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ADVANCES IN

Immunology VOLUME 79

This Page Intentionally Left Blank

ADVANCES IN

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

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

VOLUME 79

San Diego San Francisco New York Boston London Sydney

Tokyo

∞ This book is printed on acid-free paper.

Copyright C 2001 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 U.S. 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-2001 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. 0065-2776/01 $35.00 Explicit permission from Academic Press is not required to reproduce a maximum of two figures or tables from an Academic Press chapter in another scientific or research publication provided that the material has not been credited to another source and that full credit to the Academic Press chapter is given.

Academic Press A division of Harcourt Inc. 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www.academicpress.com

Academic Press Harcourt Place, 32 Jamestown Road, London NW1 7BY, UK http://www.academicpress.com International Standard Serial Number: 0065-2776 International Standard Book Number: 0-12-022479-8 PRINTED IN THE UNITED STATES OF AMERICA 01 02 03 04 05 06 SB 9 8 7 6 5 4 3

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1

CONTENTS

ix

CONTRIBUTORS Neutralizing Antiviral Antibody Responses

ROLF M. ZINKERNAGEL, ALAIN LAMARRE, ADRIAN CIUREA, LUKAS HUNZIKER, ADRIAN F. OCHSENBEIN, KATHY D. MCCOY, THOMAS FEHR, MARTIN F. BACHMANN, ULRICH KALINKE, AND HANS HENGARTNER

I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII.

Introduction Antiviral Antibody Responses Mechanisms of Neutralization by Antibodies The Role of Complement, Natural Antibodies, and Fc-Receptors in Antiviral Responses The Role of Antigen Structure, Organization, and Dose on B Cell Responses T-Cell Dependent Activation of B Cells in Viral Infections B Cell Unresponsiveness Examples of Nonclassical Virus Neutralization Virus Escape from Neutralizing Antibody Responses Immunological Memory Neutralizing Antibodies and Vaccine Strategies Conclusions References

1 2 6 9 12 16 17 21 24 28 31 34 34

Regulation of Interleukin-12 Production in Antigen-Presenting Cells

XIAOJING MA AND GIORGIO TRINCHIERI

I. II. III. IV. V. VI. VII. VIII.

Introduction The Discovery of IL-12 Molecular Structure of IL-12 Biological Functions of IL-12 Receptors of IL-12 and Signaling Producer Cells of IL-12 Pathways of IL-12 Induction Molecular Regulation of the Expression of IL-12 Genes v

55 55 56 58 58 59 62 66

vi

CONTENTS

IX. Cytokine-Mediated Regulation of IL-12 Production X. Role of Cell-Surface Receptors in the Regulation of IL-12 Production XI. Endotoxin-Tolerance-Mediated Inhibition of IL-12 XII. Inhibition of IL-12 Production by Anti-inflammatory Hormones and Small Molecules XIII. Looking to the Future References

73 77 80 80 81 82

Mechanisms of Signaling by the Hematopoietic-Specific Adaptor Proteins, SLP-76 and LAT and Their B Cell Counterpart, BLNK/SLP-65

DEBORAH YABLONSKI AND ARTHUR WEISS

I. The Role of Adaptors in Signaling by ITAM-Coupled Receptors: An Overview II. Genetic Models Reveal the Essential Roles of SLP-76, LAT, and BLNK in ITAM-Coupled Signaling Pathways III. Interactions Mediated by LAT, SLP-76, and BLNK and Their Role in Antigen Receptor Signaling IV. Mechanisms of Signaling by LAT and SLP-76 References

93 100 110 118 121

Xenotransplantation

DAVID H. SACHS, MEGAN SYKES, SIMON C. ROBSON, AND DAVID K. C. COOPER


Introduction The Need History of Clinical Xenotransplantation Choice of Species Mechanisms of Xenograft Rejection Therapeutic Approaches Nonimmunologic Barriers to Xenotransplantation Future of Xenotransplantation References

129 129 130 131 133 155 184 190 192

Regulation of Antibacterial and Antifungal Innate Immunity in Fruitflies and Humans

MICHAEL J. WILLIAMS

I. Introduction II. Recognition of Foreign Molecules III. Serine Protease Cascades and Thioester-Containing Proteins

225 228 235

CONTENTS

IV. Induction and Regulation of the Antimicrobial Genes V. Concluding Remarks References

vii 238 249 250

Functional Heavy-Chain Antibodies in Camelidae

VIET KHONG NGUYEN, ALINE DESMYTER, AND SERGE MUYLDERMANS


Introduction Natural Occurrence of Heavy-Chain Antibodies in Camelidae Adaptations in the H Chain The L Chains Heavy-Chain Antibody Generation Isolation of Antigen-Specific Heavy-Chain Antibodies, or VHHs VHH Structure and VHH–Antigen Interaction Biotechnological Importance of VHHs References

261 261 265 269 270 275 285 289 290

Uterine Natural Killer Cells in the Pregnant Uterus

CHAU-CHING LIU AND JOHN DING-E YOUNG

I. Immunobiology of Pregnancy: An Overview II. Uterine Natural Killer Cells of Rodents: Granulated Metrial Gland Cells III. Uterine Natural Killer Cells of Human and Other Mammalian Species IV. Biological Functions of Uterine Natural Killer Cells during Pregnancy V. Concluding Remarks References INDEX CONTENTS OF RECENT VOLUMES

297 299 307 310 321 321 331 341


CONTRIBUTORS

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Martin F. Bachmann (1), Institute of Experimental Immunology, Department of Pathology, University Hospital, CH-8091 Zurich, ¨ Switzerland Adrian Ciurea (1), Institute of Experimental Immunology, Department of Pathology, University Hospital, CH-8091 Zurich, ¨ Switzerland David K. C. Cooper (129), Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School, and Department of Medicine, Beth Israel Deaconess Medical Center/Harvard Medical School, Boston, Massachusetts 02129 Aline Desmyter (261), Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Sint Genesius Rode, 1050 Bruxelles, Belgium Thomas Fehr (1), Institute of Experimental Immunology, Department of Pathology, University Hospital, CH-8091 Zurich, ¨ Switzerland Hans Hengartner (1), Institute of Experimental Immunology, Department of Pathology, University Hospital, CH-8091 Zurich, ¨ Switzerland Lukas Hunziker (1), Institute of Experimental Immunology, Department of Pathology, University Hospital, CH-8091 Zurich, ¨ Switzerland Ulrich Kalinke (1), Institute of Experimental Immunology, Department of Pathology, University Hospital, CH-8091 Zurich, ¨ Switzerland Alain Lamarre (1), Institute of Experimental Immunology, Department of Pathology, University Hospital, CH-8091 Zurich, ¨ Switzerland Chau-Ching Liu (297), Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213 Xiaojing Ma (55), Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021 Kathy D. McCoy (1), Institute of Experimental Immunology, Department of Pathology, University Hospital, CH-8091 Zurich, ¨ Switzerland Serge Muyldermans (261), Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Sint Genesius Rode, 1050 Bruxelles, Belgium ix

x

CONTRIBUTORS

Viet Khong Nguyen (261), Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Sint Genesius Rode, 1050 Bruxelles, Belgium Adrian F. Ochsenbein (1), Institute of Experimental Immunology, Department of Pathology, University Hospital, CH-8091 Zurich, ¨ Switzerland Simon C. Robson (129), Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School, and Department of Medicine, Beth Israel Deaconess Medical Center/Harvard Medical School, Boston, Massachusetts 02129 David H. Sachs (129), Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School, and Department of Medicine, Beth Israel Deaconess Medical Center/Harvard Medical School, Boston, Massachusetts 02129 Megan Sykes (129), Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School, and Department of Medicine, Beth Israel Deaconess Medical Center/Harvard Medical School, Boston, Massachusetts 02129 Giorgio Trinchieri (55), Schering-Plough Laboratory of Immunological Research, 69571 Dardilly, France Arthur Weiss (93), Departments of Medicine and of Microbiology and Immunology, and the Howard Hughes Medical Institute, University of California, San Francisco, California 94143 Michael J. Williams (225), Ume˚a Centre for Molecular Pathogenesis, Ume˚a University, S-901 87 Ume˚a, Sweden Deborah Yablonski (93), Department of Pharmacology, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Bat Galim, Haifa 31096, Israel John Ding-E Young (297), Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, New York 10021 Rolf M. Zinkernagel (1), Institute of Experimental Immunology, Department of Pathology, University Hospital, CH-8091 Zurich, ¨ Switzerland

ADVANCES IN IMMUNOLOGY, VOL. 79

Neutralizing Antiviral Antibody Responses ROLF M. ZINKERNAGEL,1 ALAIN LaMARRE,1 ADRIAN CIUREA, LUKAS HUNZIKER, ADRIAN F. OCHSENBEIN, KATHY D. McCOY, THOMAS FEHR, MARTIN F. BACHMANN, ULRICH KALINKE, AND HANS HENGARTNER Institute of Experimental Immunology, Department of Pathology, University Hospital, CH-8091 Zürich, Switzerland

I. Introduction

Antibody responses have been studied in infectious diseases, particularly against toxins isolated from pathogenic bacteria. Indeed, antibody quality requirements for protection against diphtheria toxin were among the first to be elucidated (Von Behring and Kitasao, 1890; Ehrlich, 1906; Jerne, 1951). However, the inability to purify these toxins and the complexity of the biological material forced immunologists to use chemically pure substances in order to quantitatively study antigens and their reactions with antibodies (Evans, 1943). Therefore, immunochemists used haptens, usually small phenyl groups, that were easily synthesized in various configurations and could be linked to large proteins (Heidelberger, 1956; Eisen and Siskind, 1964). Because of their small size they could pass through the then-available dialysis bags to measure physicochemical parameters of antibody/antigen interactions and define binding affinities and specificities. Only recently has it become possible to express and purify biological substances sufficiently not only to repeat some of these analyses in vitro but also to correlate such measurements with protection against disease in vivo (Fazekas de St. Groth and Webster, 1961; Staudt and Gerhard, 1983; Webster and Rott, 1987; Laver et al., 1990; Bachmann et al., 1997b). Individuals with defective antibody responses, such as patients with Bruton’s disease or other antibody defects, are more susceptible to infections (Wilfert et al., 1977; Graham et al., 1983; Englund et al., 1998), as are mice devoid of B cells, such as the μMT mouse (Good and Zak, 1956; Kitamura et al., 1991; Brundler ¨ et al., 1996). Protective or neutralizing antibody responses, particularly against acute cytopathic agents, are among the most critical defense mechanisms (Sabin, 1981; Steinhoff et al., 1995; Zinkernagel et al., 1996). These responses represent the result of a long co-evolution between the host and various infectious agents. However, because of the shorter generation time and greater numbers, adaptation of infectious agents is considerably faster than that of the host. This review attempts to summarize from an evolutionary point of view evidence that neutralizing antibodies (nAbs) are key to protecting the species against many viruses, particularly cytopathic ones. Such viruses have the potential to upset 1

These authors contributed equally to this work. 1 C 2001 by Academic Press. Copyright All rights of reproduction in any form reserved. 0065-2776/01 $35.00

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ROLF M. ZINKERNAGEL ET AL.

the balance between host and pathogen; therefore, an equilibrium must have evolved to enable the survival of both. This balance is maintained by the ability of the host to mount rapid neutralizing antibody responses which is achieved by directly encoding virus-specific neutralizing antibodies in the germline. Thus, it is mandatory to assess the protective capacity of antibodies by in vivo adoptive transfer experiments or by in vitro neutralization assays, rather than measuring antigen binding in an enzyme-linked immunosorbent assay (ELISA). Interestingly, although not surprisingly, there is a certain geography of the antibody response: local IgA in the mucosa, systemic short-lived IgM and longlived IgG in blood. In addition, antibodies not only neutralize viruses and thereby prevent infection but also trap virus in the spleen and prevent it from reaching the blood. They thereby enhance immune responses in general and B cell responses in particular. This review also presents evidence that, in general, B cells are not negatively selected and are largely regulated by antigen structure and availability of cognate T help. We also review evidence supporting the notion that B cell memory, in the form of high neutralizing antibody serum levels, is the main contributor to immunological memory. Finally, both general and specific examples of neutralizing antibody responses are used to illustrate rules that govern B cell responses. These rules are also discussed with respect to their role in autoimmunity and how they can be exploited for vaccines. II. Antiviral Antibody Responses

During their development in the bone marrow, B cell precursors undergo sequential recombination-activating gene (RAG)-mediated rearrangements of D to J and V to DJ elements of the immunoglobulin (Ig) heavy-chain variable region (VH) and V to J elements of the Ig light-chain variable region (VL) (Tonegawa, 1983). Expression of functional Ig heavy and light chains promotes the transition of precursor B cells to small IgM/IgD-positive mature B cells. Autoreactive antibody specificities generated by random rearrangement are either deleted (Nemazee and Buerki, 1989; Nemazee and Buerki, 1989; Nemazee et al., 1991; Hartley et al., 1993) or edited by secondary VL and possibly VH replacements (Gay et al., 1993; Radic et al., 1993; Tiegs et al., 1993; Chen et al., 1995, 1997; Lang et al., 1996; Pelanda et al., 1997; Fang et al., 1998; Nemazee, 2000). B cells that emigrate from the bone marrow and populate secondary lymphoid organs constitute the primary B cell repertoire. The restricted number of V, D, and J gene segments that can be encoded in the genome limits the size of the potential repertoire. However, diversity of the preimmune repertoire of mice and humans can be further expanded by hypermutation, whereas other species, such as rabbits, sheep, pigs, and cattle, use gene conversion (Diaz and Flajnik, 1998). It has recently been suggested that, together with hypermutation, secondary Ig gene rearrangements may also contribute to repertoire diversification (Han

NEUTRALIZING ANTIVIRAL ANTIBODY RESPONSES

3

et al., 1997; Papavasiliou et al., 1997; Pelanda et al., 1997; Hertz et al., 1998; Meffre et al., 1998; Lopez-Macias et al., 1999; Wilson et al., 2000b). However, whether this considerably slower process confers a survival advantage to the host remains to be formally demonstrated. A. V(D)J REARRANGEMENTS IN ANTIVIRAL ANTIBODIES The primary repertoire of vesicular stomatitis virus (VSV)-specific antibodies was analyzed by isolating hybridomas 4 and 5 days after infection (Kalinke et al., 1996a). Surprisingly, 87% of clones analyzed used V genes belonging to the VHQ52 and Vκ 19–28 families. Despite their polyclonal origin, the majority of these clones expressed identical VH and VL germline segments. Because all these antibodies failed to bind and neutralize the 51.12 VSV variant (which was selected in the presence of one particular VHQ52/Vκ 19–28-positive antibody), they all appear to bind to the same subsite within the major antigenic site of the glycoprotein of VSV (VSV-G). Analysis of the primary response to influenza virus also revealed a restricted Vκ usage, with the predominance of a segment belonging to the Vκ 8 gene family (Clarke et al., 1990b). In contrast, the heavychain variable region genes displayed much greater diversity, with several VH families being used. In contrast to primary response antibodies, hybridomas isolated from secondary and hyperimmune anti-VSV responses expressed a more diverse set of V genes belonging to the VHJ558, VH7183, and VHQ52 families (Kalinke et al., 1996a). The majority of these antibodies were shown to bind a different subsite of the major antigenic site of VSV-G than the primary response antibodies. Thus, fine specificity diversification of secondary and hyperimmune responses was achieved by newly appearing V gene combinations. Similar findings were obtained from analysis of the secondary response to influenza virus where V region usage differed considerably from the primary response (Clarke et al., 1990a). B. THE ROLE OF HYPERMUTATION IN THE GENERATION OF VIRUS-NEUTRALIZING ANTIBODIES Interestingly, all anti-VSV mAbs isolated up to day 6 post infection, irrespective of which genetic elements they express, were devoid of somatic hypermutation (Kalinke et al., 1996a). This indicates the presence of VSV specificities in the mouse germline antibody repertoire. This was confirmed by the isolation of VSV-neutralizing antibody fragments from phage display libraries generated from naive mice (A. Lamarre, unpublished results). These findings are remarkable because VSV is a new-world virus of ruminants while Mus musculus is an old-world rodent. Therefore, the presence of VSV-specific, high-affinity antibodies in the germline cannot be a result of recent co-evolution; instead, this suggests that, in general, cytopathic viruses have been selected to fit the available antibody repertoire rather than vice versa (Roost et al., 1995; Bachmann et al., 1997b).

