluble IL-6WIL-6, CNTF-Ra/CNTF, and IL-11RdIL-11 complexes, would still be able to bind with high affinity to the one or inore remaining transmembrane chains of the receptor, inducing signal transduction and biological activity. If this hypothesis on the evolutionary origin of IL-12 is correct, one would assume that, analogous to the interaction of IL-6, CTNF, and IL-11 with the a chain of their receptor, the p35 and p40 chains of IL-12 have maintained a ligand-receptorlike affinity for each other, even in the absence of covalent linkage between the two chains. Indeed, when monomeric recombinant IL-12 p40 and p35 are added together to responsive cells, all the biological activities of IL-12 can be demonstrated (M. Rengaraju, A. D’Andrea, and G. Trinchieri, unpublished results), although at concentrations from two to five orders of magnitude higher than those effective for the covalently linked heterodimer. The homology of IL-12 p40 with cytokine receptors and the human growth hormone receptor, as well as that of p35 with various cytokines and with the growth hormone itself, has led to a three-dimensional

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model of IL-12 heterodimers, with the growth hormone and its receptors as reference proteins (53). This model strengthens the hypothesis of the evolutionary origin of IL-12 from a cytokine and its receptor and shows that the regions of hoinology with adhesion molecules and gastrointestinal peptides are fully exposed on the protein surface and thus are of possible functional significance. It has also been shown that a single cDNA encoding a single polypeptide formed by the p40 chain C terminus joined to the p35 N terminus by a 15 amino acid flexible linker ([Gly-Gly-Gly-Gly-SerI3), but not the joining of the p35 C terminus to the p40 N terminus, allows the refolding of a biologically active IL-12 fusion protein (61, 62). Constructs of this type may be useful not only in understanding the structure of IL-12, but also in gene therapy by overcoming the need to express two different genes in order to obtain biologically active IL-12. G. EBV-INDUCED PROTEIN 3 (EBI3):A P40-RELATED PROTEIN Devergne et al. (63) isolated a novel cDNA encoding a hematopoietic receptor family member related to the IL-12 p40 and CNTF-Ra subunits, whose expression is induced in B lymphocytes by EBV infection and is constitutive in human placenta. M. Aste, G. Gri, and G. Trinchieri (unpublished results) have shown that the EBIS gene is induced in human monocytes under similar stimulation conditions as IL-12, although with slower and more prolonged kinetics. Most newly synthesized EBIS protein is retained in the endoplasmic reticulum associated with calnexin and a novel 60-kDa protein (63). The protein encoded by the EB13 gene has a predicted M , -25,000, the characteristic amino acids at conserved positions and motifs of the hemopoietin receptor family, 30% homology with CNTFR a , and 27% homology with IL-12 p40 with conserved amino acid substitutions at many of the nonidentical residues (63). Cotransfection of cells with EBIS and IL-12 p35 cDNA results in secretion of the complex of both chains (64), although the chains are not covalently linked because EB13 lacks the cysteine involved in interchain bonding of the Cys177of p40 with p35. Similarly, a large fraction of p35 in extracts of the trophoblast component of a human placenta specifically coimmunoprecipitated with EBIS, indicating that EBIS is a heterodimer with p35, in vivo (64).Although no cytokine function has yet been defined for EBIS and it does not interact with the IL-12R (64), the ability of EBIS to associate with p35 and its expression in IL-12 producing cells at later times of stimulation raise the possibility that it competes with IL-12 p40 for association with IL-12 p35. Thus EBIS might act as an antagonist in the formation of the p70 heterodimer at later times of phagocpc cell activation.

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111. 11-12 Receptor and Signal Transduction

A. CELLULAR-BINDING SITESFOR IL-12 The binding of IL-12 to cellular receptors has been analyzed primarily in human cells, T lymphocytes, and NK cells (65, 66). Resting peripheral blood lymphocytes (PBL) did not express IL-12-binding sites, but after activation with PHA for 2 to 4 days they expressed 500-3000 binding sites/ cell with an affinity of 100 to 600 pM; the kinetics of expression of the receptor reflected the ability of the cells to proliferate in response to IL12 (65,67).The low affinity of the IL-12-binding sites originally identified could not explain the biological activity of IL-12, often observed at concentrations of a few pM or lower (19).More recent studies using '251-labeled IL-12 at higher specific activity identified three classes of IL-12-binding sites on PHA-activated blasts, with & values of 5-50 pM, 50-200 pM, and 2-6 nM (68).

B. IL-12 RECEPTOR Pl SUBUNIT A monoclonal antibody, 2-4E6, that immunoprecipitated the complex formed between radiolabeled IL-12 and IL-12-binding protein(s) on activated human T cells was produced and used in the cloning of a cDNA that encodes an IL-12R component of -100 kDa (68), corresponding to the molecular mass previously determined by cross-linking experiments for the IL-12-binding protein (67). This component of the IL-12 receptor was designated IL-RP1 (68), based on the sequence analogy of this chain with P chains of other receptors of the hemopoietin receptor family, and on the assumption that the p40 subunit of IL-12 replaced the transmembrane a chain of the receptor of other homologous cytokines, such as IL6 or CNTF, during evolution. IL-12RPl cDNA expression in COS cells led to the binding of both IL-12 and the 2-4E6 antibody (68). This cDNA encodes a mature 638 amino acid protein (calculated M , 70,426) preceded by a 24 amino acid signal peptide; a second hydrophobic area in the molecule, located between amino acids 541 and 571, represents the likely transmembrane region. Thus, the extracellular portion is 516 amino acids long and contains six predicted N-glycosylation sites; the cytoplasmic position is 91 amino acids long and contains three potential phosphorylation sites for casein kinase 11. A second cDNA clone obtained has a 13 amino acid deletion right before the stop codon, probably generated by alternate splicing, which results in a protein 2 amino acids shorter. IL-12RP1 belongs to the hemopoietin receptor family, with the two conserved pairs of cysteine residues and the WSXWS motif, and has the highest homology with gp130, followed by the G-CSF-R and LIF-R; however, unlike gp130. IL-12RP1 lacks an N-terminal immunoglobulin domain (68). The cytoplasmic do-

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main of IL-12Rp1 contains the box 1and box 2 motifs typical of members of the hemopoietin receptor families, but no tyrosine residues (68).The IL-12R composition and its signal transduction pathways are schematically represented in Fig. 2. Northern analysis of mRNA for IL-12p1 in several cell types has identified two species, the smaller one possibly corresponding to a yet uncloned alternatively spliced transcript lacking the intracellular domain (68). Expression of the IL-12Rp1 cDNA in COS cells results in the expression of binding sites with a 2-6 nM affinity corresponding to the low-affinity binding sites in PHA-activated blasts. In transfected COS cells, IL-12RP1 was present either as monomers or as covalently linked dimers or oligomers; IL-12 binds only to the preformed complexes and not to the monomers (68). Interestingly, IL-12RP1 cDNA-transfected CTLL cells express only monomers and fail to bind IL-12. Murine IL-12Rp1 was also cloned and found to encode a molecule with 54% amino acid homology with the human IL-12Rp1. When expressed in COS cells or in murine BdF3 pro-B cells, the murine IL-lBRPl also forins surface dimers/oligomers with IL-12-binding ability (69).The cytoplasmic portion of the murine /3l chain contains a tyrosine residue and has 55 additional amino acids compared to the human pl. Murine IL12Rpl-transfected B f l 3 cells bind IL-12 with affinities of 50 and 470 pM, corresponding to the medium- and high-affinity IL-12-binding

FIG.2. Schematic representation of the IL-12 receptor and of the signal transduction pathways activated by IL-12 binding.

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sites on murine lymphoblasts; however, this receptor alone is not sufficient to transduce a signal (69), suggesting the existence of another subunit of the murine IL-12R. Four different cDNAs have been identified for mouse IL-12RP1 that correspond to transcripts expressed by activated mouse T cells (69, 70). In addition to the full transcripts encoding the transmembrane region, two types of transcripts have a 97-bp deletion that eliminates the transmembrane region and results in a frameshift, modifymg the sequence of the C terminus; one of these two transcripts also has a 42-bp deletion in the extracellular portion. The products of the 97-bp deletion transcripts can bind IL-12 and appear to remain cell associated even in the absence of a transmembrane domain (69). A third transcript has an intron insert of more than 3 kb near the transmembrane domain and encodes an IL-12-binding truncated protein (70).The possibility that these transcripts reflected the production of soluble IL-12-binding proteins and their physiological significance remains to be determined. C. IL-12 RECEPTOR 02 SUBUNIT Although the IL-12RP1 chain per se is not sufficient for IL-12 signal transduction, the ability of polyclonal and monoclonal antibodies against human IL-12RP1 to specifically inhibit IL-12 functions (68, 71) and the inability of IL-lBRPl knockout mice to respond to IL-12 (72) confirmed that this chain is an essential subunit of the functional IL-12R. An additional @-typeIL-12 receptor protein, IL-12Rp2, was identified by expression cloning techniques both in mice and in humans. When coexpressed with IL-12RP1, it confers both high-affinity IL-12 binding and IL-12 responsiveness (73). The cloned cDNA contain a very long (640 bp) G + C-rich 5’noncoding region and one long open reading frame encoding a 862 amino acid class I transmembrane protein that consists of a 27 amino acid signal peptide, a 595 amino acid extracellular domain, a 24 amino acid transmembrane region, and a 216 amino acid cytoplasmic tail containing three tyrosine residues (predicted M , of the mature protein is 94,059). The mouse IL-12RP2 is very similar to the human receptor with an overall 68% amino acid sequence homology (73). IL-12RP2 is a member of the cytokine receptor superfamily with one N-terminal immunoglobulin motif and is even more homologous than IL-12RP1 to gp130 and also to G-CSF and LIF receptors (73).The cytoplasmic domain of IL-12Rj32 contains a type 1 and possibly type 2 cytokine box motifs. Two different mRNA species have been identified for IL-12RP2, but no evidence of a transcript for a possible soluble receptor lacking the transmembrane region has been obtained (73). Transfection of IL-12RP2 in COS cells generates single class of IL-12 binding sites with a & of about 5 nM; like IL-12RP1, the IL-12RP2 protein is expressed as a dimer or an oligomer, regardless of

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the presence of the ligand (73). Coexpression of both IL-12RPl and P2 chains in COS cells results in the formation of a high number of lowaffinity binding sites for IL-12 (7.5 nM) and only a low number of highaffinity (55 pM) bindmg sites, possibly due to the competition between homo- and heteroassociation of the Pl and 8 2 chains; coimmunoprecipitation of the heterocomplexes, even using chemical cross-linking, could not be demonstrated (73).Ba/F3 cells cotransfected with both Pl and P2 proliferate readily in the presence of IL-12, with maximal proliferation observed at 100 pg/ml, whereas BdF3 cells transfected with IL-12RP1 alone do not respond to IL-12; however, high doses of IL-12 (10-100 ng/ ml) induce proliferation of Ba/F3 cells transfected with IL-12RP2, suggesting that the human PZ chain may be at least partidy capable of transducing proliferation signals and that the Pl chains contribute primarily to an increased affinity of IL-12 binding (73). However, no IL-12 response, even at high concentrations, was observed in mice lacking the IL-lSRPl chain (72). The p40 homodimers bind and compete for IL-12 binding in cells transfected with the IL-lBRPl chain, but not in those transfected with the P2 chain, suggesting that Pl binds the p40 subunit of IL-12, whereas P2 binds either the p35 subunit or a structure expressed on the heterodimer (51).

D. REGULATION OF IL-12 RECEPTOR EXPRESSION Resting human T and NK cells express low but variable levels of ILl2RPl as detected by antibody staining, although no IL-12 binding can be detected. Stimulation with mitogens, anti-CD3, and anti-CD3 plus antiCD28 upregulates both the Pl and the P2 chains of IL-12R within a few days of stimulation, with a peak at days 3-4 (74,75). IL-12RPl mRNA is found in activated T and NK cells and cell lines, as well as in B cells; however, B cells and BCL are usually unable to bind IL-12, in part due to their lack of expression of the ICl2RP2 chain (71,72,76,77). In human T cells, IFN-.)Iupregulates and IL-4 downregulates the expression of highaffinity binding sites, without affecting the expression of the 01 chain, thus probably acting at the level of the P2 chain (75). If the & of the identified receptor is more than 50 pmol/liter, it must be assumed that either signal transduction occurs at minimal occupancy of the receptors or additional unidentified receptor chains are required in determining a low number of high-affinity binding sites. The other discrepancy with the functional data is that receptors cannot be identified on resting T and NK cells, whereas certain biological activities of IL-12, e.g., enhancement of cell-mediated cytotoxicity or induction of IFN-.)I production, are mediated with a similar dose-response curve on both resting and activated NK and T cells (78). This discrepancy might be

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explained in part by the observations, using analysis of IL-12 binding by cytofluorimetry or staining with anti-IL-12Rpl antibodies, that resting NK cells express low levels of IL-12R (66,71). However, in situ hybridization experiments have shown that more than 10-2096 of resting peripheral blood T cells accumulate INF-y mRNA a few hours after exposure to IL12, suggesting that a significant proportion of peripheral blood T cells also expresses functional IL-12 receptors (78). In addition to the ability of IL-12 to direct differentiation of T h l cells (79, 80), it was shown that Thl, but not Th2, clones are responsive to IL12 (81, 82). Extinction of the IL-12 signaling pathway in early mouse Th2 cells results from the selective loss of IL-12RP2 subunit expression, without alteration in pl subunit expression (83). IL-12RP2 is not expressed by mouse naive resting CD4' T cells, but is induced upon antigen activation through the T-cell receptor; IL-4 inhibits expression of IL-12RP2, leading to the loss of IL-12 signaling and thus contributing to commitment to the Th2 pathway (83).IFN-y, however, prevents the loss ofp2 chain expression and restores the ability of early Th2 cells to respond to IL-12 (83). The T-cell genetic background also contributes to the maintenance of IL-12 signaling and IL-12RP2 subunit expression: when T cells from B10.D2 mice are activated without the addition of exogenous cytokines (neutral condition), they remain responsive to IL-12, whereas activated T cells from BALB/c mice cease IL-12Rp2 expression and become unresponsive to IL12 (84),consistent with their default differentiation along the Th2 pathway (85).Similarly, the IL-12Rp2 subunit is expressed on human T h l but not Th2 clones and is induced during differentiation of human naive cells along the Thl but not the Th2 pathway. However, in contrast with the mouse system, type I ( d p ) ,but not type I1 ( y ) ,interferons induce expression of the p2 chain during T-cell differentiation following T-cell receptor triggering (86). These results are difficult to reconcile with a previous report that IFN-y induces high-affinity IL-12-binding sites on human T cells (75). E. IL-12-INDUCED SIGNAL TRANSDUCTION Early reports indicated that IL-12 induces phosphorylation of the src family lck tyrosine kinase in human NK cells (87, 88), and the ability of IL-12 to induce the expression of the CD69 activation membrane molecule on NK cells was shown to be blocked by tyrosine kinase inhibitors (89). In activated human T cells, but not in N K cells, IL-12 induces tyrosine phosphorylation of a 44-kDa protein identified as an isoform of mitogenactivated serine-threonine kinase (MAPK) (90). IL-12 treatment of both human T and NK cells induces rapid tyrosine phosphorylation of both JAK2 and Tyk2 kinases, implicating these kinases

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in the immediate biochemical response to IL-12 (91, 92). Chimeric receptors composed of the transmembrane and cytoplasmic region of IL-12Rp1 and P2 chain fused to the extracellular domain of the epidermal growth factor (EGF) have been used to show that JAKZ is phosphorylated in response to EGF in cells expressing the 61 and/or P2 cytoplasmic domains, whereas phosphorylation of Tyk2 is observed only in cells expressing the P l cytoplasmic domain; however, direct physical association has only been demonstrated between JAKZ and p 2 and between Tyk2 and 01 (93). Following activation of the two Janus family kinases, three components of the STAT family of transcription factors are phosphorylated and activated: STAT1, STAT3, and STAT4 (92,94,95). STAT1 can dimerize with either STAT3 or STAT4, and STAT4 forms either homo- or heterodimer with STAT3 (92, 94). STAT4 is both tyrosine and serine phosphorylated in response to IL-12 (96). The serine phosphorylation is not required for DNA binding, but is required for transactivation, based on the finding that the serine kinase inhibitor H7 blocks the ability of IL-12 to induce STATdependent activation of a heterologous promoter, but not its ability to bind to DNA in vitro (97). Serine phosphorylation of STAT4 involves a Pro-Met-Ser-Pro sequence in its C terminus that resembles the consensus recognition sequence for the MAPK (96) and thus may be dependent on the reported activation of the 44-kDa MAPK in T cells (90). However, the mechanism of activation of the MAPK is not clear, as the RAS activation pathway of MAPK is not involved in IL-12 signaling (97). The activation of STAT4 by IL-12 is of particular interest because this transcription factor is not activated by other cytokines (98), with the exception of IFN-a in the human system (96, 99), raising the possibility that STAT4 activation underlies the specific biological responses to IL-12. Indeed, T and NK cells of STAT4 knockout mice are unresponsive to IL-12, and these mice have a phenotype equivalent to that of IL-12 or IL-12R knockout mice (100,101).T and NK cells from STAT4 knockout mice also do not produce IFN-y in response to IL-12, suggesting that STAT4 is directly involved in IFN-y gene transcription. Two adjacent binding sites for STAT4 have been identified in the first intron of the IFN-y gene: these binding sites are variants of the consensus sequence and, alone, they bind STAT4 inefficiently. Recognition of the variations of the consensus site and STAT4 binding require cooperative interactions (102). The conserved aminoterminal domain of STAT proteins was required for cooperative DNA binding, although this domain was not necessary for dimerization or binding to a single consensus site (102). Cotransfection of the full-length STAT4, but not of an amino-terminal-deleted STAT4, induced a fourfold induction of transcription of a reporter gene construct containing the IFN-y first intron (102).

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Unlike other studies in the mouse (94, 98) and in human NK cells (92) that failed to detect activation of STAT4 by IFN-a, both tyrosine and serine phosphorylation of STAT4 in response to IFN-a was observed in human-activated T cells and the human NK cell line NK3.3 (96, 99). Although the reason for these species differences is not clear, the ability of IFN-a to mimic some of the signal transduction mechanisms of IL-12 may explain the reported effect of this cytokine on the T h l differentiation pathway. In BdF3 cells transfected with chimeric EGFIIL-12Rpl and p 2 receptors (93),phosphorylation of STAT3 in response to EGF was demonstrated in cells transfected with either the ,f3l or the /32 cytoplasmic domain, indicating that the presence and phosphorylation of a tyrosine residue (missing in the pl domain) are not absolute requirements for the activation of STAT factors. These results also show that the individual pl and p2 cytoplasmic domains are capable of signal transduction, similar to the observations with the complete receptors (73). However, proliferation of the transfected BdF3 cells in response to EGF was observed only when the p2 domain, alone or together with the pl domain, was present (93).

N. Production of 11-12 A. MEASUREMENT OF IL-12 PRODUCTION The existence of two separate genes controlling IL-12 production, the need for their simultaneous expression in the same cell type in order to produce biologically active IL-12 (21), and the production of a large excess of the p40 chain over the biologically active heterodimer have made the analysis of IL-12 production particularly complex. Transcripts for the p35 gene have been detected at very low abundance [lessthan 1fglpg total RNA in peripheral blood mononuclear cells (PBMC) or polymorphonuclear leukocytes (PMN) (32, 103)] in almost any cell type tested, including hematopoietic and solid tumor cell lines, and are upregulated on activation (46). Transcripts for the p40 gene have been detected only in cell types producing biologdly active IL-12 and their expression is highly regulated (46). Because expression of both genes is required for biologically active IL-12 expression, detection of mRNA for the tissue-specific and highly regulated p40 gene is a better indicator of IL-12 production than detection of the more ubiquitous p35 mRNA, especially when a mixture of different cell types is analyzed. However, the use of low sensitivity methods for mRNA detection, e.g., in situ hybridization on tissue sections, has led to a reported apparent dissociation of p35 and p40 mRNA expression in different cell types (104). Furthermore, upregulation and expression of the p35 gene are obviously needed for production of the biologically active

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p70 heterodimer, and p40 gene expression or p40 chain production alone can be considered only indicative for simultaneous production of the biologically active heterodimer in the absence of a direct immunological or biological demonstration of p70 secretion. Because of the extremely variable ratio between the free p40 chain and the p70 heterodimer, the level of p40 gene or protein expression cannot be extrapolated to obtain even an approximate level of heterodimer produced. The production of monoclonal and polyclonal antibodies to the p40 and p35 chains of IL-12 has facilitated analyses of IL-12 production (46, 105). Radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA) assays that detect the p40 chain (either as a single chain or as a single chain and a complex in the p70 heterodimer) and the single chain p35 have been established (46). Measurement of the p70 heterodimer proved to be more difficult. The initial RIA for detection of human IL12 p70 was cumbersome and affected by contaminant p40 (46).Now several assays are available, some commercial, that allow a sensitive detection of human or mouse p70. These assays utilize a pair of antibodies, one reacting with the p40 chain and the other with either the p35 chain or a determinant specific for the heterodimer (46, 106). An antibody capture assay using an anti-p40 antibody to capture IL-12 and quantitating either proliferation (107) or IFN-.)Iproduction (108) by IL-12-responsive indicator cells as a measure of IL-12 biological activity represents an optimal method for the determination of biologically active p70. The use of the IL-12-dependent murine T cell clone 2D6 as indicator cells in this antibody capture assay has especially contributed to the reproducibility and sensitivity of mouse IL-12 detection (109). However, because the sensitivity of the antibody capture or immunological assays for either human or mouse IL-12 p70 is on the order of a few picograms and the p40 chain is often produced at a 100-fold excess over the p70 heterodimer, it is often difficult to quantitate p70 in supernatant and biological fluids that contain less than 1 nglml of IL-12 p40.

B. IL-12 PRODUCTION BY €3 LYMPHOCYTES IL-12 was originally discovered and characterized as a product of the lymphoblastoid B-cell lines RPMI-8866, ADP, and NC37 (19, 20). EBVtransformed BCL were all found to produce at least low levels of IL-12 constitutively,and its production was enhanced by stimulation with phorbol diesters (46). Most African Burkitt’s lymphoma cell lines produced no or negligible amounts of IL-12 (46, 76). However, most EBV(+) cell lines derived from AIDS-associated B-cell lymphomas (AABCL) constitutively produce very high levels of IL-12, which are enhanced by phorbol diester stimulation to levels much higher than those observed with BCL from

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HIV-negative donors (76). In the case of AIDS-associated Burkitt’s Iymphoma (76), as well as in Hodgkin’s lymphoma (110), only EBV(+) cells were shown to produce IL-12, suggesting the EBV has a transactivating effect on IL-12 production. High levels of IL-12 were detected in the serum of SCID mice injected with human lymphocytes and in which EBV(+ ) human B-cell lymphomas were growing (R. Baiocchi, M. Caligiuri, G. Gri, and G. Trinchieri, unpublished results), suggesting that a similar production of IL-12 during the initial proliferation of EBF-transformed B cells in patients may affect the reactivity of the patient’s immune cells against the transformed cells. In addition to lymphomas, chronic B lymphocyte leukemia cells have also been shown to produce low levels of IL-12 (V. Pistoia and G. Trinchieri, unpublished results). Thus, IL-12 expression and/or secretion is not confined to in uitro-transformed normal B cells but can also be detected in malignant B-cell precursors “frozen” at early stages of B-cell differentiation pathway (early or intermediate B cells). Analysis of IL-12 mRNA accumulation has indicated that almost all cell types tested, including B cells, T cells, NK cells, and leukemic cell lines derived from these cells, as well as different malignant tumor cell lines, express transcripts of the p35 gene (46, 111).In the author’s experience, IL-12 p35 was expressed in the entire panel of the B-cell lines studied, whereas IL-12 p40 was detected only in the EBV(+) B-cell lines. The constitutive secretion of high levels of IL-12 by the majority of the EBV( +) AABCL, but only one of the non-AABCL obtained from patients with Burkitt’s lymphoma, raises the question whether IL-12 secretion by AABCL is related to HIV-1 and/or EBV. Modulation of lymphokine expression by virally encoded genes has been documented by Hsu et al. (112), who demonstrated that IL-10 has extensive homology to BCRF-1, an open reading frame in the EBV genome. In studying the B-cell lines for lymphokine expression, it was found that AABCL constitutively secrete large amounts of IL-10 (113),IL-7, and TNF (114-116). The secretion of large quantities of these lymphokines by AABCL compared with nonAABCL suggests that HIV-1 triggers B cells to secrete large amounts of IL-10, IL-7, TNF, and IL-12. However, HIV-1 does not infect B cells, and polymerase chain reaction (PCR) analysis revealed no HIV-1 transcripts in the AABCL. Moreover, lack of IL-12 expression in two EBV( -) cell lines derived from patients with AIDS and Burkitt’s lymphoma further suggests that in vivo exposure of B cells to HIV-1 alone may not induce lymphokine secretion and that both HIV-1 and EBV are required to trigger IL-12 secretion in tumor B cells. The observation that the AABCL secrete large amounts of IL-12 contrasts with data of several recent reports, which indicate that IL-12 production by monocytes from HIV-l-infected patients is impaired (117-119).

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Although any extrapolation to the in vivo situation from data obtained with established cell lines warrants caution, it seems possible that, in healthy individuals, IL-12-producing EBV-transformed cells are easily rejected by the immune response, leaving only cells that have lost the ability to produce IL-12 to give rise to Burktt’s lymphomas. In immunodeficient AIDS patients, these protective mechanisms may be inefficient, and IL-12producing EBV-transformed cells could give rise to Burkitt’s lymphomas in a relatively high proportion of patients. Although malignant or EBV-transformed cell lines produce IL-12, the physiological relevance of IL-12 production from normal B cells remains to be established. In situ detection of IL-12 mRNA in mice injected with LPS suggested that IL-12 was produced by B cells in addition to macrophages (104). In humans, only very low levels of IL-12 were found to be produced by peripheral blood B cells and by germinal center ( IgD -, CD38+) and naive (IgD+, CD38-), but not memory (IgD-, CD38-), tonsillar B lymphocytes (V. Pistoia, personal communication). Of the lowlevel IL-12 produced, most is secreted as p70 rather than the p40 chain, unlike the observations for B-cell lines and other cell types. Moreover, various stimuli that activate other functions of B cells have only a minimal effect on their ability to produce IL-12. Unlike in human B cells, no IL12 production has been demonstrated for murine B cells from immune lymph nodes (120). Thus, the physiological role of B-cell-produced IL-12 and the possible immunological effect of IL-12 produced by neoplastic B lymphocytes remain to be investigated. BY PHAGOCYTIC CELLS C. IL-12 PRODUCTION Phagocytic cells, not B cells, are probably the major physiologicalproducers of IL-12, a conclusion suggested by many in vitro and in vivo studies in infectious disease models (121). PBMC or purified monocytes produce high levels of IL-12 p40 and p70 when stimulated by bacteria, such as heat-fixed Staphylococcus aureus or Streptococcus extracts, or by bacterial products such as LPS (46). The producer cells within PBMC are mostly monocytes and other MHC class 11-positive cells, possibly dendritic cells (46). However, purified monocytes often produce lower levels of IL-12 than total PBMC, suggesting that other cells, e.g., T cells may contribute to the stimulation of monocytes to produce IL-12. In addition to monocytes, PMN also respond to LPS stimulation with the production of IL-12 p40 protein and, to a lesser extent, of the biologically active heterodimer (103). On a per cell basis, PMN produce less IL-12 p40 or p70 than monocytes (103). However, because of the large number of PMN present in the blood or in the inflammatorytissues, it is likely that IL-12 produced by PMN plays an important physiological role in the inflammatory response to infection.

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Several studies have shown that both human and mouse central nervous system (CNS) microglial cells produce both IL-12 p40 and p70 in response to LPS or IFN-7 plus LPS (122-125). The ability of astrocytes to produce IL-12 is, however, controversial: two studies indicated that astrocytes produce even higher levels of IL-12 p40 and p70 than microglia (123, 124), whereas another study demonstrated no IL-12 production from astrocytes and reported that IL-12 production by microglia is inhibited by astrocytes (125). The production of IL-12 by phagocytic cells and, at least in part, by dendritic cells is induced by a variety of mechanisms that reflect the role played by IL-12 in inflammation and immunity. These mechanisms are either T-cell independent or dependent. The T-cell-independent mechanisms are important in the proinflammatory and inimunoregulatory role of IL-12 at the interface of innate resistance and adaptive immunity. Among these mechanisms is the induction of IL-12 by infectious agents and their products, of which LPS and bacterial DNA are the most typical examples (46, 126). During inflammation, however, an important mechanism of infection-independent induction of IL-12 and other cytokines is represented by the interaction of adhesion molecules with substrates of inflammatory origin, exemplified by the interaction of CD44 adhesion molecules with low molecular weight hyaluronan (127). Thus, although infection is probably the most effective mechanism of acute induction of IL-12 production, the CD44-mediated mechanism of IL-12 induction may be operative in aseptic inflammation, contributing to macrophage activation via the proinflammatory function of IL-12 and IFN-7 induction. The Tcell-dependent mechanism of IL-12 production is dependent on the ability of the CD40 ligand (CD40L) expressed on activated T cells to induce IL12 production in monocyte/macrophages and dendritic cells by interacting with the CD40 receptor on the surface of these cells (128, 129). The Tcell-dependent mechanisms of IL-12 induction play an important role in the T-cell immunoregulatory role of IL-12, particularly in the maintenance of Thl responses; however, it should be noted that nonantigen-specific T cells also participate in the innate resistance response, alongside or independently of antigen-specific T cells. In particular, functional CD40L expression on T lymphocytes in the absence of T-cell receptor engagement has been demonstrated to be involved in IL-12 and IFN-y production induced by IL-2 (130). To fully understand the regulation of IL-12 production, it is important to remember that the ability of various stimuli to induce its production is strictly regulated by powerful positive and negative feedback mechanisms. Such mechanisms are mediated by other cytokines, by pharmacologically active mediators, and by ligands for various cellular receptors, among

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which complement components and immunoglobulins assume particular relevance in the cross-regulation between innate and adaptive immunity compartments. 1. IL-12 Induction by lnfectious Pathogens Infectious pathogens that are able to induce production of IL-12 include bacteria, both protozoan and metazoan parasites, fungi, and viruses. IL12 is often involved in the regulation of the host response to these infections and many of these pathogens will be discussed in detail. Induction of IL-12 production with bacteria is observed with avirulent or heat-killed organisms, but, in uiwo, it is more efficient when live bacteria are used (46, 131).Fixed bacteria (e.g., S. aureus) or crude bacterial extracts (e.g., OK432 from Streptococcus pyogenes) are efficient inducers of IL-12 production in monocyte/macrophages (46, 132). The question of whether phagocytosis is either necessary or sufficient for the induction of IL-12 production is a complex one. Soluble microbial products such as purified protein derivative (PPD) or heat-shock proteins from mycobacteria are poorer inducers of IL-12 than the whole organisms, although they readily induce IL-1 or TNF (131).Chitin particles but not soluble chitin (polymers of N-acetyl-D-glucosamine) induce IL-12 production via mannose receptor-mediated phagocytosis and this induction is inhibited by soluble mannan (133).Cytochalasin D blocks both phagocytosis and IL-12 induction by chitin particles or Mycobacterium tuberculosis (131,133).However, discordant results have been reported for the production of IL-12 following latex bead phagocytosis (131, 134). The reason for these contrasting results might rest in the size of particles used: beads 2 pm, which are phagocytosed, readily induce IL-12 production, probably by activating kinases associated with the cytoskeletal proteins, without triggering specific surface receptors ( 134). Several soluble microbial products have been identified that induce IL12 production. LPS from gram-negative bacteria is a potent stimulus for IL-12, although maximal activity requires priming of the producer cells, e.g., by IFN-y (32, 46, 135).The response to low-level LPS is mediated through CD14 receptors: dendritic cells, which do not express membrane CD14, produce IL-12 in response to LPS by utilizing serum-derived soluble CD14 (136). Lipoteichoic acid (LTA), a member of a class of surface glycolipids similar to LPS but present on gram-positive bacteria, is a potent inducer of IL-12 production and, like LPS, acts through a CD14-dependent pathway (137). Other microbial IL-12 inducers include trehalose dimycolate, a glycolipid from Mycobacteriurn spp. (138), glycoprotein fractions from Toxoplasma gondii (139) and Typanosomu cruzi (140), and the

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recombinant Leishmania antigen LeIF (141). Bacterial superantigens such as Staphylococcus or Streptococcus spp.-derived enterotoxins induce production of IL-12 both in vitro and in vim (142). The major mechanism of IL-12 induction by superantigens, however, appears to be induction of CD40L expression on activated T cells and not direct stimulation of macrophages (C. Son and G. Trinchieri, unpublished results). Bacterial DNA has also been shown to be an efficient inducer of IL-12 and other monokines, including IFN-a, due to the presence of immunostimulatory sequences composed of an unmethylated CpG dinucleotide flanked by two 5’ purines and two 3‘ pyrimidines (143,144).Interestingly, the presence of these immunostimulatory sequences in the plasmids has been shown to be essential for effective intradermal gene immunization (126). Another powerful inducer of IFN-a production, i.e., synthetic double-stranded RNA such as poly I-C, also induces IL-12 production (131, 145).

2. IL-12 Induction by Interaction with Injam?mtoy Extracellular Matrix The second T-cell-independent pathway of IL-12 induction is that induced by interaction of macrophages with components of the extracellular matrix selectively expressed during inflammation ( 127). Low molecular weight fragments (lo6)is ineffective (127).The induction of IL-12 production was inhibited by antibodies against the CD44 receptor that block hyaluronan binding or by competition with the high molecular weight form (127). Production of IL-12 was observed in thioglycolate-elicited macrophages or in adhesion-primed but not freshly expIanted resident macrophages and was strongly upregulated by treatment of the macrophages with IFN-y (127). The CD44-dependent mechanism of IL-12 induction might play an important role in the sterile inflamniatory response. For example, in patients with closed bone fractures and soft tissue hematomas following blunt trauma with no evidence of infection, high levels of IL-12 (>2 ng/ml) were present in the plasma and in the fracture serum from days 3 to 7 after trauma (146). Such levels of IL-12, together with other inflammatory cytokines, likely contribute to the alteration of systemic immunity observed in trauma patients. 3. T-Cell-Dependent IL-12 Induction Evidence for a T-cell-dependent pathway of induction of IL-12 production can be traced back to the work of Germann et al. (147),who reported that T-cell stimulatory factor (TSF), a cytokine required for the growth

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of T h l lymphocyte clones and later identified as IL-12 (81),was produced by accessory cells cocultured with activated T cells. The ability of murine dendritic cells to direct the development of T h l cells was demonstrated to be secondary to their ability to produce IL-12, which was induced in the dendritic cells only when exposed to T cells activated by specific antigen (148). Shu et al. (128) clarified the mechanism of T-cell-dependent IL-12 production by demonstrating that activated T cells induce IL-12 production by monocytes via CD40/CD40L interaction. Anti-CD40L antibodies inhibit IL-12 production in vivo (149),possibly explaining the ability of antiCD40L antibodies to suppress many in vivo models of pathogenesis involving T cells and also the T-cell abnormality observed in patients with X-linked hyper-IgM syndrome due to defects in the CD40L gene (150,151).However, CD40/CD40L stimulation is most likely bidirectional, with stimulation of both T cells and APC; APC produce IL-12 and upregulate B7, whereas T cells, in response to IL-12 and B7 stimulation, produce IFN-y and GM-CSF which, in turn, enhance APC ability to produce IL12 (152,153). Only one study (154) has analyzed the regulation of the two IL-12 transcripts by CD40/CD40L interaction and reported that p40, but not p35, transcripts are upregulated; however, the observation in many studies that the production of the p70 heterodimer is induced by CD40/ CD40L interaction as or more efficiently than that of the p40 chain (129, 155)points to an effective upregulation of the production of both polypeptide chains. B cells interfere with the ability of activated T cells to stimulate IL-12 production by APC, possibly because they express CD40 and thus compete for binding to CD40L on T cells (155). The ability of infectious agents to induce IL-12 production from APC is independent of CD40/CD40L interaction and of T cells, as clearly shown by the finding that LPS, S. aureus, and Listeria monocytogeneses can all induce IL-12 production in CD40 knockout mice (155)or in systems where CD40/CD40L interaction is blocked by soluble CD40L or anti-CD40L antibodies (156).However, it is likely that during an infection in viuo, the expression of CD40L on activated T cells contributes to maintaining the production of IL-12 initiated by the T-cell-independent mechanisms. Because CD40L is induced within a few hours of T-cell stimulation (157) and because stimulation of T cells by specific antigens or by other stimuli, e.g., IL-2 even in the absence of T-cell receptor engagement (130)upregulates functional CD40L expression, both antigen-specific and bystanderactivated T cells in an inflammatory response can efficiently participate in the induction of IL-12 production.

D. POSITIVE AND NEGATIVE MODULATION OF IL-12 PRODUCTION IFN-y has a powerful enhancing effect on the ability of monocytes and PMN to produce IL-12 (103,135).This observation is of particular interest

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because IL-12 is a potent inducer of IFN-y production by T and N K cells (78).Thus, IL-12-induced IFN-y acts as a potent positive feedback mechanism in inflammation by enhancing IL-12 production. Also, because IL-12 is the major cytokine responsible for the differentiation of Thl cells, which are potent producers of IFN-y (79), the enhancing effect of IFNy on IL-12 production may represent a mechanism by which T h l responses are maintained in vivo. In both monocytes and PMN, the enhancing effect of IFN-y on IL-12 production is observed when IFN-y is added simultaneously to the stimulus (e.g., LPS), but it is more effective when the producer cells are primed for several hours in the presence of IFNy (32,158).In addition to IFN-y, GM-CSF has a modest enhancing effect on IL-12 production by phagocytic cells, acting primarily at the level of the p40 gene (135, 158, 159), whereas M-CSF has no priming activity for IL-12 production (158).The ability of IFN-y to enhance IL-12 production is particularly evident in the case of infectious agents, e.g., certain mycobacteria, which are rather poor inducers of IL-12 production. In in vitro or in vivo infections with these microorganisms, IFN-y production precedes and is required for maximal IL-12 production (160). However, with many other inducers, such as LPS, toxoplasma, and S. aureus, IL-12 production in vivo and in vitro both precedes and is required for IFN-y production. For example, following injection of LPS, IL-12 is induced at 2-3 hr and IFN-y at 5-7 hr (161, 162). Neutralizing anti-IL-12 antibodies inhibit IFN-y production, but anti-IFN-y antibodies do not inhibit IL-12 production. With these potent inducers, IL-12 production is also observed in mice not expressing the IFN-y or IFN-y-receptor genes (163, 164). The role of TNF-a in the production of IL-12 is less well defined; in some experimental systems, it has been reported to enhance the ability of IFNy to prime phagocytic cells for IL-12 production (122, 1Fi6, 160). The positive feedback amplification of IL-12 production mediated by IFN-y obviously represents a potentially dangerous mechanism leading to uncontrolled cytokine production and possibly shock. There are, however, potent mechanisms that downregulate IL-12 production and the responsiveness of T and NK cells to IL-12. Some of the major positive and negative regulatory mechanisms of IL-12 production and function are schematically represented in Fig. 3. The Th2 cytokine IL-10 is a potent inhibitor of IL-12 production by phagocytic cells; the ability of IL-10 to suppress production of IFN-y and other T h l cytokines is due primarily to its inhibition of IL-12 production from APC and to inhibition of expression of other costimulatory surface molecules (e.g., B7) and soluble cytokines (e.g.,TNF-a, IL-lP) (108,165,166). However, IL-12 is able to induce IL-10 production and to prime T-cell clones for high IL-10 production both in vivo (167) and in vitro (168), indicating that IL-12 can induce factors

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FIG. 3. Major positive and negative regulatory mechanisms of IL-12 production and functions. Three major pathways of IL-12 production are effective in infection, inflammation, and immune response. Positive feedback (e.g., IFN-y) and negative feedback (e.g., IL10) mechanisms regulate IL-12 production. IL-12 production is also downregulated by pharmacological mediators such as PGE2, desensitization of the producer cells by LPS, and cross-linking of various complement and Fc receptors on the producer cells. The competition of the P40-homolog protein EBI3 with p40 for binding to p35 might represent a mechanism for downregulation of IL-12 heterodimer formation. For full biological activity on T and NK cells, IL-12 synergizes with various costimulators, including cytokines such as IL-2 and IL-18, and surface costimulatory molecules such as B7 binding to CD28 and LFA-3 binding to CD2. Competition of the p40 homodimers with the IL-12 heterodimer for receptor binding and downregulation of the IL- l2RP2 chain represent mechanisms of downregulation of the biological response of T and NK cells to IL-12. @, positive or synergistic stimulation; 8, antagonistic or inhibitory stimulation.

that enhance (e.g., IFN-y) or suppress (e.g., IL-10) IL-12 production. Another powerful inhibitor of IL-12 production is TGF-P (169). IL-4 and IL-13 can also partially inhibit IL-12 production (169, 170), suggesting the hypothesis that Th2 cells, by producing cytokines such as IL-10, IL-4, and IL-13, suppress IL-12 production and prevent the emergence of a T h l response (171,172).However, if monocytes are primed with IL-4 or IL-13 for 24 hr or longer, IL-12 production is not inhibited and instead is significantly enhanced (169, 173). The mechanism of enhancement of IL-12 production by IL-4 and IL-13 is not due to increased production of IL-10 or prostaglandin Ez (PGEJ (173, 174) and may be secondary to a differentiation effect on monocytes, which requires prolonged incubation and exposure to the cytokine, unlike the inhibitory

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effect observed when the cytokines are added simultaneously to the IL12 inducers. The priming effect of IL-4 and IL-13 is at the transcriptional level for both p40 and p35 genes. At the protein level, the effect is observed for secretion of the IL-12 p40 chain and, even more efficiently, of the p70 heterodimer (174). IL-4 and IL-13 are particularly effective in enhancing IL-12 production in response to particulate inducers (S. aureus and L. major), whereas IFN-y is most active when LPS is used as an inducer (159, 169); however, IL-4/13 and IFN-y have an additive and, in some cases, a synergistic effect on IL-12 production (159, 173). The enhancing effect of IL-4/13 on IL-12 production is accompanied by a similar increase of TNF-a, MCP-1, and, to a lesser extent, IL-6, whereas production of IL-lP, IL-8, and IL-10 is still inhibited (169, 173). Furthermore, IL-4and IL-13-treated monocytes have significantly increased expression of MHC class I1 antigens, B7.2, and CD40, and decreased CD14 (174). Some of these surface antigen changes are reminiscent of those observed in longer cultures (7 days) of monocytes in the presence of GM-CSF and IL-4 or IL-13, which generate dendritic-like cells with much enhanced antigen-presenting activity and the ability to produce elevated levels of IL-12 (129,175). Interestingly, IL-4, which inhibits IL-12 induction when added simultaneously with a bacterial stimulus, specifically upregulates IL-12 p70 production induced by CD40L through the upregulation of IL12 p35 gene expression (176). Because IL-13 receptors, unlike those for IL-4, are not expressed on T cells, and, unlike IL-4, IL-13 is not an inducer of Th2 differentiation, the ability of IL-13 to enhance IL-12 production makes it a potential inducer of T h l responses. Indeed, it was shown that injection of IL-13 in mice infected with L. rnonocytogeneses increases IL12 production, decreases IL-4 production, and enhances resistance to the infection (177). Interestingly, treatment of PBMC from HIV( +) patients with IL-4 or IL-13 almost completely corrects their inability to produce IL-12 in response to S. aureus (174) and, in part by enhancing the deficient antigen-presenting ability of the patients’ monocytes, corrects the defective proliferative responses of their T cells to recall antigens (L. J. Montaner, personal communication). IFN-y, IL-10, and other cytokines have a profound modulatory effect on the production of IL-12 by phagocytic cells, but they have no significant effect on IL-12 production by B-cell lines. It is also of interest that phorbol diesters, which enhance IL-12 production from EBV-transformed cell lines, do not induce IL-12 production in phagocytic cells, although these compounds are potent inducers of other cytokines such as TNF-a in those cells (46, 135) Other cytokines that downregulate IL-12 production are MCP-1 (178) and IL-11 (179, 180). In addition to IL-11, another cytokine of the group

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sharing the use of the gp130 subunit in their receptor, IL-6, has been shown to inhibit both T-cell independent and -dependent induction of IL-12 production (176). Among the substances with pharmacological activity that inhibit IL-12 production, and of particular interest because they may be involved in physiologic immune regulation, are PGEz (181), corticosteroids, and catecholamines (135,182- 184). The presence of high levels of PGEz in the seminal plasma may be responsible for its immunosuppressive effect (185, 186). The immunosuppressive hormone, 1,25dihydroxyvitamin D3 also inhibits IL-12 production and T h l functions (187), whereas a deficiency of vitamin A in vivo leads to constitutive IL12 expression with excessive Thl cell activation and insufficient Th2 cell development, which is corrected by dietary retinoic acid supplementation (188). The immunosuppressive calcitonin gene-related peptide released from peripheral nerves acts, in part, by suppressing production of IL-12 (189, 190); interestingly, IL-12 itself has been detected in the free nerve endings of skin, and it has been suggested that modulation of IL-12 production may be one mechanism whereby the nervous system modifies cutaneous immune responses (191). Pentoxifylline and thalidomide are inhibitors of IL-12 production, suggesting that these substances might be used pharmacologically in clinical trials of immunological disorders characterized by an inappropriate type 1 immune response (192, 193). An important mechanism of cross-talk between innate resistance and adaptive immunity is represented in the ability of complement components and immunoglobulins to react with complement or Fc receptors on the effector cells of innate resistance, modulating their functions and cytokine production. The first indication that IL-12 production is also regulated by those interactions was provided by the finding that measles virus induces a profound depression of Thl responses following infection in part by interacting with its receptor on phagocytic cells, the CD46 molecule, to inhibit IL-12 production (194). CD46, or membrane cofactor protein, is a binding site for C3b and C4b. By binding CD46, both polymeric C3b and anti-CD46 antibodies can inhibit IL-12 production, suggesting that measles virus may utilize a physiological mechanism of IL-12 downregulation induced by activated complement in its induction of immunosuppresion (194). Note that this suppressive mechanism is quite selective, as the production of many other cytokines tested is only minimally or not at all affected (194). The ability to trigger monocyte/macrophage receptors and selectively suppress IL-12 production was extended to the interaction of iC3b with complement receptor 3 ( C D l l b ) and to the interaction of immunocomplexes with Fc receptors (195,196). The mechanism of action is not clear but may depend on [Ca2+Iiflux activation by receptor crosslinking (195) and, at least in part, on inhibition of IFN-y-induced tyrosine

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phosphorylation (196). Antibodies against C D l l b in a inurine model of IL-12-dependent septic shock lead to suppression of IL-12 and IFN-y production in vizjo (196),suggesting that previous results showing suppression of DTH and response against various infectious agents by anti-CDllb in vivo might be explained in part by the inhibition of IL-12 production. C D l l b , in addition to binding iC3b, is a receptor for a variety of ligands and mehates the binding of several bacteria and intracellular parasites, either opsonized or not: thus, its ability to selectively downregulate IL12 production may have physiological importance in many infections or inflammatory situations (195, 196).

E. PRODUCTION OF IL-12 BY DENDRITIC CELLSAND OTHER CELLTYPES In addition to phagocytic cells and B lymphocytes, other cell types have been shown to produce IL-12. Mast cells derived in vitro from mouse bone marrow in the presence of mast cell growth factors and considered representative of connective-type mast cells produce IL-12, whereas IL3-derived mucosal-type mast cells produce IL-4 (197). These data, which suggest the existence of different types of mast cells that favor Thl or Th2 differentiation, await confirmation with data from in vivo-differentiated mast cells. Other cell types reported to express IL-12 mRNAs and possibly secrete minute levels of IL-12 protein are keratinocytes and epidermoid carcinoma cell lines (198, 199). Exposure to hapten or UV irradiation induces IL-12 in keratinocytes (199,200),and anti-IL-12 antibodies induce a 50% inhibition of proliferation of human allogeneic T cells stiinulated using human epidermal cells containing Langerhans cells as APC (199). However, the physiological significance of this production is doubtful; when used as APC, keratinocytes induce no stimulation of IFN-y production even when CD28 costimulation is provided by anti-CD28 antibodies unless exogenous IL-12 is added to the cultures (201). Also, analysis of IL-12 production by skin cells suggests that Langerhaiis cells rather than keratinocytes are the major IL-12 producers (202). The production of IL-12 by Langerhans cells (202) raises the issue of the ability of professional APC such as dendritic cells to produce IL-12 and the role of this production during antigen presentation and T-cell activation. Earlier data on the production of IL-12 by PBMC showed that in addition to inonocytes and B cells, other MHC class II-positive cells were also responsible for IL-12 production (46). Kanangat et al. (203) showed that LPS treatment of highly purified mouse dendritic cells induces expression of IL-12 p40 inRNA. Definitive evidence that dendritic cells are producers of functional IL-12 came from studies demonstrating that these cells, when used as APC, induce a T h l response if endogenous

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IL-4 production is blocked, and that this Thl response is prevented by neutralizing anti-IL-12 antibodies (148). The production of IL-12 by dendritic cells was directly confirmed by immunocytochemistry, and stimulation of IL-12 production was found to require exposure to T cells in the presence of the specific antigen (148), possibly via CD40/CD40L interaction. Extensive studies with both human and mouse dendritic cells have now confirmed that dendritic cells are efficient producers of the IL12 that acts in inducing Thl responses upon antigen presentation by these APC (129,148,204). Interaction of CD40L on activated T cells with CD40 ligand on dendritic cells appears to be an important mechanism of IL-12 induction during antigen presentation (129, 205, 206); in addition, ligation of MHC class I1 molecules on the dendritic cells also results in some induction of IL12, suggesting a secondary mechanism of IL-12 induction that could play a role during antigen presentation or superantigen stimulation (206). In addition to the CD40/CD40L pathway, IL-12 production in dendritic cells is also activated by LPS, with involvement of a soluble CD14-dependent pathway (136), and by phagocytosis of bacteria (175) and microparticles (207). Interestingly, uptake of microparticle-adsorbed protein antigen by mouse bone marrow-derived dendritic cells results in de nuvu synthesis of transcripts for IL-12 and MHC class I1 and triggers prolonged, efficient antigen presentation (207). Both in the mouse (208) and in humans (209), IL-10 prevents the maturatioddifferentiation of dendritic cells, inducing the generation of cells with decreased ability to produce IL-12 and to induce a Thl-type of response, leading to the development of Th2 lymphocytes. IL-10 also downregulates the production of IL-12 by purified mouse spleen dendritic cells (206). Like IL-10, the presence of PGEz in cultures induces the generation of dendritic cells defective in IL-12 production and promoting Th2-type responses in T cells (210). The production of IL-12 appears to be maximal in certain stages of maturatiodactivation of dendritic cells. In mouse spleen dendritic cells, cultured in the presence of GM-CSF, IL12 production was observed only in the mature form of dendritic cells, obtained by treatment of the culture with TNF-a, IL-1, or LPS (211). Studies in FLT3 ligand-treated mice, which have dramatically increased dendritic cell numbers of both the myeloid type (CDllb bright, F4/80+, and LyG-C+) and the lymphoid type (CDllb dull or negative, CD8a+, CDld+, CD23+), have detected IL-12 production in response to IFNy plus S. aureus in the lymphoid dendritic cells only (212). V. Molecular Control of 11-12 Gene Expression

As with immunological and biological detection studies, analysis of the molecular control of IL-12 production is complicated by the need to analyze

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the expression of two genes. At present, much more information is available on the p40 gene, which is highly inducible and expressed only in IL-12 producing cells, compared with that of the more ubiquitously expressed p35 gene. Upon activation of phagoc$c cells with LPS or S. aweus, accumulation of IL-12 p40 mRNA is observed within 2-4 hr, i.e., slightly delayed compared with that of other proinflammatory cytokines such TNFa,and subsides after several hours (46). Expression of the p35 gene is also upregulated on activation of phagocytic cells, although its ubiquitous constitutive expression has complicated analyses of its expression using nonpurified cell preparations (108, 111, 166) . In some studies (32, 103), but not in others (158),IFN-y has been shown to directly induce transcription and mRNA accumulation of the p35 gene, unlike the p40 gene on which IFN-y has a priming, but not a directly inducing effect. In activated phagocytic cells (both monocytes/macrophages and PMN) and in B-cell lines, p40 mRNA is approximately 10-fold more abundant than p35 mRNA, explaining the overproduction and secretion of the free p40 chain over the p35-containing biologically active heterodimer (21, 32, 103). The lower abundance of p35 mRNA would lead to the obvious conclusion that p35 gene expression is limiting for the production of the p70 heterodimer (213). However, a dramatic upregulation of the production of IL12 p70, e.g., in response to IFN-y and LPS, is often observed in conditions in which only a modest upregulation of p35 mRNA is evident, suggesting that translational and posttranslational mechanisms regulating the assembly of the heterodimer may play a role (32). A potential difficulty in the interpretation of data on mRNA accumulation is that the kinetics of p35 and p40 mRNA accumulation may differ; in particular, optimal priming for p35 mRNA accumulation requires longer preincubation with IFN-y (8-24 hr) than does p40 (2-8 hr) (158).Although upregulation of both p40 and p35 mRNA is observed and is probably necessary in most of the conditions in which increased production of p70 heterodimer takes place, upregulation of p40 mRNA alone, as observed in cells primed with GMCSF, is not sufficient for increased production of the p70 heterodimer (32,158,159).The enhancing effects of IFN-y and IL-13 on IL-12 production are much more relevant for the bioactive p70 heterodimer than for the p40 subunit (32,158,159,174,213), suggesting a physiological relevance in viva of the effect of these modulating cytokines. Because of the possibility that, at least in the mouse, the p40 subunit represents an antagonist of IL-12 bioactivity, the ability of IFN-y, IL-4, and IL-13 to modify the ratio between p40 and p70 is of particular interest; analogously, the inhibitory effect of IL-10 on IL-12 production was reported to be more powerful on the production of the p70 heterodimer than on the p40 subunit (213). A detailed molecular analysis that simultaneously examined nuclear transcription, steady-state mRNA, and secreted protein levels of IL-12 estab-

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lished that the human IL-12 p40 gene is primarily regulated by IFN-7 and LPS at the transcriptional level in monocpc cells (32). Both the human and the mouse IL-12 p40 gene promoters have been cloned (32, 214). The 3.3-kb human p40 promoter, linked to a luciferase reporter gene and transfected transiently into various IL-12-producing and nonproducing cell lines, largely recapitulated the cell specificity of the endogenous p40 gene, i.e., it is constitutively active in EBV-transformed B cell lines (e.g., RPMI-8866, CESS) and inducible in myeloid cell lines (e.g., THP-1 and RAW264.7), but inactive in T-cell lines (e.g., Molt-13 and Jurkat) (32). Moreover, this promoter construct responds to IFN-7 priming in monocytic cells, much like the endogenous p40 gene transcription, suggesting that it contains sufficient sequence elements to reconstitute the in vivo response. Comparison of the human and mouse IL-12 p40 promoters revealed several interesting features. A schematic representation of the IL-12 p40 promoter and of the elements involved in its regulation is depicted in Fig. 4. The promoters are well conserved up to -400 with respect to the transcription start site, where the homology breaks down with large gaps between them. Within the -400 proximal promoter region, several putative

FIG.4. cis and trans elements involved in the regulation of the IL-12 p40 promoter in quiescent phagocytx cells (top) and after activation with IFN-y and LPS (bottom).