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Similarly, anti-influenza primary response antibodies were largely devoid of somatic mutations (Clarke et al., 1990b). In contrast to primary antibodies, secondary and hyperimmune mAbs did display hypermutations (Clarke et al., 1990b; Kalinke et al., 1996a). Nevertheless, as with the primary VHQ52/Vκ 19–28positive antibodies, hypermutated anti-VSV antibodies were not able to neutralize the virus variant 51.12. However, compared to the germline antibodies, hypermutated antibodies had approximately a 50-fold increase in their ability to neutralize wild-type VSV (Kalinke et al., 1996a). Therefore, hypermutation did not alter antibody specificity, as far as can be judged by binding competition assays, but it did improve binding quality and neutralizing capacity. To verify these conclusions, another more heterogeneous group of VSVneutralizing antibodies typical of secondary and hyperimmune responses was analyzed (Kalinke et al., 2000). Although these antibodies expressed numerous different light chains, they all used the same VH germline segment belonging to the VH7183 family in combination with the JH2 segment. Again, all hyperimmune antibodies contained hypermutations. Despite most of the hypermutated antibodies not being clonally related, some of them showed the same single somatic amino acid substitutions in CDR1 and CDR2. Because no virus variant resistant to neutralization by all VH7183/JH2-positive antibodies could be generated, they apparently bound different subsites within the major antigenic site of VSV-G. To analyze the impact of the common amino acid substitutions on virus binding and neutralization, the secondary response antibody VI24 of the VH7183/JH2 group was expressed as a monovalent, recombinant, single-chain antibody consisting of the Vκ and VH regions linked to a Cκ domain (Kalinke et al., 2000). This antibody fragment expressed four somatic amino acid exchanges in VH, whereas VL was germline. The antibody fragment was mutated back to a germline configuration and mutations were introduced in VH encoding for one or both of the common amino acid substitutions in CDR1 and CDR2. The binding of the hypermutated VI24 antibody fragment was about 50- to 100-fold better than the germline antibody fragment. Expression of the CDR2 amino acid substitution alone improved binding by about 10-fold, whereas the CDR1 substitution did not affect binding. Antibody fragments were dimerized using an anti-Cκ antibody. Dimerization of the VI24 antibody fragment did not improve its binding, whereas binding of the dimerized germline antibody fragment improved by more than 10-fold. Thus, avidity effects did not contribute to the binding of the hypermutated antibody, whereas they seemed to have a major impact on the binding of the germline antibody fragment. Analysis of the second group of antibodies confirmed the previous observation that hypermutation was no prerequisite for virus neutralization. However, hypermutation improved the binding and the neutralization of VSV in vitro up to a level where avidity effects do not seem to further contribute to the quality of antibody binding.


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C. ADDITIONAL MECHANISMS OF REPERTOIRE DIVERSIFICATION Although hypermutation remains the principal mechanism of antigen-driven expansion of the primary B cell repertoire, other mechanisms have also been implicated in this process. The extent of diversification generated through secondary Ig gene rearrangements was assessed by analyzing antiviral antibody responses in the quasi-monoclonal (QM) mouse (Lopez-Macias et al., 1999). The QM mouse was generated by site-directed insertion of a rearranged VDJ region specific for the hapten (4-hydroxy-3-nitrophenyl) acetyl (NP) into the endogenous JH locus (Cascalho et al., 1996). The other heavy-chain allele was inactivated by deletion of all JH segments. In addition, QM mice have been crossed to Jκ –/– mice; therefore, theoretically only NP-specific λ+ antibodies should be generated. However, QM mice infected with VSV, lymphocytic choriomeningitis virus (LCMV) or poliovirus could mount antiviral nAbs, albeit generally with delayed kinetics, and reached titers of sufficiently high levels to confer protection against lethal viral disease. The antiviral antibodies were generated by the replacement of the targeted VH region with endogenous VH elements located upstream of the transgene by a process termed receptor editing (Gay et al., 1993; Radic et al., 1993; Tiegs et al., 1993; Chen et al., 1995, 1997; Lang et al., 1996; Pelanda et al., 1997; Fang et al., 1998; Nemazee, 2000). These experiments illustrate the potential of secondary rearrangements in the diversification of a very limited antibody repertoire and suggest that this mechanism might also play a role in expanding the normal B cell repertoire. A recent study showed that mice lacking κ light chains, the co-stimulatory molecules CD19 and CD22, or the signaling molecule Btk were not more susceptible than control mice to VSV or LCMV infections, again demonstrating the plasticity of the B cell repertoire (Fehr et al., 2000). D. AFFINITY, AVIDITY, AND CONCENTRATION PARAMETERS OF PROTECTION Molecular mechanisms of in vivo virus neutralization remain largely undefined. However, several studies have attempted to address the quantitative as well as the qualitative requirements of neutralizing mAbs for in vivo protection in passive transfer experiments. For determination of the in vivo protective capacity of VSV nAbs, SCID (severe combined immuno deficiency) mice devoid of B and T cells were passively immunized with various mAbs and challenged with VSV; 4 days later the brains of surviving mice were assessed for the presence of virus. Secondary and hyperimmune antibodies bearing hypermutations protected at concentrations between 1 and 3 μg/ml (Bachmann et al., 1997b; Kalinke et al., 2000). One of the primary response antibodies devoid of hypermutations protected at a similar concentration (4 μg/ml), whereas all others protected at concentrations ≥15 μg/ml (Kalinke et al., 2000). Thus, most primary response

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monoclonal antibodies alone protected from a lethal VSV infection at about a 10-fold higher concentration than secondary and hyperimmune antibodies, but polyclonal sera were comparably efficient, early, and late. Since at the peak of the Ig response theoretically up to 10% of the total Ig can be antigen specific (Bachmann et al., 1994b; Funk et al., 1998), protective serum levels can be rapidly obtained by germline antibodies. During later time points, when a lower percentage of serum Ig is antigen specific, an increased protective capacity generated through hypermutation would be advantageous. The VSV protection data are in accordance with the prophylactic antibody dose against numerous viruses, including rabies virus, Ebola virus, coronavirus, and mouse influenza virus (3 μg/ml, 5–15 μg/ml, 5–25 μg/ml, and 1–20 μg/ml, respectively) (Dietzschold et al., 1990; Lamarre and Talbot, 1995; Mozdzanowska et al., 1997; Wilson et al., 2000a). Surprisingly, at a concentration of 30 μg/ml, some Ebola virus-specific antibodies protected 100% of experimental animals even when treatment was started one day post virus exposure. Post-exposure protection of hamsters against rabies virus was also reported using a human G-protein-specific mAb (Dietzschold et al., 1990). The role of Fc-mediated functions and bivalency in protection against virus infection has also been investigated in several animal models. Protection from lethal murine coronavirus infection could be observed when F(ab′ )2 or Fab antibody fragments were passively transferred prior to viral challenge (Lamarre and Talbot, 1995). However, the protective capacity of Fab fragments was greatly reduced compared with bivalent molecules despite similar in vivo half-lives. The reduction in protective capacity of monovalent antibody fragments compared with F(ab′ )2 fragments correlated with a 14-fold decreased affinity constant. These results suggest that the Fc fragment and bivalency are not absolutely required for in vivo protection but that avidity might influence protective capacity. Similar conclusions were reached when recombinant single-chain antibody fragments were used in protection experiments, although the extremely short in vivo half-lives greatly reduce their efficacy (Kalinke et al., 1996b; Lamarre et al., 1997). III. Mechanisms of Neutralization by Antibodies

Studies using various viral infection models have revealed several mechanisms by which antibodies are capable of neutralizing virus. This is not unexpected given the complexity of virus–antibody interactions, alone or together with host cells. A. MECHANISMS OF NEUTRALIZATION While opsonization by antibodies seems to be the major protective mechanism against bacterial infection, prevention of attachment to, and therefore


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infection of, cells is probably the major antibody-mediated effector mechanism against viruses. However, such a general statement does not do justice to the many studies on the numerous potential effector mechanisms of nAbs. Mechanisms of neutralization have been extensively reviewed previously (DellaPorta and Westaway, 1978; Dimmock, 1984, 1993; Iorio, 1988; Bachmann and Zinkernagel, 1997; Stewart and Nemerow, 1997; Burton et al., 2000) and so will not be discussed here again in detail; nevertheless, we would like to emphasize how little we really know about these mechanisms. Various categories of neutralizing antibody activities have been described: (1) prevention of adsorption; (2) impairment of the function of viral surface proteins necessary for productive infection following virus entry (Levine et al., 1991); (3) intracellular neutralization via a pH-dependent inhibition of virus fusion to vesicles or phagosomes; and (4) aggregation of virus, or pseudo-neutralization, resulting in the reduction of replicating viral particles without necessarily changing the infectivity of each single particle (Wallis and Melnick, 1967). Interestingly, neutralization of some viruses requires binding of antibodies to more than half of the available surface determinants in order to prevent virus attachment and entry into cells. This has been shown to be required for neutralization of rhabdoviruses and may hold true for many other enveloped viruses expressing only one major neutralizing determinant (Flamand et al., 1993; Kalinke et al., 1996b; Bachmann and Zinkernagel, 1997; H.P. Roost, unpublished). For other viruses, such as poliovirus (Dulbecco et al., 1956; Mandel, 1976; Emini et al., 1983) or influenza virus (Lafferty, 1963), only a few antibody molecules (3–5) appear to suffice to inhibit infection. Such differences clearly show that distinct mechanisms may be employed by antibodies for neutralization. Whether neutralization of poliovirus requires a one- or multi-hit mechanism is still debated (Dulbecco et al., 1956; Dimmock, 1984, 1993). We believe the onehit neutralization model may be an oversimplification and is probably not a general rule. Bacterial phages have also been used to examine antibody– virus interactions; however, because phages usually have only a few possible attachment sites, contrary to animal viruses, the results may not readily be transferable. Despite effective neutralizing properties, antibodies may also enhance infection of cells bearing Fc receptors (Porterfield, 1986), including macrophages and dendritic cells, and potentially B cells, T cells, and endothelial cells (Halstead, 1988; Lewis et al., 1988; Mascola et al., 1993; Battegay et al., 1993a). Virus nAbs can be measured in vitro by assays where either plaque formation by cytopathic viruses or focus formation by noncytopathic viruses is inhibited. ELISA binding assays are less reliable in measuring nAbs except when purified intact viral particles are used. Indeed, antibodies that display neutralizing capacity bind intact viral particles, particularly when only one envelope protein is expressed such as for rhabdoviruses (Bachmann et al., 1994c).

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In addition to the ability to neutralize virus on their own, antibodies have also been shown to act in conjunction with complement. The role of complement activation in enhancement or prevention of infection has been well documented, at least for some viruses (Cooper and Nemerow, 1984; Ochsenbein et al., 1999b). In fact, complement consumption has often been used as a readout for the presence of antiviral antibodies; however, it does not necessarily indicate a role for complement in protection nor does it correlate with neutralizing capacity. Inhibition of hemagglutination correlates better with neutralization, although these two assays do not measure the same parameter. In addition, the sensitivity of these assays is quite distinct (Bachmann et al., 1999) and therefore care must be taken when interpreting these correlations. In conclusion, antibody-dependent neutralization mechanisms are probably largely due to blocking effects, but other mechanisms may apply. However, whether or not mechanisms observed solely in vitro are relevant in the living host remains to be further evaluated. B. STRUCTURAL STUDIES Recent studies have provided insight into the structural aspects of antibody inhibition of virus attachment to cells, specifically in the case of influenza, poliovirus, rhinovirus, adenovirus, and foot-and-mouth disease virus (reviewed in Stewart and Nemerow, 1997). In the case of influenza virus, the binding surface between Fab fragments or intact Ig and hemagglutinin occupies a significantly larger area than the area covered by sialic acid moieties during hemagglutination, by the order of about 1000 A2 versus 300 A2 (Stewart and Nemerow, 1997). In the case of human rhinoviruses, comprising many serotypes, ICAM-1 seems to be the major receptor for cellular entry. It was previously thought that nAbs bind to the outer rim of the canyon formed on the virus surface, thus preventing entry of the receptor. However, recent structural evidence has challenged this concept by showing that Fab fragments can actually penetrate the binding site (Smith et al., 1996). Studies with foot-and-mouth disease virus have shown that antibodies can induce a conformational change in the virus surface protein, therefore preventing exposure of the fusogenic domain normally triggered by receptor binding. Similar mechanisms have been postulated for poliovirus, human immunodeficiency virus (HIV), and other viruses. While mechanisms of neutralization that interfere with events occurring after attachment to the host cell have been postulated and experimentally illustrated, structural analyses have not been possible so far. Taken together, these structural studies have revealed that nAbs either directly block receptor binding or trigger structural changes that may influence cellular entry. Whether or not these interactions occur as such under physiological conditions in vivo, particularly in a serum-rich environment, remains to be shown. Structural mechanisms of neutralization of viruses that require complex


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stepwise structural changes in order to achieve infection, such as HIV (Robinson and Mitchell, 1990; Poignard et al., 1996), may not be so simple.

IV. The Role of Complement, Natural Antibodies, and Fc-Receptors in Antiviral Responses

A. NATURAL ANTIBODIES Natural or spontaneous antibodies are present in the sera of nonimmunized humans and mice (Avrameas, 1991; Herzenberg and Kantor, 1993; Coutinho et al., 1995). Natural antibodies are mainly of IgM isotype, but IgG and IgA have also been observed. Most natural antibodies are polyspecific, although antibodies reacting with a single antigen are also present. CD5+ peritoneal B-1 cells have been shown to be a main source of these polyreactive antibodies, although CD5– B-2 cells may also participate in the production of the natural antibody repertoire (Casali and Notkins, 1989; Herzenberg and Kantor, 1993). Natural antibodies are encoded by germline genes without, or with very few, somatic mutations. The stimulus for secretion of natural antibodies is largely unknown. It has been shown that B cells synthesizing natural antibodies are also present in newborn, nude, germ-free, and antigen-free mice with similar reactivity patterns when analyzed in a quantitative immunoblot assay (Bos and Meeuwsen, 1989; Haury et al., 1997). A possible explanation for the apparent antigen-independent stimulation of natural antibody-secreting B cells may be that such B cells can normally secrete low levels of Ig or can be triggered by cross-reactive self-antigens. It has been proposed that natural antibodies may play a role in mediating immunoregulatory functions in the hypothetical idiotypic–anti-idiotypic network theory (Jerne, 1984; Coutinho, 1989) or in autoimmune diseases such as systemic lupus erythematosus (SLE) (Cohen, 1986; Tlaskalova-Hogenova et al., 1992). However, their main physiological function seems to be in early resistance against infections. Natural antibodies together with other components of innate immunity belong to the first line of defense against microbial infections (Boes et al., 1998b; Ochsenbein et al., 1999a; Baumgarth et al., 2000). A role for natural antibodies has also been shown in protection against viral infection through multiple mechanisms. First, highly specific (serotype-specific) IgM antibodies can directly neutralize VSV (Gobet et al., 1988; Ochsenbein et al., 1999a). This normally occurs during hematogenic spread of the virus, although there are some indications in mice that natural IgM antibodies can also be transported to mucosal surfaces and protect locally against influenza virus infection (Baumgarth et al., 2000). Second, because IgM is a pentamer it can potentially bind to ten antigenic determinants per molecule and therefore form large antigen–antibody complexes that would be more efficiently retained

10


in the spleen (Lutz et al., 1987). This process controls spreading of cytopathic infectious agents through the blood, as shown for VSV, and prevents infection of vital target organs (Ochsenbein et al., 1999a). In addition, the observation that antibody-deficient mice show an increased susceptibility to viruses, such as poliovirus, that must distribute to target organs via blood circulation has provided indirect evidence for such a role (Lachmann and Davies, 1997). Third, IgM is a potent activator of complement (Fearon and Locksley, 1996; Carroll, 1998). Triggering of the complement cascade can directly lead to lysis of invading bacteria or provide protection against viral infections. The efficiency of this protection, however, may be limited due to the fact that NA titers are rather low. If the dose of the cytopathic infectious agent that reaches the systemic circulation is too high, natural antibodies, as a first line of defense, are overrun and specific nAbs have to be elicited quickly and efficiently to prevent infection of vital target organs. B. COMPLEMENT Complement components are involved in host protection against viral infection either directly or indirectly through interaction with virus-infected cells (Cooper, 1991). Enhancement of the neutralization capacity of antibodies by complement components may occur by coating of the virion with C3 and probably to a lower extent with C4 components. This was shown in very early experiments in avian infectious bronchitis where a complete disassembly of the virus was described (Berry and Almeida, 1968). In contrast, in vitro experiments using VSV showed that the active cleavage product C3b incorporated into the viral envelope prevented infection of target cells without actually destroying the viral particles (Beebe and Cooper, 1981). Enhancement of antibody-mediated neutralization by complement was recently confirmed in vivo with a panel of VSV-specific monoclonal antibodies (Ochsenbein et al., 1999b). Another way by which direct interaction of complement with virus could enhance neutralization is through the very efficient targeting of complementcoated viral particles to complement receptor (CR)-expressing cells. This effect has been documented for CD21 (CR2)- and CD35 (CR1)-expressing B cells and follicular dendritic cells (FDCs) whereby opsonization of the antigen by complement enhances antigen targeting, leading to more efficient antigen presentation and germinal center (GC) formation (Tew et al., 1990; Carroll, 1998). More recently, a direct enhancement of B-cell receptor (BCR) signal transduction has also been described. This phenomenon was called dual antigen recognition because of the simultaneous recognition of antigen by the BCR and complement-bound antigen by CR2 (van Noesel et al., 1993; Fearon and Carter, 1995). Following binding of C3b and C3d, CR2 forms a complex with CD19 and TAPA-1 which induces cross-linking of the BCR with its co-receptors and may thereby lower the threshold for B cell activation. These mechanisms have been shown to be important after immunization with various model antigens.


11

For example, hen egg lysozyme (HEL) coupled to C3d is 1000- to 10000-fold more immunogenic than HEL alone (Dempsey et al., 1996). Similarly, natural antibodies and complement were shown to be important for the induction of other T-dependent B cell responses in complement-deficient (Ahearn et al., 1996; Molina et al., 1996; Fischer et al., 1998) and soluble IgM-deficient mice (Ehrenstein et al., 1998; Boes et al., 1998a). In contrast, immunization of complement-, complement receptors-, or CD19-deficient mice with different replicating viruses resulted in the generation of neutralizing antibody comparable to controls (Fehr et al., 1998b; Ochsenbein et al., 1999b). This discrepancy suggests that lowering the threshold for B cell activation is of importance mainly for T-dependent antibody responses to limiting amounts of nonreplicating antigens. However, the generation of these T-dependent antibody responses requires at least 6 to 8 days (Oxenius et al., 1998c); therefore, early T-independent (TI) antibody responses are crucial for control of rapidly spreading viruses. Marginal zone macrophages have long been recognized as being important for the generation of TI antibody responses to bacterial antigens (Amlot et al., 1985; Buiting et al., 1996). Recently, analysis of TI-neutralizing antiviral IgM responses also revealed an important role for targeting complement-opsonized virus to marginal zone macrophages expressing CR3 and CR4 (Ochsenbein et al., 1999b). The concentration and localization of antigen were crucial for the induction of early TI antibody responses. Taken together, natural antibodies recognize the pathogen in the circulation, activate the complement cascade, and thereby target the antigen to the splenic marginal zone. This allows cross-linking of BCRs and results in early TI production of neutralizing IgM antibodies (Ochsenbein and Zinkernagel, 2000). The interaction of complement with virus-infected cells has been studied for several viruses (Welsh et al., 1975; Mills et al., 1979; Beebe and Cooper, 1981; Cooper and Nemerow, 1983, 1984; Cooper, 1991) and appears to be particularly relevant for noncytopathic viruses such as measles virus or Epstein-Barrvirus (EBV). Both viruses activate the human complement cascade via the alternative pathway, leading to lysis of infected cells (McConnell et al., 1978; Sissons et al., 1980). This lytic effect is further increased by antiviral IgG antibodies that probably bind to C3b and thereby increase its resistance to inactivation (Fries et al., 1984; Reiter and Fishelson, 1989). This allows the generation of C5 convertase and further increases lysis of target cells. However, activation of the complement cascade and generation of the lytic complex are counterbalanced by cell repair mechanisms that protect the integrity of nucleated cells through the shedding of vesicles containing pieces of damaged membrane. An important role for the complement system in antiviral protection is further suggested by the fact that several viruses have evolved strategies to evade complement-mediated lysis either by using complement receptors and complement control proteins as viral receptors or by producing complement blocking or modulating molecules (Frade et al., 1985; Lachmann and Davies, 1997). For

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example, HIV has been shown to carry the complement control proteins CD46 and CD59 in its envelope (Saifuddin et al., 1997). C. FC-RECEPTORS While complement and its receptors can interact with pathogens through natural antibodies, thus providing host protection, FcR on immune cells interact with IgG, triggering effector functions and inflammatory responses. FcR belong to the diverse multigene Ig superfamily that either activates or inhibits cellular responses (Ravetch, 1994; Daeron, 1997). Like T-cell receptor (TCR) and BCR, activating FcR are members of the immunoreceptor tyrosine-based activation motif (ITAM) family, which is composed of a ligand-binding α chain and an associated common γ chain (Reth, 1989; Kurosaki and Ravetch, 1989; Cambier, 1995). Therefore, mice with a targeted disruption of the γ chain fail to express the high-affinity IgE receptor, FcεRI, the high-affinity IgG receptor Fcγ RI, and the low-affinity activation receptor, Fcγ RIII (Miyajima et al., 1997). Analysis of these mice revealed a major role of IgG in evoking an anaphylactic response when bound to Fcγ RIII on effector cells, including mast cells, neutrophils, macrophages, and natural killer (NK) cells. The role of IgE and its receptors in the clearance of helminth infections and the role of IgG FcR in immunity to microbial pathogens such as streptococcus have been addressed by several studies. The results suggest that susceptibility to infection by these pathogens is not influenced by the presence or absence of FcR genes, as no differences are observed between knockout mice and their heterozygous littermates (summarized in Ravetch and Clynes, 1998). Similarly, analysis of different FcR–/– mice during VSV or LCMV infections also did not reveal a role for FcR in control of these viruses. Neutralizing antibody responses, virus-specific cytotoxic T cell responses, and viral clearance in infected hosts were similar in FcR–/– mice and heterozygous littermates (M. Pericin, unpublished results). Taken together, although FcRs may play a role in anaphylactic reactions, antibody-mediated autoimmune diseases, and probably even some tumor model situations, so far there is no evidence that FcR are crucially involved in protection against a variety of bacterial, fungal, protozoan, helminth, or viral pathogens (Ravetch and Clynes, 1998). V. The Role of Antigen Structure, Organization, and Dose on B Cell Responses

The neutralizing antibody response against VSV, poliovirus, and some other viruses is induced very rapidly; an IgM response is measurable by day 2, peaks by days 4 to 6, and is largely T-cell-independent during this early phase. Nude or CD4 T-cell-depleted mice mount a good IgM response if intact wild-type virions (live, UV-, or formalin-inactivated) are used for immunization. Thus, the highly organized, repetitive, densely packed, neutralizing epitopes that are usually


13

exhibited on the tip of glycoproteins of the virus envelope are able to trigger strong IgM responses in the absence of T help (T-help-independent type 1, TI-1). Moreover, these epitopes are often the only ones accessible to nAbs or B cell receptors (BCRs). This was first demonstrated for influenza viruses where it was shown that antibodies cannot squeeze in between the hemagglutinin epitopes on intact virions (Lafferty, 1963; Fazekas de St. Groth, 1981). The same has been shown for VSV-neutralizing epitopes expressed by the single-envelope glycoprotein (Lefrancois and Lyles, 1983; Lefrancois, 1984; Wagner, 1987; Luo et al., 1988; Roost et al., 1995, 1996). In contrast, low doses of monomeric glycoprotein or hemagglutinin carrying the neutralizing determinants will not induce this early and efficient IgM response (Bachmann et al., 1997a). Of course, these early antibody responses are dependent on viral dose and probably on natural antibodies and complement factors binding to complexes, as discussed above (Ochsenbein et al., 2000a). Neutralizing epitopes expressed on infected cells exhibit a less repetitive and less rigid distribution. Such cell surface multimers represent a third form of neutralizing viral epitopes and induce yet a different T-cell-dependent IgM response. In this case, the necessary T help does not have to be linked in the conventional manner and noncognate bystander T help is sufficient to help this IgM response (TI-2) (Bachmann et al., 1993; Bachmann and Zinkernagel, 1997). These results indicate that the rigid paracrystalline form of the glycoprotein tips, spaced 8 to 10 nm, cross-link specific BCRs very efficiently. This process requires at least 10 to 30 determinants to trigger B cell differentiation, activation and antibody production (Dintzis et al., 1976, 1989; Bachmann et al., 1993; Bachmann and Zinkernagel, 1996, 1997; Fehr et al., 1996, 1998a). Although efficient cross-linking of BCRs enhances B cell activation it should be noted that both TI-1 and TI-2 type IgM responses are also dose dependent, since low doses are completely T dependent (Freer et al., 1994; Ochsenbein et al., 2000a). However, with sufficient doses the IgM response is T independent and very rapid for both TI-1 and TI-2. This suggests that the distinction between TI-1 and TI-2 is probably not of great importance from a pathophysiological point of view. However, the distinction may be relevant for self-/non-self-discrimination and autoimmunity (Bachmann and Zinkernagel, 1997). The above findings support the recent model proposing that BCRs form clusters of about 10 to 20 receptors and that this clustering may be essential for B cell selection and induction (Reth et al., 2000). Because antigen-mediated cross-linking of a minimal number of BCRs is necessary and sufficient to induce B cell proliferation and antibody production, it indicates that antigen signals in the absence of so-called second signals do not anergize or delete B cells, as postulated by others (Bretscher and Cohn, 1970; Mueller et al., 1989; Schwartz, 1989; Cohn and Langman, 1990; Langman and Cohn, 1993; Matzinger, 1994). B cells seem to take antigen organization as a marker for reactivity (Bachmann et al., 1993, 1995; Bachmann and Zinkernagel, 1996, 1997; Ochsenbein and