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transcription factor-binding motifs are very well conserved: ets at -211/206, PU.1 and NFKB between -124 and -105. Functional characterization of the human p40 promoter in myeloid cell lines has identified the ets element, TTTCCT (AGGAAA for the complement), as a major response region. This element interacts with a large nuclear complex named F1 that binds to a region between -196 and -292. By electrophoretic mobility shift assay (EMSA) and DNase I footprintlmethylation interference assays, it has been established that F1 (1) is induced by LPS or IFN-y in RAW264.7; (2) interacts with the ets-2 element within the -211/-206 region in a complex way, i.e., the interaction requires substantial flanking “anchoring” space; (3) may function as a transcription activator in response to IFN-y and LPS stimulation, as loss of binding results in dramatically decreased promoter activity; (4) is composed of multiple factors including ets-2, IRF-1, c-Rel, and a novel 109-kDa protein that is highly induced by either IFN-y or LPA; and (5) closely correlates with IL-12 p40 gene expression in various cell lines and primary human monocytes (215). A second factor that also interacts with this region but requires less physical space is a complex formed with a fragment derived from -196 to -243 of the p40 promoter, named F2. F2 appears to be more responsive to IFNy stimulation than to LPS, yet its identity remains to be established. Interestingly, the ets element in unstimulated RAW264.7 cells is occupied by PU.l, which becomes displaced by F1 on IFN-y or LPS stimulation (215). The regulation of IL-12 p40 gene transcription in the EBV-transformed B cell line RPMI-8866 appears to be somewhat divergent from that of monocytic cells. The transfected p40 promoter is constitutively active, paralleling the endogenous gene. The nuclear complex F1 is also constitutively present, but its role in the regulation of p40 gene transcription does not seem to be as prominent as in monocytic cells since elimination of the F1-binding element results in only a 30-50% decrease of the promoter activity (G. Gri, G. Trinchieri, and X. Ma, unpublished results). The composition of F1 in RPMI cells also differs from that of monocytic cells in that IRF-2, instead of IRF-1, is present. The implication of the differing composition of F1 is not clear at present. Another region of potential transcriptional regulation is the NFKB half site located between -116 and -106, TGAAATTCCCC (or GGGGAATTTCA for the complement). This site in the mouse IL-12 p40 promoter has been reported to bind NFKB (p50/p65 and p50/c-Rel) in macrophages activated by a number of IL-12-activating pathogens, includmg LPS and S. aureus (214). In EBV-transformed B cells, NFKB constitutively binds to this site (G. Gri, G. Trinchieri, and X. Ma, unpublished results). The NFKBcomplex is composed of c-Re1 and p50 heterodimers. Base substitu-

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tions at this site, which abolish the NFKB binding, result in a -80% decrease in the constitutive promoter activity in B-cell lines. Cotransfection experiments with various combinations of expression vectors containing cDNAs for NFKBp65, p50, c-Rel, and ets-2 demonstrated that ets-2 and c-Re1 synergistically activate the transfected p40 promoter in both IL-12 p40-expressing cells (RPMI-8866) and nonexpressing cells such as Bjab [EBV(-)B cell line] and Jurkat (T cell line) (G. Gri, G. Trinchieri, and X. Ma, unpublished results), strongly suggesting that c-Re1 and ets-2 are the transcription factors necessary and sufficient to determine the cell typespecific expression of the p40 gene. Plevy et al. (216) identified a third element located between -96 and -88 of the murine p40 promoter that is conserved in humans (between -81 and -73) and interacts with members of the C/EBP family of transcription factors that are inducible in the murine macrophage RAW264.7 cell line by heat-killed L. mnocytogenes. The C/EBP element exhibits functional synergy with the NFKBsite upstream (216).These data are consistent with the deficient production of IL-12 in C/EBP P-deficient mice in response to Candida albicans infection (217). Mice with a disrupted gene encoding the interferon consensus-binding protein ( ICSBP) are highly susceptible to infection with intracellular pathogens such as L. mnocytogenes and T. gondii and have defective constitutive and inducible IL-12 expression due to a selective deficiency in IL-12 p40 gene expression (218, 219). ICSBP is a transcription factor of the IRF family, which is expressed exclusively in cells of the immune system, including macrophages, unlike other more ubiquitous IRF family members (220). An ICSBP consensus site is upstream of the Ets site in the IL-12 p40 promoter, and cotransfection of ICSBP and an IL-12 promoter luciferase reporter in RAW264.7 cells dramatically increases both constitutive and IFN-.)I plus LPS-induced expression of IL-12 promoter activity (221). Although evidence of IRF-1 binding to the IL-12 p40 promoter has been reported (215), cotransfection of IRF-1 with the promoter is not sufficient to enhance promoter activity (221). A role for IRF-1 in the control of IL12 is further suggested by the deficient production of IL-12 in IRF-1deficient mice, although the complex immunological alterations in those animals, including the impaired response to IFN-.)I, make it difficult to establish whether IRF-1 acts directly on the IL-12 gene promoter or is indirectly required for the action of other factors that regulate the IL-12 promoter, such as IFN-.)I itself (222, 223). Analysis of IL-12 p35 gene expression is hindered not only by its ubiquity, but also by its low activity and inducibility. Comparative studies with cycloheximide (CHX) demonstrated fundamental differences in mRNA regulation of IL-12 p40 and p35 genes. The increase in S. aureus- or LPSinduced IL-12 p40 mRNA levels was abrogated when cells were pretreated

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with CHX, suggesting that the regulation of the IL-12 p40 gene requires the induction of a CHX-sensitive, transcription activators(s). Conversely, IL-12 p35 mRNA was further upregulated by CHX, indicating that the activation of IL-12 p35 mRNAs requires only a presynthesized activator(s) that can be activated by either S. nureus or LPS at the posttranslational level. Superinduction of cytokine genes such as TNF-a, IL1-P (224), IFN7 , and IL-2 (225, 226) was observed when cells were induced in the absence of CHX for about 2 hr followed by the addition of CHX. IL-12 p40 and p35 steady-state mRNA levels also underwent superinduction when CHX was added 2 hr after S. aureus stimulation (227). The promoter of the mouse p35 gene contains putative elements, including Spl, AP1, ISRE, ICSBP, NFKB, GATA-1, and GAS (30, 31). Unlike the p40 gene, the p35 gene appears to initiate its transcription from multiple sites. The nature of these alternatively initiated transcripts with respect to their cell-type distribution and response to different stimuli remains to be investigated. Based on increasing numbers of observations, it appears that under certain conditions the p35 chain may be a rate-limiting and critical factor in determining the level of IL-12 p70 production by altering either its level of expression or its posttranslational modification in response to specific inducers of IL-12-producing cells, which would affect its association with the p40 chain (213). The Th2 cytokine IL-10 is a potent inhibitor of IL-12 production by phagocytic cells. Its ability to suppress production of IFN-.)Iand other T h l cytokines is due primarily to its inhibition of IL-12 production from APC and to inhibition of expression of other costimulatory surface molecules (e.g., B7) and soluble cytokines (e.g., TNF-a, IL-P) (108,165,166).Studies (227) on the effect of IL-10 on S. nureus- or LPS-induced IL-12 p40 and p35 gene expression in PBMCs and monocytes demonstrate that IL-10 inhibition of IL-12 production is accompanied by reduced steady-state mRNA levels of the p40 and p35 components of the heterodimeric cytokine. The mechanism(s) of IL-10 suppression of IL-12 p40 appears to be exerted mainly at the level of transcription, without significant modulation of mRNA stability. The transcriptional activity of IL-12 p35, primed by IFN-y and induced by LPS, was also substantially inhibited by IL-10. The tlp2 of S. aureus-induced IL-12 p40 was -4 hr and not altered by IL-10. It was also obsewed that CHX abolished the inhibitory effect of IL-10 on the induction of IL-12 p40, IL-12 p35, and TNF-a mRNAs. These findings, together with other reports (228-230), suggest that IL-10 may exert its negative effect through a newly synthesized repressor protein(s). VI. 11-12 Effects on Hematopietic Stem Cells

IL-12 by itself has not been described to affect the growth or differentiation of hematopoietic stem cells. However, in several experimental condi-

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tions, IL-12 reportedly synergizes with other growth factors, in particular IL-3 and stem cell factors (SCF),in supporting the formation of hematopoietic colonies. Thus, IL-12, together with IL-1, IL-6, LIF, and SCF, belongs to the group of synergistic hematopoietic growth factors that alone have little or no in vitro effect on proliferation and act predominantly to enhance the growth-promoting activity of other colony-stimulating factors (231). IL-12 at a concentration of several nanograms per milliliter with maximal activity at 10-100 ng/ml enhances both colony number and size of purified murine hematopoietic Lin- Sca-1' progenitor cells when added in culture together with other growth factors, particularly IL-3, SCF, and FLT3 ligand but also IL-4, G-CSF, and M-CSF (232-236). Single cell cloning experiments suggested that the stimulatory effects of IL-12 on the LinSca-1' cells were directly on the progenitor cells and not indirectly through cytokine production of potentially contaminant accessory cells (232). The cells that formed in response to IL-12 plus SCF were predominantly granulocytes and macrophages, although blasts were also present (232). In the presence of erythropoietin (EPO) and either SCF or IL-4, IL-12 enhances the growth of erythropoietic colonies (235). Progenitor cells enriched from bone marrow of mice treated with 5-fluorouracil in liquid cultures were used to show that IL-12 and SCF or IL-3 synergize in regulating survival and growth of myeloid stem cells and progenitor cells, including primitive long-term culture initiating cells that probably correspond to multipotent stem cells with long-term in vivo repopulating activity; lymphohematopoietic progenitor cells able to yield pre-B-cell colonies were also detected on replating in secondary culture containing SFC and IL-7 (234, 237). Very similar results were reported in the human system, where IL-12 was observed to synergize with IL-3 and SCF in enhancing the growth and colony-formingability of immature progenitor cells to differentiate into granulocytes, macrophages, or erythroid cells (238-241). The stimulatory effect of IL-12 was not observed on hematopoietic cells that were secondarily plated after a 48-hr liquid culture in the presence of IL-3, suggesting that IL-12 is active only on the most primitive progenitor cells (240). A requirement for serum or accessory cells in order to detect the hematopoietic effect of IL-12 suggests that an additional factor(s) present in the serum or produced by the accessory cells may be required for IL-12 action (240, 241). It is not known at present whether the hematopoietic progenitor cells express the IL-12 receptor. The fact that IL-12 is effective on single cells or on cultures with a very low number of cells suggests that IL-12 acts directly on the progenitor cells and not on contaminant cells, thus arguing for the presence of the receptor on these cells. However, whether the

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receptors are constitutively expressed on the progenitor cells or induced

by the synergistic growth factor required for detecting the effect of IL-12 remains to be determined. When hematopoietic colony formation is analyzed on preparations of progenitor cells containing NK cells, an inhibitory effect of IL-12 on colony formation is observed due to the secretion by the NK cells of the hematopoietic inhibitory factors IFN-y and TNF-a, which synergize in blocking colony formation (238).Elimination of NK cells from the progenitor cell preparations or neutralization of IFN-y and TNF-a eliminates this inhibitory effect and reveals the costimulatory effect of IL-12 on colony formation (238). Thus, in vitro, IL-12 has a direct stimulatory effect on hematopoietic progenitor cells and an indirect inhibitory effect mediated by the induction of suppressive factors from contaminating cells (238). Similarly, it was shown that the recovery of hematopoietic stem and progenitor cells from liquid cultures of bone marrow cells was increased by IL12; however, if the cultures were stimulated by IL-12 plus IL-2, which synergize in the induction of IFN-y and other inhibitory cytokines, an increased number of hematopoietic precursor cells was observed only at day 1, folIowed by a marked decrease in precursor number on day 7 or 14 (242). A similar dual stimulatory and inhibitory effect of IL-12 on hematopoiesis was observed in vivo. Administration of recombinant IL-12 in mice suppresses hematopoiesis in the bone marrow, with a drastic but reversible decrease in the number of colony-forming cells, especially the erythroid cells, with a concomitant mobilization of the hematopoietic progenitor cells in the circulation and enhanced peripheral hematopoiesis in the spleen (243-245). Spleen size and cellularity were increased severalfold due largely to infiltration of macrophages and NK cells, but also to an increased number of colony-forming cells (243-245). The negative effects of IL-12 on hematopoiesis in vivo are mediated mostly by IFN-y, as the decrease in bone marrow cellularity and the infiltration in the spleen of NK cells and macrophages were not observed in IFN-yR genetically deficient mice, in which IL-12 administration only promotes hematopoiesis both in bone marrow and in the spleen (245).This ability of IL-12 to promote hematopoiesis in vivo results in a powerful protective effect in mice from lethal ionizing radiation; however, IL-12, while protecting the mice from the heinatopoietic damage of the radiation, sensitized their gastrointestinal tract to radiation, inducing a severe gastrointestinal syndrome that caused their death in 4 to 6 days (246). This gastrointestinal pathology was largely abolished by anti-IFN-y antibodies, indicating a role for secondary induced cytokines in the in vivo pathological effect of IL-12 (246).

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VII. Induction of IFN-7 and Other Cytokines by 11-12

A. IL-12 INDUCED IFN-7 PRODUCTION rtv VITRO

IL-12 induces IFN-y production from resting and activated NK and T cells, with a similar dose-response relationship and a half-maximal activity at 3.5 pM (19, 78). Within T cells, both CD4' and CD8', cells with an a@ TCR, and T cells with a y6 TCR are induced to produce IFN-y (78). However, it has been reported that IL-12 induces IFN-7 through the preferential activation of CD30' T cells (247). CD30 is a transmembrane glycoprotein of the nerve growth factor receptor family, which includes CD40, Fas antigen, and the two TNF-R, and is expressed on 15-20% of anti-CD3-activated T cells, derived from the CD45 RO' memory T-cell subset (248). Not only do CD30' T cells preferentially produce IFN-y in response to IL-12, but they proliferate and expand in response to IL-12 and their generation in anti-CD3-stimulated culture is dependent on both endogenous IL-2 and IL-12, as shown by the ability of antibodies neutralizing these two cytokines to prevent the expansion of the CD30' T-cell subset (247). The induction of IFN-y by IL-12 is characterized by a powerful synergistic effect with other IFN-y inducers, in particular IL-2 and phorbol diesters (19, 78). On T cells, IL-12 also synergizes with mitogenic lectins, with stimulation of the TCR-CD3 complex by anti-CD3 antibodies or alloantigens (78), and with stimulation of the CD28 receptor by anti-CD28 antibodies or its ligand B7 (165, 166). On NK cells, IL-12 synergizes with stimulation by ligands of the CD16 receptor for IgG-Fc (anti-CD16 antibodies or immunocomplexes) and by target cells (249). IL-12 rapidly increases the transcriptional rate of the IFN-y gene; however, when IL12 and IL-2 are added together to cells, most of the synergistic effects of the two inducers are observed at the posttranscriptional level, with an increase of more than two-fold in the half-life of the IFN-y mRNA in the treated cells (250-252). Both resting and activated NK and T cells are induced by IL-12 to produce IFN-y, although maximal IFN-y mRNA accumulation is reached in 2-4 hr in activated T or NK cells and in 18-24 hr in resting PBL (78). Within PBL, IL-12 induces IFN-y mRNA accumulation, as detected by in situ hybridization, in a proportion of both NK and T cells (78); however, NK cells might be a major contributor to the early production of IFN-y in response to IL-12 or IL-2 (253). Although the production of IFN-y is usually attributed to T and NK cells, other cell types have been reported to be possible producers of IFN-y, and in particular IL-12 has been described to induce IFN-y production in B cells (254) and in peritoneal macrophages (255).These results pose interesting questions on the presence of IL-12 receptors on these two cell types and on

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the physiological relevance of their ability to produce IFN-y, all questions that have not been fully addressed yet.

B. REQUIREMENT FOR COSTIMULATION FOR IL-12-INDUCED IFN-y PRODUCTION Although N K and T cells are the IFN-y producers in PBL preparations stimulated by IL-12, an accessory cell type (MHC class II-positive, nonmonocyte, non-B cell) is required for optimal IFN-y production by resting PBL (78). These accessory cells might provide costimulatory molecules for IFN-y production. In murine spleen cells, it has been shown that IL-12 synergizes with TNF-a and IL-1 in inducing IFN-y prodiiction (256-258). This synergistic effect of TNF-a was not demonstrated with human lymphocytes, but antibodies to TNF-a or IL-IP efficiently inhibited IL-12-induced IFN-y production, suggesting that these two cytokines, endogenously produced in the PBL cultures, possibly by the class IIpositive accessory cells, act as costimulatory molecules for IFN-y production together with IL-12 (108). Another costimulatory signal possibly provided by the assessory cells is the B7 molecule, the ligand for the CD28 receptor on T cells. Stimulation of T cells with B-transfected cells or with anti-CD28 antibodies strongly synergized with IL-12 for the induction of IFN-y production (165, 166), and blocking of B7/CD28 interaction with the hybrid recombinant molecule CTLA4-Ig significantly inhibited the ability of PBL to produce IFNy in response to IL-12 (165). On human lymphocytes, CD28 is expressed on CD4' T cells and on a subset of CD8' T cells, but not on resting or activated NK cells. It is of particular interest that B7/CD28 stimulation of resting T cells, when combined with IL-12, is a strong stimulus for IFNy production, even in the absence of signaling through the TCR. Unlike human NK cells, CD28 is expressed on activated murine NK cells, on which IL-12 synergizes with B7/CD28 costimulation in inducing IFN-y production (259). Anti-CD28 antibodies have been shown to enhance the expression of functional IL-12R and of the IL-12R PI chain induced by anti-CD3 stimulation of human T cells (74). However, the effect of CD28 stimulation is not limited to the enhanced expression of IL-12R, as the combination of IL-12 and anti-CD28 results in a dramatic increase in IFNy mRNA stability, suggesting that IL-12 and CD28 synergize by affecting IFN-y gene expression through different mechanisms (S. Robertson and G. Trinchieri, unpublished results). In addition to CD28, stimulation of the surface antigen CD2 by antiCD2 antibodies or by its ligand CD58 can regulate the responsiveness of activated T cells to IL-12 (260, 261). The combination of monoclonal antibodies directed against two different CD2 determinants (T11.1 and

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T11.3) and able to deliver an activating signal to T cells (262) synergizes with IL-12 in inducing IFN-y and proliferation in T cells, whereas antibodies against T1l.l, which block the interaction of CD2 with CD58, only inhibit IFN-y induction (260). The synergistic effect of the CDgCD58 stimulation with IL-12 does not involve the regulation of IL-12R expression (260, 261). These results suggest that TNF-a, IL-lP, B7, and CD58, possibly provided to some extent by the class II-positive accessory cells (78, 261), are important costimulators for IFN-y production in response to IL-12. The ability of IL-10 to inhibit IFN-y production in T and NK cells is due primarily to its ability to suppression IL-12 production, but also, in part, to its ability to suppress expression of these costimulatory molecules on accessory cells (108, 165, 166). The presence of IL-10 during the stimulation of T cells with anti-CD3 prevents the upregulation of IL-12R, possibly by acting directly on the T cells or by preventing the production of IL12 or of costimulatory molecules required for IL-12R upregulation (74). However, on activated T and NK cells, which already express the IL-l2R, IL-10 is unable to block IFN-y production in response to IL-12 (108). TGF-6 is a potent suppressor of IFN-y production in response to IL-12. Like IL-10, TGF-/3 inhibits upregulation of IL-12R in response to antiCD3 antibodies (74),but, unlike IL-10, it also inhibits the ability of both resting and activated NK cells to respond to IL-12 (263, 264). C. IFN-y INDUCING FACTOR (IGIF, IL-18, IL-ly) A novel costimulator factor for IFN-y induction identified in the liver of mice undergoing endotoxic shock was named IFN-y inducing factor (IGIF) and was cloned both in mice and in humans (265-267). Murine and human IGIF are 157 amino acid proteins (266, 267) produced by activated macrophages and possibly other cell types such as keratinocytes (268), with structure and sequence analogy to the IL-1 cytokine family (269).The names IL-18 (267) and, based on its structure, IL-1y have been proposed for IGIF (269).Like IL-16, IGIF has an unusual leader sequence or predomain that must be cleaved by action of the protease caspase 1 or IL-lP converting enzyme for optimal secretion and biological activity (270, 271). It was originally reported that IGIF by itself was a more potent inducer of IFN-y and IL-12, and that it was effective in the absence of IL12 (266).However, it soon became clear that IGIF requires costimulation to induce IFN-y, e.g., by T-cell mitogens, IL-2, or anti-CD3 (267), and that it strongly synergizes with IL-12 in inducing IFN-y production (272). Studies with murine T-cell lines have shown an absolute requirement for coexposure or preexposure of the cells to IL-12 in inducing IGIF responsiveness and suggested that IL-12 upregulates expression of the

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IGIF receptor (273),a still unidentified molecule different from the known receptors of the IL-1 family (274). IGIF is a costiinulatory factor for the activation of T h l but not Th2 cells (275); IGIF and IL-12 synergize on Thl clones to induce IFN-y production, but only IGIF induces IL-2 production (275). However, IGIF does not promote differentiation of naive murine T cells to Thl cells, and IL-12 is required for priming T h l cells for high IFN-y production and IGIF responsiveness (276). Although IGIF does not bind to the known IL-1R (274), it activates the IL-1R associated kinase and, like IL-1, activates NFKB; however, unlike IL-12, it does not activate STAT4 (276, 277). IGIF, but not IL-1, activates NFKB on Thl clones, whereas the reverse situation is true in Th2 clones (276).The exact role of IGIF and its synergism with IL-12 remain to be defined in the in vitro and in vivo situations in which IFN-y production is induced. IGIF may be an almost absolute requirement for IFN-y production, but, as already reported for TNF-a and IL-10, at least in the human system, low concentrations of endogenous IGIF may be present in in vitro culture or in vivo to support IFN-y when other stimuli, e.g., TCR ligands or IL-12, are present. Indeed, M. Aste and G. Trinchieri (unpublished observation) have observed that, although IGIF mRNA is rapidly induced in human monocytes by the same stimuli that induce IL-12 production, a constitutive level of expression of IGIF mRNA is observed in freshly isolated monocytes, unlike the case of IL-12 p40 mRNA. Thus, the limiting roles of IL12 and IGIF in determining either acute or chronic production of IFNy remain to be established.

D. IL-12 REQUIREMENT FOR IFN-y PRODUCTION IN VITRO AND I N VIVO

Not only is IL-12 a potent inducer of IFN-y production, but it is also most likely a required factor for efficient IFN-y production, depending on accessory cells. When human PBMC were treated in vitro with stimuli, e.g., S. aureus, that induce IL-12 production, they rapidly produced large amounts of IFN-y. This production of IFN-y was almost completely inhibited by neutralizing antibodies against IL-12 (46). Even when IFN-y inducers that are not known to stimulate IL-12 production were used (e.g., IL-2, mitogens, or anti-CD3 antibodies), IFN-y production from PBMC was inhibited up to 80%, indicating that endogenously produced IL-12 is required for optimal IFN-y production (46). In these cultures, IL-12 is most likely induced by CD40L-expressing T cells, activated by stimuli with or without TCR engagement (130). However, if purified T or N K cells lacking IL-12-producing accessory cells were stimulated to produce IFNy , e.g., by IL-2 or anti-CD3 antibodies, no inhibitory effect of anti-IL-12 antibodies could be demonstrated (46).

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Injection of mice with daily intraperitoneal injections of l p g of recombinant IL-12 induced high levels of serum IFN-y, but not until 48 hr after the first injection (278). This delayed response was probably due to the lack of appropriate costimulatory signals when only recombinant IL-12 was injected. The ability of endogenous IL-12 to induce rapid production of IFN-y in wivo has been clearly shown in several experimental models of infectious diseases. A very informative experimental model for the understanding of the role of IL-12 in inducing IFN-y in vivo is provided by endotoxin-induced shock (161,162). Several cytokines, particularly TNFa and IFN-y, have been shown to be responsible for pathologic reactions that may lead to shock and death observed in infection with gram-negative bacteria and in response to endotoxins. Mice injected with LPS produced IL-12, which induced IFN-y production, as demonstrated by the ability of neutralizing anti-IL-12 antibodies to almost completely suppress IFNy production (161, 162). Studies with animals genetically deficient for the subunit of IL-12 (279,280), for the IL-12Rfll subunit (72), or for STAT4 (100, 101) have fully confirmed that IL-12 is necessary in wiwo for optimal production of IFN-y in response to bacterial products.

E. INDUCTION OF OTHERCYTOKINES BY IL-12 Although IL-12 is particularly efficient in inducing the production of IFN-y and in synergizing with other inducers, e.g., IL-2, in this effect, IL-12 also induces other cytokines and potentiates the effect of other cytokine inducers. In particular, IL-12 induces or potentiates the induction of TNF-a, GM-CSF, IL-8, IL-3, and, in certain conditions, IL-10 and IL-4 (168, 249, 281-285). IL-12 induces production of TNF-a and GMCSF from human T or NK cells at only minimal levels and is much less efficient in this effect than IL-2 or, especially in the case of GM-CSF, IL7 (249,283). When the ability of IL-12 and IL-2 together to induce IFNy and GM-CSF is compared, at both the mRNA and protein levels, a strong synergistic effect is observed for IFN-y, but only an additive effect for GM-CSF (249). However, when IL-12 is added to T or NK cells induced by other stimuli, e.g., anti-CD3, anti-CD16, anti-CD28, or phorbol diesters, IL-12 induces a strong and significant enhancement in the production of both TNF-a and GM-CSF (165, 249). The ability of IL-12 to regulate the production of type 2 cytokines is complex. Although in most instances IL-12 has an inhibitory effect on the production of IL-4 (79,286),it has also been shown to cooperate with IL4 in the generation of IL-4-producing Th2 cells (168,287,288). The effect of IL-12 on the production of IL-10 is also complex, because on the one hand, it inhibits the generation of IL-lO-producing Th2 cells and downregulates the ability of T cells from atopic patients to produce IL-

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I0 (286); on the other hand, it can directly stimulate T cells to produce IL-10 and can synergize in this effect with other stimuli such as anti-CD2, anti-CD3, IL-2, and B7/CD28 interaction (284, 289-291). IL-12 can also prime T cells for high IL-10 production, inducing the generation of clones that produce both IFN-y and IL-10 (168, 285). Indeed, IL-12 injection in vivo was shown to induce expression of mRNA for both IFN-y and IL10 (167), and although these data were orignally interpreted assuming that IL-10 was produced by macrophages rather than T cells, they should now be reinterpreted in light of in vitro data demonstrating the induction of IL-10 production by T cells in response to IL-12. Because IL-10 is a potent inhibitor of production of IL-12 and Thl-type cytokines, the ability of IL-12 to induce IL-10 production has been interpreted as a negative feedback mechanism (284). The likely importance of such a feedback mechanism is supported by findings that IL-10 knockout mice infected with T. gondii or T. cnrzi produce uncontrolled levels of proinflammatory cytokmes such as IL-12, TNF-a, and IFN-y, resulting in the death of the animals (292, 293). VIII. Mitogenic Activity of 11-12

IL-12 was originally described to have a comitogenic effect on human T cells (19). Although IL-12 was unable to directly induce proliferation of resting peripheral blood T cells, it induced a significant enhancement of proliferation that was more evident at later times of culture when added together with the mitogeii PHA or phorbol diesters (19, 281). In particular, when IL-12 was added together with PHA, it did not increase ['HITdR uptake at the peak of proliferation at day 3, but prevented the decline in proliferation and loss in viability obseived in the PHA-stimulated cultures at day 6 (19, 281). Similarly, IL-12 enhanced the proliferation of allostimulated T cells in mixed lyinphocyte cuItures (MLC) (19). Interestingly, low levels of IL-12 are produced by accessory cells in MLC, and anti-IL-I2 antibodies partially inhibit proliferation in the culture, indicating a role for endogenous IL-12 in the proliferative response of T cells to allostimulation (294). The inability of IL-12 by itself to induce proliferation of resting T ceIls is most likely due to the lack of functional IL-12R. Studies have clearly shown that T cells activated for a few days with anti-CD3 antibodies or mitogens proliferate in response to IL-12 (281, 29S, 296) and express high-affinity IL-12R (67, 74). It remains unclear why the proliferative effect of IL-12 requires the upregulation of high-affinity IL-12R on T cells, while the induction of cytokine production is observed in a large proportion of resting T cells (78), which do not express any detectable IL-12R. IL-12 induces

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proliferation of both CD4' and CD8' cells, but the maximal levels of proliferation are always lower than those induced by IL-2, even though IL-12 is active at lower molar doses than IL-2 (281, 295). The T-cell proliferation induced by IL-12 is not mediated by endogenous IL-2, as suggested by the inability of anti-IL-2AL-2R antibodies to block it and by the lack of inhibition of IL-12-induced proliferation by compounds such as cyclosporin A, which in most stimulation conditions block IL2 production (281, 295, 297). However, the mechanism of IL-2- and IL- 12-induced proliferation may have some common pathways because both are blocked by rapamycin (297). Like T cells, preactivated NK cells proliferate in response to IL-12 (281). The preferential proliferation of NK cells observed in cultures of human PBL stimulated with EBV(+) BCL is enhanced by the addition of low levels of exogenous IL-12 and is in part dependent on the production of endogenous IL-12 by the BCL, although the production of IL-2 by allostimulated T cells plays a major role in the proliferation of NK cells in these cultures (298). When IL-12 and IL-2 are added together to preactivated T cells, especially CD4+T cells, an additive effect is observed, especially at low concentrations of both cytokines, but usually not synergism (281,295). The ability of the two cytokines to affect each other's ability to induce proliferation is in part due to the induction of IL-12R by IL-2 (67, 74) and to the induction of IL-2RdCD25 by IL-12 (299, 300). However, on purified activated NK cells, Ty8 cells, and CD8+ T cells, low concentrations of both IL-2 and IL-12 had an additive effect on proliferation, but IL-12 inhibited in a dose-dependent manner the proliferation induced by high doses of IL-2 (281, 301, 302). The inhibitory effect of IL-12 on IL-2induced proliferation is dependent on the level of NK cell activation, possibly reflecting differences in the expression of the IL-12 and IL-2 receptor complex, and is blocked by anti-TNF-a antibodies (281).Although these results suggest a role for endogenous TNF-a in IL-12-mediated inhibition, TNF-a alone has no inhibitory activity when added to the cultures (281), and in one report it was shown to enhance IL-12-induced IL-2RdCD25 expression (300). Among other cytokines that have been shown to synergize with IL-12 in inducing proliferation are IL-7 on human T cells (303) and IL-4 on human N K cells (66) and on the IL-Pdependent mouse cell line CT4S (304). The synergistic proliferative effect of IL-4 and IL-12 is somewhat difficult to reconcile with the observations that IL-4 downregulates the expression of the high-affinity IL-12R and of the IL-12RP2 chain on human T cells (74, 75, 86). TGF-P also prevents expression of the high-affinity IL-12R, probably acting at the level of the IL-12RP2 chain (74,305),thus inhibiting IL-12 responsiveness in alloactivatedT cells (305).Costimulation

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of either resting or preactivated human T cells with CD28 ligands (B7 or antibodies) and IL-12 results in a powerful, IL-12-independent proliferation of T cells (165, 166). Similarly, costiinulation of preactivated human T cells with appropriate CD2 ligands (anti-CD2 antibodies or CD58) results in efficient proliferation (260). Several studies have shown that T h l but not Th2 cells are responsive to IL-12 (81, 86, 104), and this phenomenon has now been clarified with the finding that Th2 clones permanently downregulate the expression of the IL-12RP2 chain (83, 86). Interestingly, long-term established T h l clones may lose their ability to produce IL-2, and when stimulated in vitro by specific antigen and APC, their proliferative response is partially or totally dependent on the IL-12 produced by the APC (166, 306). Murine T h l clones tolerized in witro with anti-CD3 antibodies as well as anergic CD4+ T cells isolated from mice tolerized to the Mls-1" antigen demonstrated defective induction of proliferation in response to IL-12 on restimulation with antigen (307). Although IL-12 did not prevent the induction of T-cell anergy in these murine models (307), IL-12 was reported to prevent anergy in melanoma-specific CD4' T-cell clones using a melanoma cell line as APC (308). IL-12 has been reported to restore, in part, the responsiveness to recall antigens in the anergic CD4' T cells of HIV+ patients (119, 309), mostly by preventing activation-induced apoptosis in the patients' CD4+ T cells (310-313). These studies raise the possibility that, like other growth factors, IL12 may facilitate T and NK cell proliferation by preventing cell death; however, IL-12-mediated activation of NK and T cells, unlike that mediated by IL-2 and IL-7, does not upregulate the expression of the apoptosis protective gene BCL2 (314, 315). IX. Activation of Cyiotoxic Lymphocytes by 11-12

A. ACTIVATION OF NK CELLS One of the activities by which IL-12 was originally identified was its ability to enhance NK cell cytotoxic activity (19), and the two original definitions of IL-12, NKSF or CLMF, refer to the ability of IL-12 to activate NK cells and lymphokine-activated killer (LAK) cells (19, 20). Treatment of PBL with IL-12 for several hours results in an enhancement of NK-cell-mediated cytotoxic activity that can be detected against NKsensitive target cells (e.g., K562), but also against NK-resistant target cells or virus-infected cells (19, 117, 316). Mice with disrupted IL-12 p40 genes have only a modest decrease in NK cell-mediated cytotoxicity, indicating that IL-12 is not essential for differentiation of NK cells (279). However, an immature human NK cell subset expressing the

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NKR-P1A antigen, but neither CD56 nor CD16, is dependent on IL12 for differentiation in vitro to acquire the CD56' phenotype of mature NK cells (317). The enhancement of cytotoxic activity of NK cells preincubated with IL-12 is usually lower than that observed with IL2 and is comparable with that of IFN-a (19, 318); however, IL-12 enhances human NK cell cytotoxicity at concentrations between 0.1 and 10 pM, whereas IL-2 and IFN-a reached their maxiinal activity at concentrations higher than 1 nM. This sensitivity of resting NK cells to the enhancing effect of minimal concentrations of IL-12 is surprising, as no evidence of high-affinity IL-l2R has been demonstrated on these cells, although cytofluorirnetric analysis with IL-12 and IL-12 antibodies has suggested that resting NK cells, unlike most T cells, express low levels of IL-12R (66). It is also of interest that, unlike IL-12 induction of IFN-y production, the IL-12-mediated enhancement of cytotoxicity does not require tlie presence of accessory cells, which could provide costimulatory signals for upregulation of the IL-12R (316, 318). The simultaneous treatment of NK cells with IL-12 and IL-2 or IFN-a results in only an additive effect on cytotoxicity, very different from the strong synergism observed when IFN-y was induced by a combination of IL-2 and IL-12 in either NK or T cells (316). The effect of IL-12 on NK cytotoxicity is not blocked by antibodies to either IFN-dP or IL-2, suggesting that IL-12 acts directly on the cytotoxic cells and is not acting through the induction of other cytokines (318, 319). An inhibitory effect of anti-TNF-a antibody was observed when NK cells were cultured with IL-12 for 3 days (318) but not for 1 day (316), suggesting a possible requirement for this cytokine in longer term cultures only. When human PBMC are stimulated in vitro with S. aureus, a significant increase in NK cytotoxic activity is observed, which is partially inhibited by antibodies to IL-12, IFN-a, and IL-12, but is almost completely inhibited by a mixture of all three antibodies, indicating that the three cytokines may cooperate during infection in inducing NK cell activation (46). Indeed, IL-12 has been shown to be required for NK cell activation and migration in the regional lymph node in L. major infections (320). In vivo daily injections of exogenous IL-12 in mice result in an enhancement of NK cytotoxic activity in both liver and spleen: however, similar to the phenomenon described for IFN-a (321), NK activation is maximum at 2 days, declining thereafter (278). In vitro, IL-12 is chemotactic for human NK cells and stimulates their interaction with vascular endothelium via LFA-1/1CAM-1 and VLAWCAM-1 pathways (322). IL-12 activation of NK cells is accompanied by an increased expression of CD69 antigen, TNF-R p75, IL-2RaKD25, and the P2 integrin CDlla; however, IL-12, unlike IL-2, does not

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upregulate the expression of pl integrins (89, 323, 324). IL-12 treatment of human NK cells results in an increased number of granules (316) and a change in their morphology from a prevalently electron-dense inside core to a more polymorphic morphology with the formation of vesicles, indicating cellular activation (C. Grossi and V. Pistoia, personal communication). Previous exposure of N K cells to IL-12, as well as to IL-2, enhances the release of granule-derived proteins on triggering by stimuli that activate the Ca2+ and/or a protein kinase C-dependent intracellular pathway, suggesting that these cytokines confer a level of activation to the NK cells that allows them to respond with maximal granule exocytosis and cytotoxic activity to stimulation (325). Several studies (249, 326-328) have demonstrated that IL-12 enhances transcription and accumulation of m R N A for several granule-associated molecules, particularly perforin and granzyrne B, during its activation of N K cells or cytotoxic T cells. A synergistic effect of IL-12 and IL-2 in inducing the perforin and granzyme R genes is marginal and is not observed consistently (249, 327), reflecting the effect of these two cytokines on cytotoxic activity and contrasting with the powerful synergism observed for the induction of IFN-y gene expression.

B. IL-12 ACTIVITY O N CYTOTOXIC T LYMPHOCYTES IL-12 was originally reported to synergize with IL-2 in inducing the generation of LAK cells (cytotoxic cells comprising activated N K cells and non-MHC-restricted CTL) from human-purified blood lymphocytes in the presence of hydrocortisone to block endogenous cytokine production (20). In the absence of hydrocortisone, IL-12 alone induces LAK cell generation with a mechanism that requires endogenous TNF-a, (329). The ability of IL-12 to upregulate the cytotoxic mechanism of resting and noncytotoxic peripheral blood T cells has been demonstrated by its ability to endow these cells with the ability to mediate antibodyredirected lysis of anti-CD3-coated target cells on an 18-hr culture (316). IL-12 also enhances the generation of hurnan-allospecific CTL (329, 330). Notably, IL-12 has been shown to induce both proliferation and generation of CTL activity in primary mixed cultures of lymphocytes from siblings identical for MHC class I1 antigens and displaying class I disparity, suggesting that IL-12 may play a role in helper T-cellindependent CTL generation (294). In this latter system (294), but not in the generation of CTL against fully allogeneic cells (329), the effect of IL-12 on CTL generation is IL-2 independent. As with NK cells, the ability of IL-12 to enhance CTL activity is, in part, based on induction-of the expression of genes encoding cytotoxic granule-associated proteins, e.g., perforin (294, 327, 330), suggesting that IL-12 may effect

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the generation of CTL by inducing both their expansion and their differentiation in cytolytic effector cells. In vivu intraperitoneal (ip) injection of high doses of IL-12 (1 pgl mouse/day for 4-5 days) in mice immunized in the footpad with allogeneic lymphocytes induces a strong increase in the CTL activity in the draining lymph nodes, although the number of cells recovered in the lymph node is decreased by 25-50% (278). However, endogenous IL-12 is not required for the generation of allospecific CTL, as demonstrated by studies in IL12 p40 genetically deficient mice (279). In most experimental systems, no major effect of either exogenous or endogenous IL-12 in the generation of anti-viral CTL has been demonstrated (331),with a modest decrease of CTL activity in anti-IL-12-treated mice observed only in influenza virus infection (332). However, the in vitru addition of exogenous IL-12 to influenza virus-infected human dendritic cells strongly enhanced the ability of these cells to induce CD8 Tcell proliferation and generation of CTL responses, especially with lymphocytes of donors with weak reactivity to influenza virus or at a low APC : T cell ratio (333);this CTL-enhancing effect of IL-12 is not mediated by IL-12-induced IFN-y. The ability of IL-12 to facilitate CTL generation against influenza virus nucleoprotein was shown in experiments in which mice were immunized with a class I peptide in incomplete Freund adjuvant: a CTL response was observed in only a minority of mice not treated with IL-12, whereas a single 1-pg IL-12 dose at the time of immunization induced a vigorous CTL response in all animals (334). Relatively few studies have investigated the role of IL-12 in the generation of antitumor CTL. Multiple ip injections of IL-12 were shown to induce CTL activity against target cells expressing two different mutations of p53 in mice immunized either with peptides mixed with the adjuvant QS21 (335) or with peptide-pulsed dendritic cells (336). In both systems, rejection of established tumors was observed, but the optimal IL-12 doses for generation of CTL activity in the QS21-peptide experiments were found to be very low (1 nglmouse), with higher doses found to be inefficient or suppressive (335), whereas the experiments with peptide-pulsed dendritic cells used 10 injections of 300 nglmouse every other day (336). The schedule and dosage of IL-12 administration is of primary concern in possible immunotherapeutic approaches, not only in order to avoid systemic toxicity, but also because treatment with high doses of IL-12 for 2 weeks, a typical protocol for obtaining a direct antitumor effect, has a profound suppressive effect on both antitumor and allospecific CTL activity (H. Kurzawa, W. Lee, and G. Trinchieri, unpublished results).

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C. ACTIVATIONBY IL-12 OF MURINENK1 T CELLSA N D THEIRHUMAN EQUIVALENT NK1 T cells are a specialized population of T a p cells that coexpress receptors of the N K lineage and have the unique potential to very rapidly secrete huge amounts of cytokines, both T h l and Th2 type (337). They express a restricted TCR repertoire made by an invariant TCR a chain, Va14-Ja281, associated with oligoclonal TCR 0 chains (338). NK1 T cells recognize the products of the conserved family of CD1 genes, MHC class I-like molecules with a large hydrophobic-binding groove (339), consistent with their ability to present lipid antigens, including inycobacterial cell wall antigens (340). A comparable population of human T cells is, like the murine counterpart, either CD4-lCD8- or CD4+ and expresses NK cell markers, such as NKR-PIA, CD94, and CD69 (341). Injection of 0.5 pg IL-12 ip in mice enhanced the cytotoxic functions and NK1 antigen expression in hepatic NK1 T a b cells after 24 hr (342344). These IL-12-activated NK1 T cells are the major effector cells in the IL-12-mediated inhibition of experimental liver tumor metastasis (343). LPS treatment similarly induces NK1 T cells with potent cytotoxic activity and in vivo antimetastatic effects via production of IL-12, presumably from liver Kupffer cells (345). NK1 T cells are also major producers of IFN-y in response to IL-12, alone or in association with IL-2 and antiCD3 (346, 347). Although all NK1 T cells are cytotoxic, only the subset expressing the Ly-6C antigens can produce IFN-y in response to IL-12 alone or IL-2 plus IL-12 (346). It has been reported that IL-12 or IL-12 inducers such as L. monocytogenes and Bacille-Calmette-Gu6rin (BCG) reduce the pool of NK1 T cells in the liver and their cytotoxic activity after 3 days of treatment, suggesting that these spontaneously cytotoxic cells may contribute to immunosurveillance of the inflammatory process in the liver and are downregulated by IL-12 (348, 349). The reason for this major discrepancy with previous studies showing increased numbers and activation of NK1 T cells in response to IL-12 has been tentatively attributed to the different time of analysis (3 days rather than 1 day after treatment) and to the possibility that the increase in cytotoxic activity reported previously was due not to NK1 T cells but to the presence of large NK1-positive conventional NK cells in the liver (348).Ongoing studies using mice genetically deficient or transgenic for Val4 are, however, confirming that NK1 T cells are the primary responders to IL-12 in v i m ( M . Taniguchi, personal communication). Interestingly, in vitro culture of human PBL with IL-12 and IL-2 induces a selective expansion of cytotoxic CD4+ CD56' T cells, which are present in the liver of humans and may correspond to murine liver N K 1 T cells (350). This findicg raises the

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possibility that this subset of T cells may be a major responder cell type to IL-12 in humans as well. X. Effect of 11-12 on the Differentiation of T Helper Cells

A. IL-12 Is THE MAJORCYTOKINE RESPONSIBLE FOR THE GENERATION OFT^^ CELLS The requirement for IL-4 in the generation of IL-4-producing Th2 cells has been well established (351-353). More recently, the role of IL-12 for the efficient generation of IFN-y production by T h l cells has become evident, and it has been proposed that the balance between the levels of IL-4 and IL-12 early during an immune response may be responsible for the bias in the generation of Th2 and Th1 cells, respectively (354), although the presence of other cytokines and various other factors regulating the immune response also play a major role. Furthermore, the synergistic/ antagonistic interaction between IL-4 and IL-12 in regulating such responses is complex and not yet fully understood (168, 355). Stimulation in witru of PBL from atopic patients with allergens such as Dermatuphagoides pterunyssinus group 1(Der p. 1)results in the generation ofT-celllines and clones with the high IL-4 andlow IFN-y production typical of Th2 cells, whereas PBL stimulation with bacterial products [e.g.,purified protein derivative (PPD)] generates Thl-type T-cell lines and clones that produce IFN-y but not IL-4, When PBL were stimulated with Der p.1 in the presence of IL-12, T-cell lines and clones were generated that exhibited a reduced ability to produce IL-4 and an increased ability to produce IFNy (79).This Thl-inducing effect of IL-12 is not inhibited by anti-IFN-y, but is reduced by removal of NK cells from the PBL preparation. PPD-specific T-cell lines generated in the presence of anti-IL-12 antibodies during the initial antigenic stimulation produced significant levels of IL-4, unlike the cell lines generated in the absence of antibodies, and gave rise to PPDspecific CD4" cell clones showing a ThO/Th2 phenotype rather than a Thl phenotype (79).These results indicate that IL-12 not only facilitatesproliferation and activation of Thl cells in a memory response in vitru, but also that, as shown by the effect of anti-IL-12 antibodies, endogenously produced IL12 is important for T h l generation. The ability of IL-12 to directly initiate Thl cell development in native murine T cells was shown by Hsieh et al. (8O),who reported that naive CD4+ T cells derived from mice transgenic for an antiovalbumin TCR are induced by ovalbumin to develop into T h l cells in the presence of IL-12, whereas they develop into Th2 cells in the presence of IL-4. The effect of IL-4 was dominant over that of IL-12 (80).Neutralization of endogenous IFN-y also inhibited IL-12-induced generation, suggesting that the effect

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of IL-12 to induce Thl cell generation was demonstrated in inany models, in humans and in experimental animals, both in vitro and in vivo (357). The originally reported dominance of IL-4 action over IL-12 (80) was observed to be a much more complex interaction, with the two cytokines antagonizing or synergizing with each other, depending on the function analyzed (168, 355, 358).