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Zinkernagel, 2000) Indeed, accumulating evidence suggests that the more regular and rigidly ordered the identical antigenic determinants are, the less the B cells are dependent on and controlled by T cells. These multimeric paracrystalline structures are the hallmark of surface antigens on infectious agents and therefore must always be considered as non-self. This has obvious consequences for our understanding of antibody-mediated autoimmunity but also offers a new concept in self-/non-self-discrimination by B cells. Again, it needs to be emphasized that the dose and persistence of antigen are as important as antigen structure. However, the relative importance of these two factors cannot be defined because they cannot be separated experimentally; i.e., multimeric antigens are bigger than monomeric antigens which therefore affects both dose and structure (Dintzis et al., 1989; Ochsenbein and Zinkernagel, 2000). As will be pointed out later, similar structures may exist in self-antigens, but they are usually not accessible to B cells in healthy individuals. In contrast to multimeric non-self antigens, monomeric determinants are the hallmark of self-antigens in blood and the lymphatic system. B cell responses to self-antigens are therefore strictly regulated by cognate T help which is absent for self-antigens (Weigle, 1973). Efficient cross-linking of BCRs is required in order to rapidly expand B cells during the early phase of an infection and provide sufficient IgM titers to limit systemic virus distribution. Subsequently, when virus-specific T helper cells are induced around day 4 to 6 post infection they can now encounter this expanded B cell pool and may efficiently induce the switch to IgG (Roost et al., 1990; Bachmann and Zinkernagel, 1996, 1997; Baumgarth, 2000). This IgG response is longer lived (20-d half-life) and more protective than the IgM response (1- to 3-d half-life). IgG molecules also diffuse more efficiently into solid tissues and the central nervous system as they are smaller than IgM molecules. Nevertheless, IgM can very efficiently prevent hematogenic spread of virus, as has been suggested by the survival of VSV-infected CD40–/– or CD4-depleted mice which are incapable of switching to IgG (Oxenius et al., 1996). Rapid amplification of rare B cells can have both beneficial and detrimental consequences for the host. On the one hand, an efficient neutralizing or protective antibody response against acute cytopathic infections is generated, while on the other hand auto-antibodies and autoimmune disease could potentially be induced. The frequency of neutralizing antibody-producing B cells is on the order of one per 105–106 spleen cells, corresponding to about one per 104–105 B cells (Bachmann et al., 1994b). This frequency is considerably lower than frequencies determined for hapten-specific dinitrophenyl (DNP)- or trinitrophenyl (TNP)-specific B cells that are on the order of 10–2 (Bos and Meeuwsen, 1989). Similarly frequencies for complex protein antigens such as lysozyme, ovalbumin, or bovine serum albumin are about 10–100 times higher than for neutralizing epitopes, a fact that correlates with the many more antigenic determinants that can be recognized by B cells on these complex globular proteins compared to the


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usually unique neutralizing epitope exposed on intact viral surfaces (Roost et al., 1995, 1996; Bachmann and Zinkernagel, 1997; Goldbaum et al., 1999). Thus, neutralizing antiviral antibody responses protect efficiently against viral infections, whereas antibody responses against the many internal viral antigens or peptides from the surface glycoproteins are usually not protective. The reason is simply due to the structure of the limited size of the exposed determinant accessible to antibodies. In many cases, the neutralizing epitope is the sole determinant regularly accessible to B cells in the intact viral surface. Alternatively, it is the most peripheral of the available determinants (e.g., poliovirus), and antibodies may therefore efficiently prevent docking to the appropriate receptor. Neutralizing epitopes on many (if not most) viruses, therefore, have been selected by long-time co-evolution to represent single antigenic sites critical for prevention of virus infection. It is, therefore, not a surprise that very closely related viruses share greater than 95% of proteins and sequences of internal structural and nonstructural antigens but vary in single neutralizing antigenic sites forming the neutralizing epitope; these differences define what we call serotypes. By definition, serotypically distinct viruses induce no cross-protection by the respective nAbs, despite the fact that they share most if not all T helper cell and cytotoxic T cell determinants (Gupta et al., 1986; Roost et al., 1990; Zinkernagel, 2000b). It also cannot be overemphasized that antibodies against internal viral antigens in general are irrelevant and of no importance for antiviral protection. The structure of neutralizing determinants is usually defined by an area of about 500 to 1000 A2 built from several protein loops. For rotaviruses, influenza, and arenaviruses three loops seem to be involved in forming neutralizing determinants; the situation is probably in reality even more complex because the relevant glycoproteins are composed of three to four interacting units (Webster et al., 1982; Jackson et al., 1982; Burns and Buchmeier, 1993). In a few selected cases, linear epitopes have been shown to induce nAbs—for example, against foot-and-mouth disease virus (Bittle et al., 1982; Brown, 1988) or gp41 (Xiao et al., 2000) and V3 loop of HIV (Robinson and Mitchell, 1990; Poignard et al., 1996). But, in general these exceptions only confirm the rule that neutralizing sites are formed by complex loop structures. The V3 loop of HIV may be a particularly revealing example, as antibodies induced by this loop only neutralize artificial lab strains and usually do not confer protection from infection with primary isolates. In fact, the neutralizing epitope on the HIV glycoprotein seems to be extremely conformation dependent and composed of at least two separate regions of the protein engaged sequentially during infection (Cho et al., 2000). Neutralizing epitopes have been determined by X-ray crystallography for poliovirus, influenza, adenoviruses, and rhinoviruses. For many viruses, this information is still missing, but some approximations have been deduced from mutational analysis. However, the indirect relationship

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between amino acid exchanges and structural consequences renders interpretations of these results generally difficult (Laver et al., 1990). As far as analyzed, the simple notion that nAbs cover the unique antigenic sites exposed on the virus surface and/or bind to the receptor-binding structures may still be an oversimplification but remains an attractive and perhaps rather general explanation (see later). VI. T-Cell Dependent Activation of B Cells in Viral Infections

General rules of T–B cooperation and induction of T helper cells by macrophages, antigen-presenting cells (APCs), or virus-specific B cells are as described in model situations in textbooks (Paul, 1993). Nevertheless, some very interesting observations can be made by analyzing T–B cell interactions during protective neutralizing antibody responses. They involve, first, the limiting frequencies of neutralizing B cells and T helper cells and second, the relative importance of T help specific for viral envelope versus internal antigens. As pointed out above, neutralizing B cells are of very low frequency. Therefore, induction of these rare B cells must be very efficient, particularly during the early phase of a viral infection when antigen is scarce and usually strictly localized. Not surprisingly, B cells are the major limiting factor during neutralizing antibody responses, not the amount nor the activation state of T helper cells (Charan and Zinkernagel, 1986). This was documented first by immunization of mice with one serotype of VSV (VSV-IND) and after 3 months’ challenging with a second VSV serotype (VSV-NJ). The two VSV serotypes share virtually all helper and cytotoxic T cell epitopes; therefore, the first infection should prime all specific CTL and T helper cells. The challenge infection revealed, however, that the neutralizing antibody response generated was strictly of a primary type (Charan and Zinkernagel, 1986; Gupta et al., 1986). This finding suggests that priming of B cells rather than of helper T cells may be of importance for inducing protective immunity mediated by antibodies. This contrasts with the conventional view developed from model studies analyzing specific B cell responses in classical hapten carrier systems (Mitchison, 1971; Katz and Benacerraf, 1972). These early studies clearly showed that hapten-specific B cells were not limiting, whereas the amount of available and primed T help did limit the antibody response both qualitatively and quantitatively. However as pointed out above, the frequency of haptenspecific B cells is usually very high, on the order one per 102 to 103, contrasting with the much lower frequency of virus-specific neutralizing B cells (one per 104 to 105). In summary, these experiments provide strong evidence that B cells are the limiting factor in neutralizing antibody responses against many acute cytopathic viruses and presumably also against bacterial toxins or other proteins on bacteria and parasites (Charan and Zinkernagel, 1986; Roost et al., 1990; Bachmann and Zinkernagel, 1997; Zinkernagel, 2000b). This correlates well with


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clinical observations that patients having survived an infection with poliovirus 1 or influenza HA1 are not protected against a subsequent infection with a different viral serotype (Yewdell et al., 1979; Nathanson and Martin, 1979; Sabin, 1981, 1985; Fazekas de St. Groth, 1981; Webster and Rott, 1987; Laver et al., 1990; Liang et al., 1994; Nathanson and McFadden, 1997; Ada, 2000). The crucial role of neutralizing antibodies in antiviral protection does not exclude the possibility that highly activated effector cytotoxic T cells or T helper cells could also participate in protection (Zweerink et al., 1977; Effros et al., 1979). However, because antigen is eliminated quickly, this T cell effector phase is usually short (2 to 3 weeks) (Roost et al., 1990) and importantly cannot be easily induced and maintained by vaccines, as will be discussed below. T cells specific for either envelope glycoproteins or any of the internal antigens are equally efficient in their ability to help neutralizing B cells switch from IgM to IgG (Liang et al., 1994; Oxenius et al., 1998b). Interestingly, however, the switch of B cells specific for internal antigens is virtually exclusively mediated by T helper cells specific for that particular antigen. In contrast, T cells specific for any viral antigen can support switching of B cells specific for viral surface proteins. These observations strongly support the view that intact virus particles are taken up by neutralizing antibody-producing B cells, which will subsequently present any of the viral antigens on major histocompatibility complex (MHC) class II, whereas the switch of B cells specific for internal viral antigens requires that such antigens be released from infected cells or viral particles in order to be taken up.

VII. B Cell Unresponsiveness

Historically, negative selection of B cells expressing specificities for selfantigens has been postulated as a major mechanism for maintenance of tolerance. Both Ehrlich’s horror autotoxicus (Ehrlich, 1906) and Burnet’s proposal of negative selection of immune reactivity, at the time when the distinction between T and B cells was not yet known, focused exclusively on B cells (Burnet and Fenner, 1949). These notions were expanded by Nossal and coworkers to include the concept of anergy (Nossal, 1983). More recently, IgM–BCR transgenic models have been used to document negative selection as the basis for the absence of auto-antibody responses (Goodnow et al., 1989; Nemazee and Buerki, 1989; Nemazee and Buerki, 1989; Nemazee et al., 1991). Lysozymespecific IgM transgenic mice crossed to mice expressing lysozyme showed that the number of specific B cells was reduced compared to controls (Goodnow et al., 1989). Similarly, anti-H-2Kk IgM transgenic B cells were largely deleted when these transgenic mice were crossed to H-2Kk but not to H-2d mice (Nemazee and Buerki, 1989). Since the same antigen expressed as a soluble product in

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serum failed to delete the B cells to a comparable extent, the conclusion in both transgenic IgM situations was that membrane-bound self-antigens delete self-specific B cells efficiently, whereas soluble antigens in serum fail to do so (Nemazee et al., 1991; Basten et al., 1991). The conceptual views based on these experiments are in conflict with a number of clinical and other experimental observations that do not completely support negative selection or anergy models. In fact, auto-antibodies can readily be detected and have often been observed during infections, particularly chronic infections, not only against soluble self antigen but also against membrane-expressed antigens (e.g., acetylcholin receptor). The recognition that polyclonal stimulators such as lipopolysaccharides could stimulate B cells in the absence of cognate T help revealed the presence of B cells that readily secreted auto-antibodies in normal individuals (Coutinho and Moller, 1975; Moller, 1975). One inherent problem of these observations and their interpretation is that the assays measuring auto-antibodies or autoreactive T cells do not necessarily reflect in vivo reactivity. Nevertheless, most clinically relevant autoimmune diseases involve one or more auto-antibody specificities (Teale and Mackay, 1979; Rose and Mackay, 1992). This by itself is a strong indication that auto-antibodies and autoreactive B cells are not a rare exception. A. B CELL UNRESPONSIVENESS STUDIED WITH VIRUSES The data obtained from antibody transgenic models differed from those observed in a separate study of antibody responses against a VSV-neutralizing epitope expressed in mice as a membrane-associated or soluble antigen. In both mice, immunization with intact virions (live, UV-irradiated, or formalin-fixed) promptly induced a T-cell-independent IgM response (Zinkernagel et al., 1990; Bachmann et al., 1993). Since intact virions contain internal antigens offering T help, these neutralizing antibody responses were efficiently switched to IgG. In contrast, purified VSV-G delivered in adjuvant failed to induce neutralizing IgM or IgG responses. These results indicated that high-affinity B cells were still present in these VSV-G transgenic mice. In addition, they suggested that B cells may be less responsive to monomeric and oligomeric antigens, whereas they would be triggered very efficiently by highly repetitive self antigens. More recently, these findings were confirmed in anti-Kk transgenic mice that responded to a mimicking antigen coupled to bacterial viruses (Kouskoff et al., 2000). It remains to be evaluated whether these results parallel the findings obtained with VSV or reflect the presence of very low avidity antibodies assessed by ELISA. Another example that demonstrates that B cells are, in general, not tolerant or anergic, even to high levels of soluble antigen, is seen in CD8-deficient mice when infected with the noncytopathic LCMV. These mice cannot eliminate virus after acute infection and become carriers with high viral titers in most organs and 105 to 106 pfu/ml of serum. Such carrier mice possess antibodies, as detected by ELISA, specific for several viral antigens (Hotchin and Sikora, 1964; Oldstone


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and Dixon, 1967; Oldstone et al., 1980; Moskophidis et al., 1987). Interestingly, 40 to 50 days after infection of CD8+ T cell-deficient mice, high titers of antiviral nAbs are generated which are sufficient to eventually eliminate the virus even in the complete absence of cytotoxic T cells (Ciurea et al., 2000). Thus, also in this model, B cells are not tolerant and the T helper cells induced early after infection are sufficient to provide the necessary T help. This T help may eventually be exhausted if virus persists at high levels. One could still argue that even high virus titers in the blood and solid organs are not sufficient to tolerize B cells. However, mice infected in utero by carrier mothers become tolerant at helper and cytotoxic T cell levels and are unable to generate nAbs (Rowe, 1954; Hotchin, 1962; Lehmann-Grube, 1971), suggesting that, in this case, availability of T help is the limiting factor rather than potentially autoreactive B cells (Battegay et al., 1994; Matloubian et al., 1994; Oxenius et al., 1998a,b; Seiler et al., 1999). B. GENERAL CHARACTERISTICS OF AUTOIMMUNE B CELL INDUCTION Accumulating evidence suggests that B cells are not negatively selected. Instead, either they fail to be induced by low doses of monomeric antigen in the absence of T help or they only generate short-lived IgM responses that have little pathological consequences for the host (Ochsenbein et al., 2000a). Efficient antigenic cross-linking of the BCR together with the binding of complement to marginal zone macrophages are key to triggering an efficient IgM response that will switch to a long-lasting IgG response only in the presence of classical cognate T help (Odermatt et al., 1991; Seiler et al., 1997; Ochsenbein et al., 1999b). It is via these parameters—antigen structure, antigen dose, time duration, and availability of T help—that long-lasting antibody responses are regulated. What can we learn from antiviral antibody responses for the understanding of how auto-antibody responses are generated? If it is true that antigen structure, antigen amount, and availability of T help are the critical factors in regulating the usual absence of B cell and auto-antibody responses in a young host, then the induction of these responses should reflect a breakdown or defect in one or more of the regulatory requirements. From a basic point of view, it is interesting to note that auto-antibody responses and autoimmune diseases often comprise specificities for highly repetitive determinants such as collagen, DNA, acetylcholine receptors, etc. (Dintzis et al., 1976, 1989; Zinkernagel et al., 1991; Bachmann et al., 1993; Bachmann and Zinkernagel, 1996, 1997; Schulte et al., 1998). This observation supports the idea that cross-linking by polymeric determinants triggers B cells very efficiently, thus amplifying their frequency so it is more likely that they could be switched by either specific or bystander T help. It is worth emphasizing here that the initiation of many auto-antibody responses and diseases are associated with infections (Vaughn et al., 1989; Rose and Mackay, 1992; Jansen et al., 1993; Ludewig et al., 1998, 1999). Presumably, infections can lead to the release of large amounts of self-antigen, generate

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inflammatory signals, and activate antigen-presenting cells. Also it is particularly interesting to note that many chronic auto-antibody immune responses are associated with the generation of new secondary lymphoid structures in the target organ for which the auto-antibodies are specific. Haschimoto’s thyroiditis, Sjogren’s ¨ disease, and rheumatoid arthritis are excellent demonstrations of where the formation of new secondary lymphoid tissue correlates with chronic persistence of auto-antibody responses. Such neo-formation can also be induced by repetitive injection of dendritic cells presenting a neo-self-antigen (Ludewig et al., 1999). Chronic infections that persist in the periphery may also trigger formation of lymphoid structures and enhance autoimmune B cell responses. In summary, induction and maintenance of auto-antibody responses can be enhanced by repetitive auto-antigen structures and by the chronicity of the response leading to the perpetuation of inflammatory events and formation of new lymphoid tissue within target organs. To further investigate the potential of repetitive antigen structures in initiating auto-antibody responses, virus particles were used as matrix to display antibodies in a highly repetitive fashion, and the generation of anti-antibodies, similar to that found in rheumatoid arthritis, was monitored (Fehr et al., 1997). Mice immunized with either VSV particles or a monoclonal-neutralizing IgM antibody did not develop any anti-antibodies, whereas mice immunized with VSV particles complexed with anti-VSV antibodies readily mounted anti-idiotype-specific antibody responses. Similarly, when gram-negative bacteria complexed with monoclonal antibodies were used for immunization, rheumatoid factors (antibodies against constant antibody regions) were induced but not when the antibody or bacterium were injected alone. In this situation, injection of lipopolysaccharide (LPS) together with the complex was necessary in order to break B cell unresponsiveness to a self-antigen normally present in high concentration in serum. This study illustrates the potential for high concentrations of repetitive antigen structures to induce auto-antibodies. At least two other examples of autoantibody induction using a similar approach have been reported. Recombinant bovine papillomaviruses were constructed to express a peptide of the extracellular loop of the mouse chemokine receptor 5 (CCR5) within the immunodominant epitope of the main papillomavirus envelope protein L1 (Chackerian et al., 1999). Injection of this recombinant papillomavirus induced anti-CCR5 auto-antibodies which blocked regulated upon activation of normal T cell expressed and secreted (RANTES) binding and cellular entry of HIV, although this occurred without any apparent induction of autoimmune disease. However, induction of clinical autoimmunity has been described in patients with hepatitis C virus (HCV) infection. In perfect analogy to the anti-antibody study described above, serum of patients suffering from chronic HCV infection contains immune complexes of HCV–anti-HCV antibodies and cryoglobulins (a distinct type of rheumatoid factor). Interestingly, electronmicroscopic analysis revealed that these complexes have highly repetitive paracrystallin structures (Szymanski


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et al., 1994). Deposition of these complexes in tissues such as kidney or skin can lead to the autoimmune disease known as mixed cryoglobulinemia. VIII. Examples of Nonclassical Virus Neutralization

Before going into some special but revealing examples of neutralizing antibody responses against viruses, let us restate some general rules (Fenner, 1949; Sabin, 1981; Mims, 1987; Nathanson and McFadden, 1997; Zinkernagel, 2000b). Following virus infection of the mucosa, local IgA immune responses are probably important but are still poorly understood (Brandtzaeg, 1989; Macpherson et al., 2000). Viral spreading to Peyer’s patches or draining lymph nodes will induce a primary immune response of both B and helper T cells, resulting in local and systemic IgA, IgM, and IgG production. Subsequent to local infection, the virus will usually replicate in draining lymph nodes for 1 to 3 days and then spread systemically (Johnson and Mims, 1968; Mims, 1987). This leads to distribution of antigen in target organs, including skin, central nervous system, kidney, lung, and spleen. Systemic distribution is important to enable the virus to spread horizontally via aerosols, stool, or urine, but the spreading of cytopathic viruses may kill the host if not controlled early by IgM. The immune response initiated in local lymph nodes and spleen will eventually control both systemic and local infection. The sequential induction of immune responses is of key importance to control generalized infections (Fenner, 1949), although entrapment of virus within secondary lymphoid organs may also be important, particularly to rapidly induce responses against viruses that reach the blood directly, such as arthropod-borne viruses. A. VIRUS STAYS OUTSIDE OF THE IMMUNE SYSTEM Papillomaviruses exclusively infect skin or mucosal cells, usually basal cells of the epithelium, in a strictly localized fashion and they replicate productively only in terminally differentiated cells (e.g., keratinocytes) (Tindle and Frazer, 1994; Frazer et al., 1999). Effector T and B cells and even Langerhans cells cannot easily access these cells in the skin. In addition, differentiated cells do not normally release antigen or emigrate to local lymph nodes. Therefore, papillomavirus antigens are initially not transported to the lymph nodes and such infections may be ignored by the immune system for long periods of time (Kundig et al., 1995; Ochsenbein et al., 2000b). Rabies virus uses comparable tactics, but, rather than skin, it initially resides within axons. During retro-axonal flux, the virus is not accessible to T or B cells and does not reach secondary lymphoid organs. At this stage of infection rabies virus is not yet cytopathic; therefore, viral antigen will not be picked up by macrophages or other APC and thus will not reach draining lymph nodes. Once the virus has reached the neuronal body and lyses it, virus spread appears to be so rapid that the immune response is usually induced too late to be of benefit

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for the host. Post-exposure vaccination with inactivated virus seems to shorten this delay and may deliver virus antigen to draining lymph nodes. This induces nAbs that may, under favorable timing conditions, limit ongoing virus transport and prevent substantial central nervous tissue damage (Baer and Cleary, 1972; Murphy, 1977). B. ANTIBODY-DEPENDENT ENHANCEMENT OF INFECTION Normally, antibodies provide protection against viruses but in some cases they may actually enhance infection. Enhancement of viral infectivity has been mostly studied in vitro with cells susceptible to virus infections and bearing Fc and/or complement receptors. Antibody-coated virus can infect cells via FcR rather than via a specific receptor. Many non- or low-cytopathic viruses are suspected to use such antibody enhancement for infection, including HIV (Takeda et al., 1988; Robinson et al., 1988; Homsy et al., 1989), Dengue viruses (Halstead, 1988), and perhaps HCV. The first observations made with flaviviruses by Hawkes et al. (Hawkes, 1964) were largely extended by Halstead and coworkers (Halstead, 1988). They describe an antibody-dependant enhancement of Dengue virus replication of about 100-fold in cultures of peripheral blood leukocytes of humans or primates. To what extent serotype differences contribute to viral infection via FcR and whether or not antibody-dependent enhancement plays a major role in vivo is, however, still not very clear for viruses other than Dengue. However, low-avidity antibodies may enhance early uptake of a new serotype and therefore render a second infection in some immune patients more pathogenic than the first infection (Burke et al., 1988; Morens, 1994; Thein et al., 1997). This is the classical explanation for severe hemorrhagic disease after repetitive Dengue virus infections, but similar mechanisms may apply to persisting viruses that escape nAbs. It may also explain the early death of kittens inoculated with infectious feline peritonitis virus following passive transfer of antiviral antibodies (Weiss and Scott, 1981). In addition, arenavirus infections including LCMV have shown enhancement in vitro and likely also in vivo (Lewis et al., 1988; Ochsenbein et al., 1999a). C. IMMUNOSUPPRESSION Acquired immunosuppression by virus-specific CD8+ lymphocytes occurs when these cells destroy antigen-presenting cells, such as dendritic cells or macrophages, and thereby impair induction of immune responses (Mims and Wainwright, 1968; Bro-Jorgensen and Volkert, 1974; Silberman et al., 1978; Biberfeld et al., 1985; Racz et al., 1986; Leist et al., 1988; Odermatt et al., 1991; Althage et al., 1992; Tishon et al., 1993; Borrow et al., 1995; Seiler et al., 1997). Such a mechanism might help to explain the long delay in the appearance of neutralizing antibodies following noncytopathic virus infections such as HIV (Moore et al., 1994; Pilgrim et al., 1997; Carotenuto et al., 1998), HBV (Alberti


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et al., 1988) HCV in humans, or LCMV in mice (Planz et al., 1996). Detailed analyses of this process during LCMV infection has revealed the following interesting virus–host immune relationship (Zinkernagel et al., 1999). During the early phase of infection, LCMV seems to preferentially infect splenic marginal zone macrophages (Mims and Tosolini, 1969; Jacobs and Cole, 1976) and dendritic cells in the periphery and secondary lymphoid organs. These infected cells appear to be subsequently destroyed by early induced effector cytotoxic T cells (Odermatt et al., 1991; Althage et al., 1992; Tishon et al., 1993; Borrow et al., 1995; Sevilla et al., 2000). Treatment with CD8-depleting antibodies prevents acute immunopathology in lymphohematopoietic organs (Odermatt et al., 1991; Battegay et al., 1993b; Binder et al., 1997). The immunopathological destruction of antigen-presenting cells of the marginal zone hampers both humoral and T-cell-mediated immune responses (Odermatt et al., 1991; Seiler et al., 1997). This virus-induced acquired immunosuppression seems to facilitate the establishment of long-term persistence of the infecting noncytopathic virus. For LCMV it has been shown that antibody responses against the internal viral antigens, including nucleoprotein or the membrane-anchored part of the glycoprotein, are not drastically impaired by this generalized immunosuppression (Battegay et al., 1993b). However, the neutralizing antibody response is greatly reduced or even prevented completely for 70 to 150 days. This difference correlates with the observation that hybridomas specific for neutralizing determinants were frequently infected with LCMV, whereas those specific for the nucleoprotein were not (Planz et al., 1996). These findings suggest that intact virus particles were taken up by neutralizing antibody-specific B cells via the specific BCR and that virus could replicate within these cells. Infected B cells, therefore, express viral peptides in association with MHC class I antigens and become susceptible to destruction by cytotoxic T lymphocytes (CTLs). In contrast, nucleoproteinspecific B cells are not productively infected, will not express peptides on MHC class I, and are therefore not susceptible to CTL lysis (Planz et al., 1996). The parallels between the kinetics of neutralizing antibody responses of human noncytopathic or poorly cytopathic viral infections (HBV, HCV, and HIV) and LCMV are striking. In all these infections, neutralizing antibody responses become detectable only when, or a few months after, virus titers have been reduced to very low or undetectable levels. D. SENSITIVITY TO ANTIBODY NEUTRALIZATION VERSUS TROPISM As both viral sensitivity to antibody neutralization and viral tropism are determined by structures on surface envelope glycoproteins, modifications of cell tropism may alter the neutralization epitopes and vice versa. These interactions are best illustrated during murine infection with lactate dehydrogenase-elevating virus (LDV), an arterivirus which causes asymptomatic persistent infections through cytopathic replication in a renewable subpopulation of macrophages

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(Plagemann et al., 1995). Persistence is favored by specific polylactosaminoglycan chains on the ectodomain of the primary envelope glycoprotein which masks the single antibody neutralization epitope and therefore decreases viral immunogenicity (Chen et al., 2000). LDV strains that lack these specific chains are sensitive to antibody neutralization and are not able to establish lifelong infections. However, these strains have also been shown to be neuropathogenic, as the missing glycosylation allows viral interaction with a putative receptor on anterior horn neurons, causing a paralytic disease in conditions of impaired neutralizing antibody responses. A similar interaction has been suspected for HIV, where macrophage or T cell tropism is determined by the envelope glycoproteins binding to CD4 and coreceptors. As nAbs have been shown to be able to prevent the CD4-dependent association of gp120 with CCR5 (Trkola et al., 1996), changes in neutralization sensitivity could directly affect HIV tropism. Subsequent studies, however, have demonstrated that antibody-mediated neutralization of HIV is independent of co-receptor usage (Trkola et al., 1998; Montefiori et al., 1998; LaCasse et al., 1998). IX. Virus Escape from Neutralizing Antibody Responses

The molecular basis for viral immune selection lies in the extensive genetic variation of viruses generated by mutation, recombination, or reassortment of genomic segments. RNA viruses have a particularly high spontaneous mutation rate (10–3 to 10–5 substitutions per nucleotide per round of replication). This is a consequence of absence of proofreading-repair mechanisms of RNA polymerases and retroviral reverse transcriptases (Holland et al., 1982). These viruses are therefore not present as a homogenous population in their host, but circulate as quasi-species. The evolution of quasi-species depends on the population size of the virus, the competitive fitness of variants during their life cycle, and environmental factors (immune responses, particular cells or tissues in the host, drugs) acting through continuous positive selection pressure on viral recognition and tropism (Domingo and Holland, 1997). Within quasi-species, virus variants containing mutations in envelope glycoproteins that could alter recognition by nAbs would be positively selected in situations where the humoral response plays a substantial role for virus control. Several mechanisms may account for the immune escape of neutralizationresistant mutants. First, amino acid substitutions within the neutralization determinant may alter the affinity of nAb for the virion (Wiley et al., 1981). Second, mutations at distant sites may change the global conformation of the antigenic determinant (Diamond et al., 1985). Finally, mutations may allow additional glycosylation sites which may mask neutralizing epitopes (Skehel et al., 1984; Wright et al., 1989; Reitter et al., 1998; Kimata et al., 1999; Chen et al., 2000).