B. MURINESTUDIESO N THE ROLE OF IL-12 I N T h l DIFFERENTIATION 1. Identity of IL-12 and T-cell Stirnulufonj Factor While the human and murine IL-12 were being characterized and cloned, the group of Gerinann and Rude identified a inurine factor (Tcell stimulatory factor or TSF) produced by accessory cells in response to antigen-activated T cells and able to induce proliferation and IFN-y production in Thl but not in TI12 clones by a mechanism independent of TCR stimulation and Caz+flux and resistant to cyclosporin A inhibition (147, 359). As the biological activities of IL-12 on Thl cell generation and function became better understood, it was clear that TSF was indeed the same factor as IL-12 (81, 287, 360). 2. Requirenzent for IFN-y in lL-12-Induced Thl Responses

I n several in vitro experimental models in which the mechanism of IL12-induced generation of the Thl cells was studied, the major mechanism of action of IL-12 was found to be that of priming T cells for production of IFN-y and of enhancing the ability of differentiated Th1 clones to produce IFN-y (81,287,358,359).The requirement for endogenous IFNy in the mechanism of Thl generation induced by IL-12 was an almost universal finding in rnurine studies (356, 358, 360, 361), although one of these studies (358) reported that differentiation of Thl in an accessory cell-dependent system, but not in an independent one, was blocked by neutralizing anti-IFN-y antibodies. One interpretation of these results is that IFN-y acts at the level of the APC, possibly enhancing their ability to maintain a high level of production of endogenous IL-12. This hypothesis is indeed supported by studies in mice expressing a dominant negative IFN-y receptor that, depending on the promoter used for the expression of the transgene, display tissuespecific unresponsiveness in the macrophages or in the T cells; experiments with these mice identified the inacrophage as the critical responsive cell in manifesting the effect of IFN-y in regulating Th1 subset development (362). However, another study (363) demonstrated that the phenotype of CD4+ T cells was also important and that naive CD4t T cells required endogenous IFN-y for IL-12-induced Th1 generation, whereas memory CD4' T cell did not. These results are compatible with the finding that IFN-

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y upregulates the expression of the IL-12RP2 chain (83).The presence of IFN-y may be required for IL-12 activity on naive or Th2-committed CD4' cells, which do not express or have downregulated the IL-12RP2 chain, but less so on memory CD4+ T cells with an activated phenotype. A third level on which IFN-y modulates the activity of IL-12 is the production of IL-2 by T h l cells. Studies by Bradley et al. (361, 364) have shown that while the presence of IL-12 during stimulation of either naive or memory CD4+ T cells induces high level IFN-y production, it inhibits the ability of these cells to produce IL-2 compared to cells stimulated in the absence of IL-12. This negative effect of IL-12 on IL-2 production is drastically diminished by neutralization of endogenous IFN-7. 3. Relative Role of IL-12 and IL-4 in Thl Responses Although early studies indicated an almost complete dominance of IL4 over IL-12 in inducing Th2 differentiation when both cytokines were present during primary stimulation (80, 358), it became clear that the relative concentration of the two cytokines is important and that (a) IL12 does not inhibit IL-4 mediated priming for IL-4 production, but IL-4 only partially inhibits the IL-12-mediated priming for IFN-y production; (b) high amounts of IL-12 in combination with relatively low levels of IL4 give rise to a T-cell population with at ThO-like cytokine profile; and (c) in the presence of relatively high amounts of IL-4, IL-12 enhances the development of Th2 cells (287, 358). One of the major mechanisms by which IL-4 prevents IL-12 signaling is by downregulating the expression of the IL-12RP2 chain (83),an effect that is counterbalanced by IFN-y (83), thus making the balance between IL-4 and IFN-y (and its inducer, IL-12) a key factor in determining the outcome of the CD4 effector Tcell responses (365).It is also most likely that this interplay between IL12IIFN-y and IL-4 is also controlled by the genetic background of the mice; the finding (84) that in neutral conditions (i.e., with no exogenous cytokines added) T cells from B10.D2 mice maintain expression of IL12RP2, whereas those from BALB/c mice rapidly lose it, may reflect different levels of endogenous IL-4, IL-12, and IFN-y in these strains (320, 366) or different responsiveness of the IL-12RP2 gene to IFN-y and IL-4. A locus on murine chromosome 11 controlling the differential maintenance of IL-12 responsiveness has been mapped (367), and the identification of the specific gene involved should shed some light on the mechanisms of regulation of Thl/Th2 responses. 4. Stability of the ThllTh2 Phenotype

Thl and Th2 clones are quite stable in their phenotype, indicating that extremely differentiated T cells lose their plasticity to respond to polarizing

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stimuli, which are instead effective on naive or even memory T cells (361). Some of the original studies on the stability of polarized T h l or TI12 T cell populations suggested that the IL-12-induced Thl populations, after 1 week of culture, can still be induced by secondary stimulation in the presence of IL-4 to differentiate into IL-4-producing Th2 cells, whereas the Th2 population induced in the presence of IL-4 and anti-IL-12 could not be induced by IL-12 to differentiate into IFN-y-producing T h l cells (82, 368). This loss of plasticity of Th2 cells was attributed to a defined phenotypic change, i.e., the loss of the expression of the IL-12RP2 subunit (82, 83). However, the interpretation of these studies was made difficult by two factors. First, the use of polyclonal cell populations, although more reflective of the physiological in vivo conditions than the use of clones, &d not allow the investigators to distinguish between the expansion of nonterminally committed cells and the change in phenotype of individual cells. Second, the participation of endogenous cytokines, in particular IL4, IFN-y, and IL-12, could not be accurately evaluated. Indeed, Murphy et nl. (369), using single-cell analysis of cytokine expression by intracellular staining (370), showed that both T h l and TI12 polarized populations after 1 week culture can be skewed to the opposite phenotype by switching the culture conditions, most likely by expanding undifferentiated or incompletely differentiated cells, but that, after long-term stimulation for 3 weeks, this reversibility is lost. Nakamura et al. (371) addressed the same question using different approaches, i.e., analysis of IL-4 and IFN-y transcripts during stimulation and, particularly, the elimination of IL-Cproducing cells at different days of culture, utilizing the antiviral drug ganciclovir and T cells from mice transgenic for the thymidine kinase gene under the control of the IL-4 promoter (372). Those studies suggested that by day 2 of culture, a large proportion of T cells stimulated in the presence of IL-4 or IL-12 is irreversibly committed to the Th2 or T h l phenotype, respectively. The inability of Th2 cells to respond to IL-12 is due to downregulation of the IL-12RP2, but this subunit can be reexpressed if the cells are treated with IFN-y (83), an effect that is antagonized by IL4. Indeed, Nakamura et al. (365) demonstrated that the Th2 populations normally show a stable phenotype and fail to respond to IL-12 because of endogenous IL-4 production. The use of anti-IL-4 antibodies does not completely avoid this effect because IL-4 may be utilized intracellularly by the IL-4-producing cells (371). IFN-y abrogates the antagonistic effect of IL-4 and permits the conversion of Th2 populations into IFN-y producing cells. In the complete absence of IL-4, using cells from IL-4 genetically deficient mice, IFN-y is not required for this conversion and IL-12 can convert The populations in Thl cells that lose responsiveness to IL-4 due to the lack of the IL-4-mediated upregulation of IL-4R (365). The reported

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ability (82, 368) of IL-4 to revert the polarized Thl populations to a Th2 phenotype most likely reflected the presence of endogenous IL-4 during the primary stimulation, which prevented complete differentiation of the Thl cells induced by IL-12. The finding that T h l polarized populations from IL-4 knockout mice cannot be reverted by IL-4 to produce Th2 cytokines (e.g., IL-5) supports this explanation (365). Thus, in the absence of IL-4, IL-12 rapidly and irreversibly commits CD4 T cells to a Thl phenotype.

5. Is IL-12 Essential for Thl T-cell Generation? Many studies in oitro and in vizjo have clearly shown that IL-12 is a potent inducer of T h l responses. However, in IL-12 p40 genetically deficient mice, Thl responses are severely depressed, especially regarding IFN-y production, which is reduced to -10% of that of wild-type mice, but not completely absent, e.g., IL-2 production is almost normal (279, 280). In addition, the regulation of Thl response to bacterial or nominal antigen in the presence of adjuvant may be different from that observed in other experimental models: for example, the T h l response and priming for IFN-y in cardiac allograft recipients appear to be little affected in IL-12 p40 or p35 deficient mice (373), and IFN-.)Iproduction in several virus infections may be relatively independent of IL-12 (331). Although IL-12 was required in vitro for priming for optimal IFN-y production, primary antigen stimulation of TCR-transgenic CD4+ T cells, in the absence of IL-12, generated T cells with a Thl-type phenotype and able to produce significant amounts of IFN-y when challenged in a secondary stimulation in the presence of IL-12 and IGIF (276). However, IGIF, which strongly synergizes with IL-12 in inducing IFN-y production from polarized Thl cells, was unable to replace IL-12 in inducing T h l differentiation and did not represent a possible substitute for IL- 12 in supporting the modest T h l responses in IL-12 knockout mice (276). High doses of IL-2 and IL-15 are also able to induce some priming of T cells for IFN-y production, and at least the effect of IL-2 appears to be independent of endogenous I L- 12, indicating possible alternative pathways of induction of Th1 responses (374). Although Thl cells by definition are high producers of IL-2 and IFNy, most studies on the effect of IL-12 focus on IFN-y production. In one in which IL-2 production was analyzed, the addition of IL-12 to the primary stimulation of CD4 T cells did not significantly affect the ability of the cells to produce IL-2, while increasing severalfold their ability to produce IFN-y (368). In another study, IL-12 significantly inhibited the ability of the cells to produce IL-2, an effect that was mediated by IFN-y (364). Thus, IL-12 appears to be essential for the development of high IFN-y-

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producing Thl cells, but in its absence, moderate production of IFN-y and norinal production of IL-2 are still observed. 6. Ir IL-12 Necessnnj f o r Maintaining T h l Responses?

Thl cells maintain the expression of IL-12R and IL-12 responsiveness (82, 83), and IL-12, in synergy or not with costiinulatory molecules such as B7 on APC, can greatly augment cytokine production and proliferation of polarized Thl populations or clones (166, 299, 375). IGIF also strongly synergizes with IL-12 in inducing IFN-y production and proliferation of Thl cells (272,273,276).The IL-12 mediated enhancement of proliferation requires IL-2 production froin the antigen-stimulated T h l cells in most experimental conditions, and one possible mechanism of action of IL-12 is through its ability to enhance the expression of the IL-12Ra subunit, and thus to increase IL-2 responsiveness (299).However, certain terminally dfferentiated T-cell clones have lost their ability to produce IL-2 and are completely dependent on IL-12 produced by APC for proliferation when stimulated with antigen (306). Some of the studies reviewed earlier showed that polarized Th1 cells completely lose the ability to produce IL-4, whereas culture in the presence of IL-12, with or without IFN-y, can, under certain conditions, induce Th2 cells to produce IFN-7, generating cells that produce both IL-4 and IFN-7 (83, 369, 375, 376). Thus, in differentiated CD4+ T cells, IL-4 production is cytokine autonomous (although IL-4 is absolutely necessary for priming for its own production), whereas IFN-y production is cytokine dependent. This finding has led to the suggestion by IIu-Li et nl. (375) that the ability or lack thereof to produce IL-4 should be considered the defining property of Th2 and Th1 cells, respectively, rather than the ability to produce IFN-y, which either cell type can express depending on the recent exposure to a cytokine environment. Although in zjitro studies have shown that IL-12 plays a role in niaintaining and activating T h l cell function, results since the original report in T. godii-infected mice (377) in many in zjizjo infectious disease models have shown that once a powerful Thl response is established, endogenous IL12 can be neutralized without decreasing the protective response or IFNy production in animals. However, with less vigorous Th1 responses, e.g., in autoimmunity, IL-12 may be necessary for maintaining the response (378).

7. N K Cells Participate in the IL-12 Iiiduction of Thl Respniise NK cells have powerful cytotoxic activity and are efficient producers of' cytokines, particularly IFN-y (253, 379). In response to stimuli such as IL-2 (253) and IL-12 (78), not only are NK cells efficient producers of IFN-y, but they can respond at a much earlier time than antigen-specific

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T cells. An important in vivo role for NK cells as producers of IFN-y in a T-cell-independent mechanism of macrophage activation has been clearly demonstrated in L. monocytogenes (380) and T. gondii (256) infections and has been shown to depend on induction of IL-12 (257, 381). Migration of NK cells in draining lymph nodes following L. rnujor infection and their production of IFN-.)I have been demonstrated to be important for the T h l response in L. major-resistant mouse strains (382). In vivo depletion of NK cells blocks the Thl response in the resistant mouse strains (382) and also prevents the induction of a T h l response in susceptible BALBlc mice when IL-12 is used as an adjuvant in vaccination with soluble leishmania1 antigens (383). Neutralization of IL-12 in vivo during L. major infection of resistant mouse strains prevents the early migration of NK cells in the draining lymph nodes and their production of IFNy (320). NK cells also appear to play a role in human Thl response in vitro (384), and depletion of NK cells from the cultures significantly decreases the ability of IL-12 to induce a T h l response (79). Overall, data implicating proinflammatory early cytokines such as IL12, TNF-a, and IL-lP, and NK cell activation in setting the stage early in an immune response for the ensuing antigen-specific Thl-type immune response indicate a clear influence of innate resistance in directing the response of adaptive immunity. The role of NK cells might be mediated through their production of IFN-y, insofar as NK cells are the earliest IFN-y producers in an immune response. However, the requirement for NK cells in the IL-12-induced T h l response of human cells in vitro, an experimental system in which no effect of neutralization of IFN-y was observed, suggests that NK cells may favor the T h l response by mechanisms in addition to IFN-y secretion, possibly the production of other cytokines. 8. Polarization of CD8' T Cells and TyS T Cells CD8+ T cells (13, 14,385,386)and Ty6 T cells (17) can also be primed to produce Th2 cytokines and, thus, like CD4' T cells, can have a polarized Thl-type or Th2-type phenotype often referred to as TC1 and TC2. The conditions that induce polarization of CD8' T cells are similar to those inducing CD4' T cells, i.e., IL-12 is the major factor required for TC1, although differentiation of these cells is also observed in the presence of IFN-y plus anti-IL-4 or of TGF-P, whereas IL-4 is the major factor required for TC2 differentiation. TC2 cells produce nearly the same levels of IL-5 as Th2 cells, but much reduced levels of IL-4, and have much lower but not absent production of IFN-y compared with TC1 cells (387). The presence of IL-12 in addition to IL-2 during priming of TC1 cells

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results in a five-fold increase in IFN-y production, but, similar to the observation with Thl cells, a three-fold decrease in IL-2 production (385). Both TC1 and TC2 subsets are cytotoxic (386), and TC2 clones expressing CD40L may provide some B-cell help, although not efficiently because of their ability to lyse APC and B cells (386, 388). Interestingly, the IL-12 p40 homodimer, which blocks the ability of IL-12 to induce T h l differentiation in CD4' T cells, was reported to significantly enhance the priming for IFN-y production in alloreactive CD8' T cells (389). Although no conipelling evidence for a direct stimulation of the p40 homodimer on CD8' cells is provided in this study, the challenging possibility exists that the homodiiner on CD8' cells signals by binding to the IL-12Rp1 chain. Indeed, the use of chimeric receptors has shown that the cytoplasmic region of IL-12Rp1 alone can signal, transduce, and phosphorylate STAT3, although no biological activity was demonstrated in those experiments (93). Although IL-12 p40 and IL-12 p35 genetically deficient mice have a similar phenotype (280), only p35 knockout mice are resistant to Cyptococcus neoformans, and the susceptibility to infection in the p40-deficient mice can be overcome by treatment with p40 homodimer (J. Magram, personal communication). Thus, the p40 homodimer, perhaps by acting selectively on CD8' T cells, may mediate some of the functions of the p70 heterodinier. C. ROLEOF IL-12 IN DIFFERENTIATION OF HUMAN T h l CELLS Analysis of the role of IL-12-induced differentiation of human T h l cells has been obviously lianipered by the limitation of using human material, even though the original description of this activity of IL-12 was reported at the same time in human (79) and mouse (80) studies. One of the limitations of the human studies is that the recall response of memory T cells, rather than the primary response of naive T cells, is used as an indicator of antigen-specific responses. Yet, similar to observations in the murine system (361),IL-12 exerts aprofound altering effect on the cytokme production pattern of Th2-biased allergen-specificT cells by inhibiting IL4 and IL-10 production and boosting IFN-y production, but at polyclonal and clonal levels (79, 286). Furthermore, it was demonstrated that the ability of Thl-inducing recall antigens such as PPD to induce the generation of Thl-type clones is dependent on the ability of this type of antigen to induce endogenous IL-12 production from APC (79). Unlike in the murine system, IFN-y has not been generally found to play a necessary role in the IL-12-induced differentiation of human Thl cells (15, 79, 168), with the exception of one study in which anti-IFN-y antibodies were shown to partially prevent the IL-12 priming for IFN-y production in cord blood T cells (390). This species difference may be

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explained by the fact that IFN-y induces IL-12RP2 subunit upregulation on mouse T cells (83),whereas on human T cells, IFN-a, but not IFN7 , has this function (86).Thus, IFN-(r is able to induce generation of T h l cells from allergen-specific T cells (391, 392) and is responsible, together with IL-12, for the Thl differentiation effect induced by poly-1:poly-C ( 145). As in the mouse, the polarization of human T cells is regulated by IL4 produced by T cells and by IL-12 produced by dendritic cells and other APC (393). The expression of B7 costimulatory molecules on APC is required for IL-$-induced differentiation of human Th2 cells, but not for the IL-&mediated differentiation of T h l cells (394), even though IL-12 and B7/CD28 stimulation strongly synergize for transient expression of IFN-y (165). An altered TCR ligand, i.e., an analog peptide derived from an allergen and able to boost IFN-y production in a Tho clone, induces APC production of IL-12 during antigen presentation (395). Thus, the interaction between T cells and APC mediated through the TCR and an altered T-cell ligand is bidirectional, enabling APC to deliver signals to the T cells, particularly IL-12 (395). Many studies in the human system have used cord blood or thymus cells to analyze the response of naive T cells. Many of the mechanisms studied with cord blood cells can be extrapolated to adult T cells, but some important differences are present. In particular, both cord blood and thymus CD4+ T cells in default conditions differentiate to Th2-type cells (396, 397). Furthermore, IL-12, which on naive T cells from adult donors induces differentiation of Thl cells and inhibits IL-4 production (79), acts on neonatal and cord blood CD4+ T cells to promote the differentiation of cells that produce high levels of both IFN-y and IL-4 (288, 398). However, on cord blood CD8' T cells, IL-12 completely inhibits the IL4-induced capacity of CD8+ T cells to produce IL-4 (399). Analysis at the single cell level (400) and using clonal limiting dilution (15, 168) has shown that the induction of Thl differentiation by IL-12 is rapid and requires only about 4 days of priming to become stable and irreversible, whereas IL-4-induced Th2 cell differentiation requires longer time and repeated stimulation, and Th2 cells can be reverted to IFN-yproducing Thl cells by a single restimulation in the presence of IL-12. Although human Th2 clones do not express detectable IL-12RP2 chain and have not been shown to signal in response to IL-12 (86, 401), the majority of them still respond to IL-12 with a low and transient IFN-y production (15, 402). The expression of ILlO in mouse T cells is restricted to Th2 cells, with few exceptions. However, in human T cells, IL-12 induces a priming for the production of both IFN-y and IL-10, thus resulting in the generation

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of T-cell clones that produce both activators and inhibitors of macrophage activation (168, 285, 397).

DIFFERENTIAT~ON D. IL-12 AND Thl CELLGENERATION: OR SELECTION? The experimental systems used in studynig the Th response have not permitted determination of whether the different cytolanes that affect Th cell development induce differentiation of bipotential Th precursors or rather a selective priming and/or expansion of already committed Th1 and Th2 precursor cells (390, 403-405). This question is particularly relevant in human studies that have analyzed clonal expansion of memory Th cells (79, 406). However, once a Thl or Th2 response has been established, it appears to be relatively stable, and no factors capable of inducing qualitative changes in the cytokine profile of established murine or human T-cell clones have been reported. In the analysis of cytokine production froin human T cells stimulated with recall antigens (PPD) or allergens (Der p.l), the expansion of the sinall proportion of memory T cells was first obtained in polyclonal T-cell cultures, froin which single antigen-specific clones were obtained only after several weeks of culture of the polyclonal cell line (79, 406). During this culture period, emergence of Th cell subsets with characteristic cytokine production profiles could reflect differentiatioil of precursor Th cells, as well as positive selection (growth advantage) of certain Th subsets or negative selection (apoptosis, cytotoxicity, antiproliferative effects) of other subsets. Human T-cell cultures exposed to a polyclonal stiinulus that affects all T cells, such as PHA or anti-CD3, in the presence or absence of IL-12, displayed the enhanced IFN-y production and ahnost complete abrogation of IL-4 production observed in antigen-stimulated cultures (407). However, very different results were obtained when freshly isolated human peripheral blood T cells were immediately cloned by limiting dilution in cultures stimulated by PHA and IL-2, in the presence or absence of IL12 (15). When restimulated with anti-CD3 and phorbol diesters after 5 weeks of culture, the clones generated in the presence of IL-12 produced 5- to 20-fold higher levels of IFN--y than the clones generated in the absence of IL-12. This priming for IFN-y production required the addition of IL-12 within the first week, but its presence for inaxirnal priming was required only for 1 or 2 weeks (168). Once the clones were established for 2 or 3 weeks, removal or addition of IL-12 from the culture inediuin &d not significantly affect their ability to produce IFN-y (15).Because the clonal efficiency in these experiments was close to 100%,the priming effect of IL-12 was not due to selection of high IFN-y-producing clones,

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but was exerted on each single T cell, naive or memory. Furthermore, this effect was observed on both CD4+ and CD8+ cells, suggesting that IL-12 affects the differentiation of Thl-type clones from both subsets. Thus, the presence of IL-12 during the initial clonal proliferation of T cells induces an irreversible priming for high IFN-y production, which is maintained even when the clones are cultured for several weeks in the absence of IL12. However, unlike what is consistently observed in vivo and in polyclonal cultures and their derivative clones, the clones originated by limiting dilution in the presence or absence of IL-12 showed no significant difference in their average ability to produce IL-4 (15, 407). These results suggest that the ability of IL-12 to prime CD4' cells for high IFN-y production is due to a differentiation effect acting at the level of CD4+ T-cell clone precursors. The ability of IL-12 to downregulate IL-4 production, however, was not observed at the clonal level and is likely due to selective processes operative on polyclonal cultures and not to a direct effect on single clonal progenitors (15). The nature of these mechanisms remains to be investigated, although a possible selective proliferative effect of IL-12 on T h l clones or an IFN-y-mediated negative selection against IL-4-producing clones can be postulated. Alternatively, downregulation of IL-4 production might be a differentiation effect that requires cellular interaction or cell crowding (e.g.,for the production of Th2-suppressing factors such as IFNy ) during the initial phase of proliferation of the T cells; such interactions are not obtained in limiting dilution cultures, even in the presence of irradiated feeder cells. The ability of IL-12 to prime human T cells for IFN-y production has also been suggested by experiments showing that naive human cord blood T cells are unable to produce IFN-y, but acquire this ability after a few days of culture in the presence of IL-12 (390). However, selective effects and/or preferential proliferation of T-cell subsets could not be excluded in the polyclonal cultures of cord blood T cells. That the mechanisms underlying the enhancing effect of IL-12 on IFN-y and the inhibition of IL-4 production might be different is also suggested by data in the murine system, showing that the enhancement of IFN-y production is a direct effect of IL-12 on T cells, whereas the inhibition of IL-4 production is due to an indirect effect on APC or on other cell types present in the APC preparations (408). CD4' and CD8+ clones obtained by limiting dilution in the presence of IL-12 produced significantly more IL-10 than clones generated in the absence of IL-12 ( 168). However, in allergen-stimulated polyclonal T-cell cultures IL-12 was shown to downregulate both IL-10 and IL-4 (286). These apparently contradictory results are, however, consistent with the conclusion that IL-12 directly upregulates T h l cytokine production, but

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suppresses Th2 cytokine production by an indirect, possibly selective mechanism. The high variability in the production of cytokines observed in human CD4’ T-cell clones expanded in the absence of exogenous IL-12 and IL4 or in the presence of neutralizing antibodies against these two cytokines suggests that some of the cells are already primed in vivo for cytokine production. The cloning of sorted CD45RO- “naive” CD4’ cells and CD45RO’ “memory” CD4’ cells supports this interpretation (168).A high proportion of the clones generated froin CD45RO’ CD4+ cells in the presence of neutralizing antibodes to IL-12 and IL-4 produced one or a combination of IFN-7, IL-4, and IL-10, with a pattern of production that was not always consistent with the classical paradigm of T h l and Th2 cells. When CD45RO- cells were cloned in the same conditions, the clones produced only negligible amounts of the three cytokines. However, in both populations, the presence of IL-12 during cloning endowed virtually all clones with the ability to produce high levels of IFN-7 and IL-10. IL-4, either endogenously produced or exogenously added, was necessary in the limiting dilution cultures to prime T-cell clones generated from CD45RO- cells for IL-4 production, whereas approximately half of the clones generated from CD45RO’ cells produced IL-4, even when expanded in the absence of IL-4. Thus, the requirement for IL-4 in the generation of IL-4-producing cells was difficult to evaluate when total PBL were cloned. Although IL-12 is a major inducer of a T h l response, it was also shown to potentiate IL-4 production and the development of Th2 cells from naive CD4+ murine T cells (287) and froin neonatal CD4’ human T cells (288), and to potentiate a Th2 response to Schistosoim mnnsoni in IFN-7 knockout mice (409). IL-12 does not prevent IL-4 production from CD4+ clones derived from limiting dilutions of “naive” adult peripheral blood CD45RO- cells and, in fact, significantly enhances the ability of IL-4 to prime the clones for high IL-4 production (168),thus extendmg previous results (287, 288, 409) by demonstrating that IL-12 can enhance IL-4 production at the single clonal level via a differentiation effect. Furthermore, when T cells were cloned in the simultaneous presence of IL-12 and IL-4, the I F N - 7 priming effect of IL-12 was only partially and often not significantly inhibited by IL-4, whereas the priming for IL-10 production was reproducibly and almost completely blocked by IL-4 (168). Thus, paradoxically, IL-4 is more potent in inhibiting priming of Th cells for production of IL-10, a Th2-type cytokine, than for the typical Thl-type cytokine, IFN-y. Figure 5 illustrates the cytokme production pattern of clones derived from human CD4’ CD45RO- T cells in the presence of IL-12 and/or IL-4.

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'oool I* Q

I'

0

1

IL-10, pglml

IL-4, pglml

Fic. 5 . Cytokine production patterns froiu clones derived by lintiting dilution froin human CD4' CD45RO- T cells in the presence of feeder cells, PHA, and IL-2. Clones were grown for approximately 5 weeks in the presence of' the cytokines or neutralizing anticytokine antibodies shown in the legend and were then restiinulated with anti-CD3 antibody and phorbol &ester for measurement of cytokine production. Symbols represent the average cytokine production of a large number of clones (tSE): 0, IL-4 (anti-IL-12); 0, IL-12 + IL-4; A, IL-12; A,IL-12 (anti-IL-4); 0 , anti-IL-12; W, anti-IL-12 + anti-IL4. Modified froin Gerosa et ul. (1997).J. Exp. Med. 183, 2259-2269.

E. ACUTEINDUCTION VERSUS PRIMING FOR CYTOKINE PRODUCTION Froin the studies of the generation of Thl and TI12 cells, it is becoming clear that production of lymphokines, both type 1 and type 2, can be regulated through two different mechanisms. The first mechanism, observed particularly in preactivated lymphocytes, but also in resting T cells and NK cells, is the ability of various stimuli, including TCR and cytokine stimulation, to rapidly induce gene expression and cytokine production. For example, IL-12, alone or in synergywith other stimuli, induces accumulation of inRNA for IFN-y within a few hours of treatment of either resting or activated T or NK cells, followed by secretion of IFN-y. This acute induction of IFN-7 subsides within -2 days [or, in viva, even within less than 12 hr (161)]and does not induce a permanent alteration in the ability of the cells to produce IFN-y in response to IL-12 or other stimuli. The second mechanism, priming of cytokine genes, is quite different from acute induction. When T cells (and NK cells) are clonally expanded in the presence of IL-12 during the first few days of expansion, the clones are primed for high production of IFN-y and IL-10, even when cultured for several weeks in the absence of IL-12 and stimulated in the absence of IL-12; conversely, exposure of T cells to IL-4 during clonal expansion induces priming for IL-4 production and generation of IL-4-producing cells. IL-12 is particularly potent in mediating both acute induction of the

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IFN-y gene and its stable priming for response to other stimuli. IL-12 siinilarly primes the IL-10 gene, but its ability to acutely induce this gene is modest and difficult to demonstrate. Analogously, the ability of IL-4 to acutely indiice the expression of the IL-4 gene has not been demonstrated, although IL-4 is necessary and extremely potent for the priming of the IL-4 gene and the generation of IL-4 producing cells. The effect of IL-2 011 lymphokine production is different from that of IL-12 or IL-4: IL-2, alone or in synergy with other stimuli, is a potent inducer of acute expression of the several lymphokmes, including IFN-7, IL-4, and IL-10. However, altliough the presence of IL-2 may be required in the priming phenomena of all three genes, IL-2 by itself does not determine the specificity of the priming, which is instead directed by IL-12 and IL-4 (78, 253, 410). As mentioned earlier, IL-4 and IL-12 both antagonize and synergize in inducing priming of lyinphokine genes. IL-4 almost coinpletely abolishes the IL-12 priming for IL-10 production, but only partially decreases the priming for IFN-7; IL-12 potentiates rather than inhibits the IL-4 priming of T cells for high IL-4 production. The priming of lymphokine genes represents a stable modification of the inducibility of the genes, which is analogous to the stable phenotype in the pattern of cytokine production typical of Th subsets. Thus, it is likely that this priming mechanism plays a role in the determination of the Th phenotype of actitated T cells. However, certain effects of IL-12 and IL-4 on Th generation are not observed when the ability of these cytokines to induce differentiation is analyzed at the single clonal level (e.g., in clonal analyses, the powerful ability of IL-12 to block IL-4 production is not reproduced and, paradoxically, IL- 12 induces T-cell priming for production of IL-10, a prevalently type 2 cytokine). Thus, although priming of lymphoknie genes is most likely the predominant mechanism by which IL-1.2 and IL-4 induce differentiation of Th cells, the final generation of cells with Th1 and Th2 phenotype, both in vivo and in oitt-0,also depends on coinplex indrect effects of the cytohnes, including selective mechanisms. The molecular mechaniwis of both the acute induction and the priming effects remain mostly undetermined. The major signal transduction mechanisnis for IL-4 and IL-12 have been elucidated, with the former cytokine inducing activation of STAT-6 (411) and the latter of STAT 1, 3, and 4 (94, 95); however, the role of these transcription factors in the induction of expression of the IL-4, IFN-7, and IL-10 genes remains to be elucidated. The priming effects may depend on the induction of a constitutive or facilitated expression of the transcription factors responsible for the expression of the lymphokine genes or on a stalile alteration of the genes in a transcriptionally prone conformation. For example, the IFN-y gene has

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been shown to be differentially methylated in T h l and Th2 clones (412). Posttranscriptional mechanisms could also underlie the priming effect. XI. Effects of 11-12 on B-Cell Responses and Vaccination

A. POSSIBLE DIRECTEFFECTOF IL-12 ON B CELLS As discussed earlier, human BCL constitutively expresses the IL-12RP1 subunit, but only in a few cases have they been shown to bind IL-12, possibly because of lack of the IL-12RP2 chain (74,76,77).Resting murine spleen and human peripheral blood spleen cells fail to bind IL-12, but they do so after stimulation with LPS and S. aureus, respectively (77). However, peritoneal B cells, both CD5+ B1 cells and conventional B cells, bind IL-12 in the absence of stimulation (77). Expression of IL-12RP1 mRNA can be readily detected in both spleen and peritoneal B cells (77), but little information is available on the expression of IL-12R02. Early studies showed that IL-12 in vitro suppressed the synthesis of IgE by human lymphocytes stimulated by IL-4 or IL-4 plus anti-CD40 antibodies through an IFN-dependent or -independent mechanism (413). The inhibitory effect of IL-12 on IgE synthesis was most likely indirect and mediated by T or NK cells (413).However, another study (414) showed that IL-12 enhanced the growth of S. auras-stimulated human B cells and, in the presence of IL-2, potently enhanced B-cell differentiation with an increased production of both IgG and IgM (414). These results suggest a direct effect on B cells. The ability of IL-12 to enhance the growth of S. auras-stimulated human B cells was inhibited by anti-IFN-y antibodies; PCR analysis at the single cell level indicated that IL-12 induces IFN-y production in B cells and that the endogenously produced IFN-.)Iis responsible for the effect of IL-12 on B-cell growth (254). In addition, it was shown that IL-12 induces IL-10 production in CD5' B splenic B cells and IL-6 production in both CD5+ and CD5- B cells (415). IL-12 in vivo treatment of mice immunized with phosphorylcholine conjugated to keyhole limpet hemocyanin (KLH) or with S. mansoni soluble antigen resulted in a loss of peritoneal CD5' B1 cells (416,417). However, in vitro studies showed that IL-12 is required for the growth of S . aureus-stimulated peripheral blood CD5+ Bla B cells, but not of CD5- B cells (418). Overall, information on the mechanisms of action of IL-12 on B cells is still scarce, and several unanswered questions remain about the expression of functional IL-12 receptors on B cells, although increasing evidence indeed points to a direct effect of IL-12 on B-cell functions. The reported effects of IL-12 on B1 cells are also difficult to interpret because in vitro they suggest a direct effect of IL-12 on B1 cells, with increased proliferation and secretion of IL-10, which is an autocrine growth factor for these cells,

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whereas in vivo evidence suggests a depletion of peritoneal B1 cells in IL-12-treated animals. Whether this latter effect is due to actual death of the B 1 cells or to migration to different anatomical sites remains to be clarified.

B. EFFECTOF IL-12 ON ISOTYPE SELECTION AND USE AS A VACCINE ADJUVANT The ability of IL-12 to direct an immune response to the generation of Thl cells, involving a change in isotype selection, has generated interest in using it as an adjuvant in vaccination to iinprove the immune response to those pathogens for which a Th1 response is protective. The first example of such use was in the L. major infection model in the mouse (383). BALB/c mice are susceptible to L. major because they respond to the infection with a Th2 rather than a TI11 response, and different protocols of vaccination have been mostly ineffective because of their inability to shift the response from Th2 to Thl; however, vaccination of BALBlc animals with soluble Leishmania antigen plus IL-12 induced a strong and protective Thl memory response (383).Those studies raised much interest in the possible use of IL-12 as an adjuvant, but despite encouraging results, they have often been surprising and not easily interpretable within the paradigm of exclusive T h l induction by IL-12. Originally, IL-12 was shown to suppress IgG1, IgE, IgA, and, less efficiently, IgG2a and IgG3 in mice treated with anti-IgD antibodies (419). IL-12 also induced IFN-y in these animals and the role of this cytokine was complex: the suppression of IgE by IL-12 in anti-IgD-treated animals was IFN-y independent, whereas in animals treated with neutralizing antiIFN-y antibodies, IgGl was not suppressed (419). Furthermore, when IFN-y was neutralized in anti-IgD-treated animals, the production of IgG2a or IgG3 was strikingly enhanced by IL-12 (419). IL-12 did not inhibit IgE production in response to anti-IgE treatment, suggesting that IL-12 inhibits switching of B cells to cells that express IgE rather than inhibiting the differentiation of switched cells to IgE-secreting cells (419). Several later reports showed that in response to immunization with antigens such as KLH, TNP-KLH, hen egg white lysozyme (HEL), phospholipase A2 (PLA,), and alloantigens, treatment with IL-12 induced the inhibition of IgGl and IgE antibody production and upregulation of cytotoxic and complement-fixing antibody production of the IgG2a, IgGzb, and IgG3 subclasses (420-424). The timing and modality of treatment with IL-12 and antigens are obviously important, and the various studies have used different protocols, from coinjection of the IL-12 and the antigen to continuous daily treatment with high doses of IL-12. However, comparative studies to establish optimal

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protocols are missing. Nonetheless, it was of interest that an ovalbumin-IL12 fusion protein was found to be more efficient than ovalbumin plus free IL-12 in inducing a T h l response, inhibiting IgE production, and enhancing IgG2a and, less efficiently, IgGl (425).Also, absorption of both antigen (HIV gp120) and IL-12 on alum resulted in a much more efficient response in terms of Thl cytokine production and increase not only of IgG2 and IgG3, but also production (426). However, the in vitro proliferative response to gpl20 of the spleen cells of animals immunized with antigen and IL- 12 absorbed on alum was almost completely suppressed compared to animals immunized with gp12O and alum or a 1 2 0 and IL-12 not absorbed to alum (426). This immunosuppressive effect of IL-12 was inhibited by anti-IFN-y antibodies and correlated with the level of nitric oxide produced in the cultures (426). Many of the bacterial preparations used as adjuvant for vaccination, e.g., BCG or Corynebacteriurnp a m m , are good inducers of IL-12 production, but the role of adjuvant-induced IL-12 in the immune response has been analyzed in only a few cases. Iscoms formed by physical integration of Quillaria saponaria adjuvant and antigens induce IL-12 production in the serum of treated mice, and the neutralization of IL-12 in animals immunized with iscoms results in a decrease of total antigen-specific antibody response as well as IgG1, IgGBa, and IgG2b, suggesting a major role for endogenous IL-12 in the adjuvant capability of the iscoms (427). In animals immunized with ovalbumin, TNF and IL-1 were shown to mimic the adjuvant effect of LPS on accuinulation and follicular tnigration of antigenactivated T cells, whereas IL-12 mimicked the generation of Thl cells and help for IgG2a production (428). Overall, it is clear that conventional adjuvants induce several cytokines with various effects; however, IL-12 has a central role in favoring a T h l response and the production of antibodies of all subclasses, particularly the complement-fixing and cytotoxic ones. When the adjuvant effect of IL-12 was analyzed beyond the first couple of weeks after vaccination and primary response, the results became more complex. For example, in animals immunized with HEL, IgG2a was increased and IgGl decreased at 7 days, whereas both subclasses were enhanced at day 28 after immunization (421). Overall, it was observed that IL-12 as an adjuvant promoted a Thl-type response, but did not suppress a Th2-type recall response. Both IFN-y and IL-2 production were enhanced in IL-12-treated animals so that both IgGl and IgG2a were boosted following a secondary vaccination, either associated with IL-12 treatment or not, in animals primed by antigen and IL-12 (429). Administration of IL-12 during an ongoing immune response failed to permanently suppress and even enhanced antigen-specific IgE production (430), a result not completely surprising considering the ability of IL-12 in certain experimen-

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tal conditions, both in vitro (168, 287, 288) and in vivo (429), to enhance IL-4 production and The-type responses. It was also shown that administration of IL-12 together with ovalbumin antigen profoundly, but transiently, inhibited antigen-specific IgE synthesis while enhancing IgG2a: these effects were much decreased on secondary challenge, and almost absent after tertiary challenge, although the increase in IgG2a was still significant in the latter condition (431). The presence of uninethylated CpG motifs in the bacterial DNA used for vaccination has been shown to be required for induction of proinflammatory cytolanes, including IL-12, and for effective intraderind gene immunization (126). Much interest has been generated by the possibility of using the IL-12 genes in combination with DNA vaccination. Plasmid DNA immunogens encoding IL-12 and either influenza virus or HIV antigens have proven very efficient in inducing a CTL response and IFN-7 production (432,433). When either GM-CSF or IL-12 was used in DNA immunization against various HIV antigens, the former enhanced antibody formation, whereas the latter decreased antibody formation and boosted CTL activity (432). However, when vectors encoding both GM-CSF and IL-12 were used, the two cytokines d ~ not d prevent each other’s effect on boosting antibody formation and CTL generation, but rather potentiated each other (338). The use of oral vaccination holds considerable promise in inducing protective iminunity against pathogens or in inducing tolerance, e.g., in autoimmunity. Oral immunization of mice with tetanus toxoid together with the adjuvant cholera toxin induced Th2-type responses with systemic IgG1, IgE, and IgA antibodies; if IL-12 was given ip to mice immunized orally, the response shifted to production of Thl-type cytokines, increased DTH, and increased serum IgG2a and IgG3, whereas IgG1, IgE, and IgA were markedly decreased (434). Interestingly, almost identical results, except for lack of effect on serum IgA, were- observed when IL-12 was given orally complexed to liposornes, a formulation that does not result in a significant level of serum IL-12 (434). Not only did IL-12 boost DTH responses during oral immunization, but when given at the site of attempted sensitization, it also induced powerful and long-lasting DTH reactivity in mice with already fully established, orally induced tolerance to ovalbumin (435). Two of the main mechanisms by which oral tolerance develops are the production of TGF-P by T cells and clonal deletion via apoptosis; systemic administration of anti-IL-12 antibodies to animals fed high doses of ovalbumin resulted in increased TGF-,f3 production and apoptosis, suggesting that the ability of IL-12 to prevent or revert oral tolerance may be due in part to the suppression of these two mechanisms of tolerance (436).

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XII. IL- 12 in Delayed-Type Hypersensitivity, Airway Hyperresponsiveness, and Graft Rejection

A. ROLEOF IL-12 IN DTH The ability of epidermal cells, keratinocytes, and Langerhans cells (198, 199,202) to produce IL-12, constitutively or on exposure to hapten, suggests a direct role for IL-12 in the development of IFN-.), producing cells in the skin that are important for DTH and contact hypersensitivity (CHS). There is good evidence that 1L-12 acts in the induction phase of CHS. Although IL-12 induction is not detected in the skin of mice on sensitization by topical application of haptens, unlike human skin (199), significant upregulation of IL-12 is detected in dendritic cells and macrophages of the regional draining lymph nodes (437,438). The critical functional role of IL-12 during cutaneous sensitization was clearly proven by the finding that ip injection of anti-IL-12 antibodies around the time of hapten sensitization resulted in failure to induce sensitization (437, 438). Injection of anti-IL- 12 not only prevented sensitization, but also induced tolerance: animals sensitized with hapten and treated with anti-IL-12 antibodies could not be sensitized to the same hapten after a 2-week rest, but were readily sensitized to an unrelated hapten, indicating the establishment of hapten-specific tolerance (438). Treatment of mice with IL-12 was also shown to enhance the acquisition of CHS, resulting in a response of greater magnitude and duration (439, 440). IL-12 injection around the time of sensitization increased the CHS response at the challenge phase with typical participation of effector CD8' cells, possibly in part by shifting the negatively regulatory CD4' cells with a Th2 phenotype to an effector T h l phenotype; in IL-12-treated CD8-depleted mice, challenge after cutaneous sensitization resulted in a CD4-mediated response with minimal edema and acute mononuclear cell infiltration, more typical of DTH than of CHS (440). In addition to its role in the sensitization phase of CHS, IL-12 also plays an important role in the elicitation phase. IL-12 can be detected in the skin at the site of challenge and is upregulated by neutralization of IL-4 (441), suggesting that focal production of Th2-type cytokines limits the inflammatory reaction. Furthermore, neutralization of IL-12 in the elicitation phase significantly suppresses the ear-swelling response (438), indicating that IL-12 is involved in the effector phase of CHS. Irradiation of animals with high doses of UV irradiation prevents the induction of DTH or CHS to hapten applied to distant unirradiated skin areas, inducing a systemic form of immunosuppression, whereas low doses of UV irradiation block CHS sensitization in the same skin area, possibly by depleting skin Langerhans cells (442). Interestingly, UV irradiation of

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human inonocytes was shown to reduce IL-12 production, thereby limiting the activation of T h l cells (443). IL-12 treatment of the animals at the time of sensitization prevents both systemic and locally induced UV immunosuppression (437, 444, 445). The mechanism of prevention of systemic suppression may be due to the ability of IL-12 to act as an antagonist of IL-10, a likely mediator of this type of suppression (444). The ability of IL-12 to prevent local suppression is more obscure because UV irradiation induces depletion of the skin Langerhans cells, leaving open the question of which cells, in the presence of IL-12, become able to present the hapten (445). IL-12 not only prevents the induction of immunosuppression, but when administered at the time of resensitization, can overcome an established immunosuppressed state and revert it (444, 445). These results are reminiscent of the ability of IL-12, when injected at the site of sensitization, to reverse the tolerance to ovalbumin induced by oral immunization (435). Furthermore, the transfer of UV-induced tolerance by T cells to naive animals is prevented by IL-12 treatment of the recipients (444, 445). The transfer of tolerance is dependent on the presence of functional Fas and Fas ligand in the recipient, and IL-12 may act directly or indirectly on the recipients’ dendritic cells by preventing their apoptic death when presenting antigen to tolerogenic T cells (T. Schwarz, personal communication). A similar requirement for IL-12 in the induction of DTH, possibly acting directly on dendritic cells, has been reported for sensitization obtained by the transfer of dendritic cells, cells pulsed with a class I-restricted immunogenic peptide from the P815AB tumor rejection antigen of the murine mastocytoma cell line P815 (446,447).In vivo or in vitro treatment of the dendritic cells with IL-12 induces sensitization for DTH, but in the absence of IL-12, the dendritic cell transfer induces an anergic state (446, 447).

B. ROLE OF IL-12 IN GRAFT-VERSUS-HOST DISEASE The role of IL-12 in graft-versus-host disease (GVHD) has been studied primarily using mouse models, and very little information is available from human studies. Two distinct forms of GVHD have been analyzed: acute GVHD, typically observed in (C57BU6 X DBN2)Fl (BDF1) mice given parental C57B1/6 lymphocytes, and chronic GVHD observed in BDFl mice given parental DBN2 lymphocytes. Acute GVH is characterized by early lymphoid hyperplasia and increased NK cell activity, followed by generation of anti-host CTL, immunodepression, weight loss, and ultimately death. Chronic GVHD is an imrnunostiinulatory syndrome with Bcell hyperplasia, production of autoantibody, and immune complexinduced glomerulonephritis. Acute and chronic GVHD are associated with production of type 1 and type 2 cytokines, respectively.

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As expected, IL-12 stimulated the development of acute GVHD in mice that would develop chronic GVHD, an effect that was accompanied by the suppression of autoantibody formation, decreased serum immunoglobulin, generation of anti-host CTL, immunosuppression, weight loss, and death (448,449). However, results on the role of IL-12 in acute GVH are more complex. Acute GVHD is characterized by expression of type 1 cytokines, and IL-12 expression was detected in macrophages and target organs of mice (450),as well as in PBMC of patients undergoing acute GVHD (451), although serum levels of IL-12 were not associated with the development of acute GVHD in patients (452). Two studies observed that neutralization of IL-12 during induction of acute GVHD resulted in the expected polarization of the cytokine profile toward a Th2-type alloimmune response and conferred long-term protection from the disease, preventing generation of CTL, immunosuppression, weight loss, and death (449,453). Even when an enhanced Th2-type response was induced by neutralization of IL- 12, only modest activation of B cells was observed, with only moderate levels of antibodies and no gloinerulonephritis (453); thus, anti-IL-12 antibodies protect from acute GVHD without inducing chronic GVHD. However, one study (454) showed that administration of a single high dose of IL12 at the time of acute GVHD induction also significantly protected from the disease, reducing mortality and weight loss. Administration of IL-12 induced an early (days 2-3) increase in IFN-y production, which at this time derives from NK cells or NK1 T cells, followed by an inhibition of IFN-y production, prevalently from CD4' T cells, at day 4 (454). This surprising effect of IL-12 might rest in an activation of host NK cells or NK1 T cells, resulting in the suppression of iminunocompetent cells in the transplant or in the ability of a single injection of IL-12 to induce a partial unresponsiveness to IL-12 itself, as suggested by clinical trials (455) and experiments in mice (456). Another paradoxical result comes from animals in which acute GVHD was reduced by anti-B7.1 and -B7.2 antibodies; administration of anti-IL-12 antibody reversed the beneficial effect of the anti-B7 antibodies, possibly due to an impairment of natural immunity and hematopoiesis in anti-IL-&treated animals (457). Although these contradictory results remain difficult to fully interpret, the overall evidence points to the role of IL-12 as a central mediator of acute GVHD in mice. C. AIRWAYHYPERRESPONSIVENESS AND ASTHMA Asthma is characterized by increased ainvay responsiveness, elevated IgE, and chronic inflammation of the lung with infiltration of eosinophils and mast cells. This pathology is promoted by cytokines produced by Th2 CD4+ cells, particularly IL-5, which affects eosinophil infiltrates and IL4 and other cytokines, which affect mast cells. Mice of certain strains sensitized by ip immunization of antigens such as ovalbumin, sheep erythro-

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cytes, or ragweed and then challenged intratraclieally or by aerosol administration of the antigen develop eosinophilia, increased IgE, and airway hyperresponsiveness in response to cholinergic agonists. IL-12 administered at high doses around the time of either sensitization or challenge suppresses eosinophilia, IgE levels, and hyperresponsiveness (458-461). At low doses of IL-12 (0.1 p g per injection), eosinophilia but not hyperresponsiveness was completely inhibited, whereas at high doses (1 pg per injection) both phenomena were inhibited (458).However, this dissociation between eosinophilia and ainvay hyperresponsiveness remains enigmatic because both IL-4 and IL-5 production were inhibited at either dose of IL- 12 (458). Inhibition of eosinophilia and hyperresponsiveness was observed even when IL-12 was administered at the time of second antigen challenge, reflecting the ability of IL-12 to inhibit responses associated with ongoing antigen-induced pulmonary inflammation (458). However, in other studies, IL-12 administered at the time of aerosol or intratracheal challenge was sliown to inhibit eosinophilia without affecting the production of specific IgE and to have a variable effect on airway hyperresponsiveness (459, 461). These effects of IL-12 were at least in part prevented by anti-IFN-y antibodies (458, 460). A possible role of a deficient expression of IL-12 in the generation of chronic lung inflammation and asthma is supported by data showing decreased numbers of IL-12-producing cells in bronchial biopsies from asthma patients compared with healthy controls (462) and a reduced production of IL-12 in vitro from S. nurezu-stimulated blood cultures of patients with allergic asthma compared with nonatopic donors (463). The differential airway antigen-specific immune response among different mouse strains was found to correlate with the ability of the strain to produce IL-12; strains susceptible to antigen-induced ainvay hyperresponsiveness produced lower levels of IL-12 (464). The critical role of IL-12 in the regulation of airway responses to allergen is supported by the finding that the treatment of C3H mice, a strain normally resistant to the induction of airway hyperresponsiveness, with the anti-IL-12 antibody at the time of ovalbumin airway exposure results in a three-fold increase in responsiveness, concomitant with significant increases in Tl12-type cytokines and a decrease in IFN-y at the pulmonary level (M. Wills-Karp, M. Wysocka, and G. Trinchieri, unpublished results). Thus, endogenous IL-12 appears to play a central role in preventing the induction of chronic bronchial inflanirnation and asthma, and treatment with recombinant IL-12 might reverse established pulmonary inflaininatory conditions.

D. ROLE OF IL-12

IN

ALLOGRAFTREJECTION 4

The reciprocal role of Thl- and Th2-type responses and cytokines in allograft rejection is a complex question, and assessing the role of IL-12

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in such mechanisms has been a difficult task that has hitherto received only modest attention (373). Whereas T h l responses would be expected to play a determining role in graft rejection because of their ability to favor cell-mediated cytotoxicity and CTL generation, Th2-type cytokines have not been consistently shown to suppress graft rejection by antagonizing Thl response, and alternative mechanisms of graft rejection, e.g., those mediated by IL-5 and infiltrating eosinophils, may be supported by Th2 cells (373). Experiments to identify IL-12 production during graft rejection in both humans and mice have not been conclusive. Whereas the presence of IL12 p40 mRNA correlated with either rejection or viral hepatitis in human liver allografts (465), expression of IFN-y mRNA but not that of IL-12 p40 mRNA in renal allografts correlated with acute rejection (40). Rats in which donor-specific tolerance was induced by blood transfusions showed decreased expression of IFN-y mRNA in tolerized heart allografts compared to rejected allografts, but no significant difference was observed in IL-12 mRNA expression for either the p40 or the p35 subunit (466). In some experimental models, administration or blockage of IL-12 was indeed observed to correlate with graft rejection or prolongation, respectively. In the mouse model of skin graft prolongation by pretransplantation portal venous immunization with allogeneic cells, which results in preferential activation of Th2 cytokines, IL-12 in combination with anti-IL-10 antibodies reverse the graft prolongation (467), whereas anti-IL-12 in combination with IL-13 prolonged it (468). Although these results suggest an important role for IL-12 in regulating graft rejection, administering or antagonizing IL-12 by itself had only a modest effect on graft survival, and more consistent effects were observed only when the other regulatory pathways affecting Thl/Th2 balance were also modified, i.e., by blocking IL-10 or injecting IL-13. Another interesting model of IL-12 and graft rejection is the induction of high-level expression of the IL-12 p40 subunit in myoblasts by gene transfer with a retroviral vector (469);local production of excess p40 was expected to antagonize endogenous IL-12, and indeed the survival of IL-12 p40-transfected myoblasts transplanted in allogeneic recipients was substantiallyprolonged in association with impaired production of Thl cytokines, CTL generation, DTH responses, and, interestingly, a decrease in all IgG subclass antibodies (469). Unlike the previous results, Piccotti et al. (470) reported that treatment of mouse cardiac allograft recipients with either anti-IL-12 antibodies or IL-12 p40 homodimers resulted in an exacerbated graft rejection compared with control animals. Although Th2 cytokines were induced in the grafts of treated animals, IFN-y expression was not decreased and IL-12 p35 and p40 mRNA, undetectable in the control graft, became expressed (470).