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A. INFLUENZA VIRUSES Antigenic variation in viruses of the Orthomyxoviridae family, which are composed of a segmented RNA genome, have long been a paradigm for the study of escape from nAb responses (Webster et al., 1982; Wilson and Cox, 2000). First, influenza type A viruses may show genetic reassortment between human and animal strains, mainly from avian and swine reservoirs. Thus, novel pandemic strains emerge which have distinct antigenic characteristics in their surface glycoproteins, hemagglutinin (HA) or neuraminidase (NA) (Cox and Subbarao, 2000). This phenomenon is known as antigenic shift. Second, accumulation of single-point mutations within the HA and NA genes of influenza A and B viruses can occur naturally. This process, called antigenic drift, is responsible for the annual recurrence of influenza epidemics. The amino acid changes are clustered within five, but mainly in one to three, of the major antigenic sites on the surface glycoproteins (Wiley et al., 1981). Several observations indicate that these mutations are selected by an ongoing nAb response. Variants with similar mutations have been selected with monoclonal antibodies (Yewdell et al., 1979). Furthermore, there is evidence of variation in the antibody repertoire of individual mice immunized with influenza (Staudt and Gerhard, 1983). Finally, neutralization escape mutants were readily selected by sera from immune mice (Lambkin et al., 1994), indicating a restricted antibody repertoire in each animal. Although individuals are rarely able to induce an antibody response against all five neutralizing domains, a whole spectrum of anti-HA responses can be found in the human population (Wang et al., 1986). Therefore, epidemiologically significant antigenic variants may arise only through selection at the population level after accumulation of sequential mutations. In line with these observations, it has been shown that four or more amino acid changes occurring in at least two of the five antigenic sites of HA are needed for variants during antigenic drift to be of epidemiological relevance (Wiley et al., 1981). Although antigenic variants of other cytopathic RNA viruses (rabies virus, poliovirus, foot-and-mouth disease virus, measles virus) have been selected in the presence of neutralizing monoclonal antibodies (Diamond et al., 1985; Mateu et al., 1989; Schrag et al., 1999; Borrego et al., 2000), the in vivo role of escape from humoral responses for viral persistence during natural infections is unclear (Hovi et al., 1986) and may not be of importance (Gebauer et al., 1988). An alternative explanation for antigenic diversification may be that there is random occurrence of tolerated amino acid replacements within antigenic sites which may have less stringent structural requirements because they are located on the surface of envelope glycoproteins (Domingo et al., 1993). Furthermore, amino acid substitutions may occur as a consequence of other selective forces, such as changes in virus–cell receptor interactions and tropism, which would

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only secondarily affect antigenic specificity (Both et al., 1983; Kaplan et al., 1990). B. HEPATITIS B VIRUS Although hepatitis B virus (HBV) has a double-stranded DNA genome, its life cycle includes an intracellular RNA pregenomic intermediate which is reversetranscribed within the nucleocapsid (Lee, 1997). This step is responsible for the high viral mutation rate found with HBV. In association with cellular immune responses, nAbs to HBV envelope antigens play a major role in viral clearance as they are readily detectable in patients who clear the virus but not in patients with chronic HBV infection (Chisari and Ferrari, 1995). Antigenic variation in surface glycoproteins has been shown to be clinically important from several points of view. First, vaccine-induced escape mutants of HBV have been described (Carman et al., 1990) showing an incidence of 2 to 3% in endemic regions. Second, passive immunoprophylaxis with polyclonal HBV-specific Igs in order to prevent perinatal or transplantation-associated viral transmission may select nAb-escape variants (Carman et al., 1996; Hsu et al., 1997; Protzer-Knolle et al., 1998). Third, naturally occurring mutations in genes coding for surface proteins were associated with viral persistence (Ogura et al., 1999). Finally, detection of HBV in patients infected with virus containing mutations within the surface antigen (HbsAg) may not be possible using standard HBsAg assays (Carman et al., 1995), a problem which is of great public health significance. C. HEPATITIS C VIRUS Hepatitis C virus (HCV) is a positive-sense single-stranded RNA virus of the Flaviviridae family (Rice, 1996). Several lines of evidence indicate that the nAb response exerts selection pressure on the hypervariable region 1 (HVR1) of the E2 envelope glycoprotein. Emergence of autologous nAb–escape HVR1 variants have been observed during chronic HCV infection (Weiner et al., 1992; Shimizu et al., 1994). In an experimental HCV infection model of chimpanzees, anti-HVR1 antiserum induced protection against homologous HCV infection but not against the emergence of neutralization escape mutants already present in the innoculum (Farci et al., 1996). The rate of sequence variation in the HVR1 region during chronic HCV infection is lower in the absence of a humoral immune response, as demonstrated for patients with hypogammaglobulinemia (Booth et al., 1998). Furthermore, increased viral diversity in HVR1 is associated with a lack of control of HCV infection (Farci et al., 2000). D. HUMAN IMMUNODEFICIENCY VIRUS Human immunodeficiency virus (HIV) escape from humoral immunity has recently been reviewed (Parren et al., 1999), and the emergence of neutralization


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escape mutants during the course of HIV-1 and simian immunodeficiency virus (SIV) infections has been documented (Albert et al., 1990; Tremblay and Wainberg, 1990; Arendrup et al., 1992; Burns et al., 1993; Moog et al., 1997; Bradney et al., 1999). However, autologous nAb responses against primary isolates during natural HIV-1 infection are usually weak. Passive immunization with HIV Ig and several broadly neutralizing monoclonal antibodies in the experimental chimeric HIV-1/SIV infection of macaques provided protection (Mascola et al., 1999, 2000; Shibata et al., 1999; Baba et al., 2000), indicating that under certain conditions, HIV nAbs are able to provide immunological selection pressure (Poignard et al., 1999). E. LYMPHOCYTIC CHORIOMENINGITIS VIRUS While a strong CD8+ cytotoxic T cell response is responsible for acute viral clearance of LCMV (Kagi et al., 1994), indirect evidence from B-cell-deficient mice as well as passive immunoprophylaxis studies suggested an important role for nAbs for long-term viral control (Thomsen et al., 1996; Baldridge et al., 1997; Planz et al., 1997). LCMV infection in CTL-deficient mice provided an opportunity to demonstrate direct clearance by nAbs (Ciurea et al., 2000). However, viral control by the humoral response was only transient. The re-emergent virus variants were shown to be neutralization-escape mutants. They displayed one to three single amino acid substitutions within three regions of the envelope glycoprotein-1. One of the amino acid changes had previously been shown to occur following immune pressure by a neutralizing monoclonal GP1specific antibody (Seiler et al., 1999). All of the substitutions altered neutralization by polyclonal LCMV-immune sera and selected monoclonal antibodies (Ciurea et al., 2000). Interestingly, no new nAb responses against the emerging virus mutants were elicited. This was shown to be the consequence of a time-dependent induction of CD4+ T cell unresponsiveness and therefore lack of T help during high viremia (Ciurea et al., 2001). Interestingly, while the original wild-type strain induced a response against itself but not against emerging variants, the mutant viruses were able to induce nAbs that inhibited both wild-type and escape variants (Ciurea et al., 2000, 2001). Therefore, nAb responses against subsequently emerging LCMV variants seem to recapitulate their evolution in a new sort of co-evolutionarily directed “archetypical” connectivity. This phenomenon is somewhat different from that shown during influenza virus infections in the context of pre-existing immune memory called original antigenic sin (Francis et al., 1953; Fazekas de St. Groth and Webster, 1966a, b). In this case, after infection with an escape HA1* mutant (following antigenic drift), influenza HA1-immune individuals will generate higher hemagglutination-inhibiting antibody titers against the original HA1 virus than against the drifted HA1* variant.

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X. Immunological Memory

Host and virus represent two sides of an evolutionary equilibrium. Cytopathic agents usually kill immunologically low and late responders, whereas high and early responders tend to survive. Generally, the presence of neither memory B nor T cells will improve these conditions unless antibodies are preexisting or T cells are activated, neither of which are efficiently generated by standard vaccination strategies (reviewed in Ahmed, 1992; Swain and Bradley, 1992; Gray, 1993; Zinkernagel et al., 1996; Ahmed and Gray, 1996; Zinkernagel, 2000a). If an unprimed host survives a first infection, that host will not need immunological memory to survive the second infection. Of course, if the host dies due to the primary infection, immunological memory is, obsolete. Immunological memory, therefore, must confer some evolutionary advantage for the species. This advantage may stem from the fact that maternal immunological memory provides protection for immuno-incompetent newborn vertebrates (Zinkernagel et al., 1996; Zinkernagel, 2000a). Because, MHC-restriction in T cell recognition requires MHC polymorphism, immuno-incompetence of the newborn is necessary to prevent potential host-vs.-graft and graft-vs.-host reactions. In addition to immunodeficiency of the offspring, lack of reactivity is ensured by immunosuppression of the mother and absence of MHC-Ag expression at the maternal–fetal interface (Booy et al., 1992; Sarvas et al., 1992; WHO Study Group, 1995; Brent, 1997; Siegrist et al., 1998). Consequently, protection of the offspring during this critical period is mediated by maternal memory through passive transfer of soluble antibodies. An impressive example of successful transfer of maternal memory is illustrated by the fact that calves are born completely without serum Igs and must take up colostral maternal Igs within the first 24 hours after birth (reviewed by Brambell, 1970). If this does not occur, calves will remain without protective antibody and usually die of various infections during the next few weeks. Maintenance of maternal antibody memory is required to provide the offspring with protection against infectious agents because a complete primary antibody repertoire sufficient to cover all relevant infectious agents cannot be generated during the 270 days of a human pregnancy or the 20 days of a mouse pregnancy. Therefore, B and T cell memory is needed in order to accumulate immunological experience before pregnancy. In addition, hormones may well help to maintain protective antibody levels in the mother by increasing plasma cell survival. This might explain the 5 times higher incidence of auto-antibody-mediated autoimmune disease in females. Therefore, while protection of the newborn is the main and key evolutionary basis for immunological memory there are additional qualities (including herd immunity and individual fitness) that could contribute to a co-evolutionary balanced phenotype.


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A. WHAT KIND OF IMMUNOLOGICAL MEMORY IS BIOLOGICALLY RELEVANT? Immune responses and protection against cytopathic virus infections are key to species survival. Without exception, protection against these types of agents is mediated by protective antibodies. Noncytopathic viruses are usually transmitted before or at the time of birth when offspring are immuno-incompetent and this does not have any apparent disadvantage for survival of the species. Examples include LCMV (Rowe, 1954; Hotchin, 1962; Ciurea et al., 1999), mammary tumor virus (MMTV) (Acha-Orbea and Palmer, 1991), and leukemia viruses in mice and hepatitis B virus in humans (Michalak et al., 1994; Rehermann et al., 1996). Although the presence of high titers of nAbs may reduce or prevent transmission of infection from mother to offspring, overall protective immunity is not really necessary for survival of the species. Therefore, taking these considerations into account, it becomes evident that in order to understand protective immunological memory, cytopathic viruses should be studied because it is only here that memory responses are relevant. B. WHAT MAINTAINS ANTIBODY MEMORY? As discussed previously, protection against evolutionarily important lytic virus infections, including those frequently contracted in childhood, is largely mediated by nAbs, although it is well recognized that memory antibody titers tend to decrease over time. Therefore, mechanisms have evolved to help sustain antibody levels. Maternal antibodies have been shown to participate in the deposition of immune complexes on follicular dendritic cells of the offspring which can help maintain antibody memory (Nossal et al., 1965; Tew et al., 1990; Bachmann et al., 1994a). Alternatively, periodical re-infection from either external sources (poliovirus, herpesvirus, influenza, parainfluenza, and many intestinal viruses) or internal foci of persisting infectious agents (HBV, HIV, and the various herpesviruses) provides natural boosters of immunity. Some viruses do not persist in the host as intact virus particles but in a crippled form (Billeter et al., 1994). For example, measles virus persists not only in subacute sclerosing panencephalitis (SSPE) patients but also apparently in most (if not all) infected hosts (Katayama et al., 1995). This explains why in the classical epidemiological studies on the Faroes or Pacific Islands protective memory was maintained for more than 60 years in previously infected survivors but not in those born on the island after the last epidemic (Mims, 1987). Two studies have provided evidence for the presence of long-lived bone marrow plasma cells following immunization with ovalbumin or infection with LCMV (Manz et al., 1998; Slifka and Ahmed, 1998). In contrast, extensive analysis of B cell memory and long-term antibody titers post VSV and LCMV infection

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indicated that, although memory B cells may be long-lived, antibody secreting plasma cells are short-lived (Ochsenbein et al., 2000b). This study revealed that, in order to maintain long-term antibody titers, continuous antigen-driven and T-cell-dependent differentiation of B cells to plasma cells must occur. How can the differing results of these studies be explained? While the first two studies (Slifka and Ahmed, 1998) analyzed B cell memory of binding antibodies using ELISA assays, Ochsenbein et al. studied protective neutralizing antibody responses against replicating and nonreplicating antigens in addition to binding antibodies. As outlined above, protection against a primary infection and/or against re-infection with acute cytopathic viruses largely depends on nAbs (Christian et al., 1996; Bachmann and Zinkernagel, 1997; Bachmann et al., 1997b). Neutralizing antibodies also influence infection with the noncytopathic LCMV (Baldridge and Buchmeier, 1992; Thomsen et al., 1996; Planz et al., 1997) but are irrelevant for ovalbumin. In contrast, the role of non-neutralizing antibodies in host protection is negligible in both LCMV and VSV infections. The discrepancies between these studies may reflect differences in avidities of antibodies which are probably lower for ELISA and higher for neutralization (Bachmann et al., 1997b) and/or perhaps more important differences in the numbers of antigenic sites assessed (one for neutralization and many for ELISA). After antigen encounter, specific B lymphocytes undergo clonal expansion. This expanded pool of antigen-specific B cells produces a faster and stronger response upon antigen re-encounter. In a recent study, Maruyama et al. (2000) generated mice with an inducible genetic switch in the BCR to generate memory B cells in the complete absence of specific antigen. They clearly demonstrated that memory B cells can persist over a long period of time in the absence of any antigenic stimulation. However, as outlined above, from an evolutionary point of view, memory B cells are not sufficient for protective memory. In addition, they cannot differentiate into antibody-secreting plasma cells early enough following re-infection with a cytopathic virus to mount protective antibody titers. Thus, to maintain long-term protective antibody titers, long-lived and antigenindependent memory B cells have to be “reminded” by antigen to differentiate to short-lived antibody-secreting plasma cells (discussed in Ochsenbein et al., 2000b). C. A COMPARISON WITH T CELL MEMORY While T cell memory is not the focus of this review, it is interesting to note some important differences with B cell memory. Cytotoxic T cells have the key function of controlling noncytopathic viruses during acute infection. Although protective during this period, they may also be harmful to the host because they can cause immunopathological destruction of infected cells and therefore must be controlled (reviewed in Zinkernagel et al., 1996). For example, a lethal