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The number of activated CTL in the graft was decreased by in v i m blocking of IL-12, although the number of CTL precursors was not affected (470). These results point to much of the complexity of ThlR112 regulation and the role of IL-12. The induced expression of IL-12 in these experimental conditions in which both Thl and Th2 cytokines are expressed may reflect the cooperation between IFN-.)I and IL-UIL-13 in priming for IL-12 production ( 159, 169). The differentiation of IFN-y-producing T h l cells reveals the existence of IL-12-independent mechanisms of Thl differentiation in certain experimental models, but not others. The results with IL12 antagonists were reproduced using IL-12 p40 genetically deficient mice, in which the duration of graft survival was also decreased in association with an apparently unaltered Thl response (470). Interestingly, Thl-type alloresponses were observed in both p35 and p40-deficient allograft recipients, although Th1 development was enhanced in p35-deficient recipients compared with their p40-deficient counterparts (373). The possibility that endogenous p40 stimulates a Thl response in p35-deficient inice was supported by the finding that treatment of these mice with anti-p40 monoclonal antibody decreased Thl functions to the level seen in p40-deficient recipients (373). These results are consistent with previous experiments (389) suggesting that IL-12 p40 homodimers may induce T h l differentiation in CD8+ T cells, while antagonizing IL-12 action on CD4+ T cells (389).In those experiments, p40 homodimers markedly prolonged allograft survival in mice depleted of CD8' T cells while inducing accelerated cardiac allograft rejection in unmodified recipients (389). Thus, although therapeutic manipulations of IL-12 activity and of the ThltTh2 balance may have the potential to affect graft survival, the complex and redundant regulatory mechanisms and the possible role of both Th1 and Th2 cells in allograft rejection are not well understood and thus make it difficult to predict the outcome of any therapeutic manoeuvre. XIII. 11-12 in Organ-Specific Autoimmune Diseases

A. ROLE OF ThltTh.2 RESPONSESI N AUTOIMMUNITY A role for Thl cells has been demonstrated or suggested in inany organspecific autoimmune diseases, both in humans and in mice, although exactly how these cells mediate their action, and in particular the requirement for IFN-y production and the relative importance of regulation of iinmunoglobulin production, varies from disease to disease. Also, the interpretation of the pathological mechanisms of human diseases and the possible role of IL-12 have been extrapolated in many cases from data in animal models, which often poorly reproduce the clinical situation. A common observation in models of autoimmune diseases is the apparent requirement for continu-

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ous expression of IL-12 for maintenance of a pathogenic T h l response (378),whereas in infectious diseases IL-12 is usually required only in the very first few days of infection and iininunological response (377). This difference may be due to a different nature or strength of the T h l response in autoimmunity in general, but it may also reflect particular characteristics of each disease situation. For example, the continuous requirement for IL12 expression in experimental allergic encephalitis may be due to epitope spreading, with continuous activation of new T-cell clones; in experimental colitis, the role of IL-12 may be to suppress TGF-&producing downregulatory T cells or to prevent tolerance induction by apoptosis; in collageninduced arthritis, the upregulation of Th-l-dependent antibody isotypes, mediated by continuous production of IFN-y, appears to play a major role. Information on the role of IL-12 in autoimmune diseases remains very incomplete, and analyses to date have provided seemingly inconsistent results. However, improved knowledge of these regulatory mechanisms should further our understanding of the pathogenic mechanisms in experimental animals and possible therapy of human autoimmune diseases.

B. INSULIN-DEPENDENT DIABETES MELLITUS Nonobese diabetic (NOD) mice and diabetes-prone BioBreeding (DPBB) rats spontaneously develop a diabetic syndrome that resembles human type I diabetes. Islet P-cell destruction involves the participation of infiltrating mononuclear cells, with a role for both CD4' and CD8+ T cells. The initiating event precipitating insulin-dependent diabetes mellitus (IDDM)is thought to be the recognition by T h l cells of islet cell antigens, many of which have been identified (471). A role for IL-12 in the spontaneous development of IDDM in female NOD mice is suggested by the increasing expression of IL-12 p40 and p35 mRNA in mononuclear cells from islets of these animals from age 5 weeks to the onset of diabetes (472). Treatment with a single dose of cyclophosphainide,which synchronizes and accelerates the disease in NOD mice, resulted in substantially increased expression of IL-12 p40 mRNA in both pancreas and spleen of NOD mice, an effect that was not observed in cyclophosphamide-treated BALB/c mice (473). Conversely treatment of NOD mice with complete Freunds adjuvant in early life, which protects from IDDM, resulted in decreased IL-12 p40 mRNA expression in the islets (472). Daily IL-12 administration for 30 days to female NOD mice resulted in the rapid onset of IDDM, with 100% incidence by 4 weeks and massive infiltration of lymphoid cells in the pancreatic islets (474). Only 60% of control untreated female NOD mice developed IDDM and at much Iater times, whereas IL-12-injected BALB/c mice did not develop

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IDDM and their islets had a normal appearance (474). Treatment of cyclophosphamide-treated NOD mice with either IL-12 p40 hoinodimers (474) or neutralizing anti-IL-12 antibodies (L. Harrison and G. Trinchieri, unpublished results) to neutralize endogenous IL-12 resulted in a significant suppression of IDDM, associated with a reduction of islet infiltration by mononuclear cells (475). Mice deficient in IL-12 by targeted disruption of the p40 gene and backcrossed to the NOD background showed a reduced incidence of IDDM, confirming that endogenous IL-12 is required for IDDM development (476). IGIF/IL-18 was also detected in pancreas and spleen of diabetes-prone mice on treatment with cyclophosphamide and preceding the appearance of IFN-y, but not in nondiabetes-prone strains (477, 478). The IGIF gene maps within the IDD2 interval, one of the dmbetes-susceptibility loci, on mouse chromosome 9 and therefore it is a candidate for IDD2 susceptibility gene (477). Overall, these data suggest that expression of endogenous IL-12 in the islets of NOD mice parallels the progression of the inflammation and islet destruction and that IL-12 neutralization prevents the activation of the dabetogenic T cells, whereas the daily administration of excess exogenous IL- 12 hastens islet destruction. However, using a different protocol of IL-12 administration, i.e., five high-dose treatments in 2 weeks or weekly higli-dose injections, O’Hard et d.(479) showed a significant protection of IDDM development in female NOD mice. The weekly IL-12 treatment was particularly efficient in preventing IDDM, but no histological differences were observed in the islet infiltration in mice treated with IL-12 or not (479). In a model of adoptive transfer of IDDM into male NOD mice using spleen cells from diabetic female NOD mice, IL-12 treatment twice per week of the recipient mice did not prevent the transfer of the disease, suggesting that this schedule of IL-12 treatment prevents the development of diabetogenic T cells, but not their effect (479).These results are difficult to interpret and show that exposure of T cells to IL-12 at different times during their development and differentiation may lead to different outcomes. However, it is also possible that the weekly, high-dose treatments result in desensitization of the T cells to endogenous IL-12, similar to the findings of the effect of IL-12 predosing in cancer patients or in animal tumor models (455, 456). Information on the role of IL-12 in IDDM of DP-BB rats is much more limited. However, like the NOD mouse model, the pancreatic islets and thyroid of DP-BB rats showed an increase in IL-12 p40 mRNA with age of the animal and progression to disease (480). No equivalent increase in IL-12 p40 mRNA was observed in diabetes-resistant BB rats, but both IDDM development and IL-12 inRNA expression could be induced in these rats either by deletion of regulatory RT6’ T cells (480)or by infection

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with Kilham rat virus (481). Thus, in both the mouse and the rat, development of IDDM appears to correlate with IL-12 expression in the target organ. ALLERGICENCEPHALOMYELITIS C. EXPERIMENTAL AND MULTIPLESCLEROSIS Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the CNS and the most commonly used model for human multiple sclerosis (MS). Most of the EAE models used are monophasic and demyelination is minimal, but other models in which the disease is severe, protracting, and relapsing with extensive demyelination more closely resemble the clinical disease. IL-12 was shown to greatly affect a model of adoptively transferred EAE induced by injecting naive SJL/J mice with lymph node cells from mice primed with proteolipid protein (PLP) and restimulated in vitro with PLP (482). Addition of IL-12 during the in vitro restimulation resulted in a much more severe and prolonged disease after in vivo transfer; IFN-y and TNF-a were increased in the supernatant of the cells restimulated in the presence of IL-12, but neutralization of either cytokine did not decrease the severity of the transferred disease, suggesting that IL-12 directly affects the activation of the encephalitogenic T cells (482). The extent of perivascular infiltration and cytokine production was similar in animals receiving lymph node cells restimulated in the absence or the presence of IL-12, but in the later group, there was a profound increase in inducible nitric oxide synthase (iNOS) in macrophages (483), suggesting that the treatment with IL-12 enhances the ability of the transferred cells to induce the production of nitric oxide within the inflammatory foci, with a possible direct cytotoxic effect on oligodendrocytes. Unlike SJL mice, lymph node cells of BlOS mice restimulated in vitro with myelin basic protein (MBP) do not transfer EAE, unless IL-12 or IL-12 inducing microbial products such as LPS or bacterial DNA are added during the in vitro restimulation, indicating that IL-12 can unmask latent autoimmune disease in resistant mice (484, 485). In vivo treatment of the recipient SJL mice with IL-12 also increased the severity of disease, whereas treatment with anti-IL-12 antibodies for 12 days completely prevented the paralysis, with only a proportion of animals developing mild disease (482). Interestingly, if recipient mice were treated with anti-IL-12 antibodies for only 6 days, the disease was delayed, but its severity was not affected (482). Similar to the inability of anti-IFN-y to prevent the effet of IL-12 during the in vitro restimulation of lymph node cells, in vivo administration of anti-IFN-y antibodies to IL-12-treated recipient mice did not prevent

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the exacerbating effect of IL-12, but rather resulted in even more severe disease (486). These observations are consistent with previous results indicating a protective role for IFN-y in EAE (487, 488) and indicate that the ability of IL-12 to induce encephalitogenic cells or to enhance their activity is independent of its ability to enhance IFN-y production. In a model of relapsing EAE induced by MBP immunization in (SJL X PL/J) F1 mice, IL-12 treatment in vim during the remission phase induced disease relapse and strongly enhanced the severity of spontaneous relapses, whereas anti-IL-12 antibodies prevented spontaneous relapse as well as the severe relapse induced by treatment of the mice with bacterial superantigens (489). Overall, the results in transfer or relapsing models of EAE indicate that, unlike the observations in infectious disease (377), IL12 in EAE is not only needed for the initiation of the T-cell response, but also for its maintenance and for the encephalitogenic effector phase. However, it remains unknown whether IL-12 affects already differentiated T cells or contributes to the recruitment of new naive T cells, possibly specific for different epitopes of the antigen. In rats, immunization with spinal cord emulsified in complete Freunds adjuvant induces a monophasic disease in the Lewis strain, but a severe and relapsing disease in the DA strain (490). The expression of IL-12 inRNA in the spinal cord of the two strains after immunization is quite similar, whereas in DA rats the expression of other proinflammatory cytokines is prolonged and production of immunodownmodulating cytokines such as TGF-P and IL-10 was almost absent (490-492). Similar to observations in mice, administration of IL-12 to MBP-immunized Lewis rats induces EAE relapse. However, although IL-12 is effective when administered up to 1week after recovery from the primary bout of disease, it is not effective when administered at later times; the relapses were characterized histologically by greater perivascular inflammation in the CNS and the induction of iNOS-positive cells (493). In clinical MS, both IL-12 and B7.1 expression has been detected in acute MS plaques in the CNS, particularly in early disease cases (494). Elevated production of IL-12 in response to anti-CD3 stimulation was observed in PBMC of MS patients with progressive disease, whereas patients with remitting-relapsing disease produced IL-12 at levels similar to those of healthy controls (49S).The induction of elevated IL-12 production mediated by the T cells from these patients was due to an increased expression of CD40L on the anti-CD3-stimulated T cells, which induced IL-12 production from either autologous or allogeneic non-T cells (495). MS patients with progressive disease have slightly higher levels of serum IL-12 than do healthy subjects or patients with other neurological dneases

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(496); however, no correlation was observed between the presence of MS or disease severity and the level of IL-12 in cerebrospinal fluid (497). Overall, these findings raise the possibility that an early event in the initiation of MS involves upregulation of B7.1 and IL-12, resulting in conditions that synergistically stimulate T-cell activation and the effector phase of a Thl-type immune response. In addition to EAE, analysis of experimental uveoretinitis (EAU) in mice has also implicated a role for IL-12. Anti-IL-12 antibodies in v i m prevented the development of EAU induced by immunization of B1O.A mice with interphotoreceptor retinoid-binding protein and conferred resistance to subsequent antigen challenge; however, anti-IL-4 antibodies at the time of rechallenge reversed the protection induced by IL-12 (498).This finding strongly suggests the importance of the Thl/Th2 equilibrium in induction/ protection of EAU (498), a conclusion strengthened by the observations that EAU can be adoptively transferred with T cells from mice primed in v i m and restiinulated in vitru with a peptide from the retinoid-binding protein only if the in vitru restimulation is perfonned in the presence of IL-12, and that in general the ability of the primed cells to transfer EAU correlates with their level of IFN-y production (499). D. COLLAGEN-INDUCED ARTHRITIS Collagen-induced arthritis (CIA) is a murine model for human rheumatoid arthritis, characterized by a severe swelling in the joints with a massive inflammatory infiltrate, which leads to joint destruction and deformities. CIA is induced in DBA/1 mice approximately 4 weeks after immunization with type I1 collagen emulsified with Mycobncterium tuberculosis in oil (complete Freund’s adjuvant). Immunization in the absence of M . tuberculosis or immunization of other strains (e.g., C57B1/6 or Bl0.Q) does not induce CIA. Treatment with IL-12 for 5 days at doses from 50 ng to 1 pg at the time of immunization or at time of onset of the disease was shown to induce severe disease even when the mice were immunized with collagen in oil only (incomplete Freund’s adjuvant) (500, 501). The induction of CIA was associated with enhanced IFN-y synthesis and strong upregulation of anticollagen antibodies, especially of the Thl-dependent IgG2a and IgG2b isotypes (500). Neutralization of IFN-y in vivu prevented the development of arthritis, but not the priming of Thl cells by IL-12 (500,502). IL-12 treatment of collagen-immunized mice of the resistant C57B1/6 or Bl0.Q strain failed to induce CIA; in these animals, IL-12 potentiated the IFN-y production by collagen-specific T cells, but did not enhance and instead decreased the titer of anti-collagen IgG2a or IgG2b antibodies (503).

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Although those results suggested a clear role for IL-12 induced T h l cells in CIA, likely mediated through their effect on antibody formation, it was surprising to find that treatment with high doses of IL-12 (1 pg/ treatment) in complete Freund’s adjuvant for 1 or more weeks immediately after the immunization of DBAA mice efficiently protected against development of CIA (504). This protective effect was paralleled by a strong inhibition of collagen-specific IgGl antibodies, a modest decrease of IgG2b, and no significant change in IgG2a (504). These contrasting results led to the conclusion that IL-12 can both suppress and enhance CIA in DBA/1 mice, depending on the adjuvant used (504). However, in the two sets of experiments, different schedules and dosages of IL-12 administration were used, and, as in other models, it is possible that the suppressive effects of high doses reflect a desensitization to endogenous IL-12 (45,5, 456) or a reactive production of anti-inflamniatory mediators such as IL-10 or glucocorticoids (168, 486, 505). Inhibition of endogenous IL-12 by antibodies to IL-12 or byp40 homodimers only inconsistently ameliorated or delayed the onset of CIA, and in some cases, short duration anti-IL-12 treatment even enhanced the disease (M. Feldmann, personal communication). These results may be due to different requirements of IL-12 during the various stages of CIA induction, but the inconsistency of the results suggests that anti-IL-12 reagents were used that do not reproducibly and completely block IL-12 in uitro, as in other studies the use of higher affinity monoclonal antibodes or polyclonal antisera to IL-12 more consistently abrogated CIA ( M . Feldinann, personal communication, 501). Furthermore, IL-12 p40 genetically deficient mice, backcrossed into a DBN1 background, have a much reduced severity and incidence of CIA associated with a decreased antibody titer and IFN-7 production (506). Thus, these results confirm an important role for IL-12 in CIA onset, although in established disease IL-12 may have a suppressive role, as indicated in mice at late stages of CIA by an impressive exacerbation of arthritis shortly after cessation of anti-IL-12 treatment and by the ameliorating effect of IL-12 treatment of these stages (501). This antiinflammatory role of IL-12 on established CIA was associated with a 10-fold enhanced level of IL-10 in the serum of treated animals and was reversed by coadministration of anti-IL-10 antibodies (501),indicating that the ability of IL-12 to induce IL-10 may represent an important negative feedback mechanism in this elcperimental system to prevent excessive activation and tissue pathology. Very little information is available on the role of IL-12 in clinical rheumatoid arthritis, aside from the detection of IL-12 p40 inRNA and production of the IL-12 heterodimer in synovial samples from patients with this disease (507, 508).

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E. EXPERIMENTAL COLITISI N MICE AND CROHN’S DISEASE Crohn’s disease is a chronic inflammatory bowel disease that most commonly affects the terminal ileum and ascending colon. Much evidence suggests that immunological mechanisms are responsible for the disease, specificallythat Thl-type T cells possibly activated in response to microbial insults play a dominant role. In the mouse, intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS), which haptenates autologous colonic protein with trinitrophenyl (TNP), induces an intense immune response and a massive infiltration resembling the human Crohn’s disease (509). In this model of experimental colitis, administration of antibodies to IL-12 both early (at 5 days) and late (at 20 days) after induction of colitis leads to a striking improvement in both the clinical and the histopathological aspect of the established disease, often with complete regression of the pathology (509). These results are paralleled by a failure of the CD4’ T cells from the lamina propria of the anti-IL-12 treated animals to secrete IFN-.)I on in vitro stimulation (509). Injection of anti-CD40L antibodies at the time of TNBS administration also protected the mice from colitis and inhibited IL-12 production; IL-12 treatment reversed this protection effect of anti-CD40L antibodies (149). Interestingly, antiCD40L antibodies protected the animals from colitis only during the inducing phase of the disease, whereas anti-IL-12 antibodies also abrogated established disease, suggesting that the T-cell-dependent mechanism of IL-12 production is involved only at the early phases of the response and that IL-12 production is then maintained by a mechanism that depends on microbial or inflammatory products. Injection of TNBS-treated mice with anti-IL-12 antibodies also restored the T-cell tolerance against resident intestinal flora that, as in Crohn’s disease, is abrogated in experimental colitis (510). Explanation of these results may be provided by data in a different experimental model using mice transgenic for antiovalbumin TCR, in which IL-12 was shown to negatively regulate the two main mechanisms of mucosal tolerance: TGFp production and clonal deletion via apoptosis (436, 511, 512). IL-2 genetically deficient mice provide yet another experimental model of colitis; most of these animals develop colitis spontaneously, but the disease can also be experimentally induced in a time-controlled way by immunization with irrelevant exogenous antigens (e.g., TNP-KLH or TNPOVA) (513). IL-12 is abnormally expressed in the colon of immunized mice, and anti-IL-12 antibodies prevent the development of the colitis (514). In these mice, thymocyte maturation in the absence of IL-2 is abnormally directed by IL-12 toward the generation of single-positive, mature-activated Thl-type thymocytes capable of mediating colitis; these

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defects in thyinocyte maturation are prevented by treatment with anti-IL12 antibodies (513). The important role of IL-12 and Thl cells in colitis is also supported by results in Crohn’s disease patients; IL-12 production was detected from lamina propria mononuclear cells, where a predominance of T h l cells was demonstrable (515, 516), and culture of these T cells in vitro in the presence of anti-IL-12 antibodies downregulated the development of IFNy-producing CD4’ T cells (516).

F. SPONTANEOUS AUTOIMMUNE DISEASE IN MRL/lpr MICE MRL/lpr mice develop a spontaneous autoimmune disease characterized by lymphadenopathy, autoantibody production, and inflammatory manifestations, such as nephritis, vasculitis, and arthritis, and have been used extensively as a model for clinical systemic lupus erythematosus (SLE). Spleen and peritoneal cells from MRL/lpr mice produce significantly more IL-12 than do cells from MRL/+ or BALB/c mice, and this production results in increased release of IFN-y and nitric oxide (517). Sera from MRL/lpr also contain increased levels of IL-12 compared to the control mice (517), and IL-12 is upregulated in the tubular epithelium of MRL/ lpr mice with nephritis (518).Daily injections of IL-12 into MRL/lpr mice led to increased serum levels of IFN-y and nitric oxide, and to accelerated and more severe glomerulonephritis (517). Strikingly, the pyelonephritis with extensive vasculitis and infiltration of mononuclear cells at the kidney medullary region was prominent in the control animals and almost completely abrogated in IL-12 treated mice (517).This paradoxical observation likely rests in the fact that glomerulonephritis is autoimmune, whereas pyelonephritis is reactive to local infection: thus IL-12 exacerbates the former while, by increasing antimicrobial resistance, improves the latter (517). In another autoimmune manifestation of MRL/lpr mice, i.e., the Sjogren-like syndrome characterized by lymphoid infiltration in salivary and lacrimal glands, local upregulation of IL-12 and IFN-.)Iexpression was observed (519, 520). Although MRL/lpr mice are often considered a model for human SLE, the autoimmune diseases in the two situations may be very different, and the predominance of Thl-type responses observed in MRL/lpr is unlikely to be a characteristic of human SLE. Indeed, in vitro IL-12 efficiently inhibited the elevated immunoglobulin production by SLE patients’ PBMC through a mechanism independent of the observed increased IFN-y and decreased IL-10 production (521). Furthermore, PBMC from newly diagnosed SLE patients produced in vitro less IL-12 and more IL-10 than PBMC from healthy controls (D. Honvitz, personal communication).

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XIV. 11-12 in the Inflammatory Response

The ability of IL-12 to induce and regulate the production of IFN-7 and other cytokines such as GM-CSF and TNF-a, which are major stimulators of phagocytic cell activation, makes it a powerful proinflammatory cytokine, with a major role in local inflammation during infections and autoimmune responses. In addition, IL-12 plays a predominant role in the systemic inflammatory response syndrome and could play a direct or indirect role in the multiorgan failure or dysfunction observed in systemic inflammation. IL-12 also appears to be particularly sensitive to the downregulatory mechanisms (or compensatory antiresponse syndrome) activated in response to the inflammatory response. Thus, the immunosuppressive state following major infection or trauma may be due in part to IL-12 deficiency and corrected, as discussed later, by IL-12 treatment. Nakamuraet al. (522) reported that endotoxin treatment of mice induced a serum factor with a molecular mass of about 70,000 that stimulated IFN-y production. Later, these authors partially purified the factor and showed its identity with IL-12 (523). The in vivo production of IL-12 was then shown to be an absolute requirement for IFN-y production in mice treated with high (300 p g ) or low (1p g ) doses of LPS after priming with BCG infection (161, 162). IL-12 p70 is produced around 3 hr after LPS injection and reaches concentrations of up to several nanograms per milliliter in the serum, although the concentration of IL-12 p40 (both monomers and homodimers) is always manyfold higher (50, 161,162). At 2-3 hr after LPS injection, increased accumulation of niRNA for both IL-lBRPl and P2 in spleen cells is observed (L. Showe, M. Wysocka, and G. Trinchieri, unpublished results), suggesting that both IL-12 and IL-12R are expressed at this time. IFN-y is produced with a peak around 6-7 hr and its production is inhibited by neutralizing antibodies to IL-12 (161, 162), which also protect the animals from death from endotoxic shock (161). The requirement for IL-12 in IFN-7 production was confirmed in IL-12 p40, IL-12 p35, and IL-12RP1 genetically deficient mice, all of which produced 10-fold lower levels of IFN-.)Iin response to LPS than did wild-type animals (72, 279, 280). However, IL-12 alone is not sufficient for optimal IFN-y induction, and important cofactors are represented by TNF-cu (161) and IL-18 (265, 266). Although IL-12 is required for IFN-7 production, the production of IL-12 is IFN-y independent and observed in IFN-y-deficient mice (163). In baboons, IL-12 production in vivo was observed during experimental Escherichia coli-induced septic shock, but IL-12 production, unlike IFN-y, did not correlate with the dose of bacteria or with severity of shock (524). Thus, IL-12 production is likely under the control of downregulatory mechanisms, e.g., IL-10 production, and other cofactors,

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together with IL-12, are responsible for determining the level of IFN-y produced. The Shwartzman reaction in mice is elicited by two injections of LPS: the priming injection given in the footpad and the lethal LPS challenge given intravenously 24 hr later. IL-12 or IFN-y can replace LPS in the priming injection, and anti-IL-12 prevents the priming effect of LPS, suggesting that IFN-y indiiced by IL-12 following LPS injection is responsible for the priming (525). IL-12 was also shown to account for the ability of BCG to sensitize mice to the lethal effect of TNF-a and to be able to replace BCG in this sensitizing effect (526). LPS can cause tolerance to its own action both in vivo and in vitro; LPS-induced tolerance is characterized by a decreased synthesis of various cytokines, particularly TNF-a on LPS rechallenge (527). In vitro, LPS desensitization for TNF-a production can be prevented and reversed by treatment of monocytes with IFN-y or, if IFN-y-producing nonmonocytic cells are present, by IL-12 via IFN-y induction (527). IL-12, both p40 and p70, are even more sensitive than TNF-a to LPS desensitization and, unlike TNF-a, the inability of LPS-pretreated inonocytes to produce IL-12 is not readily reversed by IFN-y treatment or by using a different inducer such as S. azcreus (C. Karp, M. Wysocka, X. Ma, and G. Trinchieri, unpublished results). In chronic multisystem inflammatory disorders such as Behqet’s disease (528) or sarcoidosis (529), disease activity correlates with the expression of elevated levels of IL-12 in the plasma and in the bronchoalveolar lavage fluid, respectively. These data suggest that these syndromes are Thlmediated diseases that may be driven by chronic, dysregulated production of IL-12 at the site of disease (529). Interestingly, an elevated expression of IL-12 was also found in human atherosclerotic plaques, and IL-12 was inducible in monocytes by highly oxidized low-density lipoprotein (LDL), but not by minimally modified LDL (530). These data clearly point to the possibility that production of IL-12 and the proinflammatory network plays a major role in the pathogenesis of the atherosclerotic plaque. A state comparable to LPS tolerance is observed after severe injury by trauma or bum, manifested in part by a severely depressed ability of mononuclear cells from the patients to produce IL-12 (531, 532). This endotoxin tolerance assumes clinical relevance because it likely underlies the great susceptibility of major trauma victims to infections. In a mouse model of burn injury, in which decreased production of IL-12 was also observed (53l),treatment with low-dose IL-12 (25 ng daily for 5 days) increased survival of the burn-injured mice after cecal ligation and puncture to the same level as the sham-burn control group (533).Although IL-12 acts through IFN-y, and IFN-.)I treatment was partially effective, IL-12

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was determined to be the most effective therapy tested so far in this model of burn-induced immune unresponsiveness (533).In a similar model, IL-12 was also effective in protecting bum-injured mice from herpes simplex type 1 infection, to which they are very susceptible (534). XV. 11-12 in Infectious Diseases

A. VIRUSES

1. Experimental Viral Infections Because of its ability to induce production of IFN-y, to favor T h l responses, and to enhance CTL generation, IL-12 was expected to play an important role in viral infections. Although some of this expectation proved correct, IL-12 does not appear to play as important a role in viral infections as it does, for example, in many bacterial and intracellular parasite infections, and IL-12-independent mechanisms of IFN--y production and generation of CTL are operative in antiviral immunity (331). Early attempts to treat viral infections with IL-12 showed that low doses of IL-12 (1-10 ng daily per injection) had some protective effect on lymphocytic choriomeningitis virus (LCMV) and murine cytomegalovirus (MCMV) infections, whereas higher doses of IL-12 (10-1000 ng daily per injection) were detrimental to resistance against LCMV infection (535). These high doses of IL-12 (1)inhibited CTL activity, (2) inhibited virusinduced CD8' T-cell expansion, (3)induced necrotic lesions in splenic white pulp, (4)resulted in >2 log increase in splenic and renal viral replication, and (5)decreased body weight and thymus mass (505,535). These IL-12 toxicities were prevented by treatment with neutralizing antibodies to TNF-a: LCMV infection was shown both to synergize with IL-12 in inducing in vim TNF-a production and to sensitize target tissues, particularly CD8+ T cells, to the effect of TNF-a (505).IL-12 treatment of LCMV-infected animals also induced an increase in circulating glucocorticoid levels, which were secondary intermediaries in the dramatic thymus atrophy induced by IL-12 (505). Unlike LCMV infection, the effective defense against MCMV infection requires NK cell-produced IFN-y, and IL-12 enhances this defense pathway (536).In particular, IL-12 treatment of MCMV-infected animals increased NK cytotoxicityand IFN-y production and resulted in an improved antiviral status; virus-induced hepatitis was decreased up to 50-fold and viral burden decreased below the level of detection (536).These protective effects of IL-12 were prevented by depletion of either NK cells or IFN-y (536).Similarly, a single injection of 20 ng IL-12 18 hr before a lethal challenge with encephalomyocarditis virus (EMCV) protected all the animals from death with a mechanism mediated by IFN-y (537).

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Early expression in uivo of IL-12 inRNA or protein was observed after infection with several different viruses, including lactate dehydrogenase elevating virus, mouse hepatitis virus (MHV), mouse adenovirus (538), herpes simplex virus 1 (HSV-1) (539),MCMV (540), and influenza virus (541). The production of IL-12 in response to virus infection peaks in the first 1to 3 days after infection and is usually transient. In MCMV infection, the early IL-12 production is responsible for NK cell production of IFN-y; treatment of mice at day 3 of infection with anti-IL-12 antibodies decreased IFN-y production and resulted in a 1 log increase in virus titer (540). However, treatment with anti-IL-12 antibodies at days 7-9 after infection had no effect on IFN-y production from T cells or on virus clearance; at no time did anti-IL-12 treatment decrease NK cell cytotoxicity or T-cell functions (540). Almost identical results were obtained with infection of mice with the PR8 strain of influenza A virus: IL-12 was induced with a peak at 2-3 days after infection and anti-IL-12 antibody treatment at day 3 inhibited IFN-y production, mostly from NK cells, and resulted in a 1 log increase in lung virus titer, whereas treatment at day 7 did not affect IFN-y production or virus titer (541).Thus, in both MCMV and PR8 virus infection, the production of IL-12 and NK cell-derived IFN-y are important in early resistance to virus infection; however, IL-12 has little if any effect on the antigen-specific T-cell response (CTL activity and IFN-y production) and on the eventual virus clearance mediated by T cells. The observation that IL-12 is differentially regulated during various virus challenges, in particular the lack of production of IL-12 in LCMV infection compared to the early production in MCMV infections, moved Cousens et al. (542) to investigate the role of IFN-a/P in these infections. They reported that I F N - d P inhibited IL-12 and IFN-y production from S. aureus-stimulated mouse splenic cells, whereas TNF-(r and IL-6 were not inhibited (542).In uivo neutralization of IFN-d/3 expressed endogenously during MCMV infection enhanced early IL-12 and IFN-y protein production and, interestingly, revealed an early production of IL-12 and IFN-7 in LCMV infection that was completely undetectable in untreated LCMVinfected animals (542). These results suggest a new interplay between IFN-dP and IL-12: resistance to infection with viruses that induce high and efficient early expression of IFN-(r/P is independent of early IL-121 IFN-y production, whereas early production of IL-12 and IFN-y from NK cells is observed in infections with viruses that are poor inducers of I F N - d P production, and this early IL-12 production is important in controlling virus infection until an IL-12 independent, antigen-specific Tcell response is established.

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Infection of the central nervous system with vesicular stomatitis virus (VSV) is a particularly good example of the possibility of using IL-12 to treat viral infections. IL-12 administered intraperitoneally strongly enhanced immunity to VSV infection in the CNS, as indicated by (1)decreased viral titer, (2) increased expression of iNOS, ( 3 ) enhanced expression of class I and class I1 MHC antigens, (4)increased T-cell infiltration, and (5)decreased VSV-induced apoptosis (543).This antiviral effect of IL-12 treatment of VSV infection in the CNS was also observed in IFN-y genetically deficient mice and is therefore IFN-y independent (544). There is much clinical interest in the possibility that antiviral cytokines are effective in the therapy of chronic viral hepatitis. IL-12 was shown to be particularly effective in protection against M HV-induced hepatitis, an effect dependent on IFN-y (545).In mice transgenic for the hepatitis B antigen, IL-12 suppresses autoantibody formation by shifting the Th2 response to T h l predominance (546),and in hepatitis B virus transgenic mice, IL-12 treatment inhibits virus replication in liver and kidneys and induces clearance of the cytoplasmic hepatitis B core antigen from both tissues via IFN-.)Iinduction (547).These data suggest that IL-12 treatment suppresses the Th2-type cells that are probably involved in maintaining the infection in chronic hepatitis while inducing a Thl response that, through IFN-y production and noncytolyhc mechanisms, efficiently clears the virus. These models suggest that IL-12 may have therapeutic value for the treatment of chronic hepatitis virus infection. The ability of IL-12 to act as an adjuvant in vaccination and to enhance a Thl-type response is of obvious interest in preventing viral infections. However, to date, results have been reported only for respiratory syncyt~al virus (RSV). Because many of the clinical manifestations of RSV infection are due to the nature of the immune response to the virus, with a Th2-type response considered to be responsible for the severe lung inflammation observed in vaccinated children, a vaccination protocol that preferentially induces a Thl-type response was expected to be optimal. Indeed, immunization of mice with formalin-inactivated, alum-precipitated RSV in conjunction with IL-12 at the time of immunization resulted in inhibition of virus replication, increased IFN-7 production, and increased IgG2a antibodies on challenge (548). Thus, IL-12proved to be apowerful adjuvant for RSV vaccination, inducing a strong Thl-type response; however, no significant effect was observed on the clinical outcome or on CTL generation (548,549).In another model in which mice were vaccinated with RSV glycoprotein G as part of a recombinant vaccinia virus and then challenged with RSV, the presence of IL-12 during vaccination also determined a change in the nature but not the severity of the inflammatory lung disease observed following the RSV challenge (550).IL-12 treatment

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during vaccination determined an almost complete disappearance of eosinophils and B cells from the lung, but an increase in the lymphoid infiltration, particularly of IFN-y-producing CD4' T cells, which resulted in similar or even more severe illness (550).Thus, in this model, reversal of the Th2associated pathology does not necessarily benefit the host (550).Interestingly, the use of anti-IL-4 instead of IL-12 during vaccination with inactivated virus resulted in the predominance of IFN-y-producing CD8' T cells in the lung, which was associated with less severe illness: based on these results, it was proposed that the phenotype of effector cells involved in the iininune response to virus challenge is a more important determinant of disease than the patterns of cflokine expression classically assigned to Thl and Th2 lymphocytes (549): A particular use of IL-12 in inducing deviation of the immune response during gene therapy has been reported by Yang et al. (551).The efficacy of gene therapy in the lung using a replication-defective adenovirus vector is limited because the infected cells expressing the transgene are rejected by the host iininune system within several weeks, and reinfection is impossible because of neutralizing IgA antibodies; however, the administration of IL12 to mice at the time of initial gene therapy treatment suppresses the generation of the neutralizing IgA and allows successful reinfection of the lung and repeated gene therapy (551).

2. Measles Vinis Infection with measles virus still kills 1-2 million children annually, mostly because the infection is accompanied by a marked and prolonged abnorinality of cell-mediated immunity that contributes to increased susceptibility to secondary infections, the major cause of death. Measles infection and vaccination are characterized by a predominant expression of Th2-type cytokines, and this iininune deviation is probably responsible for the observed cellular immunodeficiency. Karp et al. (194) observed that in vitro infection of human nionocytes with measles virus resulted in a profound inhibition of the ability of the cells to produce IL-12, whereas the production of inany other proinflammatory cytokines was almost unaffected. This suppression was observed in response to various bacterial stimuli or CD40 stimulation. The effect was at the transcriptional level and, interestingly, although measles virus infection selectively affects the production of IL-12, the induced transcription of both the p40 and the p35 genes was suppressed in virus-infected inonocytes (C. Karp, X, Ma, and G. Trinchieri, unpublished results). Measles vinis inhibits IL-12 production by binding to its receptor, membrane cofactor protein or CD46, which is a natural receptor for complement factors C3b and C4b. Inhibition of IL-12 production in human inonocytes is observed on binding of CD46

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to its various ligands, i.e., measles virus, polymerized C3b, or anti-CD46 monoclonal antibodies (194). Thus, measles virus downregulates IL-12 production most likely by utilizing a physiologic mechanism of immunoregulation through complement and one of its receptors. The ability of measles virus to inhibit production of IL-12 is one of several mechanisms of induced immunosuppression (552). Measles virus replicates poorly in dendritic cells, but its replication is maximally induced when CD40 on the dendritic cells is stimulated; the infected dendritic cells are unable to stimulate T-ceI1 proliferation and are defective in their ability to produce IL-12 (553).However, a role for decreased IL-12 production was not demonstrated in other experimental systems in which immunosuppression in vitro was induced by interaction of measles virus glycoproteins with the surface of uninfected lymphocytes or by infection of resting human dendritic cells (554, 555). 3. Human Immunodeficiency Virus a. Defective Production of IL-12 by HIV-Infected Patients. As compared with PBMC from uninfected control donors, PBMC from HIVinfected individuals were found to produce very similar levels of TNF-a and IL-10, 3- to 4-fold more IL-6 (118), and 10- to 20-fold less IL-12 free p40 chain and 5-fold less biologically active p70 heterodimer when challenged in vitro with S. aureus (118,556-558). A similar defect in IL12 production was also reported by Gazzinelli et al. (559) in response to the opportunistic pathogen T. gondii. Although alveolar macrophages from HIV patients spontaneously produced low levels of IL-12, their ability to produce IL-12 in response to S. aureus was depressed (560). Lower accumulation of both p40 and particularly p35 mRNA paralleled the decreased ability of patients’ PBMC to produce IL-12 (558). Although these results need to be extended to other pathogens or stimuli able to induce IL-12 production, the specific deficiency in the production of IL-12, while other inflammatory cytokines are produced normally or at increased levels by HIV-infected patients, suggests a possible role for IL-12 deficiency in HIV disease pathogenesis. The mechanism underlying the decreased IL-12 production by PBMC from HIV-infected patients remains elusive. In vitro incubation of human monocytes with HIV for 1week resulted in decreased production of IL12 (118, 561). However, in both in vivo and in vitro HIV infection, only a small proportion of monocytes is actually infected with HIV, so that the suppression of IL-12 production is likely to be an indirect result of HIV infection. The HIV gp120 envelope protein was reported to directly stimulate production of low levels of IL-12 (562), but also to render monocytes defective in responding to S. aureus with high levels of IL-12 production,

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possibly as a function of IL-10 overproduction and secondary inhibition of IL-12 production (563). HIV-infected patients, at least in some phases of disease progression, have been described to overproduce certain Th-2 type cytokines, in particular IL-4 and IL-10 (564-566). Because Th2 cytokines and in particular IL-10 are able to suppress IL-12 production (108,169),it could be hypothesized that exposure of PBMC in vivo or in vitro to these cytokines is responsible for the decreased IL-12 production. However, these cytokines have a suppressive effect on all inflammatory cytokines, making it difficult to explain the selective IL-12 deficiency (108, 169). Furthermore, when PBMC are exposed to IL-4 or IL-13 for an extended period, their ability to produce IL-12 is boosted rather than inhibited (169) and, in PBMC from HIV patients, the deficient IL-12 production is corrected (174). Similarly, IL-12 production in patients’ PBMC was partially restored by treatment with IFN-y or IL-15 (174, 567, 568). In short-term S. aureus-stimulated cultures of PBMC, production of IL-10 but not IL-4 is observed (118, 558). Chougnet et al. (558)reported that IL-10 production is increased in PBMC cultures from HIV patients compared to healthy controls, unlike earlier studies (118) in which no significant dfference was observed. Because endogenously produced IL10 in culture is known to limit IL-12 production (108), the ability of neutralizing anti-IL-10 to correct deficient IL-12 production from HIV patients was tested (118).In the presence of antibodies, a similar increase in IL-12 production was observed in both HIV patients and healthy controls, making it unlikely that IL-10 is responsible for the differential ability of patient and control PBMC preparations to produce IL-12 (118).A significant role for PGEz in the deficient production of IL-12 in blood cells of HIV patients has also been excluded (174,557), despite the known activity of PGEz as a potent and selective inhibitor of IL-12 production (181)and the reports of enhanced PGE2production in HIV-infected monocytes (569) and of increased CAMP levels in PBMC from patients (570). Another aspect of IL-12 production that might be involved in the IL12 deficiency in HIV-infected patients and that deserves investigation is the role of T-cell signaling in IL-12 induction. Several lines of evidence suggest that, at least during antigen-specific stimulation, T cells are needed for the induction of IL-12 production in antigen-presenting cells (148, 306, 359). Thus, the IL-12 deficiency in the patients may rest in part on deficient signaling by activated T cells to the antigen-presenting cells. Because of the role of IL-12 in T-cell activation, this mechanism of immunodeficiency would be self-amplifying. Although the in vitro T-cell response and the ability of PBMC to respond to stimulation with IL-12 production are deficient, analysis of expression

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of cytokine mRNA in peripheral blood or lymph nodes of HIV patients revealed increased expression of T h l cytokines, including mRNA for IFNy. a cytokine dependent on IL-12 for optimal induction (571,572).Whereas these observations might argue against the hypothesis of a predominant Th2 response in HIV-infected patients, they are not incompatible with the deficiency of IL-12 production. Because of infections and other immunological stimulations, patients’ lymphocytes and monocyte/macrophages are thought to be in an activated state. In these conditions, a constitutive expression of mRNA for activated lymphocyte products, including IFN-y, and of proinflaminatory cytokines in macrophages and antigen-presenting cells, including IL-12, is to be expected. However, the in vivo observation of constitutive cytokine gene expression, reflecting a chronic inflammatory situation, may not be associated with an efficient acute response to stimulation or may even be responsible for a deficient production of the cytokines required for an effective immune response, e.g., IFN-y and IL-12. If this is the case, a progressive failure of antigen-specific responses over a background of chronic activation in both macrophages and lymphocytes might result which, because of homeostatic mechanisms regulating lymphocyte activation, may even lead to Th cell deletion.

b. HZV-Znfected Patients Are Responsive to ZL-12. Although HIVinfected patients are deficient in their ability to produce IL-12, their T and NK cells in vitro have been shown to respond normally to IL-12. Patients with advanced HIV disease often show a very reduced cytotoxic activity in their peripheral blood NK cells, although the number of NK cells is not decreased (573, 574). IL-12 treatment in vitro enhances the NK-mediated cytotoxic activity of peripheral blood lymphocytes from HIVinfected patients, similar to the observed effect on those of healthy donors; this NK-enhancing effect of IL-12 is particularly evident on lymphocytes from patients with advanced disease and nearly absent NK cytotoxic activity, in which IL-12 restores cytotoxic activity to levels close to those of healthy donors (117, 575, ,576). IL-12 enhances the NK cytotoxic activity of the patients’ lymphocytes against both tumor target cells and virusinfected target cells (117); interestingly, IL-12 can also boost the NK cytotoxic activity of healthy donors’ lymphocytes against HIV-infected target cells (316). On freshly purified peripheral blood lymphocytes from HIV-infected patients, IL-12 alone or in synergy with IL-2 induces IFNy production, although, in advanced patients, to levels somewhat lower than those observed with lymphocytes from healthy donors (117,577). IL12 also enhances the PHA-induced IFN-y production in lymphocytes from HIV-infected patients, almost completely correcting the low PHA-induced IFN-y production observed in some patients (568).

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The induction of IFN-7 production and the enhancement of N K cytotoxicity represent rapid and short-lived effects of IL- 12, which may not lend themselves easily to a permanent in vivo therapeutic effect. It was therefore important to deinonstrate whether IL-12 can prime T-cell clones from HIV-infected patients for high IFN-7 production, a long-lasting and possibly irreversible effect (15).Peripheral blood T cells froni 10 HIV-infected patients at different stages of disease were cloned by limiting dilution in the presence or absence of IL-12 for the first 2 weeks of culture (578). A very high efficiency of clonal expansion was obtained by culture in the presence of irradiated feeder cells, PHA, and IL-2. On average, CD4+ clones cultured in the presence of IL-12 produced lo-fold more and CD8+ clones 5-fold inore IFN-y than clones originated in the absence of IL-12 when restimulated by anti-CD3 and phorbol diester after 1 month expansion (578). This pi-iining effect, which is analogous to that observed with T cells from healthy donors (13,was observed with patients at any stage of the disease (578). IL-12 was also shown to enhance the depressed proliferation of HIV patients to recall antigens, including HIV peptide, influenza virus, Candida, tetanus toxoid, streptokinase, and alloantigens to levels close to those of healthy donors (119, 309, 568, 579). These results are consistent with the ability of IL- 12 to enhance the proliferative response to antigens, alloantigens, and initogens observed with T cells from healthy individuals (19, 281) and with the primary and, in some cases, obligatory role of IL12 in antigen-induced proliferation of ineinory T cells and differentiated Thl cells (119, 166, 294, 306). The in uitro-enhancing effect of IL-12 on HIV patient T-cell proliferation could be due to the activation of unresponsive T cells or to replacement of insufficient IL-12 produced in vitro by patients' antigen-presenting cells. However, only ininimal effects were observed in patients with less than 200 CD4' T cells/min3 (309, 579). Another important function of IL-12, shared with other Thl cytokines such as IL-2 and IFN-7, is its ability to prevent mitogen-, anti-CD3-, or CD95 (Fas)-mediatedprogramiiied cell death in T cells from HIV+ donors (311, 312). IL-12 also inhibits apoptosis induced by gp120 or CD4 crosslinking and CD3/TCR activation in a human Th1 clone (310), an in vitro mechanism of induction of apoptosis that may mimic one of the pathogenic processes in HIV infection. Because death by apoptosis is one of the mechanisms proposed for CD4' cell depletion in AIDS, which could be favored by reduced production of Th1 cytokines, including IL-12, the ability of IL-12 to prevent T-cell receptor-induced apoptosis in patients' T cells represents a potentially iinportant therapeutic function of this cytokine.

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c. IL-12 as an Adjuvant in HIV Vaccination. Several investigators have investigated the usefulness of IL-12 as an adjuvant in vaccination against HIV in experimental animals. In those studies, IL-12 was used as protein or, most often, as an expression plasmid, together with a peptide vaccine or with plasmids expressing the antigen (338, 432, 580-582). In all these studies, IL-12 proved particularly powerful in inducing specific CTL generation and DTH, whereas the effect on immunoglobulin production was less marked and usually limited to enhancement of IgG2a. However, the simultaneous use of IL-12 and GM-CSF was shown to have a cooperative effect in inducing maximum CTL and antibody generation (338,432,580). Of particular clinical interest is the finding that intranasal immunization of mice with a DNA vaccine of IL-12- and GM-CSF-expressing plasmids in liposomes induced strong mucosal and cell-mediated immune responses against HIV antigens (338).

d. Effect of IL-12 on HIV Replication. IL-12 does not enhance HIV replication in resting PBMC, but it has been reported to do so in mitogenactivated lymphocytes or in cultures depleted of CD8' T cells, especially those from infected asymptomatic donors (583-585). However, in another study (568),IL-12 was shown to have little affect by itself on HIV replication in pHA-activated human T cells and to significantly decrease HIV expression in ACH-2 cells in the presence of suboptimal concentrations of phorbol diester. Furthermore, IL-12 has been shown to inhibit IL-2-induced HIV replication in PHA-activated human T cells (568) and in CD8-depleted PBMC (584). IL-12 has been reported to be less efficient than IL-2 in inducing CD8' T-cell-mediated suppression of HIV-1 replication in CD4' T cells (586).However, in a different experimental system, IL-12 decreased HIV-1 replication in human inacrophages cocultured with autologous peripheral blood mononuclear cells (587);in this latter system, the IL-12 antiviral effect was mediated by IFN-y produced by NK cells, making it possible that IL-2 and IL-12 exert an antiviral effect acting through different mechanisms and effector cell types, CD8' T cells for IL-2 and primarily NK cells or possibly CD4' T cells for IL-12. 4. Murine Retroviruses

Murine AIDS (MAIDS) is a syndrome of progressive lymphoproliferation and increasingly severe immunodeficiency that develops in mice of certain strains, e.g., C57BU6, following infection with a retrovirus mixture containing a replication-defective pathogenic murine leukemia virus (MuLV) designated BM5def and a helper MuLV (588).IL-12 and IFNy are produced in the first week of infection of C57BW6 mice, and IFNhas an important, although not essential, role in inducing proliferation

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of the MuLV-infected B cells (589). The IFN-y mRNA level was maintained or continued to increase at a later time of infection, but production of IFN-y protein declined with the progression of the immunodeficient state and of T-cell anergy (589). At 4 weeks of infection, IFN-y could still be induced in T cells by LPS, IL-12, and/or anti-CD28 antibodies, but at later times the CD4+ T-cell anergy became irreversible (589, 590). Although IL-12 and IFN-y appear to have a pathogenetic role in the lymphoproliferation and immunodeficiency of MAIDS, treatment of the animals with 100-250 ng of IL-12 per mouse, 5 times a week, starting at the time of infection or up to 9 weeks after infection, markedly inhibited the development of splenoinegaly and lymphadenopathy, B-cell activation, Ig secretion, and T-cell immunodeficiency (591). The expression of the pathogenic BM5def was almost completely suppressed, whereas the expression of the helper MuLV was reduced to one-third (591). This effect of IL-12 is dependent on IFN-y production and is likely due to the inhibitory effect of high doses of IFN-y on viral replication in B cells; indeed, the use of lower concentrations of IL-12 resulted in exacerbation of the disease, probably because lower levels of IFN-y enhance the proliferation of B cells, whereas higher doses resulted in systemic toxicity, similar to what is observed in LCMV infection (588). Rauscher leukemia virus infection in mice is also very irnmunosuppressive, causing a block in dendritic cells but not T-cell functions (592). Treatment with five daily doses of 100 ng of IL-12 per mouse at the time of exposure resulted in an improveinent in the capacity of lymph node dendritic cells to stimulate allogeneic responses and in the restoration of DTH (592).Whether these data reflect a dxect effect of IL-12 on dendritic cells or an indirect effect mediated by IFN-y or other cytokines produced by T and NK cells remains to be determined.

B. BACTERIA 1. Listeria Monocytogenes

After the role of IL-12 as a mediator of IFN-y production in bacterial infection was demonstrated in human peripheral blood mononuclear cells (46), L. nzonocytogenes (257, 593) and 7'. gondii (256, 377) infections were the first two experimental murine models in which such a role was confirmed first in vitro and then in tjivo. Heat-killed L. rrwnocytogenes (HKLM) in vitro induces production of IFN-y from SCID splenocytes via a mechanism that involves IL-12 production, and IL-12 synergizes with TNF-a, and IL-2 in inducing IFN-y production (257). In vim, L. monocytogenes infection induces mRNA and protein production of both IL-12 and IFN-.)I begnning around 24 hr from infection, and anti-IL-12

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antibodies efficiently block IFN-y production and resistance to infection in both SCID and wild-type aniinals (593, 594). A single dose of IL-12 at the time of infection significantly enhanced resistance of mice to L. rnoriocytogenes infection (595). Although NK cells appear to be the major producers of IFN-y in the primary response to L. monocytogenes, their ability to produce IFN-y is defective in mice lacking Ty6 cells, perhaps due to deficient TNF-a, production (596); production of IFN-y by Ty6 cells was shown to be induced by IL-12 in synergy with IL-1(597).Although the primary response to L. wwnoqtogenes required IL-12 in uivo for IFNy production, resistance to secondary challenge was blocked by anti-IFNy antibodies, but not by anti-IL-12 antibodies (598), indicating that, as previously shown in T. gondii infection (377), an established Th1 response is independent of IL-12 for IFN-y production. However, in vitro production of IFN-y by spleen cells from immune mice in response to HKLM, but not in response to anti-CD3 antibodies, was at least partially dependent on IL-12 (598). The requirement for IFN-7 in the resistance to listeriosis was clearly shown by the rapid death of Listeria-infected IFN-yR-deficient mice (599). IL- 10-deficient mice, conversely, have an increased resistance to listeriosis, with a dramatically enhanced proinflainmatory cytokine production and Thl response that was protective and did not result, as in the case of 7'. gondii or T. cruxi infection (292,293), in systemic toxicity (600). Interestingly, IL-13 treatment of mice resulted in an enhanced resistance to listeriosis, most likely by acting indirectly through stimulation of IL-12 production (177). Whereas in tiitro heat-killed L. rnonocytogenes was a potent inducer of IL-12 production, only the live bacteria were efficient in vivo (131, 601). However, HKLM, soluble listerial antigen preparations, or a synthetic peptide corresponding to a dominant MHC class II-restricted listerial determinant, which by themselves are inefficient vaccines, when coinjected with IL-12, elicited a potent antigen-specific iinmune response that conferred protective iininunity against L. monocytogenes (602-604). 2. Mycobacteriu Mycobucteriurn tuberculosis induces IL-12 production in both human and murine phagocytic and dendritic cells (46, 605, 606). Administration of IL-12 to inice enhances their resistance to M . tuberculosis infections, especially in the susceptible BALB/c strain (606,607),whereas the requirement for IL-12 in the resistance to the infection was shown by neutralization of endogenous IL-12 with monoclonal antibodies (606) or, more convincingly, in IL-12 p40 genetically deficient inice (608). The ability of M. tuberculosis to induce IL-12 production was in part dependent on phagocytosis because phagocytosis of large latex beads induced similar

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production of IL-12, but not, however, TNF-a (134, 609). Furthermore, arabinofuranosyl-terminated lipoarabinornannan derived from rapidly growing Mycohacteriuni sp., but not the extensively mannosylated form, is capable of inducing IL-12 (610). IL-12 used as an adjuvant for an experimental subunit vaccine based on secreted antigens from M. tuberculosis was able to accelerate the development of an efficient immune response, but not to change the final outcome of a full vaccination regime (611). In humans, IL-12 was produced by pleural fluid cells of patients with tuberculosis pleuritis, and anti-IL-12 antibodies suppressed the proliferation of these cells in response to M . tuberculosis (612). Using in situ hybridization, it was found that the percentage of bronchoalveolar lavage cells expressing IL-12 p40 mRNA was much higher in patients with active compared to inactive tuberculosis (613) and by Elispot, the number of IL12-producing cells in the peripheral blood was found to be increased in patients with tuberculosis compared to healthy donors (614). In patients infected with M. leprae, it was observed that the tuberculoid lesions, characterized by a CD4' type 1 response, express 10 times more IL-12 mRNA and protein coinpared to leproniatous lesions, characterized by a CD8' type 2 response (FilFj).Anti-IL-12 antibodies blocked M. lepraeinduced T-cell proliferation in tuberculoid patients and IL-12 induced proliferation of CD4+ type 1 T cell clones from tuberculoid patients but not in CD8' type 2 T-cell clones from lepromatous patients (615). However, IL-12, in synergy with IL-2, restores both IFN-.)I production and proliferation in response to M . leprae in T cells from nonresponder patients almost to the level of responder patients (616). In M. aviuna infection of mice, endogenous IL-12 is required for resistance, and neutralization of IL-12 resulted in a several hundredfold increase in bacterial load (617),whereas treatment with recombinant IL-12 induced protection of susceptible BALB/c mice, which have a decreased IL- 12 production in response to infection (618,619). In a family in which several members have disseminated M. avium complex infection, it was observed that adherent cells from patients and their unaffected mothers produced abnorrnally low levels of IL-12 following stimulation with S. aureus, although they produced normal levels in response to S. aureus plus IFN-.)I (620). Thus, members of this fainily have a defect in IL-12 production that closely resembles that observed in HIV-infected patients (118) who also are susceptible to M . nviurn complex infection.

3. Other Bacterial Species Salmonella dublin infection of inice or of inacrophages in vitro induces the production of IL-12 (621-623), and the important role of IL-12 in the

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resistance to the infection is indicated by the increased salmonellosis and reduced survival time in mice orally challenged with S. dublin and treated with anti-IL-12 antibodies (624). Similarly, in S. thyphimurium infection, neutralization of IL-12 not only decreased the ability of mice to resist the infection, but also prevented the immunosuppression that accompanies the acute stages of the disease (625) showing a dual role of IL-12 in enhancing resistance and inducing immunosuppression. The role of IL-12 in murine Lyme borreliosis is complex; anti-IL-12treated mice infected with Borrelia burgdorferi had an increased number of spirocheta, but a significant decrease in peak arthritis severity, accompanied by a reduction in Thl response (626).Thus, the IL-12-induced innate immunity and T h l response is effective in preventing spirocheta growth, but it is also involved in the generation of the arthritis. Vitamin A deficiency is associated with higher IL-12 and IFN-.)I production and exacerbates murine Lyme arthritis (627). In humans, dendritic cells isolated from the dermis or from peripheral blood phagocytose B. burgdorferi and produce IL-12, thus probably playing a role in the initial T h l response to the bacteria, whereas Langerhans cells from the epidermis are unable to do so (628). Endogenous IL-12 is required for resistance to Brucella abortus infection in mice: neutralization of IL-12 at the time of infection prevents the generation of a protective T h l response, and the effects of this treatment in terms of exacerbation of the infection and inhibition of splenomegaly and granuloma formation in the liver are still evident 6 weeks after the treatment (629, 630). The ability of B. abortus to efficiently induce IL-12 production makes it a potential vaccine candidate (631). IL-12 is produced in vivo by animals immunized with live or killed Bordetella pertussis; however, an acellular vaccine constituted of various bacterial components adsorbed to alum induced a Th2 response and was unable to induce protective immunity (632). The same level of protection obtained with the IL-12-inducing whole cell vaccine can, however, be obtained by adding recombinant IL-12 to the acellular vaccine (632). The requirement for endogenous IL-12 in resistance to bacterial infections has also been shown with several other species, including Yersenia enterocolitica (633),Chlamydia tracomatis (634),Helicobacterpylori (635), and group B streptococci (636). Exogenous IL-12 was shown to be protective in mice for infections with Klebsiella pneumonia (637), group A and B streptococci (636, 638). IL-12 was also shown to be effective as an adjuvant in a vaccine against Y. enterocolitica (639). In humans, macrophages from patients with Whipple’s disease, a systemic infection in which the causative bacteria, Tropheryma tohippelii, accumulate within macrophages, have been found to be severely defective

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in IL-12 production in response to various stimuli (640). The production of other cytokmes, with the exception of IFN-y, was within the normal values (640). Because a similar, although less severe, deficiency in IL-12 production was observed in two relatives of the patients, it is possible that a genetic defect in IL-12 production is responsible for susceptibility to Wliipple’s disease (640).

PARASITES C. PROTOZOAN 1. Leishmania Species

Treatment of the susceptible strain BALB/c with IL-12 during the first week following cutaneous infection with Leishmania nujor induces resistance to the infection with a shift from a Th2 to a T h l response (641, 642). The cured animals are resistant to a subsequent challenge (641), but if the IL-12 treatment is delayed more than 1 week, it is ineffective in curing the animals (642). Vaccination of BALB/c with soluble leishmania antigens (383) or the recombinant leishmania LACK antigen (643, 644), together with IL-12, also induces a protective T h l immunization. In addition to IL-12, anti-IL-4 antibodies or DNA immunization with the LACK gene in a bacterial plasmid able to induce IL-12 also generates protective immunity (644,645).Although IL-12 alone cannot cure BALB/c mice when administered 1 week after L. major infection, the combined treatment of BALB/c mice 3 weeks after infection with antimony-based leishmanicidal drugs and IL-12 or IFN-y can effectively cure them, shifting the response from Th2 to T h l (646, 647); the effectiveness of the therapy based on IFN-y is dependent on endogenous IL-12 and its effect is prevented by anti-IL-12 antibodies (647). The switch from a Th2 to a T h l response most likely does not involve a phenotypic change in already differentiated Th2 cells, but the IL-12-induced generation of T h l cells from yet uncommitted T cells or de n o m thymus emigrants (648). The role of endogenous IL-12 early in L. major infection has been an element of controversy. Reiner et al. (649) could not demonstrate IL-12 mRNA until 7 day after infection and proposed that amastigotes, which do not mature in vivo until about a week from infection, and not the infective metacyclic promastigotes, are able to induce IL-12 production. Indeed, proinastigotes have been shown to be able to efficiently suppress IL-12 production induced by various stimuli (159,650),although promastigotes, especially the immature procyclic forms, have been shown to have some ability to induce IL-12 in vitro in human monocytes (159). However, other studies have shown that IL-12 is already produced at 24 hr from infection in the lymph nodes of infected animals (or in peritoneal macrophages, followingip injection of the parasite) (320,651,652).The important

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role of this early IL-12 production was clearly proven by the observation that anti-IL-12 antibodies injected at the time of infection blocked IFNy production and NK cell activation in the popliteal lymph node 2 days after infection (320). The essential role of endogenous IL-12 in resistance to L. major infection has been confirmed by the inability of both IL-12 p40 and p35 genetically deficient mice to resist the infection (652, 653). Mice lacking the expression of CD40L are also unable to produce IL-12 and to resist infection, in part because of lack of IL-12 production at a late time of infection (654, 655). The early production of IL-12 is comparable between resistant (C3H) and susceptible strains (BALB/c), although a delay in IL-12 production was observed in the resistant C57BU6 strain (320). However, whereas T cells from C3H mice rapidly upregulated both the IL-12RP1 and the p2 chain within 1 or 2 days of infection, this upregulation was minimal in BALB/c mice (D. Jones, M. M. Elloso, L. Showe, D. Williams, G. Trinchieri, and P. Scott, unpublished results), possibly due to the downregulatory effect of IL-4 on IL-12 responsiveness (330, 656). In experimental visceral leishmaniasis induced by L. donooani infection, IL-12 treatment both before infection or 2 weeks after challenge improves resistance: in this model, however, unlike in L. major infection in BALB/c mice, the disease is characterized not by a Th2 response, but by an ineffective Thl response (657),which was boosted by IL-12 treatment. Vaccination with heat-killed L. major in BALB/c mice has been used to obtain a cross-reactive Th2 response to L. donovnni: the animals so vaccinated were unable to resist a challenge with L. donooani, but treatment with IL-12 successfully induced antileishmanial activity (658) by inducing a switch from a Th2 to a Thl response. Although these effects of IL-12 are mostly mediated by induction of a Thl response and production of IFN-y in IFN-y gene-disrupted mice, IL-12 still enhances resistance to L. donovani by a mechanism that involves production of TNF-a and activation of iNOS (659). The essential role of IL-12 both early and late in the resistance to L. donooani infection was shown by the ability of antiIL-12 antibodies to exacerbate infection when administered the first week of infection or from the second to the fourth weeks (660). In humans, IL-12 expression was detected in most lesions of individuals with cutaneous leishmaniasis and correlated strongly with the level of IFNy expression (661). The addition of IL-12 to culture of lymphocytes of patients with active visceral leishmaniasis restored their ability to proliferate and produce IFN-y in response to leishmanial antigens (662, 663). A gene from L. braziliensis was cloned and termed LeIF because of its homology with the eukaryotic ribosomal protein eIF4A: LeIF is a potent inducer of

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IL-12 production and possibly one of the important antigens recognized by the host immune response to Leishrnnniu infection (141).

2. Toroplusmu gonclii IL-12 is required for the T-cell-independent induction of IFN-y in SCID mice in response to T. gondii infection (256).The optimal production of IFN-y by NK cells requires not only IL-12, but also TNF-a, IL-1, and B7/CD28 interaction (256, 258, 259, 664). The expression of CD28 on NK cells is probably upregulated by IL-15 (259). IL-10 and TGF-/3 are negative regulators of IL-12 production and responsiveness (263,377,664, 665). IL-10 genetically deficient mice rapidly die of T. gonclii infection, although they control parasite growth more efficiently than wild-type mice (377, 66.5): the reason for death is an uncontrolled production of proinflainmatory cytokines, including IL-12 and IFN-y, and a pathology reminiscent of a systemic inflammatory response syndrome (377). However, the toxic response is in part dependent on T lymphocytes, and IL-10 genetically deficient SCID mice survive T. gondii infection longer than wild-type SCID mice (665). IL-12 is produced within the first few days of toxoplasma infection and its production is unimpaired in IFN-y-deficient mice (164). In these latter animals, neutralization of IL-12 blocks the cytotoxic NK cell response, but does not decrease survival, suggesting that IL-12-induced IFN-y is necessary for the control of parasite growth (164). In normal mice, treatment with either anti-IL-12 or anti-IFN-y antibodies at the time of infection blocks the induction of resistance to T. gondii and all animals die within 2 weeks (377).However, once a chronic infection controlled by a Thl response is established at 4 weeks, treatment with anti-IFN-y induces the death of the animals, but the treatment with anti-IL-12 antibodies is ineffective, indicating that whereas IFN-y is still required for the antiparasite inacrophage activity, the maintenance of the established T h l response is IL-12 independent (377). The requirement for IL-12 in the resistance to T. gondiii infection has been confirmed using IL-12 p40 genetically deficient mice (666). 3. Tnjpnnosomn cruzi T. cmai, a hemoflagellate protozoan parasite that is the causative agent of human Chagas’ disease, is a potent inducer of IL-12 production in macrophages (667, 668). Only live, UV-, or gamma-irradiated trypomastigote forms are able to induce IL-12, but not the heat-killed parasites or lysates, or the epimastigote forms (667, 668). A glycosylphosphatidylinositol-anchored mucin-like glycoprotein isolated from T. cruzi has been shown to be able to initiate in macrophages the synthesis of proinflammatory

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cytokines, including IL-12 (140). Endogenous IL-12 is required for both innate and acquired immunity to T. cmzi infection, and its effect requires the participation of TNF-a and IFN-y (669,670).Administration of recombinant IL-12 enhances the resistance of the mice to T. cmzi (670).Endogenous IL-10 downregulates IL-12 production and limits the resistance to the infection; however, although IL-10-deficient SCID mice survive a T. cruzi infection longer than wild-type SCID mice, IL-10-deficient C57BL/ 6 mice died earlier of infection than wild-type C57BLJ6 mice (293, 669). The IL-10-deficient mice had a lower parasite burden, but much higher serum levels of IL-12, TNF-a, and IFN-y, and mortality was prevented by neutralizing anti-IL-12 antibodies, suggesting that there is a critical requirement for IL-10 to prevent the development of a systemic immune inflammatory response associated with activation of CD4' T cells and overproduction of IL-12 (293). In humans, IL-12 has been shown to potentiate both proliferative response and cytotoxicityin response to T. cmzi antigen stimulation in PBMC from patients with the different forms of Chagas' disease (671, 672). 4. Plasmodium Species

Because of the high worldwide incidence of malaria and the emergence of plasmodium strains resistant to many antimalarial drugs, there is much interest in new methods of prevention that could destroy the plasmodium during the intrahepatic cycle. Intraperitoneal injection of a single dose of 150 ng of IL-12 2 days before challenge of mice with Plasmodium yoelii protected 100% of mice against hepatic malaria via a mechanism that required IFN-y and iNOS (673). Similarly, a single subcutaneous injection of 10 pg/kg of recombinant human IL-12 in seven rhesus monkeys 2 days before challenge with P. cynomolgi sporozoites induced an increase in plasma levels of IFN-y and protected the monkeys against malaria (674). IL-12 treatment is effective not only against the hepatic stage of malaria infection, but it also induces protection against blood-stage P. chubaudi AS with a mechanism that requires IFN-y, TNF-a, and iNOS activation (675). PATHOGENS D. FUNGAL 1. Candida albicans The outcome of systemic challenge of mice with the fungus C. albicans is determined in part by immunological events occurring shortly after infection and leading, in resistant strains of mice challenged with live vaccine strains of the yeast, to a protective T h l response, whereas the mice challenged with a virulent strain have an exacerbative Th2 response (676). IL-12 is readily induced in mice infected with the vaccine strains and its level, rather than the production of IFN-y, correlatedwith induction

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of T h l response (677).Treatment with anti-IL-12 antibodies of mice undergoing a healing infection with a vaccine strain ablates the development of acquired anticandidal resistance and switches the response from T h l to Th2 (678). Surprisingly, however, IL-12 treatment does not improve, but rather worsens the resistance to virulent C. albicans infection in mice and high doses of IL-12 have a toxic effect, possibly by inducing a fungal-type systemic inflammatory syndrome, on mice infected with the vaccine strains (676, 678). The failure of IL-12 to protect mice from C. albicans infection is, at least in part, explained by the particular physiological role played by neutrophils in C. albicans infection (679). Neutrophils have a major role in providing a first line of defense against Candida. In vitro neutrophils in response to C. albicans produced both IL-12 and IL-10: IL-12 was predominantly produced in response to the vaccine strains of C. albicans, whereas IL-10 was produced in response to the virulent strains (680). Similarly, in vivo IL-12 production from neutrophils was found to be associated with healing infection, whereas IL-10 production with progressive infection, indicating that neutrophils have not only an effector role in C. albicans infection, but also an immunomodulatory one, regulating T h l and Th2 differentiation (679, 680). IL-12 treatment of mice with progressive infection increased the production of IL-10 from neutrophils, and it was therefore unable to prevent the exacerbating Th2 response; however, if mice were made neutropenic, IL-12 treatment induced IFN-.)Iproduction and the generation of a T h l response that resulted in protection of the animals against the C. albicans infection (680).

2. Crzjptococcus neoformans High-dose IL-12 treatment, combined or not with the antifungal agent fluconazole,of mice infected intravenouslywith C. n e o f o m n s dramatically reduced the level of infection in the brain and liver, but had no effect on spleens or lungs (681). However, in a model of intratracheal infection, IL-12 induced a strong response against the pulmonary infection and completely prevented the dissemination to the brain (682).The protective effect of IL-12 was associated with an increase in Thl-type cytokines and iNOS in the lungs of infected mice (683) and was almost abolished by neutralization of TNF-a (684). Because C. neoformans is an opportunistic pathogen that causes serious life-threatening disease in both healthy and immunocompromised persons, the strong activity of IL-12 against this fungal pathogen and its cooperativitywith antifungal drugs have an interesting therapeutic potential. 3. Histoplasma capsulaturn H . cap.sulatum is another fungal pathogen that is emerging as a serious opportunistic infection in immunocompromised hosts. In mice, IL-12 has

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been shown to lower the mortality rate of both norinal and SCID mice infected with H . capsulatum through the induction of IFN-.)/ (685-687). Inhibition of endogenous IL-12 with anti-IL-12 antibodies induced accelerated mortality, an effect that is, however, reversed if the animals were treated with anti-IL-4 (685, 687). In reinfection histoplasmosis, treatment with anti-IL-12 antibodies did not alter survival (687). Thus, endogenous IL-12 is necessary for the establishment of a protective T h l response to H . capsulatum and treatment with exogenous IL-12 enhances the resistance to the infection. 4 . Coccidioides immitis

Coccidioidomycosis is a mycotic disease endemic to the southwestern United States and Central and South America that can cause severe and fatal pulmonary and disseminated infections. In mice, the DBM2 strain is resistant to the infection with a prevalent T h l response, whereas BALBk mice are susceptible with a prevalent Th2 response. IL-12 treatment around the time of infection protects BALBk inice by inducing a Th2 to Thl switch, whereas anti-IL-12 treatment prevents the ability of DBA/2 mice to resist the infection, inducing a Th2 response (688).

E. HELMINTHIC PARASITES I. Schistosoma mansoni Injection of S. munsoni eggs intravenously results in the formation of pulmonary granulomas that are initially characterized by a ThO/Thl type of response, but that rapidly switch to the expression of prevalently Th2

type cytokines (689). Anti-IL-12 or anti-IFN-y antibody treatment of the animals enhances the Th2 response and the granuloma formation, whereas treatment with IL-12 prevents the switch to a Th2-type response and decreases the granulomas (689). In IFN-y genetically deficient mice, IL12 treatment exacerbates the Th2-dependent pathology by failing to suppress the production of Th2 cytokines and boosting IgE levels, while enhancing lymphocyte proliferation (409). However, in B-cell-deficient mice, higher levels of IL-12 are produced with maintenance of a T h l response, but, even in the absence of a Th2 response, the size and the number of egg granulomas in the liver of infected animals are unchanged (450). Not only can IL-12 treatment prevent the formation of lung granulomas, but vaccination with eggs in combination with IL-12 commits the mice toward a T h l response, such that they develop only minimal granulomas and lung fibrosis on subsequent egg challenge (689, 690). In a prophylactic vaccine model, IL-12 has been shown to enhance the protective effect of immunization with either a soluble lung-stage larval antigen preparation or irradiated cercariae (691, 692). It is of interest that

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inultiple vaccinations with irradiated cercariae without IL-12 resulted in a Th2-dominant response, whereas mice vaccinated in the presence of IL12, in addition to a strongly polarized Thl response, also showed a significant increase in parasite-specific IgG antibodies that were able to protect naive recipients in transfer experiinents (692).

2. Filarial H e h i n t s The filarial helinint Bnrgia nzalayi induces Th2 response both in mice (693) and in hurnans (694). In mice, IL-12 treatment in vivo or in vitro induces a Thl response to B. inalayi infection and, even if administered after a Th2 response has already been established, profoundly inhibits the production of Th2 cytokines (693). However, the elimination of blood bone microfilariae was not altered by IL-12 treatment (693). 3. Intestinal Nematodes

Resistance and expulsion of intestinal nematodes are primarily mediated by a Th2-type response, and particularly by the production of IL-4 (695). Treatment with IL-12 before or during the infection has been shown to downregulate Th2 responses while promoting Th1 responses and to eliininate or decrease the protective immune response to Nyppostrongylus brasiliensis (167,696), Stroiigyloides stereoralis (697),and Trichuris muris (698). In general, IL-12 administered during the initiation of an iinrnune response to nematodes can, through the induction of IFN-y, change the predominant response froin Th2 to Thl, whereas IL-12 treatment has less effect once the production of Th2 cytokines has become established (167). These effects of IL-12 are consistent with the hypothesis that Th2associated responses protect against and Thl responses exacerbate nematode infections (167, 695).

XVI. Antitumor Effects of 11-12 A. TOXICITY OF SYSTEMIC IL-12 TREATMENT In order to obtain an efficient antituinor effect of IL-12 in v i m , high doses of recombinant IL-12 need to be injected for an extended period of time. The most commonly used protocol in inice involved five daily injections of IL-12, inost usually ip, followed by a 2-day rest: although this protocol was initiated for empirical reasons, it proved necessary to avoid the formation of pulinonary edema, a toxicity observed when mice are treated without interruption even with low concentrations of IL-12 (P. Bouchard, personal communication). The half-life of injected recombinant IL-12 is approximately 3.5 hr in inice (161), 18 hr in rhesus inonkeys (38), and between 5 and 10 hr in hurnans (699). Most mouse strains tolerate

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repeated injections of up to 1 pglmouselday, but some mouse strains, e.g., C3H and A/J, show lethal toxicity at such doses and need to be treated at IL-12 doses 5-10 lower in tumor treatment experiments (456). The primary toxicities observed in normal mice treated with IL-12 were hematological alterations, hepatotoxicity,and skeletal muscle degeneration. Daily administration of IL-12 for 7 days led to severe anemia with red blood cells dropping to about half of the normal values (245, 700). Both lymphopenia and neutropenia, possibly due to emargination of leukocytes onto vascular endothelium and liver, were also observed (700).Splenomegaly was an early and constant finding, largely caused by extramedullar hematopoiesis involving the erythroid, myeloid, and megakaryotic lineages (245, 700). The bone marrow, however, was hypoplastic, with a loss of mature neutrophils and precursor cells of all lineages (243, 244). The anemia in IL-12-treated mice appears too early to be uniquely due to decreased generation of erythrocytes, and it has been hypothesized that increased tissue erythrophagocytosis, as suggested by the marked activation of Kupffer cells in the liver, may play a role (700). The hematological toxicities in IL-12-treated mice are mediated by IFN-y: in IFN-yRdeficient mice, spleen cellularity was less increased, there were fewer infiltrating NK cells, but a strong extramedullary hematopoiesis was still induced, showing that, in the absence of IFN-7, IL-12 promotes hematopoiesis, consistent with its in vitro activities (245). Significant elevation in transaminases and mildly increased liver weights were observed in IL-12-treated mice (701). Intense macrophage infiltrates as well as of NK and CD8' T cells were localized around central veins and terminal portal vessels, associated with occasional necrosis of isolated hepatocytes observed after 4- 10 days of treatment, followed by progression with continued administration of IL-12 to areas of coagulative necrosis with marked elevation of serum transaminases (700, 701). In IFN-yR genetically deficient mice the activation of macrophages was still observed, often associated with an increased number of eosinophils (701). Skeletal muscle toxicity was seen in mice treated with doses of IL-12 of more than 1 p g and consisted in visibly white muscle at necropsy, muscle necrosis and calcification, and elevation of serum muscle enzymes beginning after about 5 days of treatment (700). Normal mice given high doses of IL-12 displayed ascites and pleural effusion, but pulmonary edema was observed only in IFN-yR-deficient mice or in mice treated with uninterrupted continuous treatment for more than 10 days (700, 701). IL-12 damage to the intestinal tract was particularly evident from its ability to sensitize it to radiation: in mice treated with IL-12 doses as low as 40 ng for 3 or 4 days and then given 1200 cGy radiation, the lumen of small intestine was distended with fluid ingest and displayed severe mucosal damage with

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marked shortening of villi or villi fusion and loss of epithelial lining cells (246). This sensitization of the intestinal tract to ionizing radiation, which resulted in the death of' the animals within 4 to 6 days after irradiation, was prevented by neutralization of IFN-y (246). A heinatological toxicity similar to that described in the mouse was also observed in nonhuman primates treated with IL-12 (39, 702); however, although signs of extramedullary hernatopoiesis were evident in these animals, the bone marrow was characterized by hypercellularity with increase of all three lineages rather than by the hypocellularity observed in mice (39). Squirrel monkeys (Sciureus suimiri) receiving daily subcutaneous injections of human rIL-12 in doses ranging from 0.1 to 50 pg/kg/day for 14 days showed dose-related fever, mild to moderate anemia, leukocytosis, hypoproteinemia, hypoalbuininernia, hypophosphatemia, hypocalcemia, generalized lymph node enlargement, splenomegaly, and thymic cortical atrophy (39).Two of the six high-dose animals developed pulmonary edema and ascites; neither hepatoxicity, besides evidence of Kupffer cell hypertrophy and hyperplasia, nor muscle degeneration was observed in IL-12treated squirrel monkeys (39). Phase I clinical trials were initiated in 1994 in cancer patients and identified a maximum tolerated dose of 500 ng/kg with reversible side effects consisting of fever, mild anemia, neutropenia, thrombocytopenia, fatigue, and myalgia. Oral stomatitis of unknown origin and elevation of transaminases were observed at the 1000-ng/kg dose (699). Biological effects included elevations in IFN-7 in the serum that peaked in the first 3 to 4 days, decreasing thereafter despite continuing dosing (699). Phase I1 trials were initiated in 17 patients with daily 500-ng/kg iv injections, but it was interrupted after a few injections because of profound neurastenia requiring admission for 9 patients and associated with two treatmentrelated deaths with multiorgan toxicity, including intestinal bleeding (455, 699). This difference in toxicity between the phase I and the phase I1 trials was attributed to the fact that in phase I a single predose was given and then the daily treatment was initiated after 2 weeks, whereas in the phase I1 trials daily injections were immediately started (455, 699). Indeed, in mice, it was shown that a single predose 1 week before initiation of daily treatment effectively protected the animals from IL-12 toxicity, allowing treatment with higher doses (J. Leonard, personal communication; 455, 456).

B. ANTITUMOREFFECTS OF IL-12 I N EXPEHIMENTAL ANIMALS IL-12 was shown to have a powerful antitumor and antimetastatic activity against many murine tumors (703-707). With several tumors, IL-12 sys-

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temic treatment was effective even when initiated up to 2 to 7 weeks after tumor transfer (703, 705). Often, IL-12 induced only a temporary tumor regression and tumor growth resumed when the treatment was interrupted; in some cases, e.g., following peritumord IL-2 inoculation of the RenCd tumor or systemic treatment of the CSAlM fibrosarcoma, complete regression was observed, and the animals that rejected the tumor were specifically resistant to challenge with the same tumor type (703, 705). The depletion of CD8' T cells or both CD8' and CD4' T cells was most often required to suppress the antitumor effect of IL-12, whereas depletion of CD4' T cells or NK cells had only a marginal effect (703, 704). In all tumor models analyzed, the antitumor effect of IL-12 was at least in part mediated by IFN-y (704,705,708,709).However, IFN-y production is clearly not the only antitumor mechanism mediated by IL-12 because the antitumor effect of IL-12 is reduced in nude mice compared to euthymic mice, but a -10fold higher level of IFN-y was induced in nude mice compared to euthymic mice (708). Endogenous IL-12 is induced in response to tumor cell transplantation (710, 711), and it was demonstrated to be necessary in vivo for rejection of spontaneously regressing P815 tumor variants (711). IL-12 treatment also delayed tumor appearance and decreased tumor incidence induced by the carcinogen 3-methylcholanthrene (712); interestingly, in contrast to the characteristically round, hard, well-circumscribed, and protruding tumors normally induced by the carcinogen, most tumors induced in IL-12-treated mice were atypical with flat, soft, and invasive characteristics (712). The ability of IL-12-induced IFN-y to affect tumor growth may be due to direct effects on the tumor or indirect ones on the host cells. Many of the tumors affected by IL-12 in vim are sensitivein vitro to the antiproliferative effect of IFN-7, suggesting a direct effect on the tumor cells (708). In addition, in tumor cells and/or in host cells IFN-y induces activation of iNOS and production of nitric oxide, which inhibits tumor growth (713715). Another mechanism by which IL-12 induced-IFN-y can prevent tumor growth is its ability to inhibit angiogenesis in vivo (716, 717). The antiangiogenic effect of IL-12 is not direct, but mediated, through IFNy, by the production of the chemokine interferon-inducible protein- 10 ( IP-lo), which, in addition to being a chemoattractant for lymphocytes, has a powerful inhibitory effect on proliferation and hfferentiation of endothelial cells (718). Because IL-12 can effectively inhibit angiogenesis induced in vivo by basic fibroblast growth factor mixed in a gel pellet (718, 719), the antiangiogenic effect of IL-12 on tumors couId be attributed to induction of IP-10 from host cells; however, production of IP-10 was also observed in tumor tissues from IL-12 treated mice (709). By rendering tumor cells unresponsive to IFN-y by overexpression of a dominant nega-

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tive truncated IFN-yR construct (720),it was demonstrated that the ability of tumors cells to respond to IFN-y was necessary for both the antitumor effect of IL-12 and its ability to inhibit angiogenesis induced by the tumor cells (C. Couglilan, W. Lee, and G. Trinchieri, unpublished results). Thus, the ability of IFN-y to induce the production of antiangiogenic factors in the tumor cells themselves appears to play a major role in the antitumor effect of IL-12. In order to itnprove the local delivery of IL-12 to the tumor sites, several viral vectors have been prepared. Because IL-12, unlike other cytokines, is encoded by two separated genes, bicistronic vectors (721) or vectors encoding fusion proteins (61, 62) have been utilized. Different types of viral vectors, including retroviral vectors (721-723), adenovirus vectors (724, 725), and vaccinia virus vectors (726, 727), have been prepared for expression of IL-12, and overall promising antitumor activity has been obtained by injecting them peri- or intratuinorally. Interestingly, the use of adenoviral vectors producing the IL-12 p40 homodimers inhibited the antitumor effect of injection of a bicistronic vector encoding IL-12 heterodiiners in a inurine bladder carcinoma model (723). In addition to the use of viral vectors, injection of cDNA encodmg IL-12 at distant sites from the tumor injection site or gene gun-mediated transfection with IL-12 genes on the skin overlaying intradermal tumors were shown to result in regression of established primary and metastatic tumors (728, 729). IL-12-transfected fibroblasts were also used to deliver IL-12 locally in the proximity of tumors with good antitumor activity against different tumors (730, 731). The regression of the murine BL-6 melanoma was IL12 dose dependent, with better regression obtained by injecting the highest proportion of IL-12-producing fibroblasts, whereas the most efficient antitumor immunity to subsequent challenges was observed at intermediate doses of IL-12 (730). The first use of transfected tumor cells producing IL-12 was reported for the C-26 murine colon carcinoma cells (722); these transfected cells produced only 30-80 pg/inl of IL-12 and were able to induce tumor rejection, which was mediated by CD8+T cells, only when inhibitory CD4' T cells were eliminated by antibody treatment (722).However, C-26 tumor cells engineered to express much higher levels of IL-12 induced a significant infiltration of CD8' T cells and NK cells and induced an efficient tumor rejection, even when CD4' cells were not depleted (732). Effective eradication of established murine tumors and induction of an efficient antitumor immunity were obtained using other IL-12-producing tumor cells of different origin (721, 733). The toxicity of IL-12 treatment alone makes particularly attractive the possibility to combine its use with other antitumor agents with which IL-12

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may synergize for antitumor activity without increased toxicity. Combined treatment of Lewis lung tumors with IL-12 and fractionated radiation therapy was shown to result in a synergistic antitumor response (734). Administration of IL-12 with pulse IL-2 was reported to induce rapid and complete eradication of murine renal cell carcinoma with a greater effect than with each cytokine separately and with a tolerable toxicity (735). Furthermore, systemic or local IL-2 was shown to enhance the antitumor effect of IL-12 gene therapy (736) and IL-12 to potentiate the curative effect of a vaccine based on IL-2-transduced tumor cells (737). A strong synergistic antitumor effect against poorly immunogenic tumors and induction of antitumor immunity was observed by utilizing systemic IL-12 therapy and induction of expression on the tumor cells by gene therapy of the costimulatory molecule B7.1 (738, 739). A strong synergistic antitumor effect on contralateral wild-type tumor growth was observed when tumor cells transfected with IL-12 and with the gene encoding IGIFAL-18 were coinjected in mice (C. Coughlan, W. Lee, and G. Trinchieri, unpublished results). The combined use of IL-12 and IL-18-transfected tumor cells induced a potent antiangiogenic effect that was mediated by IFN--y and required IFN-7 responsiveness in the tumor cells. An effective therapeutical vaccination against Meth-A tumor, able to induce regression of established tumors, was obtained by immunizing mice with a mutated p53 peptide and IL-12 in the presence of the QS2l adjuvant (335);interestingly, a good CTL response and antitumor effect were observed only with low doses of IL-12, whereas the high doses normally utilized for obtaining an in vivo antitumor effect were immunosuppressive (335).IL-12 has also been shown to enhance the specific immunotherapy of cancer mediated by dendritic cells pulsed with a mutated p53 peptide (336)or a class I-restricted tumor peptide derived from the P815AB antigen of P815 tumor cells (446). Interestingly, injected dendritic cells pulsed with the P815 peptide induced T-cell anergy in v i m , but IL-12 could prevent or revert the anergic state (447). Because in vitro treatment of the dendritic cells with IL-12 rendered them effective in inducing an antitumor response, these data raise the possibility of a direct effect of IL-12 on dendritic cells (446, 447). Tumor cells transfected with IL-2 or IL-12 were compared for their ability to induce vaccination against established tumors: although both cell types induced similar CTL generation, the IL-12-transfected ones were more efficient in inducing tumor regression because of their ability to induce complement-hng antibodies and systemic activation of T h l rather than Th2 cells (740).

c. ANTITUMOR EFFECTSOF IL-12 O N HUMAN TUMORS The majority of data on the antitumor effects of IL-12 in humans are obviously based on in vitro experiments. After the original report by Lieber-

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inan et al. (741)that IL-12 increases NK and antibody-dependent cytotoxicity of human PBL against tumor cells, many papers have reported an activity of IL-12 on the NK or CTL activity of cancer patients PBL or tumorinfiltrating lymphocytes against autologous or allogeneic tumors (742-750). IL-12 was shown in vivo to enhance the antitumor activity of human NK cells or PBL coinjectedwith human tumor cells into SCID mice (751,752). Malignant CD4' T cells from patients with Sezary syndrome, a forin of cutaneous T-cell lymphomas that produce prevalently Th2-type cytokines and PBMC from patients, were reported to produce lower levels of IL12 than those from healthy donors (753, 754). IL-12 treatment of the malignant lymphocytes in vitro resulted in a significant decrease in the production of IL-4 (753).Because of the poor prognosis and high degree of fatality associated with Sezary syndrome, on the basis of these results, trials with IL-12 have been initiated (A. Rook, personal communication). It is too early to evaluate the results of the clinical trials of IL-12 in cancer patients; however, a stable partial response in a patient with renal cell carcinoma, a transient complete remission in a melanoma patient, and four patients with stable disease have been reported in the first phase I trial (699). XVII. Concluding Remarks

Although IL-12 has been discovered only relatively recently, a remarkable level of knowledge has been reached for this cytokine, and many possible therapeutic applications are being tested or have been proposed. The biological interest for this cytokine stems from its dual function as a proinflammatory cytokme and as an iminunomodulatory molecule. Being produced at the early times of inflammation in response to infection or other stimuli, it contributes via the production of IFN-y and other cytokines to the inflammatory process itself and particularly to the activation of macrophages, while setting the stage for the ensuing antigen-specific adaptive immune response by directing CD4' and CD8' T cells to differentiate into Thl or TC1 effector and memory cells. Thus, IL-12 represents a bridge between innate resistance and adaptive immunity, with a central role in the regulation of the response to infection and tumor cells, as well as in autoimmunity and allergy. Because of these activities, its clinical use has been proposed in infections, both as an immunopotentiating agent or as an adjuvant in vaccination, against tumors, and for the prevention of severe allergic syndromes. Conversely, the use of IL-12 antagonists has been proposed in autoimmunity and in systemic inflammatory responses. The early reports of toxicities in the initial clinical trials, although not unexpected and directly mediated by the physiological functions of this molecule, have dampened some of the early enthusiasm and forced a more

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thoughtful approach to its planned use. Undoubtedly, the study of IL-12 has taught us much about the physiology of innate and adaptive immunity: whether we will be able to harness its potent biological activities for efficient therapeutical applications is now the challenge before us.

ACKNOWLEDGMENTS The author thanks Drs. Ellen PurC, Louise Showe, and Jihed Chehimi for critical review of the manuscript, Mrs. Marion Sacks for typing, and Ms. Marina Hoffman for editing. The author was supported in part by NIH Grants CA 10815,CA 20833, CA 32898, and A1 34412.

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614. Munk, M. E., Mayer, P., Anding P., Feldman, K., and Kaufmann, S. H. E. (1996). Increased nnmbers of interleitkin-12-producing cells in human tuberculosis. Infect. Zntmun. 64, 1078-1080. 615. Sieling, P. A,, Wang, X. H., Gately, M. K., Oliveros, J. L., McHugh, T., Barnes, P. F., Wolf, S. F., Golkar, L., Yaniamura, M., and Yo@, Y. (1994). IL-12 regulates T helper type 1cytokine responses in human infectious disease.]. Immnnol. 153,36393647. 616. de Jong, R., Janson, A. A. M., Faller, W. R., Nzafs, B., and Ottenhoff, T. H. M. (1997). IL-2 and IL-12 act in synergy to overcome antigen-specific T cell unresponsiveness in mycobactend disease. J. Irnmund. 159, 786-793. 617. Saunders, B. M., Zhan, Y., and Cheers, C. (1995). Endogenous interleukin-12 is involved in resistance of mice of Mycohacterizim nvium complex infection. Infect. Immnun. 63, 4011-4015. 618. Kobayashi, K., Kasama, T.. Yamazaki, J., Hosaka, M . , Katsura, T., Mochizuki, T., Soejima, K., and Nakamura, R. M. (1995). Protection of mice from Mycobacterium nvium infection by recombinant interleukin-12. Antimicro. Agents Chernother. 39, 1369-1371. 619. Kobayashi, K., Yamazaki, J., Kasama, T., Katsura, T., Kasahara, K., Wolf, S. F., and Shimainura, T. (1996). Interleukin (1L)-12 deficiency in susceptible mice infected with Mycobacterium auium and amelioration of established infection by IL-12 replacement therapy. 1.Infect. Dis. 174, ,564-573. 620. Frucht, D. M., and Holland, S. M. (1996). Defective inonocyte costiinulation for IFN-y production in familial disseminated Mycobncteriicrn auiiim complex infection: Abnorinal IL-12 regulation. J . Inzrnunol. 157, 411-416. 621. Chong, C., Bost, K. L., and Clements, J. D. (1996). Differential production of interleukin-12 mRNA by rnurine macrophages in response to viable or killed Salmonella spp. Infect. Immun. 64, 1154-1160. 622. Bost, K. L., and Clements, J. D. (1997). Iiitracellular Salmonella dublin induces substantial secretion of the 40-kilodaton subunit of interleukin-12 (IL-12) but minimal scretion of IL-12 as a 70-kilodalton protein in murine macrophages. Infect. Irnmun. 65, 3186-3192. 623. Bost, K. L., and Clements, J. D. (1995). In vivo induction of interleukin-12 mRNA expression after oral immunization with Salmonella dublin or the B subunit of Escherichia coli heat-labile enterotoxin. hfect. bnniun. 63, 1076-1083. 624. Kincy-Cain, T., Clements, J. D., and Bost, K. L. (1996). Endogenous and exogenous interleukin-12 augment the protective immune response in mice orally challenged with Sabnunelln dsiblirt. Infect. lmniun. 64, 1437-1440. 625. Mastroeni, P., Harrison, J. A., Chabalgoity, J. A,, and Hormaeche, C. E. (1996). Effect of interleukin 12 neutralization on host resistance and gamma interferon production in mouse typhoid. Infect. Immnuti. 64, 189-196. 626. Anguita, J., Persing, D. H., Rinch, M., Barthold, S. W., and Fikrig, E. (1996).Effect of anti-interleukin 12 treatment of murine lyme borreliosis. J. Clin. Invest. 97,1028-1034. 627. Cantorna, M. T., and Hayes, C. E. (1996). Vitamin A deficiency exacerbates murine Lyme arthritis. J . Infect. Dis. 174, 747-751. 628. Filgueira, L., NestlC, F. O., Rittig, M., Joller, H. I., and Groscurth, P. (1996). Human dendritic cells phagocytose and process Borrelia hzirgrlorferi. J. Onmunol. 157,29983005. 629. Zhan, Y., and Cheers, C. (1995). Endogenous interleukin-12 is involved in resistance to Brucella abortus infection. Infect. Immun. 63, 1387-1390.

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630. Zhaii, Y., Liu, Z., and Cheers, C. (1996).Tuinor necrosis factor alpha and interleukin12 contribute to resistaiice to the intracelhilar bacteiium Brucella nhortu.~by different mechanisms. Iifecf. I r i i i r ~ t t r i ,64, 2782-2786. 631. Zaitseva, M., Golding, H., Manischewitz, J., Wehb, D., and Golding, B. (1996).Bmcellri C L I I O ~ ~ T as ~ S a potential vaccine candidate: Induction of interleukin-12 secretion and enhanced B7.1 and B7.2 and intercellular adhesion inolecule 1 surface expression in ehitriatetl hiinian monocytes stinialated by heat-inactivated B. nhortu.u. Iilfect. Ziniituri. 64,3109-3117. 632. Malion, R. P., Ryan, M. S., Griffin, F., and Mills, K. H. (1996). Interleukin-12 is produced by tnacrophages in response to live or killed Bordetelln pertussis and enhances the efficacy of an acellular peitu vaccine by promoting induction of Thl cells. Infect. Itiitnun. 64, 5295-5301. 633. Bohn, E., and Autenrieth, 1. B. (1996). IL-12 is essential for resistance against Yesinin enterocoliticn by triggering IFN-y production in NK cells and CD4+T ce1ls.J. Itmnunol. 156, 1458-1468. 634. Perry, L. L., Feilzer, K., and Chldwell, H. D. (1997).Inllnunitj to Chlaniydiin trtlclzotiintis is inediated by T helper 1 cells through IFN-gannna -dependent and -independent pathways. /, I I I L ~ I L Z L158, ~ W ~ .3344-3352. 63.5. Karttunen, R. A,, Karttuiien. T. J., Yousfi, M. M., El-Zimaity. H. M., Graham, D. Y., and El-Zaatari, F. A. (1997). Expression of inRNA for- interferon-gamma, interleukin10. and interleulan-12 (p40) in normal gastric inucosa and in inucosa infected with Helicobacter pyliiri. Scanrl. J. Gn.striieiitero/. 32, 22-27. 636. Mancuso, G., Cusumano, V., Genovese, F., Gambuzza, M., Beninati, C., and Teti, C . (1997). Role of interleuhi1 12 in experiinental iieonatal sepsis caused by group B streptococci. Infect. I ~ ~ L I I L W65, I . 3731-3735. 637. Creenberger, M. J., Kunkel, S. L., Strieter, R. M., Lukacs, N. W., Brainson, J., Caddie, J., Graham, F. L., Hitt, M., Danforth, J. M., and Standiford, T. J. (1996). IL-12 gene therapy protects mice in lethal Klebsielln pneumonia. J. Iiniiiutiol. 157, 3006-3012. 638. Metzger, D. W., Raeder, R., Van Cleave, V. II., and Royle, M. D. P. (1995). Protection of mice from group A streptococcal skin infection by interleukin-12. 1. Itfect. Dis. 171, 1643-1645. 639. Noll, A., and Autenrieth, I. B. (1996). Iininiinity against Yersinia enterocolitiua by vaccination with Yersinia HSP60 iminunostimulating coinplexes or Yersinia HSPGO plus interleukin-12. Iifect. Z w i ~ t m64, 2955-2961. 640. Marth, T., Neurath. M., Cuccherini, B. A,, and Strolier, W. (1997).Defects ofnionocyte interleulaii 12 production and humoral immunity in \VIiipple’s disease. Grrstroetiterology 113,442-448. 641. Ijeinzel, F. P., Schoenhaut, D. S., Rerko, R. M., Rosser, L. E., and Gately, M. K. (1993). Reconihiiiaiit interleukin 12 cures mice infected with Leishmania major. /. Exp. Me(!. 177, 1,505-1509. 642. Sypek, J. P., Chung, C. L., Major S. E. H., Subramanyain. J. M., Goldlnan, S. J., Sieburth, D. S., Wolf, S. F., and Schaub, R. G. (1993). Resolution of cutaneous leishmaniasis: Interleukin-12 initiates a protective T helper type 1 inmune response. J . Exp. Med. 177, 179771802, 643. Mougneaii, E., Altare, F., Wakil, A. E., Zheng. S., Chppola, T., Wang. Z.. Wddiiiann, R., Locksley, R. M., and Glaichenhaus. N. (199s). Expression cloning of a protective Leishrimnia antigen. Scierice 268, 563-566. 644. N’akil, A. E., Wang, Z. E., and Locksley, R. M. (1996). Leislziiu~niaiiwjor: Targeting IL-4 in successfulimmunomodulation ofmiirine infection. Exp. Prirmital. 84,214-222.

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645. Gumnathm, S., Sacks, D. L., Brown, D. R., Reiner, S. L., Charest, H., Glaichenhaus, N., and Seder, R. A. (1997). Vaccination with DNA encoding the irninunodominant LACK parasite antigen confers protective immunity to mice infected with Leishnlania major. J. Exp. Med. 186, 1137-1147. 646. Nabors, G. S., Afonso, L. C., Farrell, J. P., and Scott, P. (1995). Switch from a type 2 to a type 1T helper cell response and cure of established Leishinunia major infection in mice is induced by combined therapy with interleukin 12 and Pentostam. Proc. Natt!. Acad. Sci. USA 92, 3142-3146. 647. Li, J., Suttenvala, S., and Farrell, J. P. (1997).Successful therapy ofchronic, nonhealing murine cutaneous leishmaniasis with sodium stibogluconate and gamma interferon depends on continued interleukin-12 production. Infect. Inmiun. 65, 3225-3230. 648. Mocci, S., and Coffman, R. L. (1997). The mechanism of in uitro T helper cell type 1 to T helper cell type 2 switching in highly polarized Leishmania major-specific T cell populations. J. Immunol. 158, 1559-1564. 649. Reiner, L. S., Zheng, S., Wang, Z., Stowring,L., and Locksley,R. M. (1994).Leishmania promastigotes evade interleukin 12 (IL-12) induction by macrophages and stimulate a broad range of cytokines from CD4+ T cells during initiation of infection. J. Exp. Med. 179,447-456. 650. Carrera, L., Gazzinelli, R. T., Badolato, R., Hieny, S., Muller, W., Kuhn, R., and Sacks, D. L. (1996). Leishmania promastigotes selectively inhibit interleukin 12 induction in macrophages from susceptible and resistant mice. J . Exp. Med. 183, 515-526. 651. Vieira, L. Q., Hondowicz, B. D., Afonso, L. C. C., Wysocka, M., Trinchieri, G., and Scott, P. (1994). Infection with Leishinunia mujor induces interleukin-12 production in vivo. Iinmunol. Lett. 40, 157-161. 652. Mattner, F., Alber, G., Magram, J., and Kopf, M. (1997). The role of IL-12 and IL4 in Leishmania major infection. Chem. Immunol. 68, 86-109. 653. Mattner, F., Magram, J., Ferrante, J., Launois, P., Di Padova, K., Behin, R., Gately, M. K., Louis, J. A,, and Alber, G. (1996). Genetically resistant mice lacldiig interleukin~ mount a polarized Th2 cell 12 are susceptible to infection with Leishmania V X Z ~ C Jand response. Eur. J. Immunol. 26, 1553-1559. 654. Campbell, K. A., Ovendale, P. J., Kennedy, M. K., Fanslow, W. C., Reed, S. G., and Maliszewski, C. R. (1996). CD40 ligand is required for protective cell-mediated immunity to Leishmania nmjor. Iminunity 4, 283-289. 655. Kamanaka, M., Yu, P., Yasui, T., Yoshida, K., Kawabe, T., Horii, T., Kishimoto, T., and Kikutani, H. (1996). Protective role of CD40 in Leishmuiiia major infection at two distinct phases of cell-mediated immunity. Immunity 4, 275-281. 656. Launois, P., Swihart, K. G., MiIon, G., and Louis, J. A. (1997). Early production of IL-4 in susceptible mice infected with Leishmania major rapidly induces IL-12 unresponsiveness. J. Immunol. 158, 3317-3324. 657. Murray, H. W., and Hariprashad, J. (1995). Interleukin 12 is effective treatment for an established systemic intracellular infection: Experimental visceral leishmaniasis. J. Exp. Med. 181,387-391. 658. Murray, H. W., Hariprashad, J., and Coffman, R. L. (1997). Behavior of visceral Leishmania donouani in an experimentally induced T helper cell 2 (Th2)-associated response model. J. Exp. Med. 185, 867-874. 659. Taylor, A. P., and Murray, H. W. (1997). Intracellular antimicrobial activity in the absence of interferon-y: Effect of interleukin-12 experimental visceral leishrnaniasis in interferon-? gene-disrupted mice. J. Exp. Med. 185, 1231-1239. 660. Murray, H. W. (1997). Endogenous interleukin-12 regulates required resistance in experimental visceral leishmaniasis. J. Infect. Dis. 175, 1477-1479.