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graft-vs.-host-like immunopathology can be induced by injection of a high dose of viral peptide into a primed mouse that has a high precursor frequency of virusspecific CTLs. The immunopathology probably occurs as a result of peptide loading of many host cells, including cells of the immune system which are then killed by primed CTLs (Oehen et al., 1991; Aichele et al., 1997). Therefore, while memory nAb responses are required to protect against cytopathic infections, CTLs protect against noncytopathic acute infections, and increased precursor frequencies may actually be harmful. In contrast to maternal antibodies, CTL responses are not transmitted to offspring because transplantation antigen differences between mother and offspring can potentially cause graft-vs.-host reactions. In addition, the specificity of maternal T cells will not recognize the paternal MHC–peptide configuration of the offspring and would therefore be useless. Therefore, primed CTLs may function primarily to prevent virus from spreading again within the same host, so as to limit or prevent immunopathological disease (Kundig et al., 1996; Bachmann et al., 1997c). An example illustrating this point is the spectrum of virus–host relationships found after HBV or HIV infections in humans. If virus is controlled down to low levels, then chronic disease either does not develop or develops only very late. However, if the virus is not controlled, a severe autoaggressive disease (aggressive form of HBV-hepatitis) may develop. A similar balance exists in lepra or tuberculosis infections. In all these examples low-level infection maintained protective immunity (reviewed in Bianchi, 1981; Mondelli and Eddleston, 1984; Chisari and Ferrari, 1995). Mackaness introduced the term infectious immunity, or infection immunity, to describe this important co-evolutionary equilibrium (Mackaness, 1964, 1969). We conclude that although immunological memory has been considered to be an indispensable component of an individual’s immune system, this may be more idea than fact. Protective immunological memory most likely reflects a lowlevel response driven and maintained by persisting or re-encountered antigen. An antigen-driven memory response would protect the host against both direct damage by infections and indirect (immunopathologically mediated) damage mediated by infectious agent-specific T cells and antibodies. Most importantly, from an evolutionary point of view, antibody memory protects offspring during the physiological phase of immunodeficiency. XI. Neutralizing Antibodies and Vaccine Strategies

When we consider successful vaccines and compare them with those infectious diseases where efficient protective vaccines are lacking (reviewed in Nossal, 1998), it is striking to note that all successful vaccines induce high levels of nAbs that are both necessary and sufficient to protect the host from disease. Successful vaccination against infectious diseases such as tuberculosis, leprosy,

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or HIV would require induction of additional long-lasting T cell responses to control infection. Although long-lasting nAb responses can be efficiently generated by classical vaccination strategies we are not yet able to generate vaccines that mimic infections that persist at very low levels, providing the necessary long-term T cell immunity. A. SUCCESSFUL VACCINES Generation of neutralizing antibodies is central to successful vaccine development. Poliovirus vaccines serve as a good example (Nathanson and Martin, 1979; Sabin, 1985). Neutralizing antibodies in the form of mucosal IgA, or serum IgG, can prevent or reduce infection at a very early stage. Both the Sabin and the inactivated Salk vaccine induce long-lasting IgG responses in serum that prevent circulating virus from reaching the central nervous system. Since none of the successful vaccines is able to completely prevent re-infection, the generally accepted mode of action is to greatly reduce systemic spread of the infectious agent through induction of nAbs. B. INEFFECTIVE VACCINES Analysis of successful vaccine strategies may perhaps reveal why certain vaccines, including mumps (Arya, 1994; Strohle et al., 1997) and measles (OfosuAmaah, 1983; Garenne et al., 1991; Malfait et al., 1994; Wild, 1999; Bennett et al., 1999), both of which are paramyxoviruses, are less effective and therefore subject to criticism by opponents of vaccination. Mumps vaccines cannot always prevent re-infection of the salivary gland (Germann et al., 1996; Strohle et al., 1997); however, severe forms of disease such as orchitis and encephalitis do not occur in vaccinated children. This finding may indicate that, following systemic vaccination with a low-dose vaccine, local mucosal immunity may not be sufficiently high enough to protect against re-infection. However, similar to the Sabin vaccine, even though a local breakthrough can occur in the mucosa, systemic disease does not develop because virus is trapped by neutralizing IgG in the blood. Similar considerations may apply to measles vaccines, although the consequences of re-infection are more severe. Once again, systemic measles immunization may not provide sufficient titers of local antibodies to prevent infection of throat and tonsils. In addition, measles virus has the capacity to cause immunosuppression by interfering with APC and B cell function (reviewed in McChesney and Oldstone, 1989. Interestingly, this may also apply to the attenuated vaccine strains. Increased incidences of insufficient vaccine protection, together with the possibility that maternal antibodies may interfere with efficient vaccination during early childhood (Sabin et al., 1983; Englund et al., 1998; Siegrist et al., 1998), are of great concern, particularly for third-world countries (Gwatkin, 2000). In an attempt to enhance measles vaccine efficacy, the innoculum dose was increased


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by a factor of 10 to 100, but this led to late disease development in children and therefore has been stopped (Gwatkin, 2000). This example shows that even attenuated viruses, when given in high doses, can cause disease comparable to low-dose infections with wild-type viruses. The details of these complications with measles are still unknown and require further evaluation. Nevertheless, this example demonstrates that attenuation of a virus is not going to yield optimal vaccines in all instances. Wild-type measles is also interesting because it may persist in the host for a very long time, possibly until death (Katayama et al., 1995). Whether this also applies to vaccine strains is not yet known. However, it is important to determine where measles virus persists and for how long. In addition, whether such persisting virus is necessary and sufficient to maintain long-term protection and whether vaccine strains can also cause SSPE must be investigated. C. ATTENUATED VACCINES THAT PROVIDE INSUFFICIENT PROTECTION OR CAUSE DISEASE Whether or not attenuated viruses are always good vaccine candidates is debatable. This question has recently been discussed using naturally occurring nef-deficient HIV in humans and attenuated SIV strains in monkeys as examples (Ruprecht, 1999). Although both attenuated strains did not initially cause immunodeficiency, eventually both humans and monkeys developed disease and some of them died. Similarly to HIV infection, successful long-term protective vaccines against tuberculosis or leprosy are still not available (Bloom and Ahmed, 1998; Bloom and McKinney, 1999). In contrast to wild-type tuberculosis, bacillus Calmette-Guerin (BCG) apparently does not persist in humans for more than a few years and this correlates with the time of protection against tuberculosis in young children. This suggests that persistent infection is required for maintenance of an activated T cell response which limits granuloma size in order to avoid immunopathology yet sustains long-lasting immune responses (Bloom and McKinney, 1999). In summary, attenuated vaccines strains cannot yet imitate that optimal mixture of “infection immunity” as defined by Mackaness (Mackaness, 1962, 1969). Similarly, when CTL responses are necessary to control infection, such as for HIV, HCV, and herpesviruses, attenuated viruses have not been successful. At this time, DNA vaccination may be the most promising candidate to provide a low-dose persistent antigen source that guarantees long-term activation of both T and B cells. D. LINEAR EPITOPES FOR INDUCTION OF NEUTRALIZING ANTIBODIES A discussion of vaccine strategies should also include the old hope of mimicking neutralizing epitopes using linear peptides that would provide a safe, nonreplicating vaccine that induces long-lasting titers of nAbs. Theoretically, such a

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strategy should be successful and has been shown to be effective in rare examples such as with foot-and-mouth disease virus (Bittle et al., 1982). In addition, use of linear peptides of the V3 loop of HIV has provided limited success in vitro but has not yet yielded success in vivo (Conley et al., 1994). In general, it is very difficult to imitate the serotype-specific three-dimensional structure of neutralizing epitopes. This is particularly true for high avidity-neutralizing antibody/antigen interactions. The reasons probably rest in the simple fact that neutralizing epitopes are, in general, not linear but are composed of two or more loops of protein chains. XII. Conclusions

Studies of neutralizing antibodies against viruses, bacteria, and toxins have largely been neglected in the past 30 years. However, numerous studies have focused on induction of binding antibodies against soluble protein antigens such as ovalbumin or against haptens. Although these classical studies have provided some insights into B cell and antibody function, re-evaluation of the conclusions reached is timely and necessary. One major reason for neglecting to study antiviral antibody responses has been perhaps that T-cell-mediated immunity occupied much of the interest of immunologists. Recently, many new tools have been developed to allow analysis of B cell and neutralizing antibody responses at a level that was not imaginable 30 years ago. Antibodies can now be sequenced rapidly, transgenic and gene-knockout mice permit the evaluation of precise immunological functions, and advances in molecular virology provide many new and fascinating research perspectives. Neutralizing antibodies are evolutionarily important effectors of immunity against viruses. Their evaluation has revealed a number of basic insights into specificity, rules of reactivity (tolerance), and memory: (1) Specificity of neutralizing antibodies is defined by their capacity to distinguish between virus serotypes; (2) B cell reactivity is determined by antigen structure, concentration, and time of availability in secondary lymphoid organs; and (3) B cell memory is provided by elevated protective antibody titers in serum that are depending on antigen stimulation. These perhaps slightly overstated rules are simple, correlate with in vivo evidence as well as clinical observations, and appear to largely demystify many speculations about antibodies and B cell physiology. The implications for vaccines are obvious; therefore, careful and critical assessment of the reviewed experimental evidence and concepts will be of great importance. REFERENCES Acha-Orbea, H., and Palmer, E. (1991). Mls—a retrovirus exploits the immune system. Immunol. Today 12, 356–361.


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Regulation of Interleukin-12 Production in Antigen-Presenting Cells XIAOJING MA∗ AND GIORGIO TRINCHIERI† ∗Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021; and †Schering-Plough Laboratory of Immunological Research, 69571 Dardilly, France

Interleukin-12 is a cytokine produced by antigen-presenting cells that is essential for host defense against intracellular microbial infection and control of malignancy by virtue of its ability to stimulate both innate and adaptive immune effector cells. The immune potentiating capacity of IL-12 and its mandatory requirement in host defense predispose it to rigorous regulation. The time, localization, and magnitude of IL-12 production during an immune response strongly influence the type, extent, and, ultimately, the fate of the response. Disturbance of this evolutionarily maintained “balance of power” frequently leads to immunologic disorders. This article reviews the intricate pathways that have been uncovered in which IL-12 production is modulated by numerous pathogens and immunological regulators. The understanding of IL-12 regulation in physiological settings will undoubtedly C 2001 Academic Press. lend valuable support to the design of therapeutic applications of IL-12.

I. Introduction

Interleukin-12 (IL-12) was originally identified as a molecule able to stimulate natural killer (NK) cell activity, to generate lymphokine-activated killer (LAK) cells, and to induce the production of interferon-γ (IFN-γ ) from NK and T cells. Since its discovery in the late 1980s, it has been catapulted to the center stage of immunology because of its essential role in both innate and adaptive immune responses against infectious pathogens and malignant growth. The potency and obligatory requirement of IL-12 in host defense make it a target for stringent regulation. Indeed, the temporal, spatial, and quantitative expression of IL-12 during an immune response in a microenvironment contributes critically to the determination of the type, extent, and ultimate resolution of the response. Many immunologic disorders result from breaches of such a fine balance. This article seeks to review comprehensively the intricate pathways that have been uncovered in which IL-12 production is modulated by numerous pathogens and immunological regulators. II. The Discovery of IL-12

Activities that stimulate NK cytolytic capacity and IFN-γ production in response to bacterial infection were observed as early as the mid-1980s both in vivo 55 C 2001 by Academic Press. Copyright All rights of reproduction in any form reserved. 0065-2776/01 $35.00

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and in vitro (Kearns and Leu, 1984; Tarkkanen et al., 1986). In 1989, Nakamura et al. identified a soluble activity of around 70,000 kDa in the serum of mice infected with Mycobacterium bovis BCG and challenged with lipopolysaccharide (LPS) that was able to potently stimulate IFN-γ production (Nakamura et al., 1989). In the meantime, Epstein–Barr virus (EBV)-transformed lymphoblastoid cell lines had been used as feeder cells for their ability to support the growth of T and NK cells (Hercend et al., 1982) as well as to secrete an activity capable of inducing IFN-γ production (Reem et al., 1982). Kobayashi et al. (1989) analyzed factors secreted by EBV-transformed B cell lines and identified an activity, which was named natural killer cell stimulatory factor (NKSF), that mediated several biological activities on human T and NK cells, including induction of IFN-γ production, enhancement of cell-mediated cytotoxicity, and comitogenic effects on resting T cells. NKSF was purified to homogeneity from the conditioned medium of phorbodiester-stimulated, EBV-transformed B cell line RPMI-8866 and was found to be a heterodimeric cytokine. Subsequently, the genes encoding the two-subunit chains of NKSF were cloned, and biologically active recombinant NKSF was expressed in mammalian cells cotransfected with cDNA for both chains of NKSF (Wolf et al., 1991). Independently, Stern and coworkers later identified an activity named cytotoxic lymphocyte maturation factor (CLMF) in the conditioned medium of another EBV-transformed B cell line, NC37, on the basis of its ability to synergize with IL-2 in inducing the generation of LAK cells from hydrocortisone-treated T cells (Stern et al., 1990). Cloning of the genes encoding CLMF revealed its identity with NKSF (Gubler et al., 1991), hence the unifying term of interleukin-12 (IL-12) was introduced. The small subunit is named IL-12 p35 (α chain) and the large subunit IL-12 p40 (β chain). The heterodimer is also referred to as IL-12 p70. III. Molecular Structure of IL-12

The two genes encoding IL-12 p40 and p35 are located on separate chromosomes (5q31–33 and 3p12–q13.2, respectively, in humans; chromosomes 11 and 6, respectively, in the mouse) (Noben-Trauth et al., 1996; Sieburth et al., 1992; Tone et al., 1996; Yoshimoto et al., 1996) and do not share sequence homology. The p35 cDNA encodes a 209-amino-acid polypeptide corresponding to a mature protein of 27.5 kDa. It contains seven cysteine residues and three potential N-glycosylation sites. The p40 cDNA sequence encodes a 328-amino-acid polypeptide with a 22-amino-acid signal peptide generating a mature protein of 34.7 kDa. It contains ten cysteine residues, four potential N-glycosylation sites, and one consensus heparin-binding site (Gubler et al., 1991; Wolf et al., 1991). The p35 gene has some homology to IL-6 and G-CSF (Merberg et al., 1992), whereas the p40 chain is homologous to the extracellular portion of the α chain of ciliary neurotropic factor (CNTF) receptor as well as to those of the IL-6

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and granulocyte colony-stimulating factor (G-CSF) receptors (Gearing and Cosman, 1991; Schoenhaut et al., 1992). The p40 chain bears the hallmarks of the hematopoietin receptor family: one tryptophan and four cysteine residues in conserved positions and a WSEWAS sequence, similar to the consensus motif WSXWS in the hematopoietin receptor family (Taga and Kishimoto, 1992). Most of the members of this transmembrane receptor family can be released from the producer cells in soluble forms containing the extracellular portion truncated after the WSXWS motif by either alternative splicing of the mRNA transcripts or by proteolytic digestion of the receptor (Taga and Kishimoto, 1992). Heterodimers of p40 and p35 are formed via disulfide bonds and secreted, usually upon stimulation of producer cells. However, IL-12 may also exist as a performed membrane-associated molecule that can be quickly released (within minutes) from phagocytic cells on contact with intracellular microbes such as Leishmania species in the absence of de novo transcription (Quinones et al., 2000), in contrast to the production of IL-12 induced by bacterial products such as LPS, which takes place with a much slower kinetics (several hours) and which depends on de novo transcription. In addition to forming heterodimers with p35, both mouse and human p40 are secreted in large excess as free p40 monomers and can also form homodimers (p402), which exhibit biological activities antagonistic to the heterodimeric IL-12 p70 (Gillessen et al., 1995; Ling et al., 1995). Homodimers of p35 have not been reported to date. However, p35, which is not secreted in the absence of a second chain, may heterodimerize and be secreted together with a second cellular protein, EBV-induced gene 3 (EBI-3), although no biological function of this novel heterodimer has been demonstrated yet (Devergne et al., 1997). A novel sequence discovered in a computational screen of genomic databases showed distant homology to the p35-chain of human and mouse IL-12. It was named p19 for its apparent molecular weight (Oppmann et al., 2000). p19 has no detectable biological activity by itself and is not secreted from transfected cells; instead, it can dimerize and be secreted together with the IL-12 p40 subunit to form a novel disulfide-bridged, biologically active composite cytokine. The p19/p40 complex has been named interleukin-23 (IL-23). IL-23 appears to have at least some of the biological activities of IL-12. However, it also induces strong proliferation of mouse memory T cells, a unique activity not shared with IL-12, which preferentially induces proliferation of na¨ıve T cells. Production of natural IL-23 heterodimers has been shown both in the mouse and in humans; although the full spectrum of cell types producing IL-23 is not known yet, dendritic cells (DCs) that are potent producers of IL-12 are also able to produce IL-23. Because of the necessity of simultaneous expression of IL-12 p40 and p35 chains in the same cell in order to form IL-12 p70, it is always important to analyze the production of biologically active heterodimer in order to obtain meaningful information on the production of IL-12 and its functional role in vivo.