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661. Melby, P. C., Andrade-Narvaez, F., Darnell, B. I., and Valencia-Pacheco, G. (1996). In situ expression of interleukin-10 and interleukin-12 in active human cutaneous leishmaniasis. FEMS Iinmunol. Mecl. Microbial. 15, 101-107. 662. Bacellar, O., Brodskyn, C., Gerreiro, J., Barral-Netto, M., Costa, C. H., Coffman, R. L., Johnson, W. D., and Carvalho, E. M. (1996). Interleukin-12 restores interferongamma production and cytotoxic responses in visceral leishmaniasis. J. Iifect. Dis. 173, 1515-1518. 663. Ghalib, H. W., Whittle, J. A,, Kubin, M., Hashim, F. A,, El-Hassai, A. M., Grabstein, K. H., Trinchieri, G., and Reed, S. G. (1995). IL-12 enhances Thl-type responses in human Leishrnanio donouani infections. f. lnm2~moE.54, 4623-4629. 664. Hunter, C. A,, Subauste, C. S., van Cleave, V. H., and Reinington, J. S. (1994). Production of gamma interferon by natural killer cells from Toxoplusnln gondii-infected SCID mice: Regulation by interleukin-10, interleukin-12, and tumor necrosis factor alpha. Infecf. Iinnzun. 62, 2818-2824. 665. Neyer, L. E., Grunig, G., Fort, M., Remington, J. S., Rennick, D., and Hunter, C. A. (1997).Role of interleukin-10 in regulation ofT-cell-dependent andT-cell-independent mechanisms of resistance to Toxoplusina gondii. Irfect. lminun. 65, 1675-1682. 666. Scharton-Kersten, T. M., Yap, G., Magram, J., and Sher, A. (1997). Inducible nitric oxide is essential for host control of persistent but not acute infection with the intracellular pathogen Toxoplusmn gondii, J . Exp. Med. 185, 1261-1273. 667. Aliberti, J. C., Cardoso, M. A,, Martins, G. A,, Gazzinelli, R. T., Vieira, L. Q., and Silva, J. S. (1996). Interleukin-12 inediates resistance to Typanosomu cruzi in mice and is produced by inurine macrophages in response to live trypomastigotes. Infect. I I ~ W L U64, ~ . 1961-1967. 668. Frosch, S., Krans, S., and Fleischer, B. (1996). Trr~pnnosomacruzi is a potent inducer of interleukin-12 production in macrophages. Med. Microbiol. Imniunol. 185,189-193. 669. Abraliamsohn, I. A,, and Coffinan, R. L. (1996). Tnjpanosoia cruzi: IL-10, TNF, IFN-gamma, and IL-12 regulate innate and acquired immunity to infection. Exp. Parasitol. 84, 231-234. 670. Hunter, C. A,, Slifer, T., and Araujo, F. (1996). Interleuldn-12-mediated resistance to Trypanosonm cmzi is dependent on tumor necrosis factor alpha and gamma interferon. fn$e&. fmmun. 64, 2381-2386. 671. Brodskyn, C. I., Barral, A,, Bulhoes, M. A., Souto, T., Machado, W. C., and BarralNetto, M. (1996). Cytotoxicity in patients with different clinical forms of Chagas’ disease. Clin. Exp. Inimunol. 105, 450-455. 672. de Barros-Muon, S., Guariento, M. E., and Abrahamsolin, I. (1997). IL-12 enhances proliferation of peripheral blood mononuclear cells from Chagas’ disease patients to Typanosoinn cruzi antigen. Imrrrunol. Lett. 57, 39-45. 673. Sedegah, M., Finkelman, F., and Hoffman, S. L. (1994). Interleukin 12 induction of interferon gamma-dependent protection against malaria. Proc. Natl. Acad. Sci. USA 91, 10700-10702. 674. Hoffman, S. L., Crutcher. J. M., Pun, S. K., Ansari, A. A., Villinger. F., Franke, E. D., Singh, P. P., Finkelman, F., Gately, M. K., Dutta, G. P., and Sedegah, M. (1997).Sterile protection of monkeys against malaria after administration of interleukin12. Nature Med. 3, 80-83. 675 Stevenson, M. M., Tam, M. F., Wolf, S. F., and Sher, A. (1995). IL-12-induced protection against blood-stage Plnsmodiuin chabaudi AS requires IFN-y and TNF-a and occurs via a nitric oxide-dependent mechanism. J . Imniunol. 155, 2545-2556. 676. Roniani, L., Bistoni, F., and Puccetti, P. (1997). Initiation of T-helper cell immunity to Candirla albicans by IL-12: The role of neutrophils. Chenr. Imniunol. 68,110-135.

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677. Romani, L., Mencacci, A., Tonnetti, L., Spaccapelo, R., Cenci, E., Wolf, S., Puccetti, P., and Bistoni, F. (1994). Interleukin-12 but not interferon-y production correlates with induction of T helper type-1 phenotype in murine candidiasis. Eur. J. Irnnwnol. 24, 909-915. 678. Romani, L., Mencacci, A,, Tonnetti, L., Spaccapelo, R., Cenci, E., Puccetti, P., Wolf, S. F., and Bistoni, F. (1994). Interieukin-12 is both required and prognostic in vivo for T helper type 1 differentiation in murine candidiasis.]. h n u n o l . 153,5167-5157. 679. Romani, L., Mencacci, A,, Cenci, E., Del Sero, G., Bistoni, F., and Puccetti, P. (1997). An immunoregulatory role for neutrophils in CD4+ T helper subset selection in mice with canddiasis. ]. Imrriunol. 158,2356-2362. 680. Romani, L., Meneacci, A,, Cenci, E., SpaccapeIo, R., Del Sero, G., Nicoletti, I., Trinchieri, G., Bistoni, F., and Puccetti, P. (1997). Neutrophil production of IL-12 and IL-10 in candidiasisand efficacy of IL-12 therapy in neutropenic mice.]. Ininwnol. 158, 5349-5356. 681. Clemons, K. V., Brummer, E., and Stevens, D. A. (1994). Cytokine treatment of central nervous system infection: Efficacy of interleukin-12 alone and synergy with conventional antifungal therapy in experimental cryptococcosis. Antimicrob. Agents. Chevnother. 38, 460-464. 682. Kawakami, K., Tohyama, M., Xie, Q., and Saito, A. (1996). IL-12 protects mice against pulmonary and disseminated infection caused by Cnjptococcus neofonnans. C h i . Exp. Inzmunol. 104, 208-214. 683. Kawakami, K., Tohyama, M., Qifeng, X., and Saito, A. (1997). Expression of cytokines and inducible nitric oxide synthase mRNA in the lungs of mice infected with Cyptococcus neofonnans: Effects of interleukin-12. Infect. Imrnun. 65, 1307-1312. 684. Kawakami, K., Qifeng, T., Tohyama, M., Qureshi, M. H., and Saito,A. (1996). Contribution of tumor necrosis factor-alpha (TNF-alpha) in host defence mechanism against CnjptococnLs neoformans. Clin. Exp. Imniunol. 106, 468-474. 685. Zhou, P., Sieve, M. C., Bennett, J,, Kwon-Chung, K. J., Tewari, R. P., Gazzinelli, R. T., Sher, A,, and Seder, R. A. (1995). IL-12 prevents mortality in mice infected with Histoplama capszllatum through induction of IFN-y.]. Inununol. 155,785-795. 686. Zhou, P., Sieve, M. C., Tewari, R. P., and Seder, R. A. (1997).Interleukin-12 modulates the protective immune response in SCID mice infected with Histoplasnw capsulatum. Infect. Immun. 65, 936-942. 687. Allendoerfer, R., Biovin, G. P., and Deepe, G. S., Jr. (1997). Modulation of immune responses in murine pulmonary histoplasmosis. 1.Infect. Dis. 175, 905-914. 688. Magee, D. M., and Cox, R. A. (1996). Interleukin-12 regulation of host defenses against Coccirlivides iwintitis. Infect. bnniun. 64, 3609-3613. 689. Wynn, T. A., Eltoum, I., Oswald, 1. P., Cheever, A. W., and Sher, A. (1994). Endogenous interleukin 12 (IL-12) regulates granuloma formation induced by eggs of Schistosonui inansoni and exogenous IL-12 both inhibits and prophylactically immunizes against Exp. Med. 179, 1551-1561. egg pathology. I. 690. Wynn, T. A., Cheever, A. W., Jankovic, D., Poindexter, R. W., Caspar, P., Lewis, F. A., and Sher, A. (1995). An IL-12-based vaccination method for preventing fibrosis induced by schistosome infection. Nature 376, 594-596. 691. Mountford, A. P., Anderson, S., and Wilson, R. A. (1996). Induction of Thl cellmediated protective immunity to Schistosomnu mansoni by co-administration of larval antigens and IL-12 as an adjuvant. J. Imnzunol. 156, 4739-4745. 692. Wynn, T. A., Reynolds, A., James, S., Cheever, A. W., Caspar, P., Hieny, S., Jankovic, D., Strand, M., and Sher, A. (1996). IL-12 enhances vaccine-induced immunity to

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702.

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scliistosonies by augmenting both liuniord and cell-mediated imniune responses against the parasite. J . Z t t i t n i i t d . 157, 4068-4078. Pearlman, E., Heinzel, F. P., Hazlett, F. E., Jr., and Kazura, J. W. (1995). 1L-12 modillation of T helper responses to the filarial helmintli, Bnigici itwlnyi. J . Z t t m i i n d 154,4658-4664. Mahanty, S., Ravicliandran, M., Ranian, U., Jayarainan, K.. Kuniaraswaini. V.. and Nutnian, T. B. (1997). Regulation of parasite antigen-driven iinniune responses by interleukin-10 (IL-10)and IL-12 in lyrnphatic filariasis.Irfect. I n ~ t m i n65, . 1742-1747. Locksley, R. M. (1994). Th2 cells: Help for helminths. j . Exp, Mcd. 179, 1405-1407. Urban, J. F., Jr., Fayer, R.. Sullivan, C., Goldhill, J., Shea-Donohue, T., Madden, K., Morris, S. C., Katona, I., Cause, W., Ruff, M., Mansfield, L. S., and Finkelinan, F. 11. (1996). Local T H l and TH2 responses to parasitic infection in the intestine: Regulation by IFN-gainina and 11,-4. Vet. I t t i t r u m d . Zmttiuiiopnthol. 54, 337-344. Rotinan, H. L., Sclinyder-Candrian, S., Scott, P.. Nolan. T. J., Schad, G. A,, and Abrahani, 13.(1997).IL-12 eliminates the Th-2 dependent protective inunune response of inice to larval Strongyloicks .stereoralis. Parosite Irrmunol. 19, 29-39. Bancroft, A. J., Else. K. J., Sypek, J. P., and Grencis, R. K. (1997). Interleukin-12 proniotes a chronic iutestind nematode infection. Eur. J. Znurruiiol. 27, 866-870. Lotze, M. T., Zitvogel, L., Cainpht.11, R., Robbiiis, P. D., Elder, E., Haluszczak, C., Martin, D., Wliiteside, T. L., Storkus, W. J., and Tahara, H. (1997). Cytokine gene therapy of caiicer using interleukin-12: Murine and clinical trials. Ann. N.Y. Acud. Sci. 795,440-454. Brunda, M. J., and Gately, M. K. (1995). Interleukin-12: Potential role in cancer therapy. Import. Arlo. Oncol. 1, 3-18. Car, B. D., Eng, V. M., Schnyder, B., LeHir, M., Shakhov, A. N., \Voerly, G., Huang, S., Agriet, M., Anderson, T. D.. and Ryffel, B. (1995). Role of interferon-y in IL-12induced pathology in mice. Am. /. Pcithol. 147, 1693- 1707. Bree, A. G . .Schlerman, F. J., Kaviani, M. D., Hastings, R. C., Hitz, S. L.. a i d Goltlman, S. 1. (1994). Multiple effects on peripheral hematology following administration of recombinant human interleukin 12 to nonhuman primates. Biochett~Biophys. Kes. C ( J I ~ I ~204, ~ ~ U1150-1 I I . 157. Brnnda, M. J,, Luistro, L., Warner, R. R., Wright. II0 kb

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FIG.28. Cluster and translocon arrangements present in teleost and chondrostian fishes. The catfish gene arrangements are shown for the teleostei and sturgeon for chondrostei,

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manners. The G isotype has one constant segment and one J segment but is associated with at least three V regions (Ghaffari and Lobb, 1993).Two V segments are 5’ of the C segment and one is 3 ’. Interestingly, these V regions are in opposite transcriptional orientation (Ghaffari and Lobb, 1993, 1997), a feature which distinguishes fish and elasmobranch clusters. The only functional significance of this orientation is that rearrangement would occur by inversion rather than deletion (Max, 1993). These loci were characterized using phage A clones and so there is the possibility that the 3’ V segment found on the approximately 20-kb genomic fragments is the proximal 5’ V segment belonging to the next downstream cluster. In which case, the distance between clusters could be as little as 7 kb. The F type light chain clusters are strikingly similar to shark clusters except that the V segments are in opposite transcriptional orientation (Ghaffari and Lobb, 1997). However, these clusters are only approximately 9 kb apart (Fig. 28), raising the possibility that intercluster rearrangement may occur. There are greater than 50 F clusters and 15 G clusters (Ghaffari and Lobb, 1997), numbers consistent with estimates of light chain genes in other species (Pilstrom and Bengten, 1996). Sturgeons constitute a branch of fish between sharks and teleosts and may represent a transitional species. Therefore, it is interesting that the light chain gene in sturgeons (Fig. 28) have a mammalian K-like translocon arrangement with many V segments and at least seven J segments upstream of the constant region (Lundqvist et al., 1996). This result suggests that the translocon organization arose early in evolution after the elasmobranchs and, therefore, the light chain cluster pattern in teleost fish was independently derived. Ig gene organization in amphibians appears to fit the typical mammalian translocon model. This has been best demonstrated in the frog Xenopus (Haire et al., 1991, 1996; Picker and Siegelman, 1993; Schwager et al., 1988, 1991). Three isotypes, IgM, IgY, and IgX, are found in Xenopus. Isotype switching from IgM to IgY is observed during an immune response (Du Pasquier, 1993; Du Pasquier et al., 1989),and the IgX heavy chain has been shown by Southern analysis to be linked to the p gene (Mussmann et al., 1996). Thus, the Y and IgX heavy chain genes appear to be downstream of the p gene in a normal translocon pattern and to be expressed by isotype switching. However, the exact position of these genes has not been fully characterized. Some species have a modification of the basic translocon arrangement, which is characterized by a reduced capacity for combinatorial diversity, and therefore can be classified as limited or restricted translocon (Fig. 29). These translocons have one J, limited numbers of Ds in heavy chains, and low numbers of functional V regions (Butler, 1997; Butler et al., 1996).

484

JOHN J. MARCHALONIS et nl.

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Ch &-

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I

b-+-b

A

* -

-20 kb

Jh

Vhl

2.8 kb

1.8kb 1.6 kb

FIG.29. The limited translocon type arrangement is illustrated with examples from the pig and chicken. The arrangement and order of heavy chain isotype genes shown in brackets are unknown. $I indicates pseudogenes.

An extreme example of limited translocon are chicken heavy and light chain genes (Reynaud et al., 1987, 1989). Only one functional V region is found a short distance upstream of J (Fig. 29). However, there are numerous pseudo V regions in the locus, and a large secondary repertoire is generated by templated mutations (gene conversion from these pseudogenes). The IgY and IgA heavy chain genes are probably located downstream of the p gene, although the exact organization is not known. An intermediate type of limited translocon is present in the pig (Fig. 29). There are less than 20 VH genes, which all belong to the same family (Butler et al., 1996). Newborn piglets show a preferential usage of VH and DHgenes (Sun and Butler, 1996), and most of the repertoire is generated by gene conversion and hypermutation. Similar patterns are found in rabbits, sheep, and cattle (Dufour et al., 1996; Dufour and Nau, 1997; Hedrick and Eidelman, 1993). A rabbit VHlocus, for example, has 200 VH

EMERCENCE AND EVOLUTION OF THE IMMUNOGLOBULIN FAMILY

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genes, all belonging to the same VHfamily, of which half are pseudogenes (Knight and Crane, 1994; Knight and Winstead, 1997). In about 90% of rearrangements, the proximal VHis utilized and diversity is generated by gene conversion (Berens et al., 1997; Knight and Crane, 1994; Knight and Winstead, 1997). Generation of dwersity by gene conversion and hypermutation is a common feature of animals with the limited type of translocon. The distinguishing feature of the immune system in these animals is that the primary repertoire is generated in gut-associated lymphoid tissue rather than in the bone marrow (Weill and Raynaud, 1996). TCR loci in mice and humans have a translocon arrangement similar to the A light chain loci (Hedrick and Eidelman, 1993). Large numbers of diverse V regions are located upstream of J(D)C cassettes, which may be duplicated in tandem array. For example, in the mouse p chain locus there are two copies of the DJC cassette and four copies of the y JC cluster. Although it is now clear that TCR genes are present in all jawed vertebrates, most work has been done with cDNA and there is very little characterization of genoinic loci structure (Chretien et al., 1997; Fellah et al., 1993a; Hawke et al., 1996; Partula et al., 1995, 1996; Rast et al., 1995, 1997; Rast and Litman, 1994). Initial work in the horned shark suggested that the TCR p locus was in a cluster organization similar to the Ig genes (Rast and Litinan, 1994). However, subsequent work in the skate demonstrates that the TCR constant region exons are present in single copy, although a diverse array of V regions exists (Rast et al., 1997). This result suggests a translocon arrangement, and the results in the horned shark can also be interpreted to reflect a translocon arrangement (Litman and Rast, 1996). XI. Molecular Events Underlying the Explosive Emergence of Immunoglobulins and Their Initial Phases of Evolution

Possibly the most striking feature of the vertebrate combinatorial immune system is its apparent origin as a single burst with the emergence of gnathostomes. Agnathans have complement components and apparent lymphocytes, but all attempts thus far to detect genes for Igs, TCRs, MHC, or RAG proteins have been unsuccessful. Although absence of proof is not proof of absence, these negative results are puzzling because all of these genes that are requisite for the function of the combinatorial immune system are present in the most primitive extant jawed vertebrates, the chondrichthyes. These molecules in chondrichthytes comprise a diverse set but are clearly homologous to their counterparts in mammals. Thus, if the coinbinatorial immune system arose following the ancestral divergence of agnathans and chondrichthytes, it occurred as an explosive event within a relatively short evolutionary span of 20 million years or less.

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A speculative model for the emergence of the antigen-specific elements of the vertebrate immune system is shown in Fig. 30. The precursor gene ancestral to both V and C genes duplicated to generate these segments, which remained closely linked but separated by an intron of 1-4 kb consistent with the separation distance within shark clusters (Hohman et al., 1993). It has been speculated that the ancestral Ig domain was related to cell adhesion molecules (Matsunaga and Mori, 1987), although these contain C2 domains rather than the bonajde Ig C1 domains found in MHC and immunoglobulins. The actual ancestor has not been identified and the search must continue in agnathans, protochordates, and deuterostomes. Following the generation of the V-intron-C cassette, the C domain duplicated to generate C1 domains that translocated out of this cluster and evolved into the closely related MHC Ig-C domains and p2microglobulins (see Fig. 3). The next step was the insertion of a J minigene and recombination signal sequences into the intron separating the V and C gene segments. The origin of these is unknown. Once this insertion had taken place, the Vrss-rssJ-C cluster could be acted on by products of RAG genes to rearrange and to be expressed. It has been proposed that the segment of DNA containing RAG-1 and RAG-2 was introduced into the genome of primitive vertebrates by horizontal transfer mediated by retroviruses (Schatz et al., 1992).This hypothesis gained support from the homologies between RAG-1 and site-specific microbial recombinases (Bernstein et al., 1996a; Hughes and Yeager, 1997), by similarities in mechanisms of retroviral recombination and VDJ recombination (Dik et al., 1996), by findings of retrotransposons of the type lacking LTR in sharks, and by potential homology of the shark RAG-1 5'-untranslated region to a rat adenovirus right junction sequence (R. M. Bernstein, S. F. Schluter, and J. J. Marchalonis, unpublished observations). Although the homology to microbial recombinases does not by itself establish that horizontal transfer occurred, it may prove significant that these types of site-specific recombinases (integrases and integration host factors) are present in bacteria, but not in protostomes, lower deuterostomes, or agnathan vertebrates. Their appearance in gnathostomes is either the result of convergent evolution from other systems or the product of horizontal transfer. A number of examples from plants, eukaryotes, and lower deuterostomes provide support for the conjecture that transposable elements introduced a substantial amount of regulatory variation in evolution (Britten, 1996; Kidwell and Lisch, 1997).Whatever the explanation, this is a dear example of the combinatorial immune system coopting elements of other genetic systems. The Vrss-rssJ-C cluster at this stage resembles that of shark type I light chains (Rast et al., 1994). A second major type of light chains (h-like) that

I

VJ fused V

J

V

VJ separated C

Insertion of J minigene and HSS

I

I

C a l C&? C03 C& I

1 Prototype V-intron-C

J

C

J

V

D

-

1

-

*A

1

\ /

V/C Precursor lg domain (unknown)

Agnathadchondrichthyian Ancestor

FIG.30. Hypothetical scheme of the genetic events underlying the generation of the combinatorial immune system of jawed vertebrates. Solid lines represent Ig domains and J segments; transmembrane (TM) and secretory (S) exons are open boxes and solid ellipses, respectively; solid boxes are D segments; and shaded triangles are recombination signal sequences. Introns are indicated by wavy lines.

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is dominant in carcharhines has the V and J segments fused in register in the germline (Hohman et al., 1993). The orthologous light chains of rays are also fused in the germline (Anderson et al., 1995). The authors suggest that the VJ fusion was a secondary event at this stage. The ancestral heavy chain gene cluster was formed by a duplication of this cassette followed by the tandem duplication of the C segment into four closely linked domains that evolved into the C p l , Cp2, Cp3, Cp4 structure. In addition, a diversity (D) segment utilizing comparable RSS was inserted between the V and the J segments. The primordial light and heavy chain clusters duplicated subsequently to form hundreds of similar cassettes with mutation and selection of V, J, and C segments occurring. The process was a rapid one in that the characteristic distinctions in these segments apparently became stabilized in the 20 million year period followingthe divergence of gnathostomes and ancestral chondrichthytes. It can be proposed that this original burst of duplication and mutation was an explosive event, but selective pressures involved in forming heterodimers and in recognition of antigen resulted rapidly in the stabilization of canonical sequences followed by the domains evolving at moderate constant rates after their appearance in primordial form in the chondrichthytes. Another crucial event was the incorporation of the transmembrane/cytoplasmic exon(s) into the heavy chain cluster that enabled the (lightheavy) 2 structure to serve as a membrane receptor for antigen on B cells. The emergence of the full range of TCR chains likewise preceded the appearance of the ancestors of contemporary elasmobranchs and mammals, approximately 430 million years ago (Rast et al., 1997).These developments for a,0, -y, and 6 gene clusters incorporated the insertion of TM/CYTO exons for all the chains and also D segments into the P and 6 gene array in positions similar to their occurrence in heavy chains. Although it has been proposed that membrane associated TCRs arose prior to secreted immunoglobulins, the alternative should also be considered. Early cladistic analyses of Ig and TCR V-C segments indicated that TCR V domains could have diverged from the light chains following their separation from heavy chains (Beaman et al., 1987). Quantitative comparisons of the rates of evolution of TCR and Ig C domains from sequence data presented earlier indicate that the average rate of TCR C domain evolution, 2.8 0.2 X lo-', was significantly faster than that of Ig light and p chain C domains, 1.1 ? 0.3 X lo-'. Assuming that the rates for each are constant and that 20% identity is the cutoff point for positive identification ofbonufide Ig C domains, TCR C domains appeared between 420 and 540 million years ago, but Ig C domains emerged approximateIy 1000 million years ago. The latter figure (900-1290 million years) is also supported by consideration of the rate of evolution of C p domains, C p l ,

*


489

Cp2, Cp3, Cp4, within individual species, including humans, mice, chickens, and sharks. Interestingly, these calculations suggest that TCR arose at approximately the time of the origm of gnathostomes but that the emergence of recognizable Ig C domain precursors preceded this event in evolution. Although the early emergence of TCR as cell surface receptors has been an attractive h,ypothesis, the prior appearance of Ig light and heavy chains is not unreasonable because at least two additional systems-proteosomes and MHC-would have to evolve or be coopted for functional antigen-specific T-cell immunity to arise. The possibilities must be considered either that refinements in the computations will yield Ig values comparable to those for TCR or that, in line with their estimated ages, bona$de Ig constant domains will be found in agnathans, protochordates, or deuterostomes. If we accept the hypothesis that the cluster or cassette organization of Ig segments characteristic of chondrichthtylans is the primordial arrangement, it is not difficult to envision mechanisms for the origin of distinct immunoglobulin isotypes and also to explain why all immunoglobulin domains seem to show 30-40% identity to one another when considered over large evolutionary distances. Possibly in the development of an ancestral osteichthyian species, the chromosomal arrangement was such that the entire array of elasmobranch type clusters was not transmitted to the newly formed species. In the simplest case, one light chain cluster and one heavy chain cluster might be inherited. The heavy chain gene locus would be a p chain, and the light chain could be K-like, h-like, or one of the other types. The transferred clusters probably had the V and J separated with RSS sequences because rearrangement is an essential part of immunoglobulin and TCR activation in species more advanced than chondrichthytes. Light chain clusters occur in teleost, but these contain two VL segments, thereby indicating tandem duplication of these gene segments. Furthermore, in chondrostian fishes such as the sturgeon, an array of distinct VI, genes comparable to that found in mammalian light chain translocons occurs (Lundqvist et al., 1996). The heavy chains of all higher vertebrate species also occur in translocon arrangements. Thus, a second event that followed the inheritance of a p chain cluster was the duplication of variable region segments with these remaining linked to the D, J, and C segments, although these arrangements can cover hundreds of kilobases (Lai et al., 1989). Individual light chain isotypes might have arisen depending on the number of gene clusters inherited from the chondrichthtyian ancestors or, alternatively, they may have arisen by duplication within the phylogenetic development of individual vertebrate classes. All of the vertebrates derived from the ancestral chondrichthtyans would possess IgM heavy chains, and these would be expected to show an evolu-

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tion in p chains consistent with time and speciation. Heavy chains distinct from the p chain would arise via duplications of the C p cluster and subsequent independent evolution. The o (Bernstein et al., 19968) or NARC (Greenberg et al., 1996) heavy chains of sharks occur in clusters separate and unlinked to those of the p, chain and apparently have not been passed on to higher vertebrates in evolution (Schluter et al., 1997). It is possible that non-p heavy chains forming soluble immunoglobulin dimers of the form (LH)zmay have arisen independently in each vertebrate class (AtweU and Marchalonis, 1975) or, alternatively, there may be a lineal descent of these molecules to some extant in mammals. For example, Warr et al. (1995) argue that the heavy chain of IgY may have been ancestral to mammalian y and E heavy chains. The TCR chains appear to have occurred in stable configuration by the time of the ancestral divergence between chondrichthytes and osteoichtyes and have been maintained in substantially similar form throughout subsequent vertebrate evolution. A major event in the evolution of immunoglobulins was the appearance of IgG immunoglobulins in eutherian mammals, if not all mammals (Marchalonis, 1977), and the capacity for affinity maturation following somatic mutation and selection occurring in B cells in the germinal centers of lymph nodes (Han et al., 1996; Liu et al., 1992). This event required the appearance of histological structures, the emergence of IgG as the dominant immunoglobulin class, and the reappearance of RAG genes to facilitate recombination in adult cells. Despite the unequivocal homologies among, respectively,the V domains, C domains,joining segments, and transmembrane segments in Igs and TCRs of gnathostomes, there has been a tremendous plasticity in the organization of these segments and in the relative dependence on different mechanisms for the generation of diversity in both primary and secondary responses. The chicken, for example, has both a A light chain and a heavy chain gene system that are degenerate forms of the translocon in which there is only one functional Vh or VHand one functional JHor Jh, but a number of pseudogenes in a tandem array with these. Diversity is generated by templated hypermutation (gene conversion) using these pseudogenes (Reynaud et al., 1987, 1989). Primates and rodents have comparable translocon arrangements in both their immunoglobulin and their TCR gene arrangements. In the immunoglobulins,they have relatively large numbers of V regions and these are combinatorially assorted to give diversity. In addition, somatic mutation and selection occur, which are reflected with affinity maturation in the secondary response. The rabbit has a large array of VHgenes comparable in magnitude to those of humans and mice, but utilizes predominantly the VH segment most proximal to the D elements and copies by gene conversion from the other VHsegments (Knight and Crane, 1994).


491

Heavy chains of artiodactyls such as pigs (Butler et al., 1996) and sheep (Reynaud et aE., 1991) have only a single J H segment and a small number of VHgenes. The pig depends on gene conversion for generation of dversity, and the sheep relies heavily on nontemplated somatic hypermutation. In broad perspective, the combinatonal immune system of gnathostomes arose in a burst of gene duplication and cooption of genes and mechanism of cell differentiation from other systems and was established prior to the divergence of ancestral chondrichthtians and higher vertebrates. The immunoglobulins underwent evolutionary changes at a relatively conserved rate by comparison with other protein families, and variation in mechanisms for the generation of diversity indicated great plasticity in the organization of the gene segments and a requirement for special processes such as junctional diversification, somatic hypermutation, or gene conversion. XII. Conclusions

The combinatorial immune system defined by the presence of antigenspecific recognition units from the immunoglobulin family (heterodimeric immunoglobulins and T-cell receptors), the genetic machinery necessary for recombination, and cells of the lymphoid series expressing these receptors is fully functional from the earliest extant gnathostomes (sharks and their kin) to mammals. Despite efforts by a number of workers, definitive evidence for genes specifylng immunoglobulins, T-cell receptors, or recombination activating genes has not been documented for more primitive agnathan vertebrates (lampreys and hagfish), for lower deuterostomes such as tunicates and starfish, or for protostome or acoelomate invertebrates. Light chain type molecules form a large and diverse array within cartilaginous fishes, and the complete panoply of T-cell receptor chains (a,p, y , S) has been identified in these species. The combinatorial immune system of jawed vertebrates apparently arose as an evolutionary “big bang” involving the generation and duplication of V and C domains from an unknown precursor and the incorporation of joining segment genes, recombination signal sequences, and transrnembrane/cytoplasmic segments within the brief evolutionary span of 10-20 million years. Mechanisms for cell activation and division were coopted from existing systems widespread in evolution that can be termed inflammatory mechanisms. Orthologs of light chain variable segments may be shared among sharks, mice, and humans, and orthologous relationships among T-cell receptor variable segments of cartilaginous fishes and higher vertebrates have been found. Constant domains of K and y light chains and p heavy chains and Cn, Cp, C y , and CS TCR chains have been identified in diverse vertebrate species. From presently available sequence data, these most probably

492


evolve at constant rates, with the rate of evolution of TCR C domains being more rapid than that of the constant domains of immunoglobulins. Despite the clear homology among members of the immunoglobulin family, particularly among orthologous V domain sequences, there is a marked plasticity in the organization of gene segments; the most extreme differences occur between those of sharks, which are distributed as individual clusters or cassettes, each of which contains a single VL,JL, and CLelement or a single VH,a few Ds, possibly a few JHs, and one C p as opposed to that of mammals where a large number of V segments and a few Js or Ds are associatedwith one CLor a set of heavy chain C segments in a translocon arrangement. Organizational flexibility can differ considerably even within a single vertebrate class such as mammals, where swine have relatively simple heavy chain translocons compared to those of humans or mice and depend on templated hypermutation (gene conversion), whereas primates and rodents generate diversity via rearrangement mechanisms. Chickens also have restricted light chain (A) and heavy chain translocons and utilize gene conversion to V segment pseudogenes in the generation of the primary antibody repertoire. The most recent major step in the evolution of the immune system was the emergence in mammals of germinal centers within the lymph nodes, correlating with the IgM to IgG switch and affinity maturation following from somatic mutation and antigenic selection. The y heavy chain definitive of IgG is present in examples of all mammals, but has not been identified in more primitive vertebrates. Distinct heavy chain isotypes such as IgY of chickens, reptiles, and amphibians and IgW of sharks have also arisen through gene duplication in evolution and these may show distant relationships to mammalian immunoglobulins such as IgD and IgE.

ACKNOWLEDGMENTS This work was supported in part by Grant MCB 9631846 from the National Science Foundation to JJM and Grant CA72803 from the National Cancer Institute, USPHS to ABE. We thank Ms. Diana Humphreys for valuable assistance in the preparation of the manuscript.

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Anderson, M. K., Shamblott, M. J., Litman, R. T., and Litman, G. W. (1995). Generation of immunoglobulin light chain gene diversity in Raja erinacea is not associated with somatic rearrangment an exception to a central paradigm of B cell immunity. /. Exp. Men. 182, 109. Andersson, E., and Matsunaga, T. (1993). Complete cDNA sequence of a rainbow trout IgM gene and evolution of vertebrate IgM constant domains. Zmniunogenetics 38, 243. Andersson, E., and Matsunaga, T. (19954 Evolution ofimrnunoglobulin heavy chainvariable region genes: A VH family can last for 150-200 million years or longer. Zozmunogenetic.9 41, 18. Anderson, E., and Matsunaga, T. (1995b). Evolutionary stability of iininunoglobulin heavy chain variable gene family: A VH family can last for 150-200 million years or longer. Inmunogenetics 41, 18. Andersson, E., Peixoto, B., Tormanen, V., and Matsunaga, T. (1995). The evolution of the immunoglobulin M constant regon genes of Salmonid fish, rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Soltielinus alpinus): Implications on the divergence time of species. Iminunogenetics 41, 312. Arden, B., Clark, S. P., Kabelitz, D., and Mak, T. W. (1995a). Human T-cell receptor variable gene segment families. lmtnunogenetics 42, 455. Arden, B., Clark, S. P., Kabelitz, D., and Mak, T. W. (1995b). Mouse T-cell receptor variable gene segment families. Zmmunogenetics 42, 501. Atwell, J. L., and Marchalonis, J. J. (1975). Phylogenetic emergence of immunoglobulin classes dstinct from IgM. J. Zmmunogenetics 1, 367. AtweU, J. L., and Marchalonis,J. J. (1976).In “Comparative Immunology” (J. J. Marchdonis, ed.), p. 276. Blackwell Press, Oxford. Atwell, J. L., Marchdonis, J. J., and Edey, E. H. M. (1973). Major immunoglobulin classes of the echidna (Tuchyglossus aculeatus). Zinmunolugy 25, 835. Ayme-Southgate, A., Vigoreauz, J., Benian, G., and Pardue, M. L. (1991). Drososphila has a twitcllidtitin-related gene that appears to encode projectin. Proc. Natl. Acad. Sci. USA 88, 7973. Baeuerle, P. A,, and Henkel, T. (1994). Function and activation of NK-KB in the immune system. Annu. Reu. Zmmurzol. 12, 141. Barker, W. C., Ketcham, L. K., and Dayhoff, M. 0. (1978). In “Atlas of Protein Sequence and Structure” (M. 0. Dayhoff, ed.), p. 197. National Biomedical Research Foundation, Washington, DC. Bartl, S., Baltimore, D., and Weissman, I. L. (1994). Molecular evolution of the vertebrate immune system. Proc. Natl. Acad. Sci. USA 91, 10769. B a d , S., and Weissman, I. L. (1994).Isolation and characterization of major histocompatibility complex class IIB genes from the nurse shark. Proc. Natl. Acad. Sci. USA 91, 262. Beaman, K. D., Barker, W. C., and Marchalonis, J. J. (1987). In “Antigen Specific T Cell Receptors and Factors” (J. J. Marchalonis, ed.), p. 105. CRC Press, Boca Raton, FL. Beard, J. (1894).The development and probable function of the thymus. Anat. Amy. 9,476. Beck, G., Cooper, E. L., Habicht, G. S., and Marchalonis,J. J. (1994). “Primordial Immunity: Foundations for the Vertebrate Immune System.” New York Academy of Sciences, New York. Beck, G., Vasta, G. R., Marchalonis, J. J., and Habicht, G. S. (1989). Characterization of interleukin-a activity in tunicates. Coinp. Biochem. Physiol. B 92, 93. Bell, J. I., Owen, M. J., and Simpson, E. (1995). “T Cell Receptors.” Oxford University Press, Oxford. Bengten, E. (1994). “The Immunoglobulin Genes in Atlantic Cod (Gadus morhua) and Rainbow Trout (Oncorhynchus ntykiss).” Sweden Uppsda University, Uppsda, Sweden.

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ADVANCES IN IMMUNOLOGY,VOL. 70

Current Insights into the ”Antiphospholipid” Syndrome: Clinical, Immunological, and Molecular Aspects DAVID A. KANDIAH,’ ANDREJSAU,~YONGHUA SHENG,’ EDWARDJ. VICTORIA,* DAVID M. MARQUIS,* STEPHEN M. COVrrS,* and STEVEN A. KRIUS’ ‘Depadment of Immunology, Allergy, and lnfeciiaus Disease, Universiiy of New h u h Wales School of Medicine, St. George Haspipol, Kogarah 2217, Austmlia; tRackefeller Universiiy, New York, New Yark 1002 I; and *la Jolh Pharmaceutical Company, k n Diego, California 92 12 1

1. Introduction

In 1983, a distinct syndrome consisting of vascular thrombosis, livedo reticularis, thrombocytopenia, and movement disorders associated with “antiphospholipid (aPL) antibodies was first described (Hughes, 1983). Early studies on aPL antibodies were on patients with systemic lupus erythematosus (SLE) and it was in a subset of patients with SLE that the “antiphospholipid syndrome” (APS) came to be recognized (Hughes, 1985). The association of vascular thrombosis and autoimmune disease was found in the 1960s (Bowie et al., 1963; Alarcon-Segovia and Osmundson, 1965)and laid the foundation for studies to discover the pathogenesis and immunological features of this distinct group of individuals. It was soon noted that a subset of patients had the clinical manifestations of APS without sufficient clinical and immunological criteria to satisfy the 1982 American College of Rheumatology (ACR) diagnostic criteria for SLE (Tan et al., 1982).The definition and criteria for a “primary antiphospholipid syndrome” (PAPS) was first proposed (Asherson, 1988) and the first series was documented the following year (Alarcon-Segovia and Sanchez-Guerrero, 1989). In a 2-year multicenter follow-up study of patients with PAPS and secondary APS (SAPS)in other autoimmune diseases, a lower female/male sex ratio in PAPS was found compared to that in patients with SAPS. Patients with SAPS had more neutropenia, autoimmune hemolpc anemia, endocardial vegetations, and low levels of complements compared to PAPS patients. The incidence of thrombosis was no different in the two groups (Vianna et al., 1994). Although the original concept of the APS was shown to comprise one or more of the clinical manifestations of venous thrombosis, arterial thrombosis, recurrent fetal loss, and thrombocytopenia, more diverse clinical manifestations are now recognized, such as cardiac valvular lesions, adrenal insufficiency, and multiorgan thrombotic complications known as “catastrophic” APS (Asherson, 1992).Despite better understanding of the target antigens of aPL antibodies, original laboratory criteria of moderate to high 507

Copyright 0 1998 by Acditernic Pres.; All nghts of reproduction in any form reserved 0065-2776/98 $25 00

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levels of “anticardiolipin” (aCL) antibodies and/or lupus anticoagulant (LA) antibodies are still being used and have now replaced the ACR criteria listing of the lupus erythematosus (LE) cell in the revised list for the diagnosis of SLE (Hochberg, 1997). Current guidelines for the diagnosis of APS are the presence of at least one of the clinical criteria of venous thrombosis, arterial thrombosis, recurrent pregnancy loss, and thrombocytopenia, with one or more of the laboratory criteria of moderate to high levels of IgG and/or IgM aCL antibodies and detection of LA activity in a clotting assay. Current criteria for the detection of LA activity in plasma will be discussed later in Section XI. In a previous review of this subject, the emphasis was on antibody interactions with phospholipids, and a brief introduction was made on the role of P2-glycoprotein I (P2GPI) (McNeil et al., 1991). This review develops on the current insights on antibody interactions with phospholipid-binding plasma proteins, in particular P2GP1, and covers currently recognized clinical associations, immunological aspects, molecular studies, and therapeutic interventions. II. “Antiphospholipid Antibodies

aPL antibodies are a heterogeneous group of autoantibodies that have specificity for a number of phospholipid-binding proteins, phospholipid molecules, and phospholipid-protein complexes. A number of phospholipid-binding proteins have been implicated in APS, including PBGPI, prothrombin, protein C, protein S, kininogens, thrombomodulin, and annexin V. Traditionally, aPL antibodies are detected in LA assays and in solid-phase immunoassays using cardiolipin as the target antigen. The target antigen detected in clotting assays is still unclear, and a number of coagulation proteins have been implicated, particularly PZGPI and prothrombin. It appears, however, that the complexes assembled on the phospholipid surfaces in functional clotting assays are more important than any one protein. The paradoxical phenomenon of prolongation of in vitro phospholipid-dependent clotting tests to detect LA antibodies while being associated in vivo with vascular thrombosis has intrigued scientists and clinicians for years. Studies of APS may throw some light on the pathophysiology of the mode of action of these autoantibodies. Because the original immunoassays for the detection of aPL antibodies used coated cardiolipin on microtiter plates, they were called anticardiolipin antibodies (Loizou et al., 1985). It has been shown that the target antigen in this assay is P2GPI (McNeil et al., 1990; Galli d al., 1990), the original nomenclature is a misnomer, and the antibodies should be known as anti-p2GPI antibodies. For the purpose of this review, antibodies detected on assays employing P2GPI as the coated antigen in the absence

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of phospholipids are referred to as anti-P2GPI antibodies and antibodies detected in a cardiolipin ELISA are referred to as aCL antibodies. These two populations of antibodies in autoimmune patients are identical with few exceptions. As a generic term for anti-P2GPI antibodies and LA antibodies, the term aPL antibodies will be employed. The units of antibody levels in sera expressed in the standard CL-ELISA have been calibrated to known sera from the R a p e Institute, London. One GPIJMPL unit each represents one microgram of affinity-purified antibody per milliliter of serum. Antiphospholipid antibodies are found in “normal” individuals. In a population of 499 blood donors, the prevalence of LA antibodies was 8% and anticardiolipin antibodies was 4.6 (IgG aCL), 4.6 (IgM aCL), and 5.6% (for polyvalent aCL antibodies) (Shi et nl., 1990).When these samples were stratified and the demographics of the blood donors studied, LA antibodies were found in young females. Prospective studies need to be done on these individuals to detect if any clinical problems had developed in subsequent years. As aPL antibodies are not normally distributed with most individuals having undetectable levels, it is more appropriate to use a nonparametric definition of the normal range, such as the 95% central tendency. Antiphospholipid antibodies can be divided into two main groups, classified according to their association with autoimmune or infective conditions. Traditionally these antibodies are termed autoimmune and alloimmune, respectively. Until the discovery that autoimmune antibodies are generally directed to the phospholipid-binding protein p2GPI instead of to the phospholipid molecule itself, the difference between these antibodies was unclear. Although there are exceptions to this rule, autoimmune aPL antibodies detected in solid-phase immunoassays with anionic phospholipids, such as cardiolipin as the coated antigen, are directed to P2GPI captured on a negatively charged surface. Alloimmune aPL antibodies found in chronic infections such as malaria, syphilis, leprosy, tuberculosis, and parvovirus infections do not bind P2GPI but are directed against the anionic phospholipid with p2GPI competing for binding with these antibodies (Hunt et nl., 1992).The binding of this latter group of antibodies has been shown to be charge dependent as high salt-containing buffers abolish binding to cardiolipin (Monestier et al., 1996). A. LUPUS ANTICOAGULANT ANTIBODIES

Plasma that contained proteins that prolonged phospholipid-dependent in vitro clotting assays were first described in SLE in 1952 (Conley and

Hartmann, 1952).Increasing the phospholipid in the assay system appeared to neutralize this LA reaction (Yin and Gaston, 1965). LA activity was

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found in the IgG fraction of serum. It was shown that immunoglobulins with LA activity react with anionic phospholipid but not with zwitterionic phospholipids. The phospholipid configuration appeared important with LA antibodies directed to the hexagonal phase rather than lamellar-phase phospholipids (Rauch et al., 1989). Current evidence would suggest that the antibodies may actually interfere with the assembly of enzymatic procoagulant and anticoagulant complexes on phospholipid surfaces, resulting in clinical vascular complications. Although these complications are predominantly thromboses, reports of hemorrhagic diatheses in patients with LA activity have been described. One of the probable causes of this is the presence of high-affinity antiprothrombin antibodies that complex with prothrombin, resulting in removal of the immune complexes by the reticuloendothelial system. This creates a situation of functional hypoprothrombinemia and bleeding. The dilute Russell’s viper venom time (dRVVT), the dilute activated partial thromboplastin time (dAPTT),and the dilute kaolin clotting time (dKCT) are the most frequently used tests for the detection of LA antibodies in routine practice and for research papers. However, up to 53% of patients with LA antibodies have a prolonged prothrombin time (Horellou et aZ.,1987). Although this may sometimes be due to low factor I1 levels, most of the patients studied have been shown to have normal antigenic levels of prothrombin (Horellou et al., 1987; Fleck et al., 1988). B. ANTI@ GLYCOPROTEIN 1ANTIBODIES Autoantibodies can be produced in response to tissue breakdown as a result of exposure of a target antigen not usually in contact with immunemediated cells. They can also arise after altered expression of cell surface proteins due to external stimuli or to translocation of intracellular antigens to cell surface membranes. The ability of a particular host to handle specific antibody-antigen complexes also predisposes to tissue and organ damage. These factors may all interact to suggest a pathogenic mechanism for the generation of autoantibodies in APS. Following purification of aCL antibodies by ion-exchange chromatography or phospholipid-polyacrylamide affinity chromatography, these antibodies failed to bind to the same phospholipid affinity column unless native or bovine plasma was also present (McNeil et al., 1989). Hence a plasma cofactor had been separated from the antibodies during the purification process that formed part of the antigenic target for these antibodies. The purified antibodies were able to bind in a cardiolipin ELISA where bovine serum is used in diluent and blocking buffers (called a standard CLELISA). This plasma cofactor was purified to homogeneity, sequenced, and identified as P2GPI (McNeil et al., 1990). This phospholipid-binding

FIG. 7-21A. Four-stranded P-pleated sheet (red arrows) of a V, domain, taken from the structure of a human Fab with V, of subgroup I11 (A. B. Edmundson et al., unpublished data). Totally conserved residue positions are colored blue and those 90% conserved are represented as blue chevrons. Residues conserved at lower levels are designated by pale red (75%)or red striped bands (60%).Strategically located residues are numbered to allow correlation of this model with the sequence presented in Fig. 20. This figure was devised by Benjamin Goldsteen and Allen Edmundson, using the program MOLMOL (Koradi et d.,1996).

FIG. 7-21B. Five-stranded P-pleated sheet (white arrows) of the same V, domain. Color coding for the conserved residues and the numbering follow the patterns for A. This figure was devised by Benjamin Goldsteen and Allen Edmundson, using MOLMOL (Koradi et al., 1996).

FIG.8-3. Distribution of charges in the three-dimensional model of human p2GPI-5. Main chain trace of the three-dimensional inodel of P2GPI-5. The positively charged side chains ( h i s , Arg) are shown in blue. The His side chains are not shown, but their main chain is colored blue. The main chains of the negatively charged residues (Asp, Clrr) are shown in red. The phospholipidbinding site is indicated by an arrow. The figure was prepared by program QUANTA (MSI, Sari Diego, CA). Reproduced with permission from Sheng et 01. (1996). 01996. The American Association of Immunologists.

FIG. 8-4. Distribution of charges in the three-dimensional model of human p2GPI-5. Electrostatic potential at the phospholipid-binding region of native and mutant p2GPI-5. (A) Native p2GPI-5 at neutral pH. (B) Lys 42/44/45 + Glu triple mutant at neutral pH. The molecular surfaces of the models are colored by the electrostatic potential, as shown by the color bar on each panel (in units of kT; 1 kT unit = 0.58 kcal/electron mol). The figures were prepared by program GRASP (Nicholls et al., 1991), using the relative dielectric constants of 2 and 78 for protein and solvent, respectively, and the salt concentration of 150 mM. The positively and negatively charged residues are numbered in yellow. Relative to Fig. 3, p2GPI-5 is viewed from the top. Reproduced with permission from Sheng et al. (1996).0 1996.The American Association of Immunologists.

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plasma protein is found in relatively high concentrations of 4 p M in plasma or sera and appeared to have a role in the coagulation cascade as a natural anticoagulant based on in vitru experiments. This has led to the proposed theory that anti-P2GPI antibodies found in APS interfere with the natural procoagulant-anticoagulant homeostatic mechanisms, resulting in a procoagulant tendency and clinical thrombosis and atherogenesis. The first clinical study to investigate the role of anti-bZGPI antibodies with thrombosis found that 36% of patients with SLE had these antibodies (Viard et al., 1992). If these antibodies were found with LA antibodies, there was a strong association with thrombosis. Other small retrospective studies were performed to confirm an association of anti-P2GPI antibodies, but were fraught with technical problems in patient selection, retrospective analyses, and the ELISA methods used to detect these antibodies (Martinuzzo et al., 1995; Balestrieri et al., 1995; Cabiedes et al., 1995; Pengo et al., 1996). In the last paper the population of patients selected was done on the basis of their reactivity in a cardiolipin ELISA. It is therefore not surprising that anti-PeGPI antibodies were found in all the patients who had thrombosis, as the target antigen in the standard CL-ELISA is bovine PSGPI, which supports the binding of most human aPL antibodies. There is still some controversy as to whether most patients with autoimmune APS have both anti-pZGPI and antibodies that bind anionic phospholipids directly. Examining the sera of patients with both PAPS and SLE/APS, 68% were found to have true aCL antibodies as demonstrated by reactivity to CL on thin-layer chromatography plates, independent of the presence of PZGPI (Sorice et al., 1996). Using delipidated P2GPI as the antigen, 22.6% of the CL-ELISA positive sera bound P2GPI on immunoblotting. There may also be a population of antibodies that require the complex of PZGPI and phospholipid. Sixteen out of 18 patients with SLE and clinical manifestations of APS with negative IgG and IgM standard CLELISA reactivity had IgG anti-62GPI antibodies (Cabiedes et al., 1995). In a study of 97 patients with IgG and/or IgM anti-02GPI reactivity in a PBGPI-ELISA, 43% of IgM and 8% of IgG antihuman P2GPI antibodies did not bind to purified bovine PBGPI, explaining a negative aCLELISA where bovine PZGPI is the major source of P2GPI (Arvieux et al., 1996). The initial concentration of P2GPI in the patients' sera and the dilution of sera used could determine whether binding occurs in the CL-ELISA. The discovery that PZGPI exhibits genetically determined structural polymorphism with the occurrence of four alleles is another potential source of confusion in the conventional CL-ELISA. PZGPI from certain individuals, hoinozygous for the APOH"3 allele, is unable to bind anionic phospholipid (Kamboh et al., 1995). This group has also found two structural mutations

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at codons 306 and 316 in the fifth domain of 02GPI. These inissense mutations affect the structural integrity of the fifth domain of /32GPI affecting phospholipid binding. The authors suggest that there are individuals who are compound heterozygotes for the two mutations, who may be precluded from producing anti-b2GPI autoantibodies (Sanghera et al., 1997).Antibodies with P2GPI reactivity and true CL reactivity exist in the same patient population, with IgG2 subclass restriction of anti-P2GPI antibodies in patients with autoimmune disease (Arvieux et al., 1994). These factors all contribute to the differences in assay results for patients with APS in a conventional CL-ELISA. 111. Clinical Features of the "Antiphospholipid Syndrome

A. CARDIOVASCULAR MANIFESTATIONS APS is associated with a number of clinical manifestations affecting multiple organs. Although the common pathophysiological theme for organ damage appears to be thrombotic microangiopathy, there are other clinical manifestations that cannot be explained by this, e.g., cardiac valvular abnormalities. Patients with APS can have both arterial and venous thrombosis, the only clinical condition that predisposes to this without any structural vascular anomalies. Other inherited conditions tend to predispose to thrombosis in one vascular bed, e.g., homocystinemia and arterial thrombosis, and a number of familial protein deficiencies and genetic mutations that predispose to venous thrombosis. In younger patients and patients with PAPS, the vascular event is often an acute occlusive vascular event instead of being secondary to atherosclerosis. Patients with SLE and secondary APS, however, may have a combination of thrombotic diathesis and atherosclerosis related to other factors, such as long-term steroid administration, hyperlipidemia, and hypertension. Coronary vasculitis is less frequent. The prevalence rates in APS for myocardial infarction have been reported between 0 and 7% (Asherson et al., 1985). A Finnish study showed that the presence of high antibody levels in a standard CL-ELISA was an independent risk factor for myocardial infarction. Subjects with aCL levels in the highest quartile of distribution had a relative risk of myocardial infarction of 2.0 (95% confidence interval, 1.1to 3.5) independent of confounding factors normally associated with coronary vascular disease, such as smoking, age, systolic blood pressure, and hyperlipidemia (Vaaralaet al., 1995).In a series of 83 patients who had undergone coronary artery bypass graft surgery, autoantibodies detected in CL-ELISA were elevated in late bypass graft occlusions (Morton et al., 1986). A placebo group not treated with aspirin with these autoantibodies


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had a high rate of coronary artery bypass graft occlusion (Gavaghan et nl., 1987). aPL antibodies may be associated with acute and chronic myocardial dysfunction. The clinical findings of left ventricular isolated and global dysfunction with the presence of insignificant coronary vessel disease as seen on angiography may be associated with aPL antibodies predisposing to coronary microangiopathy (Leung et al., 1990). This can also be found in the context of valvular heart disease in particular mitral regurgitation. The link between aPL antibodies and aseptic vegetations in patients with autoimmune disease was recognized in the 1980s (Anderson et al., 1987; Ford et al., 1988). Studies using echocardiography have shown that autoimmune patients with aPL antibodies have a higher prevalence of valvular vegetations (Khamashta et al., 1990; Cervera et al., 1992, Roldan et al., 1992).Although patients with SLE, especially if they are immunosuppressed with medication to control the disease activity, may have infective endocarditis, it appears that these patients have a higher prevalence of noninfective, thrombotic endocarditis. Linear deposition of IgG aCL antibodies in the subendothelial layer of heart valves in patients with APS has been demonstrated (Ziporen et al., 1996),suggesting a possible pathogenic role of these antibodies in valvular abnormalities. This would need to be studied more extensively in the future. B. NEUROLOGICAL MANIFESTATIONS The cerebral arterial circulation appears to be the most common site for arterial thrombotic episodes in patients with aPL antibodies (Harris et al., 1984). The Antiphospholipid Antibodes in the Stroke Study Group (APASS)found that the presence of autoantibodies above 10 GPL or MPL units in a standard CL-ELISA to be an independent risk factor for a first ischemic stroke in an elderly population without SLE (APASS, 1993). In a prospective study of stroke in patients below the age of 50 years, the risk of stroke recurrence was eight times higher in patients with aPL antibodies than those without (Brey et nl., 1990). It therefore appears that while the presence of aPL antibodies is an important factor to be evaluated for in the context of cerebrovascular events, the positive predictive value is greatest in patients below the age of 50 years. As in other vascular beds, the presence of cigarette smoking and hypercholesterolemia may independently increase the risk of recurrent cerebral ischemia in patients with aPL antibodies (Levine et al., 1990). In another prospective study of patients who presented with focal cerebral ischemia without any prior autoimmune disease, a titer of > 40 GPL units in a standard CL-ELISA conferred a twofold increased risk for a further thromboocclusive event (peripheral or central) or death. This is despite more of these individuals

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receiving antiplatelet or anticoagulant therapy or both at the time of followup. These results imply that more specific methods need to be derived to stratify these high-risk patients and to maintain them on suitable therapy after a first vascular occlusive event (Levine et al., 1997). Limited cerebrovascular histopathological data suggest that the vascular abnormality in aPL syndrome is increased fibrin thrombi formation in small- and medium-sized vessels in the absence of vasculitis (Woodard et aZ.,1991).As discussed earlier in the context of cardiac valvular abnormalities, another source of vascular occlusion in patients with aPL antibodies is cardiac emboli, and one-third of the 72 patients studied by the APASS group found cardiac abnormalities in patients with aPL antibodies and cerebral ischemia, predominantly mitral valve abnormalities (APASS, 1990). Although epilepsy is a recognized clinical event in SLE patients, the etiology of this is multifactorial. Hypertension, infection, cerebral ischemia, and vasculitis have all been implicated in patients who develop epilepsy. Epilepsy as a primary neurological event in SLE patients was associated with a high prevalence of aPL antibodies (Herranz et al., 1994). In a study by Verrot et al. (1997), 163 patients with epilepsy were evaluated for autoantibodies in a standard CL-ELISA. The authors found 31 (19%) patients with IgG aCL antibodies of moderate to high titers. None of these patients had any previous clinical events to suggest APS. Brain imagmgs in these patients showed no significant difference in those who were aCL positive and negative (Verrot et al., 1997). Hence there appears to be a group of patients who have epilepsy as a primary clinical manifestation of the presence of aPL antibodies, independent of possible cerebral ischemia. Movement disorders have been associated with SLE initially (Lusins and Szilagyi, 1975) and subsequently with aPL antibodies (Asherson and Hughes, 1988). These movement disorders may be brought out in an estrogen-related hormonal environment, e.g.,in pregnancy, or if the patient was taking the oral contraceptive pill (Asherson et al., 1986a). Movement disorders in patients with aPL antibodies may also follow cerebral infarctions. Another neurological manifestation of aPL antibodies is migraine (Hughes et al., 1986),although the association at the moment is considered tenuous (Hering et al., 1991). Transverse myelopathy has also been described in autoimmune patients with aPL antibodies (Adrianakos et al., 1975).Although there have been a number of case reports of aPL antibodies being found in patients with transverse myelitis (Lavalle et al., 1990), the presence of these antibodies in patients with autoimmune disease may just be part of the spectrum of autoantibodies found in these patients and not be directly responsible for the clinical problem.


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C. OCULAR ISCHEMIA aPL antibodies may be associated with thromboembolic disease in the visual pathway. A study of patients with cerebrovascular disease and APS found 19% (9/48) had clinical features of amaurosis fugax, ischemic optic neuropathy, and retinal artery occlusions (Levine et al., 1990). Various other studies and case reports have also described patients with aPL antibodies and ocular ischemia, often in the context of more generalized cerebral ischemia (APASS, 1990; Briley et al., 1989). The occurrence of amaurosis fugax in patients under the age of 50 or in patients with frequent episodes ranging from 2 to inore than 100 episodes a week may indicate that APS and aPL antibodies should be screened in these patients (APASS, 1993). A prospective study of 550 patients with SLE revealed that 7.5% of these patients had occlusive ocular vascular disease, and 38% of these patients had LA antibodies investigated by one clotting test only (APT") (Stafford-Brady et al., 1988). Patients presenting with headaches and found to have papilloedema in the presence of a normal cerebral CT scan may have cerebral venous thrombosis. This condition has been associated with aPL antibodies and should be screened for with multiple sensitive tests (Levine et al., 1987). The true incidence of aPL antibodies in optic ischemia and cerebral venous thrombosis has yet to be clearly defined, as more information is available on the detection methods for aPL antibodies. It appears, however, that in patients below the age of 50 years who present with ocular symptoms and have been found to have vasoocclusive disease should be screened for these antibodies by multiple tests.

D. PULMONARY MANIFESTATIONS Recurrent deep venous thromboses are the most common vasoocclusive events that occur in patients with aPL antibodies (Boey et al., 1983). Subsequent pulmonary emboli are not infrequent and often occur in the absence of symptomatic deep venous thromboses (DVTs) (Asherson and Cervera, 1992). Pulmonary hypertension in patients with APS has been documented, but this appears to be mainly associated with SLE and not with thrombotic disease (Asherson, 1990). It is therefore unclear whether SLE patients with pulmonary hypertension and aPL antibodies have the two conditions related or whether the high frequency of aPL antibodies in SLE may mask their true clinical relevance. Intraalveolar pulmonary hemorrhage has been described in patients with SLE and aPL antibodies (Howe et al., 1988). In a retrospective review of inpatients with intraalveolar pulmonary hemorrhage and SLE, six of the eight patients were found to have aCL antibodies (Schwab et al., 1993).

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This study identifies the problems encountered in retrospective clinical studies of patients with aPL antibodies as the current battery of tests for LA and anti-02GPI antibodies should be performed so as not to miss patients with aPL antibodies. This is particularly true of hemorrhage associated with aPL antibodies where antiprothrombin antibodies should be investigated to detect those high-affinity antibodies that form prothrombin-antiprothrombin complexes that are removed by the reticuloendothelial system. This results in liypoprothrombinemia and hemorrhage in some patients with these antibodies. Hence, pulmonary complications in APS are common but are often related to macrovascular thromboses as part of a systemic hypercoagulable state. Patients may also have adult respiratory distress syndrome as part of multiple organ involvement with extensive microangiopathic thromboses (Ghosh et al., 1993).

MANIFESTATIONS E. RENAL Primary renal disease is increasingly recognized in APS. Antiphospholipid antibody-related intrarenal thromboses may present with systemic hypertension, proteinuria, hematuria, and progressive renal failure, especially in the context of severe thrombotic microangiopathy in catastrophic APS (Asherson, 1993; Piette et al., 1994). Glomerular capillary thrombosis has been found to have a strong association with LA antibodies and predisposes to glomerular sclerosis independent of immune complex disease (Kant et nl., 1981). Renal disease may also arise in the APS with renal artery stenosis. The nature of this vascular occlusion is unclear and may arise as a primary thrombotic phenomenon in the context of aPL antibodies (Ostuni et al., 1990) or may be secondary to previous damage to the renal vessels from atheromatous degeneration in the blood vessel walls or prior renal artery fibromuscular dysplasia (Mandreoli et al., 1992). Renal vein thrombosis may be the cause and result of a thrombotic tendency in the APS. Thrombotic microangiopathy predisposing to the nephrotic syndrome can result in the loss of circulating natural anticoagulants predisposing to vascular thrombosis. However, the thrombophilia associated with aPL antibodies has been shown to occur in the absence of previous renal disease. Comparing two matched groups of SLE patients with renal disease, with and without LA antibodies, no differences were found in their renal biochemical and histological features except in a higher prevalence of intrarenal thromboses in patients with LA antibodies (Farrugia et al., 1992).

F. ADRENAL MANIFESTATIONS The link between adrenal gland hypofunction and aPL antibodies was first recognized in the late 1980s (Grottolo et al., 1988; Asherson and


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Hughes, 1989). A number of possible theories exist for this association, including the primary occlusion of the adrenal veins leading to glandular edema and compression of the arterial blood supply and adrenal infarction. Asherson (1994) reviewed 38 cases with adrenal hypofunction and aPL antibodies and showed that 31 of the 38 patients had PAPS and 7 had SLE-associated APS. In 20 of these patients, vascular occlusive events preceded the adrenal hypofunction, whereas in 10 patients, concurrent events occurred in the context of acute adrenal failure. These events were predominantly venous in particular pulmonary emboli (Asherson, 1994). Therefore, patients with aPL antibodies who suddenly develop circulatory collapse need to be investigated for electrolyte disturbances and adrenal structure and function and treatment with adequate and prompt fluid replacement is essential. Marie et al. (1997)identified adrenal failure secondary to bilateral adrenal hemorrhagic infarctions in a 70-yearold patient as a first clinical manifestation of the PAPS who then went on to develop extensive upper limb deep venous thrombosis while on aspirin.

G. HEPATIC MANIFESTATIONS Structural and functional obstruction of venous blood flow in the liver may lead to Budd-Chiari syndrome. In the context of aPL antibodies, this may be the result of thrombosis in the hepatic veins extending to the inferior vena caw. The occurrence of Budd-Chiari syndrome and aPL antibodies was first reported in 1984 (Pomeroy et al., 1984). The majority of patients described with this syndrome in the presence of aPL antibodies have PAPS and have had previous venous occlusive disease and concurrent thrombocytopenia. Hepatic venoocclusive disease resulting in hepatomegaly and ascites secondary to central and sublobular vein occlusions occurs with aPL antibodies. The venous occlusions lead to hepatic sinusoidal congestion, hepatocellular necrosis, and finally fibrosis. The liver has a dual blood supply from the systemic and portal circulation. As such, hepatic infarction is rare unless the patient has generalized thrornbophilia. The first case of hepatic infarction in association with aPL antibodies was described in 1989 (Mor et al., 1989), and primary portal hypertension has been described in APS (Mackworth-Younget al., 1984). Thrombosis of mesenteric vessels has been described in APS resulting in intestinal infarction. Patients may have both arterial and venous occlusions. The presentation is usually with acute abdominal pain or “intestinal angina” (Asherson et al., 1986b). Other abdominal organs that have been described with vascular occlusion and infarction include the spleen (Arnold and Schrieber, 1988) and pancreas (Wang et al., 1992).

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H. DERMATOLOGICAL MANIFESTATIONS In line with the common theme of vascular occlusion and insufficiency in end organ disease in APS, skin changes are fairly common in APS. Livedo reticularis, a mottled violaceous discoloration of the skin in a netlike pattern, is found frequently in APS. In patients with livedo reticularis and aPL antibodies, recurrent episodes of cerebral ischemia have been described (Sneddon, 1965).These patients may have a range of neurological manifestations from headache and dizziness, focal neurological deficits, and progressive cognitive deficits (from loss of concentration and memory loss to severe dementia). Livedo reticularis is found sufficiently frequently to be included in the clinical diagnostic criteria proposed (Alarcon-Segovia et al., 1992). Necrotic skin ulcers have been reported since 1963 in association with circulating LA antibodies (Bowieet al., 1963). Superficial cutaneous necrosis has also been described with aPL antibodies. This condition is also found in deficiencies of natural anticoagulants, e.g., protein C and protein S, or in cryoglobulineinia and cryofibrinogenemia,and these abnormalities may be found concurrently with aPL antibodies, with additive risks for the underlying superficial thrombosis. Digital gangrene may also be seen in APS (Alegre et nl., 1989). The histological features of small vessel disease producing the skin and soft tissue manifestations in APS appear to be a noninflammatory thrombosis of small arteries and veins throughout the dermis and subcutaneous fat tissue, occasionally accompanied by endarteritis obliterans. This condition is characterized by narrowing of the vascular lumen with endothelial cell proliferation and fibrohyalinization of the vessel wall (Alegre and Winkelmann, 1988). In livedo reticularis, skin biopsies rarely reveal thrombosis of the small vessels, and vessel wall hyperplasia may be the only histological feature seen. These skin lesions may frequently be the first sign of APS; up to 37% of patients with skin lesions and aPL antibodies develop multisystem thrombotic phenomena in the course of their disease (Alegre et al., 1989). This observation, performed before the antigen specificities of aPL antibodies were better defined, should lead to prospective studies investigating this association with multiple screening tests for aPL antibodies. This could determine the subset of patients who present with skin lesions, who could well go on to develop more severe systemic manifestations that may be prevented or &minished with specific antithrombotic therapy. I. AVASCULAR NECROSISOF BONE aPL antibodies may be associated with clinical avascular necrosis (AVN) of bone. There have been a number of cases in the literature of

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patients with PAPS who have developed clinical AVN adjacent to various joints (Vela et al., 1991; Seleznick et al., 1991). The prevalence of AVN in patients with aPL antibodies is difficult to ascertain, as patients with SLE are often on corticosteroid therapy, which predisposes to this condition.

J. OBSTETRIC MANIFESTATIONS Lupus anticoagulant antibodies have been associated with pregnancy loss and intrauterine death (Nilsson et al., 1975). Others have shown the same association with aCL antibodies (Lockshin et al., 1985; Harris et al., 1986). It is often suggested that patients with aPL antibodies exist in a prothrombotic state that need some other trigger to precipitate a clinical event. This could well include surgery, oral contraceptive use, and pregnancy. Previous pregnancy failures are an important feature for predicting subsequent pregnancy failure. Autoimmune disease has a variable effect on pregnancy. It has been suggested that the underlying immune abnormality that permitted the development of the autoimmune disease or the autoantibodies that arise may be directly responsible for the fetal loss (Gleicher, 1994). The maternal effects of aPL antibodies in pregnancy are uncommon, but have been reported, including preeclampsia (Scott, 1987), chorea gravidarum (Lubbe and Walker, 1983), and cardopulmonary distress (Branch, 1990). Severe early onset preeclampsia and abruptio placentae may predispose to fetal complications in late pregnancy. However, the fetus appears to be at risk throughout the pregnancy, and detection of aPL antibodies appears to be a useful test in the investigation of autoimmune reproductive failure (Aoki et al., 1995). Abnormal uterine artery flow velocity may predict a poor outcome in cases of aPL antibodies (Caruso et al., 1993). The most common cause of pregnancy loss in the first trimester is chromosomal abnormalities, and this has not been adequately studied in patients with aPL antibodies. Cross-reactivity of aPL antibodies with villous trophoblast cell membrane phospholipids may expose these cells to cytotoxic maternal immune effector cells (Hasegawa et al., 1990; McCrae et al., 1993). One of the essential considerations in pregnancy loss associated with aPL antibodies is whether the aPL-related pregnancy loss(es) may have been triggered by a nonrelated earlier miscarriage in an immunologically susceptible individual. There may be a biphasic pattern of pregnancy loss with embryonal death by 8.5 weeks and fetal complications from week 14 (Goldstein, 1994). Animal models have suggested that aPL antibodes per se appear to predispose to increased fetal resorption (Blank et al., 1991). However, the results of these studies are not conclusive (Blank et al., 1994a and Silver et al., 1997). Late fetal death is the most commonly found obstetric complication in

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APS. This has been attributed by early (Nilsson et al., 1975) and later studies (de Wolf et al., 1982) to placental infarction. Immune complex deposition on the trophoblast basement membrane has also been implicated in SLE-related fetal loss (Grennan et al., 1978). Elevated aPL levels have been associated with chronic uteroplacental vasculitis in the placental bed (Erlendsson et al., 1993).The occurrence of thrombosis and infarction in non-aPL fetal death, as well as inflammatory changes in aPL-related fetal death, suggests that the end-organ damage (placenta) in aPL disease is multifactorial and may have complex humoral and cellular interactions together with coagulation pathway abnormalities in the maternal circulation, villous trophoblast surface, and within the fetoplacental circulation. Antitrophoblast cytotoxicity initiated by maternally derived aPL antibodies cross-reactive with fetal trophoblast phospholipid epitopes and phosphatidylserine may induce chronic inflammation in the villi (Hasegawa et al., 1990). IV. p2-Glycoprotein I

PSGPI, a plasma protein,was first described in 1961 (Schultze et al., 1961) and has been the subject of extensive research in autoimmune disease. P2GPI is associated with different lipoprotein fractions in plasma and is also designated apolipoprotein H (Lee et at., 1983). PZGPI is a single-chain polypeptide of 326 amino acids with an apparent molecular mass of 50 kDa and is highly gIycosylated (Lozier et al., 1984).The carbohydrate content of P2GPI has been reported as being approximately 18%of its molecular mass (Schultze et al., 1961) and, when tested in phosphate buffer at pH 7.4, exists as 40% P sheet, 30% P turn, and 30% random coil (Walsh et at., 1990). P2GPI is a member of the complement control protein repeat (CCP) or short consensus repeat (SCR) superfamily (Reid and Day, 1989). The SCR is found in proteins involved in the regulation of the complement system (e.g., C4b-binding protein and factor H) and in some noncomplement proteins (selectin family and factor XIII). Although the first four of the five domains are typical examples of this CCP superfamily, the fifth domain is aberrant, containing an additional disulfide bond and a long C-terminal tail. P2GPI is highly conserved among mammalian species, suggesting that it plays an important physiological role (Kandiali and Krilis, 1994). Haptoglobin and factor H, two other members of this superfamily, are not bound by anti-P2GPI antibodies. Haptoglobin is used routinely as a control protein for antibody binding to PBGPI, as nonspecific binding can be detected when compared to p2GPI. p2GPI could not bind complement C3b coated on both activator (zymosan) and nonactivator (sheep

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erythrocytes) of the alternative complement pathway, whereas factor H bound to both surfaces coated with C3b, suggesting that despite structural similarities, these proteins had distinct nonoverlapping functions (Puurunen et nl., 1995). Although P2GPI has been characterized structurally, the tertiary structure of p2GPI and its biological function are not clear. p2GPI has a highly conserved pattern of cysteine residues (Steinkasserer et nl., 1991). Molecular modeling has suggested that a highly positively charged sequence in the fifth domain of P2GPI is surface exposed (Steinkasserer et nl., 1992; Sheng et nl., 1996). This had been predicted previously using the known tertiary structure of factor H, another CCP protein. This surfaceexposed net positive charge could well explain the binding of P2GPI to negatively charged surfaces, e.g., anionic phospholipids (Wurin, 1984), heparin (Polz, 1979), and DNA (Kroll et nl., 1976). Although PZGPI has been shown to be an absolute requirement for autoimmune aPL antibodies to bind in CL-ELISA, a preparation of P2GP1, proteolytically cleaved predominately between Lys317 and Thr318 in the fifth domain, lacked binding to anionic phospholipid (Hunt et al., 1993). This led to further work to map the major phospholipid-binding site on P2GPI initially with peptide inhibition studies (Hunt and Ki-ilis, 1994) and then with sitedirected niutagenesis (Sheng et al., 1996).The lysine-rich segment in the fifth domain ( Lyszxz-Lys2si)has been shown to be the major phospholipidbinding site on PZGPI. Modification of amino acid residues on P2GPI by potassium thiocyanate treatment completely destroys binding capacity, indicating the crucial involvement of lysine residues in the binding of P2GPI to anionic phospholipids (Kertesz et al., 1995). Many of the proposed physiological functions of p2GPI involve its phospholipid-binding properties. The binding of P2GPI to anionic phospholipids had been assessed using multilamellar, predominantly anionic phospholipid vesicles under nonequilibrium conditions (Wurm, 1984). Data suggested a high-affinity interaction of p2GPI with phospholipid in the 10-20 nM range. Zn vivo, however, physiological membranes contain significantly lower concentrations of anionic phospholipids, and normal plasma levels of P2GPI could easily displace Gla-containing proteins from cell membranes disrupting normal homeostatic mechanisms. Moreover, normal plasma concentrations of sodium and divalent cations would inarkedly inhibit this charge-dependent interaction. Physiological concentrations of PZGPI do not have much effect in in vitro coagulation tests unless anti-P2GPI antibodies are also present (Oosting et al., 1992; Roubey et al., 1992; Galli et al., 1992; Matsuda et al., 1993). Extrapolation of the calculation of the apparent dissociation constant for P2GPI binding with physiological anionic phospholipid membranes, to M, suggests that

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P2GPI alone may not be able to displace other coagulation proteins from these membranes (Willems et al., 1996).However, the binding of a complex of an IgG anti-P2GPI molecule bivalently to two P2GPI molecules could have a markedly higher affinity for that anionic membrane in vivo and hence displace other coagulation proteins. In vitro, the presence of less procoagulant proteins will result in a delay in the clotting time, a plausible explanation for the lupus anticoagulant phenomenon. Excess phospholipid will allow the capture of other coaguIation proteins again and restore the clotting time back to normal levels. As the target antigen of pathogenic antibodies in APS, much research has gone into studying the interaction of these antibodies with P2GPI. Three hypotheses have been proposed to explain the interactions between P2GPI and anionic phospholipids, allowing subsequent binding of aPL antibodies. They are (1) a shared epitope on the phospholipid-PZGPI complex (McNeil et al., 1990), ( 2 ) a cryptic epitope exposed on P2GPI when it interacts with anionic phospholipids (McNeil et al., 1990), and ( 3 ) increased density of PZGPI captured on the anionic phospholipid (Roubey et al., 1995). There has been increasing evidence to suggest that aPL antibodies bind preferentially to P2GPI immobilized on anionic phospholipids or certain synthetic surfaces (irradiated plates), whereas binding in the fluid phase is weak and often nondetectable. It is unlikely that the same epitope on P2GPI is exposed by different surface interactions, as shown by work done with monoclonal antibodies that are immunoreactive with p2GPI bound to anionic phospholipids and to irradiated plastic wells (Wang et al., 1995). The intrinsic low affinity of anti-P2GPI antibodies in APS is significantly enhanced when there is an increased density of P2GPI bound to a negatively charged surface. It has been found that the binding of purified monoclonal anti-p2GPI antibodies on y-irradiated polystyrene wells was higher than on untreated wells. This binding is most likely due to increased density of BSGPI, as the amount of iodinated p2GPI retained on irradiated wells after the same amount of protein was coated overnight was 200% higher than untreated wells (Kandiah and Krilis, 1996a). Matsuura and colleagues ( 1994) have reported that polyclonal human aCL antibodies and a monoclonal murine aCL antibody bound PZGPI coated on electron or y-irradiated microtiter wells but not on untreated wells. The degree of binding depended on the irradiation dose, and aCL binding to PZGPI adsorbed to these wells correlated well with that of P2GPI complexed to solid-phase CL. Antibodies binding to P2GPI on these irradiated wells were only competitively inhibited by the simultaneous addition of CL-coated latex beads mixed together with P2GP1, but were

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unaffected by the addition of excess PBGPI, CL micelles, or CL-coated latex beads. These findings again support the hypothesis that aCL antibodies are low-affinity antibodies and that interaction of antibodies to P2GP1, the target antigen, requires capture of this protein to an appropriately charged surfice. Roubey et al. (1995)have has shown that Fab’ fragments of patient IgG demonstrated little or no binding to PZGPI on y-irradiated polystyrene wells, whereas the whole molecule bound to P2GP1, suggesting a critical role for antibody bivalency. A. EPITOPEMAPPINGOF PHOSPHOLIPIDA N D ANTIBODY-BINDING SITES ON 02-GLYCOPROTEIN 1 Using synthetic peptides spanning the fifth domain of PSGPI, the peptide sequence Cys281-Lys-Asn-Lys-Asp-Lys-Lys-Cys288 inhibited binding of P2GPI to anionic phospholipid in a dose-dependent manner (Hunt and Krilis, 1994). Removal of the flanking cysteines abolished the ability of the peptide to inhibit phospholipid binding of native PSGPI, suggesting that the tertiary structure of P2GPI is important for phospholipid binding. By site-directed mutagenesis of the Lys residues in this amino acid sequence, binding of P2GPI to anionic phospholipid was reduced to about 50% by substituting one Lys residue with an Asp residue and abolished binding with two and three substitutions in this amino acid sequence (Sheng et at., 1996). Using monoclonal antibodies derived from patients with APS and peptide inhibition studies, linear epitopes in the C-terminal end of the fifth domain of P2GPI were recognized by these antibodies (Wang et al., 1995).Constructing two kinds of plasmid expression vectors that express P2GPI and the fifth domain of P2GPI only, polyclonal human anti-PSGPI antibodies were shown to bind the fifth domain of P2GPI directly and could inhibit, in a dose-dependent manner, the binding of these polyclonal antibodies to whole molecule P2GPI coated on irradiated microtiter wells (Yang et al., 1997). Their results suggest that the antigenic epitope for antibody binding is in the fifth domain. B. MOLECULARMODELINGOF P2GPI MODULES 1. Introduction To determine and understand the function of PSGPI, it would be useful to know its three-dimensional (3D) structure. Regrettably, no experimentally determined structure of P2GPI is available. However, it has been shown that the 3D structure of a protein may be calculated with useful accuracy if its amino acid sequence is sufficiently simi!,i to that of a protein with a known 3D structure (Sanchez and Sali, 199713). This comparative or homology modeling technique is particularly

524

DAVID A. KANDIAH ct

01.

useful when only low to medium resolution results are required, such as prediction of exposed regions that may interact with antibodies (de la Paz et al., 1986; Sali et al., 1993) and models of interaction based on electrostatic complementarity (Salemine, 1976; Sali et al., 1993). This review describes a comparative modeling study of the “sushi” domains of P2GPIs from five mammalian species. In particular, the authors review their previously published model of the fifth module in human P2GPI (P2GPI-5) and data on the cardiolipin (CL)-binding site on its surface (Sheng et al., 1996). The relationship between the various P2GPI modules is also discussed.

2. Alignment of SCR Modules in P2GPl and Factor H Amino acid sequences are known for 24 modules in P2GPIs from five mammalian species, including human, bovine, dog, mouse, and rat (Table I). Each P2GPI consists of 5 modules, except for rat PSGPI, which consists of only 4 modules. It has been suggested that the p2GPI modules are related to the SCR modules of factor H (Reid and Day, 1989). Four medium-resolution 3D structures of two different SCR modules from factor H (Table I) have been determined by solution nuclear magnetic resonance ( N M R ) and deposited in the Brookhaven Protein Databank (PDB) (Abola et al., 1987). Structures of the factor H modules and sequences of the 24 P2GPI modules were compared manually to obtain the

TABLE I SOURCESOF STRUCTURAL AND SEQUENCE DATAUSEDIN COMPARATIVE MODELINGOF /32GPI MODULES‘ Name Factor H modules with 3D structures determined by NMR Factor H, module 16 Factor H , module 15 Factor H, modules 15-16 P2GPI sequence Human Bovine Rat Mouse DO%

PDB Code

lHCC lHFI lHFH

Reference

Norinan et al. (1991) Barlow et al. (1993) Barlow et nl. (1993) Kristensen et al. (1991) Bendixen et al. (1992) Aoyama et ril. (1989) Nonaka et nl. (1992) Sellar et al. (1985)

‘ Structures were obtained from the sunliner 1993 release of the Brookhaven Protein Databank (Abola et al., 1987). The deduced amino acid sequences of /32GPIs were obtained from the GenBaik database (Bilofsky and Burks, 1988),except for dog PZGPI, which was obtained from the original paper.


525

alignment in Fig. 1. Even though sequence identity between factor H modules and P2GPI modules 1-4 is only approximately 20%, their alignment appears to be relatively accurate because there are few gaps and because of the invariability of the two disulfide bonds. Similarly, the alignment of the core regions of factor H and the fifth modules in P2GPIs is strongly determined by the assumption that disulfide bonds 5-50 and 36-61 in H-15 are equivalent to disulfide bonds 3-54 and 39-64 in /32 GPI-5 (Steinkasserer et al., 1993), even though only 9-13 residues out of 62 are identical between factor H modules and p2GP1-5~.The only major ambiguity arises around the 5-residue insertion at residue 21 in human P2GPI-5. There are also three single residue insertions in human P2GPI5 relative to factor H-15 at positions 47 (loop), 51 (loop), and 57 (extended chain). A major difference between factor H modules and 62GPI-Tj modules is that the latter has a 19 residue addition at the C terminus.

3. Overall Sirnilarities anzong Factor H and p2GPl Modules To find the clustering of the modules in factor H and the five /32GPIs, the table of percentage sequence identities for all pairs of the modules was calculated from their alignment (Fig. 2). This matrix was used with the Kitsch computer program (Felsenstein, 1985) to calculate a tree that expresses the relationships among the sequences of the modules, similar to the trees used to deduce the evolution of protein families. In this tree, differences between two groups of sequences are approximated by a vertical distance from the top of the tree to the highest node from which the two groups of sequences branch off. The sequences cluster in six groups. There is one group with the factor H modules H-15 and H-16 and five groups each containing the modules with the same relative position in the five P2GPI sequences. This arrangement suggests that the missing module in rat PZGPI is probably the first module because the group of the first modules does not contain a member from the rat P2GPI. The tree indicates that the first event in evolution of a multidomain PZGPI was gene duplication, which separated P2GPI module 5 from the predecessor of the rest of the modules. This may have been followed by the consecutive appearances of modules 1 and 3, with the final duplication resulting in modules 2 and 4. 4. The Three-Dirnensional Model of the FiBh Domain of Hurnan /32GPI The template structure for comparative modeling of P2GPI-5 was that of the 15th domain of human factor H. H-15 conformation has been determined by solution NMR (Barlow et al., 1993) (PDB code 1HFH). The alignment between P2GPI-5 and H-15 (Fig. 1)was used as input for MODELLER-11 (Saliand Blundell, 1993; Sanchez and Sali, 19974,which

lHCC

"

*o

20

lHFl

60

EKIPCSQPPQIEHGTINSSRSSQ-- ---ESYAHGTKLSYTCEGGFR-ISEENETTCYM-GKWSS-PPQCE EGLPCKSPPEISHGVVAHM--SD------SYQYGEEVTYKCFEGFG-IDGPAIAKCLG-EKWSH-PPSCI

Bovine-1

GRTCPKPDDLPFSTVVPLKT--------FYEPGEEITYSCKPGYVSRGGMRKFICPLTGLWPINTLKCT GRTCPKPDELPFSTVVPLKR--------TYEPGEQIVFSCQPGYVSRGGIRRFTCPLTGLWPINTLKCM

Dog-l

GRTCPKPDDIPFATVVPLKT--------FYDPGEQIAYTCQPGYVFRGLTRRFTCPLTGVWPrNTVRCE

Mouse-1

GRICPKPDDLPFATVVPLKT---------SYDPGEQlVYSCKPGYVSRGGMRRFTCPLTG~lNTLRCV

Human-2

PRVCPFAGILENGAVRYT----------TFEYPNTISFSCNTGFYLNG-ADSAKCTEEGKWSPELPVCA

Bovine2

PRVCPFAGILENGTVRYT----------TFEYPNTISFSCHTGFYLKG-ASSAKCTEEGKWSPDLPVCA PRVCPFAGILENGAVRYT----------TFEYPNTISFACNTGFYLNG-SSSAKCTEEGKWSVDLPVCT PRVCPFAGILENGVVRYT----------TFEYPNTIGFACNPGYYLNG-TSSSKCTEEGKWSE-LPVCA PRVCPFAGILENGIVRYT----------SFEYPKNISFACNPGFFLNG-TSSSKCTEEGKWSPDIPACA PIICPPPSIPTFATLRVYKPSAGN~---NSLYRDTAVFECLPQHAMFG-NDTITCTTHGNWTK-LPECR PITCPPPPIPKFASLSVYKPLAGN----NSFYGSKAVFKCLPHHAMFG-NDTVTCTEHGNWTQ-LPECR RVTCPPPSVPKFATLSVFKPLATN----NSLYGNKAVFECLPHYAMFG-NDTITCTAHGNWTT-LPECR

Human-l

Dog-2 Rat-2 Mouse-2

Human-3 Bovine3

Dog-3 Rat-3

RlTCPPPPlPKFAALKEYKTSVGN---.SSFYQDTVVFKCLPHFAMFG-NDTVTCTAHGNWTQ-LPECR

Mouse-3

RlTCPPPPVPKFALLKDYRPSAGN----NSLYQDTVVFKCLPHFAMIG-NDTVMCTEQGN~R-LPECL

Human-4

EVKCPFPSRPDNGFVNYPAKP-------TLYYKDKATFGCHDGYSLDG-PEEIECTKLGNWSA-MPSCK EVRCPFPSRPDNGFVNHPANP--------VLYYKDTATFGCHETYSLDG-PEEVECSKFGN~A-QPSCK EVKCPFPSRPDNGFVNYPAKQ-------ILYYKDKAMYGCHDTYTLDG-PEVVECNKFGN~A-QPSCK EVKCPFPSRPDNGFVNYPAKP-------VLSYKDKAVFGCHETYKLDG-PEEVECTKTGN~A-LPSCK EVKCPFPPRPENGYVNYPAKP-------VLLYKDKATFGCHETYKLDG-PEEAECTKTGAWSF-LPTCR

B0"l"ed

Dog-4 Rat4 Moue-4

111

Human-5 Bovine5

Dog-5 Rat-5 Mouse-5 PDB-lH(C :

BBB

AF

BBBB

PhD

BBBBBBBBBBB

JMC

BBBB

Homolog GOR

a0

30

ZO

* **

so

70

60

80

ASCKLPVKKATVVYQGERVKIQEKFKNGMLHGDKVSFFCKNKEKKCSYTEDAQCID-GTIE--VPKCFKEHSSLAF~TOASDVKPC ASCKLSIKRATVIYEGERVAIQNKFKNGMLHGQKVSFFCKHKEKKCSYTEDAQCID-GTIE--IPKCFKEHSSLAF~TDASDVKPC ASCKLSVKKATVLYQGERVKLQE~FKDGMLHGQKVSFYCKNKEKKCSYTEDAECID-GTIE--IPKCFKEHSSLAF~TDASDVKPC ASCKLSVKKATVLYQGQRVKIQDQFKNGMMHGDKVHFYCKNKEKKCSYTEEAQCID-GTIE--IPKCFKEHSSLAFWKTDASDVTPC ESCKLPVKKATVLYQGNRVKIQEQFKNGMMHGDKIHFYCKNKEKKCSYTVEAHCRD-GTIE--IPSCFKEHSSLAFVM(TDASELTPC

BBBB

B

HH

H

EBB

BBBBBBBB

B BB

BBBBBB

BBBBB BBBBBBBBB

BBBBB

BBB EBB

HHHHH

BB

HHH

BBB

HHHHHHHHHH H

HH

H

BBBB

BHHHHH HHHHH

HHHHHHHHHH

B

BBB

B

BBBBBB HHHH

BB

BB H H

HHHHHH

527


100 L

80 k

z w

P

zz w

3

40

2 u)

s

I

15

FIG. 2. Clustering of the SCR modules from human factor H and five mammalian P2GPIs.

produced a model of P2GPI-5 containing all main chain and side chain nonhydrogen atoms. [Modeller is available at URLhttp://guitar.rockefeller.edu:pub/modeller and also as part of Quanta InsightII, and GeneExplorer (MSI, San Diego, CA, USA; e-mail [email protected])]. The standard automated modeling procedure was used, except that additional distance restraints were imposed on the 19 residue extension at the C terminus. These restraints were obtained as follows. First, four secondary structure prediction methods were applied to human P2GPI-5 (Fig. 1).Three of the four methods resulted in an approximately correct

Frc;. 1. Alignment of the amino acid sequences of the modules from five P2GPIs and modules 15 and 16 from human factor H. The top line refers to the residues in 1HFI. The stars indicate the Lys residues that were mutated to the Glu residues. The line PDB-1HFI contains the secondary structure assignments for lHCC from the corresponding PDB (protein data bank) file. The predictions by the following secondary structure prediction methods are shown: AF, a method based on the physicochemical properties of the residues (Ptitsyn and Finkelstein, 1983); PhD, a neural network method (Rost and Sander, 1993); JMC, a neural network method (Chandonia and Karplus, 1996); Homolog, a method based on residue statistics (Biou et al., 1988); and GOR, a method based on the residue statistics (Biou et ul., 1988). The secondary structure predictions are indicated by H for helix and B for strand.

528

DAVID A. KANDIAH et a[

P

structure content, and all three of these predicted a strand starting at position 71 within the 19 residue C-terminal extension. The method that did not predict a P strand in this region also gave an incorrect secondary structure composition, with many residues predicted as helical. On the basis of these results, the authors predicted that region 71-75 in p2GPI5 was indeed a p strand. The partner of this strand must be one of the known strands in the rest of the molecule. An examination of the preliminary model suggested that the only two possible partners are strands from residue 4-10 and from residue 57-63, with strand 71-75 lying between and/or on top of these two strands. Any other possibility would have involved main chain knots or breaking the disulfide bond to the C-terniinaI Cys residue. Given the assumption that strand 71-75 interacts with strands 4-10 and/or 57-63, there are still four possibilities for the register of the predicted strand with the existing strands. These were explored by generating 3D models that satisfied as well as possible 24 lower distance bounds on the interstrand C ff distances, each restraint set corresponding to one of the four strand registers. The best model was then identified as the model that had minimal restraint violations, best 3D model quality index of Eisenberg and co-workers (Luthy et al., 1992), and the smallest number of residues other than Gly and Asn that had positive angles (Fig. 83, see color insert). The 3D-PROFILES quality index of this representative P2GPI-5 model is 21.5, which is within the allowed range for the protein of the same size as 62GPI (Luthy et al., 1992).This quality index can be compared with the quality indices for the experimental structures of the H-15 and H-16 modules in PDB files lHFI, lHCC, 1HFH-15, and 1HFH16, which are generally, but not always, higher at 30.3, 21.0, 30.1, and 24.4, respectively. The fold of the p2GPI-5 model consists of eight strands, organized in two distorted P sheets with long coiled regions connecting the strands (Fig. 3). There are no helices.

5. Electrostatic Properties of Human P2GPI-5 Electrostatic terms in the potential energy often give rise to specific interactions in complexes (e.g., that between a Lys and a sulfate at contact distance). However, in order to understand or to predict the nature of a complex between two molecules, it is often useful to look at their global electrostatic potential. If the structure of only one ligand is known, it is particularly helpful to examine its electrostatic potential for possible binding sites of the other ligand. This is true in the present case where the interaction between a positive (the protein) and a negative (cardiolipin) ligand is considered and the detailed structure of cardiolipin is not available. Thus, to investigate more closely which particular amino acid residues are critical for phospholipid binding by the intact fifth domain of P2GP1,


529

electrostatic properties of the 3D model of the fifth domain of human P2GPI (Fig. 8-4, see color insert) were examined. The electrostatic potential on the surface of P2GPI-5 and its mutants was calculated with GRASP (Nicholls et al., 1991), a computer program that uses the finite difference method to solve the linearized PoissonBoltzmann equation. A net charge of -1 was assigned to each Asp and Glu residue and a net charge of +1 to each Lys and Arg residue. Each His was assigned a neutral charge because P2GPI is active in plasma at a pH of about 7.2. Models with all hydrogen atoms and partial charges from the CHARMM-22 force field were used for electrostatic calculations. Although there are considerable uncertainties in the positions of positive charges at the end of long Lys and Arg side chains on the protein surface, these have a small effect on the global features of the electrostatic potential considered below (Sali et nl., 1993). Most of the positively charged side chains (14 out of 16) are located on the surface of two regions. The first of these regions is defined by segments 40-46,63-66, and 81-84 (top face of the module in Fig. 4A). The second region is defined by one long and wide omega loop 3-28 (left face in Fig. 4A). Most of the negatively charged residues (8 out of 11) are located in segments 33, 50-62, and 67-80 (right face in Fig. 4A). The pronounced positive electrostatic potential above the top region in P2GPI-5 is predicted to be significantly reduced if any one of the three Lys residues in the center of the top region is mutated to the glutamic acid residue (Fig. 4B). The sequences of the fifth modules from the five species are highly similar (- 80%),which is reflected in the similarities of the charge distribution and of the electrostatic potentials. For example, the central segment Cys39Cys46 of the top region is identical in all five species, as is Lys66, whereas Lys82 is present in three of the five species.

6. Location uf the Cardiolipin-Binding Region in Human P2GPl-5 Both positively charged faces on P2GPI-5 are likely to attract negatively charged ligands such as cardiolipin (Fig. 4A). However, because the top positively charged face contains peptide 40-46, which is known to bind cardiolipin (Hunt and Krilis, 1994),the three central charges in this particular region are predicted to be part of the binding site for cardiolipin in the intact P2GPI-5 domain. Moreover, the mutation of these three Lys residues to Glu residues is predicted to prevent the interaction between CL and P2GPI-5 (Fig. 4B). These predictions are similar to those made based on homology modeling alone (Steinkasserer et al., 1991, 1992). To test this prediction, the cDNA for human P2GPI was inserted into the baculovirus viral DNA BacPAK 6 for expression in insect cells (Sf21) (Sheng et al., 1996). As discussed previously, site-directed mutagenesis

530


was then performed to assess the role of the individual amino acids in the Lys40-Lys45 loop in phospholipid binding and anti-P2GPI activity. It was found that residues Lys42, Lys44, and Lys45 were indeed critical for p2GPI binding to anionic phospholipids, but not crucial for direct binding of P2GPI by anti-P2GPI antibodies. As mentioned earlier, it has been shown that cardiolipin binds to an isolated peptide Cys39-Cys46 with the two flanking Cys residues, but not to Lys40-Lys45 or to Ser39-Ser46, where the flanking Cys residues were replaced by Ser (Hunt and Krilis, 1994).Thus, the conformation of segment 40-46 is likely to be critical for phospholipid binding. It appears that the flanking Cys residues form a disulfide bond that favors the peptide conformation in the peptide-phospholipid complex, thus increasing the free energy of binding via reducing its entropy. This is explained by the 3D model ofP2GPI-5 as follows. Even though the two flanking Cys residues are not disulfide bonded to each other in the native molecule, their relative position in the model is consistent with such a bond (Fig. 3). As a consequence, a nonnative disulfide bond between Cys 39 and Cys 47 is expected to favor the native conformation for the intervening peptide segment. The model for interaction is not sufficiently detailed to distinguish between a specific electrostatic interaction that requires a certain peptide sequence and an interaction that relies on charge density without many steric restrictions. Nevertheless, the model did serve as the basis for informed sitedirected mutagenesis experiments that provided more information on the binding of phospholipids to P2GPI. AND CHARACTERIZATION OF THE GENEENCODING C. CLONING MOUSEP2GPI

A mouse ES genomic library in the bacteriophage P1 cloning system was screened using polymerase chain reaction (PCR). A Hind111 fragment was shown to contain the entire mouse P2GPI gene and was ligated into the pBluescript SK vector for further analysis and sequencing. This plasmid clone was digested with different restriction enzymes, and some fragments were further subcloned into pBluescript SK vectors for sequencing. The mouse P2GPI gene was subsequently found to be encoded by eight exons spread over about 18 kb of genomic DNA. Exon 1 contained the 5’untranslated region, the 19 amino acid long signal peptide, and the first 2 amino acids of the mature p2GPI protein. Exons 2-7 contain the rest of the protein-coding sequences. Exon 8 contained the last 19 codons and the entire 3’-untranslated region. The exons correlate well with the structural domains. CCPl is encoded by exon 11, CCP I1 by exons 111 and IV, CCP I11 by exon V, CCP IV by exon VI, and CCP V by exons VII and

531


VIII (Sheng et al., 1997) (Fig. 5). The mouse PZGPI gene has been localized to distal chromosome 11 (Nonaka et al., 1992), whereas the human P2GPI gene has been assigned to chromosome 17 (Haagerup et al., 1991). However, comparative mapping of human and mouse genomes has shown that the mouse distal chromosome 11 has extensive homology with human chromosome 17 (Buchberg et al., 1989). The amino acid sequence of p2GPI for mammalian species dwovered so far reveals a large degree of homology to the human sequence: mouse (76.1%), bovine (83%), and rat (80%).Alignment of these sequences shows that the fifth domain is the most highly conserved, suggesting that the main functional activities of the protein are present here. ' V. lmmunogenicity and Animal Models

In order to determine if autoantibodies are pathogenic in vivo, suitable animal models need to be studied. A murine model of autoimmune vascular disease (NZW X BXSB/F1) was first described (Hang et al., 1981). Other autoimmune strains predisposed to lupus were studied and showed autoantibodies reactive in a standard CL-ELISA (Gharavi et al., 1989). NZW X BXSB/Fl mice also have thrombocytopenia and were found to have anti-

A

I

ATG

I

AATAAA

STOP

FIG.5. Organization of the mouse PZGPI gene. (A) The structure of the mouse PZGPI gene is shown with restriction enzyme sites. The positions of exons are shown as boxes, and the introns are shown as lines connecting the exons. Restriction sites indicated: X (XhoI), B (BamHI), E (EcoRV), and Xb (XbaI). (B) SCR repeat domain structure of P2GPI. The positions of the translation initiation site (ATG), the polyadenylation site (AATAAA),and the termination codon are indicated. S, signal peptide (Sheng et al., 1997)

532

DAVID A. KANDlAH et 01.

bodies to P2GPI similar to that seen in autoimmune human APS patients (Hashimoto et al., 1992). Two groups have suggested that aPL antibodies have a direct role in causing pregnancy loss in vivo. Passive immunization of normal pregnant mice with human polyclonal (Branch et al., 1990; Blank et al., 1991) antibodies or monoclonal aPL antibodies (Bakimer et al., 1992) have been shown to result in increased fetal loss and fetal resorption and lower mean weights of embryos and placentas compared with mice immunized with normal immunoglobulins. However, one of these groups have since suggested that passive immunization with human IgG polyclonal aPL antibodies had variable effects on murine pregnancy outcome. The rate of fetal death did not increase uniformly with increasing doses of IgG and was unrelated to the individual patient’s medical history (Silver et al., 1997). BALBk mice immunized with CL mixed with PZGPI, CL alone, PZGPI alone, or buffer alone were studied. Mice immunized with CL mixed with PSGPI produced high levels of anti-P2GPI antibodies and antibodies reactive in a standard CL-ELISA. Mice immunized with CL alone did not produce aPL antibodies, and mice immunized with PSGPI alone produced anti-02GPI antibodies (Rauch and Janoff, 1992). Another group suggested that immunization of mice with P2GPI produced a high percentage of fetal resorption in utero when the mice were mated, suggesting an induced model of the APS (Blank et al., 1994b).To study the issue of pathogenesis and thrombosis in an animal model, ~nechanicalstimulus of exposed femoral veins in CD-1 mice was used to promote clot formation. Mice actively immunized with PZGPI and human IgG aPL antibodies from patients with APS developed propagation of the clot and slower dissolution (Pierangeli et al., 1996). Immunization of MLW+ + mice with PZGPI produced aPL antibodies. The development of neurological dysfunction and production of antinuclear and anti-DNA antibodies was controversial, with two opposing conclusions (Cote et al., 1994; Aron et al., 1995). Further research in this area needs to be done as another study has suggested that immunization of BALB/c mice with a monoclonal human aPL antibody (H-3) induces neurological and behavioral defects (Ziporen et al., 1997). Polyclonal antibodies purified from patients with APS, with a PZGPI affinity column, have binding characteristics similar to anti-PZGPI antibodies induced by immunization of a rabbit with human PZGPI. Some of the polyclonal human autoantibodies bound both PZGPI and anionic phospholipids. The binding to anionic phospholipids involves ionic interactions as the binding was reduced significantlyin the presence of high ionic strength buffers (Kouts et al., 1995). Nine monoclonal antibodies derived from NZW X BXSBF1 mice had two populations of antibodies with P2GPI reactivity and anionic phospholipid reactivity in the absence of p2GPI.


533

These latter antibodies, as with the polyclonal human antibodies, had charge-dependent binding to the anionic phospholipid with abolishment of binding in the presence of high ionic strength buffers (Monestier et al., 1996). Anti-P2GPI antibodies had a clear preference for purified murine P2GPI in a fashion similar to the preference human polyclonal antibodies from patients with APS had for purified human PSGPI. The analysis of the V region sequences of these antibodies suggest that cationic residues in the H chain CDR3 are important for their charge-dependent phospholipid reactivity. Sequence analysis of one of the monoclonal antibodies that recognized P2GPI in a phospholipid-free system, with little change in binding in the presence of high ionic strength buffers, did not reveal any cationic amino acid residues. The structural features of the VH-D-JH junctions of these monoclonal autoantibodies further support the view that an increased frequency of unusual V(D )J rearrangements contribute directly to the development of murine autoimmunity (Monestier et nl., 1996). The presence of these animal models of APS allow for the comparative study of the mechanisms of action of autoantibodies and induced antibodies. This will allow the continuing study of therapeutic interventions that may prevent the clinical manifestations of APS. VI. Prothrombin

Lupus anticoagulant antibodies could potentially inhibit any of four procoagulant phospholipid complexes or two anticoagulant phospholipid reactions. A number of early reports suggested a role for plasma proteins in the activity of lupus anticoagulants. The lupus anticoagulant “cofactor” phenomenon, i.e., the addition of normal plasma to patient plasma, increasing the inhibition of coagulation, was first attributed to the presence of prothrombin (Loeliger, 1959).Autoantibodies to prothrombin were shown in two patients with the lupus anticoagulant-hypoprothrombinemiasyndrome (Bajaj et al., 1983). Circulating prothrombin complexes were found in 74% of patients with LA antibodies and normal prothrombin levels (Fleck et al., 1988). The authors and others have shown that LA can react with human prothrombin directly on phospholipid-free, high-binding (irradiated) ELISA plates (Arvieux et al., 1995, Kandiah and Krilis, 19974, phospholipid-bound prothrombin (Bevers et al., 1991), and phospholipid alone (McNeil et al., 1989; Pierangeli et al., 1993; Kandiah and Krilis, 199713). Antiprothrombin antibodies have been found to have immunological prediction of myocardial infarction in men (Vaarala et al., 1996). In a study of 233 patients with aPL antibodies, 26% had IgG and/or IgM

534


antiprothrombin antibodies. There was poor correlation between antiprothrombin and anticardiolipin and anti-P2GPI antibodies in this same patient population. Univariate analysis suggested that antiprothrombin IgG correlated well with a history of venous thrombosis, but this effect was lost in the multivariate analysis, whereas anti-PBGPI IgG was the only variable that showed statistical significance ( Forastiero et al., 1997). In another retrospective study of SLE patients with aPL antibodies, the presence of LA antibodies was the only variable that had statistical significance in the multivariate analysis of association with venous thrombosis (Horbach et al., 1996). The varying results obtained by different groups on the prevalence and pathological links of antiprothrombin antibodies cannot be explained by the patient population studied alone. The method of performance of the antiprothrombin-ELISA is different in different studies, varying from the buffers used to dilute the samples and block the prothrombin-coated wells, to the cutoff levels determined. Some investigators use buffer-only wells as controls, which may be important, as it deducts nonspecific binding that can occur with the high binding plates used. If any meta-analysis is to be performed on these studies, to make generalizable deductions on the role of antiprothrombin antibodies, this important variable would need to be considered. It has been shown that LA antibodies in some patients with APS can be separated into antibodies positive in the d R W clotting assay and the dKCT. The immunoreactivity of these separate populations of autoantibodies cannot be explained by their immunoreactivity to P2GPI or prothrombin (Kandiah and Krilis, 1997b). Affinity-purified antiprothrombin antibodies from different patients had different reactivities in these two clotting assays (Kandiah and Krilis, 1997a).This observation was in variance with indirect studies on plasma reactivities, which suggested that anti-P2GPI reactivity corresponded to a prolongation in the d R W assay and antiprothrombin reactivity with prolongation in the dKCT assay (Gdli et al., 1995), but was supported by another study on a large population of patients that did not find a difference in the plasma reactivities in the dRWT and dKCT clotting assays and their anti-p2GPI and antiprothrombin reactivities ( Forastiero et al., 1997). The anticoagulant activity of antiprothrombin antibodies appears to be dependent on their recognition of a phospholipid-human prothrombin complex that inhibits both the conversion of prothrombin into thrombin in the prothrombinase complex (Bevers et al., 1991) and the tenase complex (Permpikul et al., 1994). There appears to be a high species specificity to human prothrombin in the functional assays (Bevers et al., 1991; Rao et al., 1995). LA IgG from two patients inhibited the activation of human but not bovine prothrombin in a purified prothrombin activation system

ANTIPHOSPIIOLIPID SYNDROME

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(Bevers et al., 1991), whereas LA IgG from a third patient inhibited the activation of both human and bovine prothrombin (Galli et al., 1993). This is in variance to the immunoreactivity of the purified antibodies that recognized both human and bovine prothrombin coated on microtiter wells and on a Western blot, although the binding to human prothrombin was substantially higher in 6 of the 14 preparations studied. Twelve of the preparations showed a significantly increased binding to human prothrombin and 9 to bovine prothrombin in the presence of phosphatidylserine and calcium ions. Further experiments with phosphatidylserine/phosphatidylcholine ( P S R C ) vesicles, soluble prothrombin, and LA IgG failed to explain why LA IgG inhibits human prothrombin activation more effectively than it inhibits bovine prothrombin activation (Rao et al., 1995). In a study of 59 patient plasmas with aPL antibodies, 90% showed reactivity to prothrombin bound to phosphatidylserine in the presence of calcium, whereas only 58% of these plasmas had reactivity to prothrombin coated directly on high binding wells (Galli et al., 1997). These authors suggested that the mode of presentation of prothrombin in solid-phase influenced its recognition by antiprothrombin antibodies. They postulated that these differences were produced either due to clustering and conformational orientation of the prothrombin bound to phosphatidylserine, allowing better capture of the antibodies, or that the capture of prothrombin-antiprothrombin complexes may be better in the presence of calcium ions. This may also be due to the patient population studied, as in patients with antiprothrombin antibodies, the binding to prothrombin coated on irradiated surfaces in the absence of calcium ions was significantly higher than for prothrombin bound to phosphatidylserine in the presence of calcium. This applied to both plasma samples, as in the previous study as well as to affinity-purified antiprothrombin antibodies through a prothrombin column. The dissociation constant calculated for these antibodies was in the region of 200 nM, which showed about 10 times higher affinity than anti-/32GPI antibodies purified from a P2GPI column (Kandiah and Krilis, 1997a). Both studies, however, confirmed the heterogeneity of antiprothrombin antibodies in coagulation assays with no one assay detecting these antibodies consistently. VII. lupus Anticoagulant Antibodies and Protein C Activation

LA antibodies have been shown to have multiple effects on protein C. Results in the literature have been contradictory, with some researchers finding a significant inhibition on the rate of activation of protein C by thrombin on endothelid cells by purified LA IgG (Cariou et al., 1988), whereas others could not confirm this (Oosting et al., 1991; Keeling et al.,

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1993). LA antibodies have also been shown to prevent the inactivation of factor Va by protein C. These appeared to be IgGs directed against negatively charged phospholipid-protein complexes of either protein C or protein S (Oosting et al., 1993). APC resistance (i.e., the association of dysfunctional APC with a venous thrombotic tendency) predominates in LA plasma, but is not restricted to the presence of the Arg506-Gln point mutation on factor V (Bokarewa et al., 1995). VIII. lupus Anticoagulant Antibadies and Phosphaiidylethanolamine

The presence of phosphatidylethanolamine (PE) has been shown to augment LA activity and inhibit the anticoagulant effect of activated protein C (APC) in vitro. This effect appeared to arise from interference of LA antibodies with APC activity by binding to PE or the complex of APC and PE (Smirnov et nl., 1995). aPE antibodies have also been shown to require plasma cofactors in their binding to PE, including high and low molecular weight kininogens (HMWK and LMWK) and, less frequently, prekallikrein and factor XI (Sugi and McIntyre, 1995). Kininogens inhibit thrombin-induced platelet aggregation. Kininogendependent IgG aPE markedly increased thrombin-induced platelet aggregation in vitro, whereas kininogen-independent IgG aPE did not (Sugi and McIntyre, 1996). Hence, kininogen-dependent aPE could cause thrombosis in vivo by disrupting the antithroinbotic effects of kininogen. As PE can increase the procoagulant activity of vesicles containing PS and PC, and aPE has been associated with thromboembolic events, the pathogenesis of the thrombosis in these patients is multifactorial. PE undergoes the transition from lamellar to hexagonal I1 phase under certain physiological conditions, and mice immunized with hexagonal PE develop phospholipid-dependent inhibitors of coagulation (LA antibodies) (Rauch and Janoff, 1990). Preincubation of aPL-positive plasma with hexagonal-phase I1 PE has been shown to reduce or even abolish the prolongation of clotting times in phospholipid-dependent coagulation assays, suggesting that this phospholipid is the target antigen for some LA antibodies (Rauch et al., 1989). These authors have suggested that the target antigen for these antibodies may be a complex of PE and human prothrombin (Rauch et al., 1997), although these experiments have been performed in clotting assays and not in a purified system. IX. Antiphospholipid Antibodies and Endothelial Cells

When a more physiological surface, such as endothelial cells, is used for the assembly of the prothrombinase complex, only 18% of IgG fractions


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with LA activity were able to inhibit prothrombinase activity (Oosting et al., 1993).Endothelial cells play a central role in the prevention of unwanted activation of the coagulation cascade in intact vessels, This antithrombogenic property of endothelial cell surfaces responds to physiological stimuli and is therefore susceptible to injur>i.aPL antibody-positive SLE sera, but not purified antibody, in the presence of low doses of tumor necrosis factor (TNF)stimulated procoagulant activity by cultured endothelial cells. However, no association was found with clinical thrombotic events (Hasselaar et al., 1989). A high prevalence of antiendothelial cell (AECA)-binding activity is found in sera from patients with APS (Hasselaar et al., 1990; Del Papa et al., 1992). P2GPI is able to bind resting endothelial cells and be recognized by monoclonal and polyclonal anti-P2GPI antibodies (Del Papa et al., 1995; Le Tonqueze et al., 1995). Although platelet binding has been related to the expression of anionic phospholipids on their cell membranes after activation, a comparable phenomenon is unlikely on resting endothelial cells that do not display such phospholipid distribution changes (Del Papa et al., 1992). Both polyclonal and monoclonal antiPSGPI antibodies can upregulate adhesion molecule expression after endothelial cell binding (Del Papa et al., 199s; Del Papa et al., 1997). Del Papa et nl. (1998) showed that P2GPI binds endothelial cell membranes through its fifth domain. The major phospholipid-binding site that mediates the binding of PZGPI to anionic phospholipids is also involved in endothelial binding (Fig. 6). Human umbilical vein endothelial cell (HUVEC) monolayers provide a suitable surface for P2GPI binding comparable to that displayed by anionic phospholipids dried on microtiter wells. The formation of p2GPI and anti-P2GPI complexes induces endothelial activation as supported by E-selectin expression and IL-6 secretion (Del Papa et d., 1998) (Fig. 6). X. Pathogenesis of the Antiphospholipid Syndrome

Patients with APS have a tendency to atherogenesis that is likely related to the multiple immunological abnormalities that occur in this condition. The oxidation of plastic microtiter plates that increases the capture of the target antigens for aPL antibodies may be an in vitro model of the vascular inflammatory processes that result in a high oxidative capacity in vascular walls. Oxidation of plasma proteins and oxygen-mediated endothelial injury decrease the physiological anticoagulant function of endothelium (Vaarala, 1997). Antibodies to oxidized low density lipoproteins (LDL) are associated with carotid atherosclerosis (Salonen et nl., 1992) and myocardial infarction (Puurunen et al., 1994). In patients with SLE, these antibodies have been

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FIG.6. PZGPI, anti-PZGPI, and endothelial cells. Anti-PSGPI antibodies bind a synthetic peptide spanning the fifth domain of PZGPI after capture on activated endothelial cells. PZGPI binds activated endothelial cells through the major phospholipid-binding site, KNKEKK (Del Papa et al., 1998). Human polyclonal anti-pSGPI antibodies bind to the same peptide sequence previously shown to support human monoclonal antibody binding after PSGPI bound to a negatively charged surface (Wang et al., 1995). Antibody binding upregulates adhesion molecule (E-selectin) expression and IL-6 secretion (Del Papa et al., 1995, 1998).

shown to cross-react with autoantibodies detected in the standard cardiolipin ELISA to PZGPI (Vaarala et al., 1993). Monoclonal anti-/32GPI antibodies derived from NZW x BXSB/F, mice also cross-react with oxidized LDL (Mizutani et al., 1995). Lipids are transported in blood as lipoproteins, macromolecular complexes of lipids and proteins (apolipoproteins). The properties and functions of apolipoproteins include being structural components of lipoproteins, the regulation of enzyme activity, and binding of lipoproteins to cell surface receptors for internalization and catabolism (Laker and Evans, 1996).Lipoprotein(a), which consists of an LDL particle and apolipoprotein(a), has been shown to be a strong independent risk factor for coronary heart disease (Mbewu and Durrington, 1990; Scott, 1991). Lipoprotein(a) has physiological interactions with coagulation and fibrinolytic systems (Hajjar et al., 1989; Miles et al., 1989). By studying its cDNA sequence, apolipoprotein(a) has been shown to have marked similarities in structure with plas-

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minogen (McLean et al., 1987). Elevated levels of lipoprotein(a) have been reported in patients with APS, with significantly higher levels in patients with arterial than venous thrombosis (Yamazaki et al., 1994). It has been shown that a protein ligand for apolipoprotein(a) is P2GPI. Using the repetitive apolipoprotein(a) kringle IV type 2 domain as bait to screen a human liver cDNA library by the yeast two-hybrid interaction trap system, 11 clones were identified, of which 8 were P2GPI (Kochl et al., 1997). Coimmunoprecipitation experiments showed specific binding of P2GPI to immobilized apolipoprotein(a), lipoprotein( a), and low density lipoproteins (which had been shown previously).The binding of P2GPI to lipoprotein(a) is via domains 2-4. These observations will lead to further investigations into the role of this P2GPI-apolipoprotein(a) interaction and its role in a prothrombotic tendency. Apolipoprotein(a) may form a multimeric complex with PBGPI, which would be cleared from the circulation by macrophages, a process that could be affected by anti-P2GPI antibodies. In a study of middle-aged men with elevated lipids but no autoimmune disease or history of thrombosis, antiprothrombin antibodies were significantly higher in men who developed myocardial infarctions or cardiac deaths than in controls. When all variables were analyzed, there was an interactive effect of antiprothrombin antibodies with smoking and triglyceride levels independently. Autoantibodes detected in the standard CLELISA and antibodies to oxidized low-density lipoproteins had an additive effect with antiprothrombin antibodies to the risk of cardiac events (Vaarala et al., 1996). The possible link between antibodies to P2GPI and the pathogenesis of thrombosis has been studied extensively. Anionic PLs promote initiation of the contact activation system in blood coagulation, which is inhibited in vitro by physiological concentrations of P2GPI (Schousboe, 1988). The autoactivation of factor XI1 in prekallikrein-deficient plasma in the presence of anionic PLs and cationic zinc is inhibited by PZGPI and the anti-p2GPI antibodieslP2GPI complex, which could behave as a LA (Schousboe and Rasmussen, 1995). One group classified aPLs into two types, depending on their sedimentation characteristics after adsorption with cardiolipin liposomes (Galli et al., 1992). If LA activity cosedimented with the liposomal pellet, antibodies eluted from the liposomes were P2GPI dependent in prolonging dRWT. However, the LA antibody present in the supernatant prolonged dRVVT, irrespective of the presence of PBGPI. This group subsequently suggested that antibodies positive in a d R W T clotting assay were more likely to be associated with clinical thrombosis. This study was done indirectly with patient plasma and retrospective analysis of the clinical histories (Galli et al., 1995). P2GPI at physiological concentrations has been shown to inhibit the generation of factor Xa in the presence of

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activated gel-filtered platelets (Shi et al., 1993). aCL antibodies interfered with this inhibition, whereas LA antibodies inhibited this process in a manner similar to PZGPI without any additive effect shown. P2GPI also appears to inhibit the prothrombinase activity of resting nonactivated platelets, lysed platelets, and phosphatidylserine/phosphatidylcholinevesicles (Nimpf et al., 1986), although it is unclear whether it requires small amounts of anti-PZGPI antibodies for this activity (Galliet al., 1993).PZGPI at physiological levels inhibits the factor Va-dependent prothrombinase complex (Mori et aE., 1996). However, in the same system, it potentiates thrombin generation in the presence of activated protein C (APC). This inhibitory effect was diminished by the addition of increasing concentrations of cephalin, suggesting that PZGPI competitively inhibits the binding of APC to the phospholipid surface. This group also showed that the anticoagulant activity of APC was significantly potentiated in P2GPIdepleted plasma, an effect that was reduced with the addition of increasing concentrations of p2GPI. (These in d t r o reactions are illustrated in Fig. 7.) The affinity of P2GPI for phospholipid is increased in the presence of anti-P2GPI antibodies (Willems et al., 1996). Hence in individuals with these antibodies, the P2GPI-anti-PZGPI complex may well displace other coagulation proteins affecting homeostatic mechanisms. Although the affinity constant for polyclonal anti-/32GPI to PZGPI is low at 3.4-7.2 pM (Tincani et al., 1996), this affinity constant is increased with dimerization of P2GPI (Sheng et al., 1998). In a study of 46 patients with SLE and autoantibodies detected in a standard CL-ELISA, comparisons of binding specificities and avidity of binding of the 22 patients with APS and the 24 patients without clinical manifestations of the APS were made. The authors found that while all patients had a positive result in the standard CL-ELISA, the absorbance values were higher in the group with APS. Reactivity in an anti-fi2GPIELISA was significantly more in patients with APS. Urea, a chaotropic agent interfering with electrostatic interactions, was able to reduce binding of the antibodies to p2GPI in both the standard CL-ELISA and the anti-P2GPI-ELISA, mainly in the group without clinical manifestations (Vlachoyiannopoulos et al., 1998). This suggested that patients with APS have a significant increase in autoantibodies detected in an anti-PZGPI ELISA and that these antibodies are generally of high avidity as compared to the autoantibodies found in individuals without clinical manifestations. Auger et al. ( 1995) reported that heparin-induced thrombocytopenia developed in 56.5% of patients with LA antibodies with an increased occurrence of thrombotic events. Although the mechanism is not clear, this could be due to heparin binding to P2GP1, enhancing the antibody binding to anionic phospholipids, e.g., activated platelets. Unfractionated

FIG.7. PZGPI effects in coagulation reactions. In oitro experiments with p2GPI suggest that it inhibits activation of the intrinsic pathway (Schousboe, 1988); inhibits Xa generation (Shi et al., 1993), inhibits prothrombinase activity of human platelets (Nimpf et al., 1986), and modulates the anticoagulant activity of activated protein C on phospholipid (Mori et d.,1995).

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heparin has been shown to enhance aPL binding to P2GPI in the presence of phosphatidylserine as well as changing the electrophoretic mobility of P2GP1, which did not occur with low molecular weight heparin (McNally et al., 1994). Hence the perceived anticoagulant properties of PZGPI may be altered by unfractionated heparin and may influence its use in clinical practice for the prophylaxis and treatment of thrombosis associated with these autoantibodies. Another group suggested that heparin reversibly bound aPL antibodies in vitro as shown by depletion of aPL antibodies after passage through a heparin affinity column and by dose-response heparin inhibition of aPL to CL in the presence of PZGPI (standard CLELISA) (Ermel et al., 1995).This effect would be mediated by the binding of PZGPI to heparin. The activation of human umbilical vein endothelial cells by IgG autoantibodies from patients with APS, as measured by increased monocyte adhesion, has been shown to be PZGPI dependent. Interestingly, rabbit polyclonal anti-PSGPI antibodies also activated endothelial cells (Simantov et al., 1995). p2GPI adhesion to endothelium has been described in normal placental vessels. In the placental samples studied, increased P2GPI deposition was found by indirect immunofluorescence on the trophoblast surfaces of placentas from patients with persistently raised titers of aPL antibodies (La Rosa et al., 1994). aPL antibodies have been eluted from placentas of women with elevated serum aPL antibodies. PZGPI was present in placental eluates from both control and aPL affected pregnancies (Chamley et al., 1993). Using reverse transcriptase polymerase chain reaction (RT-PCR),placental cells were shown to synthesize p2GPI transcripts. Immunoblotting experiments suggested that P2GPI is localized in syncytiotrophoblast and extravillous cytotrophoblast. Anti-P2GPI antibodies may therefore bind placental PZGPI, inhibiting their function in vivo (Chamley et al., 1997). The function of placental p2GPI is unclear at the moment, but may have an effect on placental circulatory hemostatic mechanisms. The binding of P2GPI to endothelial cells, via the cluster of Lys residues (Del Papa et al., 1998), suggests the same possibilities as for anionic phospholipid binding. This binding may increase antigen density and/or induce conforinational changes, alIowing for the capture of circulating antiP2GPI antibodies. Anti-peGPI monoclonal antibodies have been shown to exert LA activity in vitro by enhancing the binding of PZGPI to phospholipids (Takeya et al., 1997).This clustering may hinder the lateral mobility of coagulation proteins, affecting the fine balance between their procoagulant and anticoagulant activities. This effect may also occur in PZGPI binding to endothelial cell membranes perturbing endothelial function. It is therefore possible in vivo that autoantibodies directed against P2GPI induce endothelial cell activation, which in the presence of some other


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insult may trigger a thrombotic event. Hence aPL antibodies may independently influence atherogenesis by moderating coagulation reactions toward hypercoagulation by an as yet unexplained mechanism. XI. Laboratory Investigations of the Antiphospholipid Syndrome

As aPL antibodies are increasingly shown to be a heterogeneous group of autoantibodies, the need to perform multiple immunoassays and coagulation tests has become imperative. This is especially important in prospective studies of patients with APS as subsets of patients may be identified who are at particular risk of clinical events and may be identified by certain laboratory tests or a combination of tests. Standardization of assays for anticardiolipin antibodies and lupus anticoagulants have been fraught with difficulty, despite numerous attempts to perform this by international standardization workshops and committees. A. ANTICARDIOLIPIN ANTIBODIES At the third international workshop held in 1992,the delegates confirmed that bovine P2 glycoprotein I (PSGPI) supported the binding of purified aPL antibodies to CL (Harris et al., 1994). The authors and others have now found that there are patients who have selective binding to human P2GPI and may therefore have persistent negative aCL titers in conventional immunoassays. The workshop delegates also found binding of purified antibodies to ovalbumin and casein used in the blocking and diluting buffers, which suggest that any protein solutions used in an ELISA system need to be checked for contamination with PSGPI, even in small concentrations (Harris et al., 1994). It has also been found that non-fatty acid-free bovine serum albumin used in a number of laboratory experiments contains sufficient P2GPI to support binding of anti-P2GPI antibodies. The current standard ELISA kits are increasingly using P2GPI as a discriminator of autoimmune aCL antibodies from true aCL antibodies that do not require PZGPI for direct binding to CL. Nine commonly used commercial kits for measuring aCL antibodies were compared with a standardized in-house method. The authors found marked differences in the positivity rate between kits rangng from 31 to 60% for IgG and from 6 to 50% for IgM (Reber et al., 1995). The P2GPI content of the dilution buffers and the wells supplied with the lats were significantly different. Despite extensive efforts over the years to achieve standardization, these results suggest that some technical aspects need to be reevaluated, including cutoff points and the use of controlled amounts of P2GPI or incubation times. P2GPI has been shown to be inhibitory to the binding of antibodies from patients with chronic infections (Hunt et al., 1992). This is due to

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the competition of PZGPI with the positively charged antibodies for binding to anionic phospholipids (Monestier et al., 1996). Hence high positivity of aCL titer may be found in some patients with chronic infections, and in the right clinical setting, further tests need to be performed to identify the binding specificities of these antibodies. SLE patients with clinical manifestations of APS but negative for conventional tests in the aCL assay and LA clotting tests were tested for immunoreactivity to various phospholipids, including phosphatidylserine, phosphatidylinositol, phosphatic acid, phosphatidylcholine and phosphatidylethanolamine (Roch et al., 1997). No correlation was found between detected antibodies to phosphatidylethanolamine and clinical manifestations of APS. Overall, in all patient groups, the authors found no additional benefit from testing for immunoreactivity to other phospholipids other than CL (Roch et at., 1997). The use of these new PZGPI immunoassays may supplant the conventional aCL iminunoassays in use because of their improved specificities but not completely replace them because of their relative costs. Clinical studies highlight the importance of detecting aPL antibodies and quantitating their levels and therefore stratifylng the risk for each patient so that optimum treatments could be developed. B. LUPUS ANTICOAGULANT ANTIBODIES The LA/aPL antibody subcommittee has met annually since 1988 to update the nomenclature, methods, and standardization practices of LA testing. Screening tests for LA need to be sensitive, and the amount of phospholipid in the test system is a critical determinant of sensitivity. The reactivity of a particular patient LA antibody is also important. Hence the use of at least two sensitive screening tests is important in detecting the LA antibody. The combination of tests also needs to detect reactivity to different parts of the clotting cascade (Kandiah and Krilis, 1996; Triplett, 1995). Current criteria for the diagnosis of LA antibodies are:

1. Prolongation of at least one PL-dependent clotting test. 2. Evidence of inhibitory activity shown by the effect of patient plasma on pooled normal plasma. 3. Evidence that the inhibitory activity is dependent on PL by the addition or alteration of PL, hexagonal-phase PL, platelets, or platelet vesicles in the test system originally used. 4. LAs must be carefully distinguished from other coagulopathies. Specific factor assays and the clinical history may be helpful in these situations (Brandt et al., 1995).


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Predictive tests that identify patients most at risk of the clinical manifestations of APS have not yet been developed. More recent test systems have looked at dot blots to various protein antigens, in vitro thrombin generation, functional assays to detect acquired APC resistance, and inhibition of downregulation of factors Va and VIIIa (Triplett, 1996). Laboratory tests for the detection of aPL antibodies have become more specialized. Hence, clinical and research studies looking at aPL antibodies and clinical features need to be precise in the performance and reporting of the methodology of their tests, which will promote the reproducibility of results across different population groups and allow accurate interpretation of data obtained. XII. Antiphospholipid Syndrome and Future Therapies

Antiphospholipid syndrome belongs to a wide spectrum of clinical disorders that are categorized as autoimmune disorders based on the presence of autoantibodies and/or the finding of lymphocytic infiltrates in the target organs. As in most autoimmune disease, the particular role of the immune system in the initiation and progression of the disease remains uncertain. The finding that plasma proteins are the target antigens for some of the aPL antibodies has gone a long way in investigating the potential pathogenesis in APS, especially in relation to thrombosis and atherogenesis. Some patients respond well to aspirin alone, whereas other patients require high doses of anticoagulation to prevent recurrent vascular events. Autoantibodies in APS have increasingly been shown to be heterogeneous, and the combination of antibodies may be the precipitant for the diverse clinical manifestations of these patients. Not only does the combination of binding specificities of the antibodies need to be identified, but also those patients who respond to existing therapies. In disease in general, and autoimmune disease in particular, the nature of the inciting antigen is central to the definition of the disease and therapeutic interventions. In APS, as in other autoimmune diseases, the three processes that interact to produce disease need to be studied. These are (a) selection in the thymus of a repertoire of T cells that discriminates self from nonself, i.e., tolerance; (b) lymphocyte activation after a potential autoreactive T cell has emerged from the thymus and entered the peripheral circulation; and (c) preprogrammed cell death that eliminates T and B cells with particular autoreactive properties, i.e., apoptosis. In PAPS and APS associated with SLE, there is considerable diversity in the spectrum of autoantibodies and severity of disease among affected indwiduals. One theory proposed for the diversity of autoantibodies is the presence of common structural motifs found in many diverse implicated

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molecules, e.g., phosphodiester groups found in single-stranded DNA and phospholipids, and P2GPI- and DR9-binding motif (Fujisao et al., 1996). “Antigenic spreading,” in which the T-cell response to a particular peptide antigen leads to the involvement of other T cells with a progressively wider spectrum of activity (Lehmann et al., 1993),has also been proposed. Thymic tolerance failure may also explain the development of autoimmune disease. This may arise by (a) anatomical sequestration of self-antigens, not exposing developing T cells in the thymus to these self-antigens during their maturation; (b) formation of neoantigens or cryptic antigens when the target antigen is conformationally changed, thus not being recognized by T cells (Fatenejad et al., 1993); and (c) failure to suppress autoreactive T cells in the periphery by some as yet unexplained mechanism (Clark and Ledbetter, 1994). The ability of B cells to internalize antigens and present them as peptide/ class I1 complexes on their cell surface may play a critical role in “antigenic spreading,” promoting a progressively more polyclonal T-cell response against an autoantigen (Mamula and Janeway, 1993). In addition to the crucial processes generating autoreactive T cells, apoptosis and factors leading to the death of cells and their removal in the thymus and the periphery may be important in pathogenesis. It has been shown that M R U p r autoimmune mice with T-cell antigen receptor (Y chain knockout (which lack ap T cells) lack IgG and IgM aCL antibodies compared to their wild-type controls and that CD40 ligand-deficient M R U lpr mice lack IgG aCL antibodies compared to their wild-type controls. These observations suggest that the development of aCL antibodies in these autoimmune mice is dependent on cognate T/B-cell interaction (augmented by the presence of (YPT cells) and is facilitated by the binding of CD40 ligand on activated T cells to CD40 on B cells (Kang et al., 1997). Multiple genes are involved in autoimmune disease pathogenesis. The strongest argument for genes predisposing to disease is increased disease frequency in monozygotic twins. Disease concordance in this twin population, however, is generally less than 30%, and other factors such as random events occurring during the maturation of antigen receptors on T and B cells and environmental factors must also play a significant part. The detection of viral protein epitopes similar to that found in PZGPI may promote further insights into the mechanism of initiation of pathological events and clinical manifestations in APS (Celli et al., 1997). P2GPI is in the CCP family. The complement system and its receptors play an important role in immune defense, linking humoral and cellmediated responses. Complement receptor (CRI), through its binding to activation products of complement C3, can activate cells and induce chemotaxis (Fearon, 1991). B lymphocytes have complement receptor

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CR2, which may form complexes with CD19 that are important in B-cell activation (Matsumoto et al., 1991). Complement receptors also serve to concentrate immune complexes on the surface of B cells, leading to antigen presentation by the B cell (Tuveson et al., 1991). In deriving therapy for an autoimmune disease such as APS, the immune system needs to be modulated so as not to result in total immunosuppression. This modulation may be performed at the level of autoimmune T-cell generation in the thymus or their clonal expansion in the periphery. Current treatment appears to only address the end result of the autoimmune process, i.e., treatment of the thrombosis. Synthetic miniotope peptides, characterized by (a) the inability to activate T cells while (b) retaining the ability to bind immune B cells, may be used to tolerize B cells in an antigen-specific manner. B-cell tolerance entails administering such peptides conjugated to multivalent, stable, nonimmunogenic valency platforms in order to abrogate antibody production via B-cell anergy or clonal deletion after the cross-linking of surface immunoglobulin. The ability to modulate B-cell activity in humans on an antigenspecific basis, using single signal inactivation of target B cells, has been identified as a means of pharmacological intervention in antibody-mediated pathologies (Coutts et al., 1996). Although the exact molecular nature of the target epitopes recognized by aPL antibodies is unknown, the use of peptides derived from epitope libraries may allow for the construction of successful tolerogens. XIII. Summary and Conclusions

Advances in defining the target antigen(s) for the autoantibodies in the APS highlight the inadequacies of the current classification of these autoantibodies into anticardiolipin and LA antibodies. The discovery that p2GPI is the target antigen for the autoantibodies detected in solid-phase immunoassays has opened a number of areas of research linking these autoantibodies to atherogenesis and thrombus formation. Although the role of p2GPI in the regulation of blood coagulation in unclear, current evidence suggests that anti-fi2GPI antibodies interfere with its “normal” role and appear to promote a procoagulant tendency. The expansion of research in this area and the diversity of the clinical manifestations of patients with APS have resulted in the inclusion of molecular biologists and pharmaceutical companies joining immunologists, hematologists, rheumatologists, obstetricians, neurologists, vascular surgeons, and protein and lipid biochemists in attempting to understand the pathophysiology of this condition. Although the published literature may result in conflicting results and introduce new controversies, developing standard-

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TABLE I1 VASCULAR THROMBOSIS Presence of Antiphospholipid antibodies Homocystinemia Protein deficiencies Protein C Protein S Antithrombin 111 APC resistance Genetic point mutations

Arterial Thrombosis

Venous Thrombosis

Yes

Yes

Yes

Yes Yes Yes Yes Yes

ized laboratory methods and extrapolation of in vitro experimental results to the in vivo situation will advance our understanding of the regulation of the immune system and its interaction with normal hemostatic mechanisms. Since the authors’ last review in 1991, the study and understanding of the pathophysiology of APS have evolved from lipid biochemistry to molecular techniques that may eventually provide specific therapies for the clinical manifestations of this condition. Although current treatment has improved the morbidity associated with this condition, especially in improving pregnancy outcomes, future therapies, as outlined in this review, may specifically address the biological abnormalities and have fewer side effects. Better diagnostic tools, such as magnetic resonance imaging with perfusion studies, will allow the study of the true incidence and prevalence of vascular flow changeshssue ischemia and infarction associated with aPL antibodies and help determine treatment and prophylaxis for APS patients. APS is still the only hypercoagulable condition where both arterial and venous beds can be affected independently or in the same individual (Table 11).

ACKNOWLEDGMENTS Work from the authors’ laboratories was supported by grants from the National Health and Medical Research Council (NH&MRC) of Australia and the National Institutes of Health. DAK was supported by a NH&MRC Postgraduate Biomedical Scholarship. AS is a Sinsheimer Scholar and was a Fellow of the Jane Coffin Childs Memorial Fund for Medical Research at the Department of Chemistry, Harvard University.

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INDEX

A Activation-induced cell death cbaracterization, 15-19 T cell deletion, 62 Adaptive immunity, 83-84 ADP-iibosylation Factor. see GTPases Adrenal gland, liypofiinction, 516-517 AICD, see Activation-induced cell death Aiway h~erresponsiveness,154-1.55 Allograft rejection. 155-157 Antibodies anticardiolipin, 543-544 antiprothrombin. 534-535 anti-02-glycoprotein I, 510-512 antiphospholipid, 508-509, 52-537 lupus anticoagiilant efrects. 535-536 laboratory studies, 544-545 location, 509-510 Anticardiolipin antibodies, 543-544 Antigenic spreading, 546 Antigen-presenting cells location, 315 MCH I1 loading, 320-321 targeting soluble antigens, 321-323 Antiphospholipid antibodies characterization, 508-509 endothelial cells, 536-537 A~itjphospholipidlsyndrome adrenal manifestations, 516-5 17 animal models, 531-533 anticardiolipin antihodies, 543-544 antiphospholipid antibodies, 536-537 APS-associated. 515 AVN-associated, 5 1R-5 19 cardiovascular manifestations, 512-513

characterization. 507-508 dermal nranifestations, 518 endothelial cells, 536-537 fuhire therapies, 545-547 02-glycoprotein I antihody binding sites, 523 characterization, 520-523 gene encoding characterization, 530-531 cloning, 530-531 molecular modeling alignment, 524-525 cardiolipin-binding region, 529-530 comparisons, 524-525 electrostatic properties. 528-529 fifth domain, 525, 527-528 structure, 523-524 phospholipid sites. 523 thrombosis link, 539-540 hepatic manifestations, 517 laboratory studies, 543-548 lripiis anticoagulant antibodies, 535-536 laboratmy studies, 544-545 neurological manifestations, 513-514 obstetric manifestations, 519-520 pathogenesis, 537-543 phos~~hatidyletlianolainine, 536 protein C activation. 535-536 piilmonary manifestations, 5 15-5 16 renal rnanifestations. 516 sllnlnlaly, 547-548 APC, we Antigen-presenting cells Apical surface. 399-400 Apoptosis, see dso Programmed cell death AICD, 15-19 IL-2, 14-19 inhibition, 251-253 initiating factor, 258-259

INDEX

mechanism, 269-270 mitochondria1 control, 257-259 promotion, 253-255 protein inhibition, 251-253 APS, see Antiphospholipid syndrome Arterial thrombosis, 513 Arthritis, collagen-induced, 162-164 Autoimmune disease organ-specific, 157-158 pregnancy effects, 519 spontaneous, 165-166 Autoimmune vascular disease, 531-532 Avascular necrosis of bone, 518-519 AVN, see Avascular necrosis of bone

B Bacteria, see also specijic species Bcl-2 homology, 263-264 IL-12 inhibition, 177-181 Basolateral surface, 399 B cells antigenic spreading, 546 CD4' expression, 327-328 CD4+ priming, 325-326 development, 42-47 1L-18 effect, 289 IL-12 effects, 148-151 IL-12 production, 102-104 IL-18R expression, 296-297 tolerizing, 547 BcI-2 apoptosis inhibition, 251-253 promotion, 253-255 cell physiology, 261 cell sunival, 266-269 characterization, 250-251 function, 256-257 ion channels, 265-266 bcalization, 255-256 structure, 263-264 Bcl-x,, CED, 268-269 ion channels, 265-266 structure, 261-263 Bone, avascular necrosis of, 518-519 Bordetelln pertusssis, 180 Borrelia hrgdorferi, 180

Brucelln abortus, 180 Brugin inalnyi, 187

C CnndicZu albicans, 184-185 Cardiolipin, 529-530 Cardiovascular disease, 512-513 Caspase-1, 286-287 CED Bd-x,,, 268-269 characterization, 248-250 mammalian homologs, 260-261 Cell cycle regulation, 11-12 Cell death, see Apoptosis Cell survival, 266-269 Cell viability pathways, 41-42 c-~).Y> 26-27, 41 Chaperones, protein assembly, 385-387 Chinese hamster ovary (CHO) cells, 91 CIA, see Collagen-induced arthritis Ciliary neurotrophic factor, 92-94 c-jttn, 41 c-myc, 26-27, 41 CNTF, see Ciliary neurotrophic factor Coat proteins families, 374-375 functions, 376-377 GTPases regulating, 377-381 Coccidioides inmitis, 186 Colitis, experimental, 164-165 Collagen-induced arthritis, 162-164 Crohn's disease, 164-165 Cyptococms neofonnans, 185 Cutaneous necrosis, superficial, 518 Cytokines, see ulso specijic cytokine CD4+ secretion, 332-334 IL-12-induced, 122-126 production, 146-148 Cytomegalovirus, 168-171

D Delayed-type hypersensitivity, 152-153 Dendritic cells, 113-114 Diabetes mellitus, 303-304 Dilysine motif, 391-392 Diphtheria toxin, 264

567

INDEX

E EAE, see Experiinental-allergic encephalomyelitis EAU, see Experimental uveoretinitis EBV-induced protein 3, 94 Ectrornelia, 344-346 Encephaloinyelitis, experimental-allergic, 160-162 Endocytosis, 39,3-394 Endoplasmic reticulum bidirectional transport, 389-390 degradation studies, 387-389 description, 369-372 MHC 11, 391 retention studies, 387-389 Endosomal system, 372 Endothelid cells, 536-537, 542 Endotoxin-induced liver injury, 301-303 Epidermal growth Factor receptor, 396 Epilepsy, APS-associated, 514 Epitope mapping, 523 ER, see Eiidoplasmic reticulum N-Ethylinaleimide, 381-382 Evolution coinbinatorial system, 418 foundations gene specificity, 421-424 mechanism, 418-420 superfamilies, 420-421 immunoglobulins amphibians. 437 birds, 437-438 bony fishes, 436-438 chondrichthytes, 433-436 emergence, 425-430 gene organization arrangement, 479-480 basic elements, 475-476 intron removal, 476-477 promoters, 478-479 species comparisons, 480-485 transcription, 477-478 heavy chains isotypes, 470-475 universal p, 465, 467-470 variable domains, 460, 463-465 jawed vertebrates, 430-433 light chains, 451-460 mammals, 438-439

molecular events, 485, 488-491 reptiles, 437-438 overview, 417 T-cell receptors, 425-430 jawed vertebrates, 430-433 species comparisons, 441-451 variable domain, 439, 441 Experimental-allergic encephalomyelitis, 160-162 Experimental colitis, 164-165 Experimental uveoretinitis, 162 Experimental viral infections, 168-171

F Factor H, 524-525 Fas receptors, 63

G PZ-Glycoprotein I antibody binding sites, 52-3 characterization. 520-523 gene encoding characterization, 530-531 cloning, 530-531 inoleciilar modeling alignment, 524-525 car&olipin-bin&ng region, 529-530 comparisons, 524-525 electrostatic properties, 528-529 fifth domain, 525, 527-528 structure, 523-524 phospholipid sites, 523 thrombosis link, 539-540 Anti-p2-Glycoprotein I antibodies, 510-512 Golgi network &-, 391 trans-, 371 Graft-uerms-host disease, 153-154 Growth factors activation, model, 19-20 IL-2, 10-11 GTPases subfamilies, 377-381 GTP-binding protein, 393-394

H Hematopoietic stein cells, 119-121 Hemorrhages, intraalveolar pulmonary, 515

568

INDEX

Hepatitis B virus, 340-341 Herpes simplex virus, 348-349 Histoplasma capstdatum, 185-186 HIV, see Human immunodeficiency v i m Human immunodeficiency virus IL-12 production, 172-176 pathological changes, 339-340 Hypercholesteroleinia, APS-associated, 513 Hyperresponsiveness,airway, 154-155 Hypersensitivity, delayed-type, 152-153 Hypoprothrombineriiia, APS-associated, 516

I IDDM, see Insulin-dependent diabetes melIitus Iininature dendritic cells, 316 Iininune system adaptive responses, 83-84 evolution combinatorial system, 418 foundations gene specificity, 421-424 mechanism, 418-420 superfamilies, 420-421 immunoglobulins, 425-430, 463-475 amphibians, 437 birds, 437-438 bony fishes, 436-438 chondrichthytes, 433-436 gene organization, 475-491 heavy chains, 460 jawed vertebrates, 430-439 light chains, 451-460 mammals, 438-439 molecular events, 485, 488-491 reptiles, 437-438 overview, 417,491-492 T-cell receptors, 425-430 jawed vertebrates, 430-439 species comparisons, 441-451 variable domain, 439. 441 function, 313-314 IL-2R role, 58-60 infectious agents and, 85, 351-352 Immunogenicity, 531-533 Immunoglobulin-binding protein, 385-387 Iminunoglobulins evolution amphibians, 437

birds, 437-438 bony fishes, 436-438 chondrichthytes, 433-436 emergence, 425-430 gene organization arrangement, 479-480 basic elements, 475-476 intron removal, 476-477 promoters, 478-479 species comparisons, 480-485 transcription, 477-478 heavy chains isotypes, 470-475 universal p, 465, 467-470 variable domains, 460, 463-465 jawed vertebrates, 430-439 light chains, 451-460 molecular events, 488-491 reptiles, 437-438 IgE, IL-18 effect, 292-294 intracehlar transport, 400-402 Infections, experimental viral infections, 168-171 Inflammatttion, 166-168 Influenza, 341-344 Insulin-dependent diabetes mellitus, 303-304 Insulin receptor substrate-1, 33 Interleukin-12 allograft rejection, 155-157 antitumor effects, 187-193 bacteria, 177-181 B cell response, 148-151 CHO cells, 91 CIA, 162-164 cloning, 86 colitis, 164-165 Crohn’s disease, 164-165 discovery, 85-86 EAE, 160-162 EB13, 94 expression, 114-1 19 fungal pathogens, 184-186 graft rejection, 153-154 helminthic parasites, 186-187 heterodimers, 90-92 homodimers, 90-92 IiyI)erresponsiveness,154-155 hypersensitivity, 152-153 IL-18-regulated, 294-295

INDEX

induction cytoldne, 122-126 dendritic cells, 113-114 infectious pathogens, 106- 107 inflammatory extrucelhlar matrix, 107 modulation, 108-113 T-cell dependent, 107-108 inflainmation. 166-168 mitogenic activity, 127-129 midtiple sclerosis, 160-162 NK cell-induction, 129-131 NK1 T cell induction, 133-134 nonhuinan sources, 89-90 organ-specific autoiininune disease, 157-158 production B cell, 102-104 measurement, 101-102 phagocytic cells. 104-106 protozoan parasites. 181-184 p35 subunit, 87-89 p40 subunit, 86-87, 90-92, 94 purification. 8G receptor. 95- 101 signaling, 95-101 spontaneous autoimmune disease, 165-166 stem cell effects, 119-121 structure, 92-94 T-cell induction, 131-133 Thl cells differentiation. 141-143 generation importance, 138-140 N K cells role, 139-140 process, 143-146 role, 134-136 polarization, 140-141 vaccinations, 148-151 viruses experimental, 168-171 HIV, 172-176 MAIDS, 176-177 ineasles, 171- I72 Interleukin- 18 B cell effect, 289 biologv, 287-294 characterization, 304-305 history, 282-283 host defenses, 298-301 IgE effect, 292-294

IL-12 regidation, 294-295 NK cell effects, 291-292 osteoclast effects, 294 pathological roles, 301-303 processing, 286-287 producing cells, 285-286 receptors, 294-298 role, 281-282 structure, 283-285 T cell effect, 287-289 Interleuldn-2 receptors binding, 3-4 cell cycle regulation, 11-12 cell survival regulation, 14-19 expression, 7-10 fiinction, 2 iinmune fimction, 58-60 lyniphocytes characterization, 42-47 genetic studies, 47-49 stnicture, 53-58 mechanism, 19-21 PHOX region, 24-26 PRHIII, 29-30 signaling cell viability, 41-42 characteristics, 21-22 downstream factors, 37-38 downstream pathways, 24-27 generation, 1-2 intracellular, 21-24 Stat proteins, 27-30 IRS-I, 33 JAK 3 defects, 52-53 lymphocyte effector function, 13-14 M A P kinase pathways, 30-32 mitogenic, 34-37 P13 kinase, 38-39 responses to, 10-19 'SHPS, 33-34 Src fantily kinases, 32-33 STAM role, 40-41 Stat& 38 target genes, 41 TOR role, 39-40 subunit chains cloning, 4-5 sharing, 5-6 striictiise, 4-5

569

570

INDEX

T cells deletion, 61-63 growth, 63-64 IntraceUular transport coat proteins, 374-377 endocytic pathways, 372 description, 392-393 mechanisms, 393-394 MHC 11, 396-398 T-cell activation, 394-396 GTPases regulation description, 377-381 SNARE mediation, 381-384 subfamilies, 377-381 mechanism, 373-374 overview, 369, 402 polarized cells apical surface, 399-400 basolateral surface, 399 description, 398-399 polymeric immunoglobulin receptor, 400-402 secretory pathway bidirectional transport, 389-390 characterization, 385 immunoglobulin-binding protein, 385-387 MHC 11, 390-392 T-cell antigen receptor, 387-389 secretory pathways characterization, 369-372 in vivo cytoskeleton role, 384-385 models, 383-384 organelle structure, 385 Ion channels, 265-266 Ischemia focal cerebral, 513 ocular, 515

J Janus tyrosine kinases mitogenic signaling, 34-35 signaling pathways, 24-27

KDEL proteins, 380, 390 Kidneys disease, 516 PI3 Kinase, 38-39

L LCMV, see Lymphocytic choriomeningitis viiruS Leishmania major, 299 Leishmania spp., 181-183 Lipoproteins, low-density, 537-539 Listeria mononjtogenes, 177-181 Livedo reticularis, 518 Liver diseases, 517 injury, 301-303 Lung diseases, 515-516 Lupus anticoagulant antibodies, 535-536 characterization, 509-510 laboratory studies, 544-545 phosphatidylethanolamine and, 536 LY294002, 39 Lymphocyte effector function, 13-14 Lymphocytes, see also specijic lymphocytes development characterization. 42-47 genetic studies, 47-49 role, 49-52 yL deficiency, 52-53 peripheral, structure, 53-58 Lymphocytic choriomeningitis virus, 168-171, 336,338-339 Lysosomal associated membrane protein-1, 397

x

MAIDS, see Murine AIDS Major histocompatibility complex class I1 antigen presentation, 317-321 endocytic compartments, 396-398 secretory system, 390-392 Malaria, 299-300 MAP kinase pathways, 30-32 MCMV, see Murine cytornegalovims Measles virus CD4+ control, 344 immune deviation, 171-172 Mesenteric vessels, thrombosis, 517 Meth-A tumor, 192 Microangiopathy, 516 Migraines, 514

571

INDEX

Mouse mammary tumor virus. 350-351 Mulitiple organ failure, 303-304 Multiple sclerosis, 160-162 Murine AIDS, 176-177 Murine cytomegalovirus, 168- 171 Mycohacterium auiurn, 179 Mycohacteriurn leprae, 179 Mycobadenurn tuberculosis, 178-179 Myocardial infarction, 533-534

Natural killer cells development, 46-47 IL-18 effect, 291-292 IL-12-induced, 129-131 Th1 generation, 139-140 Natural killer cell stimulatory factor, see Interleukin- 12 NEM-sensitive factor, 381-382 Nyppostrungyliis brasiliensis, 187

0 Organ-specific autoimmune disease, 157-158 Osteoclast. 294

P p35, 87-89 p40, 86-87, 90-92, 94 Permeability transition, 257-259 Phagocytic cells, 298 IL-2 production, 84 Phosphatidylethanolamine, 536 Phospholipids, 523 Phygocytic cells, 104-106 Picornavimses, 349-350 Pla.srorliurn spp., 184 Polarized cells apical surface, 399-400 basolateral surface, 399 characterization. 398-399 polymeric imrnunoglobulin receptor, 400-402 Polio virus, 349-350 Polymeric iniinunoglobulin receptor, 400-402

Poxvinises, 344-346 Pregnancy, failures, 519 Producing cells, 285-286 Programmed cell death, see also Apoptosis genetics, 247-250 pathway, 251 significance, 245-247 Protein assembly, 385-387 Protein C, 535-536 Prothronibin, 533-535 p40 subunit, 90-92, 94

Recombinase activating gene 1, 421-424

Snlnwnellri dublin, 179-180 Scliistosoma IruLnsoni, 186- 187 Serine/threonine kinases, 38-39 Severe combined immunodeficiency IL-2 defective, 59-60 IL-2 factors, 48-49 XSCID, 49-50 Signal recognition particle, 369 Skin lesions, 518 SNARE hypothesis, 373 vesicle fusion, 382-383 Spontaneous autoimmune disease, 165-166 Src family Idnases, 32-33 SRP, see Signal recognition particle STAM protein, 40-41 Stat transcription factors description, 27-30 IL-ZR, 27-30 IL-lZR, 100-101 Stem cells, hematopoietic, 119-121 Struiigyloi&s stercoralis, 187 Synthetic mimotope peptides, 547

T Target of rapamycin, 39-40 T-cell receptors antigen, 387-389

572 evolution emergence, 425-430 jawed vertebrates, 430-433 species comparisons, 441-451 T cells antigenic spreading, 546 CD4' activation administration, 314-319 APC targeting, 321-323 dosage, 314-319 location, 314-319 MHC I1 molecules, 317-321 Th cells, 324-327 B cell priming, 325-326 characterization, 330 effector functions cytokines, 332-334 cytomegalovirus, 347-348 hepatitis B virus, 340-341 herpes simplex virus, 348-349 HIV, 339-340 influenza, 341-344 kinetics, 332 LCMV, 336, 338-339 measles virus, 344 mechanism, 330-331 mouse mammary tumor virus, 350-351 picornaviruses, 349-350 poxviruses, 344-346 vesicular stomatitis virus, 346-347 function, 313-314 overview, 351-352 presentation, 314-319 soluble antigens MHC class 11, 317-319 development, 42-47 growth L 2 R , 63-64 IL-18 effects, 287-289 IL-18 expression, 294-295 IL-12 induced, 107-108 IL-12-induced, 131-133 IL-18R expression, 296-297

INDEX

NK1

IL- l2-induced, 133- 134 proliferation, 10-13 regulation, 394-396 Theiler's virus, 349-350 T helper cells activation, 324-327 autoimmunity, 157-158 differentiation. 141-143, 141-146 effector functions, 330-336 generation, 134-136 IL-12 role, 134-140 NK cells role, 139-140 IL-18 effect, 289-291 polarization, 140-141 stability, 136-138 Thrombosis anitprothrornbin-link, 534 deep venous APS-associated, 515 glomerular capillary, 516 P2-glycoprotein I link, 539-540 mesenteric vessels, 517 TN thymocytes, 46 Toxoplasina gondii, 183 Transporter of antigenic peptides, 390-391 Trichuris muris, 187 Ttypanosom cmzi, 183-184 Tumors, 187-193 Tyrosine kinases, 34-35 Tyrosine phosphatases, 33-34

Uveoretinitis, experimental, 162

v Vaccinations, 148-151 Vaccinia, 344-346 Venoocclusive disease, hepatic, 517 Vesicle fusion, 382-383 Vesicular stomatitis virus, 346-347

CONTENTS OF RECENT VOLUMES

Volume 68

Volume 66

Peptide Bindii'g Specificity"Id HLA 'lass Molecular and Cellular Mechanisms of T Autoimmunity Lymphocyte Apoptosis JUERCXN HAMMER. TIZ~ANA STURNIOI.O, JOSEF M PENNINCER A N D GUIDO A N D FRANCESCO SINIGAGLIA KHOEMEH Prenylation of R a GTPase Superfamily Proteins and Their Function in 1111111unobi 01 ogy ROBERTB. L O ~ E L I .

Role of Cytoldnes in Sepsis C. ERIKHACK, LUCIEN A. AARDEN, ~NI) LAMBERTUS G. TIIIJS Role of Macrophage Migration Inhibitory Factor in the Regulation of the Iininune Response CHRISTINE N. ME= AND R I C H A R J I BUCAL.A

Generation and TAP-Mediated Transport of Peptides for Major Histocompatibility Complex Class I Molecules MOMBURG A N D GUNTHER J. FRANK HAMMERLING

The Intrinsic Coagulation/Kinin-Fori~li~~~ Cascade: Assembly in Plasma and Cell Surfaces in InAainination ALLENP. KAPLAN,KLl5UMAM JOSEPH, YOJI SHIBAYAMA, SESIIA REDDIGARI, BEIIIHANE

Adoptive Tumor Iininunity Mediated by Lymphocytes Bearing Modified AntigenSpecific Receptors BROCKEH AND KLA~JS THOMAS KARJALAINEN

GHEBRESIIWET, A N D M I C H A E L

SILVEHBERC

CDB' Cells in Human Ininiunodeficiency

Membrane Molecules as Differentiation Antigens of Murine Macrophages ANDREWJ. M c K ~ i

Advances in Immunology Volume 70

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Advances in Immunology Volume 64

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Advances in Immunology Volume 28

Advances in Immunology Volume 70

Advances in Immunology Volume 45

Advances in Immunology Volume 34

Advances in Immunology Volume 1

Advances in Immunology Volume 10

Advances in Immunology Volume 30

Advances in Immunology Volume 82

Advances in Immunology Volume 31

Advances in Immunology Volume 52

Advances in Immunology Volume 48

Advances in Immunology Volume 56

Advances in Immunology Volume 12

Advances in Immunology Volume 21

Advances in Immunology Volume 41

Advances in Immunology Volume 71

Advances in Immunology Volume 84

Advances in Immunology Volume 43

Advances in Immunology Volume 73

Advances in Immunology, Volume 61

Advances in Immunology Volume 14

Advances in Immunology Volume 46

Advances in Immunology, Volume 106

Advances in Immunology Volume 19

Advances in Immunology Volume 99

Advances in Immunology Volume 81

Advances in Immunology Volume 4

Advances in Immunology Volume 80

Advances in Immunology Volume 64

Advances in Immunology Volume 62

Advances in Immunology Volume 28

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