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Disassociated measurement of the single chains can sometimes mislead investigators, considering the facts that IL-12 p40 and p35 gene expression can be found in different cell types, that both of them have alternate dimerization partners, that their relative expression is not constant, and that the rate of dimerization and secretion can be controlled post-translationally. IV. Biological Functions of IL-12

Interleukin-12 is produced by phagocytic and antigen-presenting cells in response to intracellular bacterial and parasitic infections. It is a proinflammatory cytokine able to activate NK and T cells for the induction of IFN-γ production and cytolytic activity. In part through its induction of INF-γ production, IL-12 enhances the phagocytic and bactericidal potential of phagocytes and their ability to release proinflammatory cytokines such as GM-CSF, IL-1β, IL-6, TNF-α and IL-12 itself (Nagayama et al., 2000). In addition to its role in mobilizing the first line of defense, IL-12 produced during the early phases of an infection also lays the groundwork for the ensuing antigen-specific immune response by promoting the differentiation and function of T helper 1 (Th1) cells that support cell-mediated immunity, cytotoxic T cell generation, induction of opsonizing antibodies, and activation of phagocytic cells, at the expense of Th2 cell differentiation (Hsieh et al., 1993; Manetti et al., 1993; Trinchieri, 1993). Particularly in the presence of costimulation, IL-12 is also a potent mitogen for T and NK cells (Kobayashi et al., 1989; Kubin et al., 1994). Although the majority of the biological activities of IL-12 in directing innate and adaptive immunity against self (tumor) and non-self antigens are mediated by IFN-γ , the latter cytokine does not replace IL-12 in all systems. For example, IFN-γ is absolutely required for IL-12-induced resistance to Leishmania major infection. However, IL-12 is also able to induce IL-10 production and suppress IL-4 synthesis in IFN-γ knockout mice during acute or established infection (Wang et al., 1994). The rejection of IL-12-transduced carcinoma cells that takes place in IFN-γ -null mice is mediated apparently by a CD4-T-cell-dependent production of GMCSF, which helps to maintain the CD8–polymorphonuclear leukocyte cross talk in the absence of IFN-γ (Zilocchi et al., 1998). V. Receptors of IL-12 and Signaling

Interleukin-12 receptors (IL-12R) are primarily expressed on activated T and NK cells (Desai et al., 1992). Expression of IL-12R on other cell types such as DCs (Braun et al., 1999; Grohmann et al., 1998), melanocytes (Lasek et al., 1999; Yue et al., 1999), and B cell lines (Airoldi et al., 2000; Wu et al., 1996) has also been shown. Both high-affinity (Kd = 5–20 pM, 100–1000 sites per cell) and lowaffinity (Kd = 2–6 nM, 1000–5000 sites per cell) IL-12 binding sites have been


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detected on human phytohemagglutinin (PHA)-activated lymphoblasts. The cDNAs for two IL-12R subunits have been cloned from human and mouse T cells and designated as IL-12Rβ1 (Chua et al., 1994, 1995) and IL-12Rβ2 (Presky et al., 1996). Both of these subunits have extensive homology with gp130, the common receptor β chain of the IL-6-like cytokine superfamily. They are type I transmembrane glycoproteins, with molecular weight of about 100 kDa (IL-12Rβ1) and 130 kDa (IL-12Rβ2). On the cell surface, each of the two recombinant IL-12R subunits occurs as dimers/oligomers (Gately et al., 1998). Coexpression of IL-12Rβ1 and IL-12Rβ2 is required for the generation of high-affinity (pM) IL-12 binding sites (Presky et al., 1996). For both human and mouse, the IL-12Rβ2 subunit appears to function as the signal-transducing component of the high-affinity receptor complex (Presky et al., 1996; Zou et al., 1997). This is suggested by the absence of tyrosine residues in the cytoplasmic domain of human IL-12Rβ1 and the presence of conserved tyrosine residues in the cytoplasmic portion of IL-12Rβ2. Recent evidence suggests that within T cells the expression of both the human and mouse IL-12Rβ2 proteins may be confined to Th1 cells and that IL-12Rβ2 expression correlates with IL-12 responsiveness in these cells (Rogge et al., 1997; Szabo et al., 1997). Signal transduction through the IL-12R induces tyrosine phosphorylation of primarily the Janus family kinases JAK2 and TYK2 (Bacon et al., 1995a), which in turn phosphorylate and activate STAT4 (Bacon et al., 1995b). In the mouse system, IL-12 is the only stimulus known to activate STAT4. Unlike in the mouse, in humans type I interferons induce Th1 development and can activate STAT4 by recruitment to the IFN-α receptor complex specifically via the carboxy-terminus of STAT2; the mouse Stat2 gene harbors a minisatellite insertion that has altered the carboxy-terminus and selectively disrupted its ability to activate STAT4 but not other STATs (Farrar et al., 2000). STAT4, through the production of IFN-γ , mediates most but not all of the biological activities of IL-12 in host defense against microbial infection. IL-12 also induces tyrosine phosphorylation and activation of 44-kD mitogen-activated protein kinase (ERK1) in human T cells (Pignata et al., 1994). The involvement of MAP kinases in the alternative, Stat4independent IL-12 signaling responsible for residual type 1 immunity in patients genetically deficient for IL-12Rβ1 chain has been reported (Verhagen et al., 2000), implicating IL-12Rβ2 in this role. VI. Producer Cells of IL-12

Interleukin-12 was originally identified and purified from EBV-transformed B cell lines, which constitutively produce low levels of IL-12 that can be slightly enhanced upon treatment with phorbol diesters (D’Andrea et al., 1992). Although most African Burkitt’s lymphoma cell lines produce no or negligible amounts of IL-12, most AIDS-associated EBV(+) Burkitt’s lymphoma cell lines (AABCL)

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constitutively produce IL-12, which can be further enhanced by phorbol diesters to levels much higher than in normal B cell lines (Benjamin et al., 1996). A lack of IL-12 expression of EBV(–) AABCL suggests that in vivo exposure of B cells to only HIV-1 does not induce IL-12 secretion and that both HIV-1 and EBV exposures are required (Benjamin et al., 1996). The production of IL-12 in normal B cells has been questioned ever since the discovery of IL-12 in EBV-transformed B cell lines. In mice infected with LPS or staphylococcal enterotoxin B (SEB), only IL-12 p35 mRNA was detectable by in situ hybridization in the B cells area of the spleen, whereas the p40 mRNA was found in T cell areas, indicating a disparate expression pattern of the two chains (Bette et al., 1994). Recently, Schultze et al. (1999) demonstrated that a subset of human tonsillar B cells can be induced to secrete bioactive IL-12 mainly via CD40 ligation facilitated by activated Th1 cells. Expression after CD40 activation is restricted to CD38–IgD ± , non-germinal center (GC) B cells. IL-12 produced from these cells is postulated to provide a positive feedback during T–B interactions, thereby maintaining the differentiation pattern of the T cells during amplification of the immune response (Schultze et al., 1999). Bioactive IL-12 production has never been detected in T cells. However, Nagayama et al. (2000) reported that in concanavalin-A-activated human peripheral blood T cells stimulated simultaneously with IL-12, both IL-12 p40 and p35 mRNA were detected by reverse transcription–polymerase chain reaction (RT–PCR). No bioactive IL-12 was measured in this study (Nagayama et al., 2000). Many studies, however, established that the major physiological cell types that produce IL-12 p70 rather than B or T cells are phagocytic cells such as monocytes/macrophages (D’Andrea et al., 1992) and neutrophils (Cassatella et al., 1995), as well as DCs (Macatonia et al., 1995). On a per-cell basis, polymorphonuclear cells (PMN) produce less IL-12 than monocytes in response to LPS stimulation (Cassatella et al., 1995); however, because of the large number of PMN present in the blood or in inflammatory tissues, it is likely that IL-12 produced from these cells plays a physiological role in the inflammatory response to bacterial or parasitic infection. A recent study (Bliss et al., 2000) demonstrated that neutrophils responding to in vivo infection with the protozoan pathogen Toxoplamsa gondii are capable of IL-12 production. Moreover, IL-12 production by neutrophils mobilized rapidly to the site of infection appears to be derived from prestored pools (Bliss et al., 2000), analogous to the in vitro study with Leishmania species (Quinones et al., 2000). During the course of inflammatory diseases of the central nervous system (CNS), including multiple sclerosis (MS) and HIV-encephalopathy, microglial cells undergo a morphologic change into an activated phenotype (Suzumura et al., 1991) expressing an array of cell-surface molecules and cytokines which can participate in regulating immune reactivity or effecting tissue injury within


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the CNS (Becher and Antel, 1996). Human CNS-derived microglia, but not astroglia, can produce IL-12 p70 in vitro following activation with LPS (Becher et al., 1996). Murine bone-marrow-derived mast cells cultured in either IL-3 or mast cell growth factor (MGF; also known as c-kit ligand and stem cell factor) differentially express cytokine genes. Bone marrow cells cultured in IL-3 proliferate and differentiate to a mucosal mast-cell-like phenotype. These cells express the IL-4 gene. Bone marrow cells cultured in MGF develop a connective tissue mast-cell-like phenotype and possess transcripts for both of the subunits of the IL-12 cytokine in quantities comparable to those in the control splenocyte or macrophage cultures (Smith et al., 1994). Mucosal mast cells have been previously implicated in the development of the Th2 cell phenotype via their expression of IL-4. The finding that the MGF-derived connective-tissue-like mast cells possess IL-12 transcripts suggests that the development of the Th1 cell pathway may be positively influenced by this type of mast cells. The first line of evidence that DCs, the most powerful antigen-presenting cells, produce IL-12 was obtained in studies showing that in the skin-resident DCs, the Langerhans cells, rather than keratinocytes, are the major IL-12 producers (Kang et al., 1996). It was also shown that LPS treatment of highly purified mouse DCs induces expression of IL-12 p40 mRNA (Kanangat et al., 1995). The definitive evidence that DCs are producers of functional IL-12 comes from studies demonstrating that these cells, when used as antigen-presenting cells, induce a Th1 response if endogenous IL-4 is blocked and that this Th1 response is prevented by neutralizing anti-IL-12 antibodies (Macatonia et al., 1995). Extensive studies with both human and mouse DCs have now clearly confirmed that DCs are efficient producers of the IL-12 that acts in inducing Th1 responses upon antigen presentation by these antigen-presenting cells (Cella et al., 1996; Heufler et al., 1996). However, due to the heterogeneity of both human and mouse DCs it is still unclear which type of DC is the major producer of IL-12, and there are apparent discrepancies between the human and mouse data. Besides Langerhans cells, which produce only low levels of IL-12, human monocyte-derived DCs, obtained from cultures in GM-CSF and IL-4, are very potent producers of IL-12 p70 in response to various stimuli (Cella et al., 1996). Although the ability of these cells to produce high levels of IL-12 may in part be due to their culture in the presence of IL-4, a potent primer for IL-12 production in monocytes and other cell types (D’Andrea et al., 1995), these data are generally interpreted to indicate that human DCs of myeloid origin may be the major IL-12 producer cells also in vivo. Plasmacytoid DCs, present in human peripheral blood and other lymphoid organs, are of possible lymphoid origin and correspond to the cells previously described as natural IFN-α producer cells (Perussia et al., 1985; Rissoan et al., 1999). These cells have been reported to produce no IL-12 (Rissoan et al., 1999) or only low levels of it (Cella et al., 2000), although the ability of virus-activated

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plasmacytoid DCs to produce IL-12 may be important in their ability to induce a Th1 response. In the mouse the equivalent of the human plasmacytoid DCs has not been described yet, but a novel subset of DCs of possible lymphoid origin that express a CD8α/α homodimer and limit the proliferation of CD4+ T cells in vitro by Fas-mediated death has been described (Suss and Shortman, 1996; Vremec et al., 1992). Studies in mice infected with Toxoplasma gondii have clearly shown that the CD8α-positive DCs and not the myeloid DCs are the producers of IL-12 in vivo early after infection (Reishe Sousa et al., 1999; Sousa et al., 1997). Thus, the unambiguous identification of the DC type responsible for IL-12 production during infections is hinged on a better understanding of the correspondence between the different DC subsets identified in humans and mouse. The early induction of IL-12 is a critical event in determining the development of both innate resistance and adaptive immunity to many intracellular pathogens. Numerous earlier in vitro studies have suggested that the macrophage is a major source of the initial IL-12 produced upon microbial stimulation and that this response promotes the differentiation of protective Th1 CD4+ lymphocytes from precursors that are primed on antigen-bearing DCs. Sousa et al. (1997) showed by immunolocalization experiments and flow cytometric analysis that DCs, not macrophages, are the initial cells to synthesize IL-12 in the spleens of mice exposed in vivo to an extract of T. gondii or to LPS. This production of IL-12 occurs very rapidly and is independent of signals from T cells, such as CD40 ligand. In light of the finding of preformed pool of membrane-associated IL-12 that is released rapidly upon DC contact with Leishmania species (Quinones et al., 2000), it would not be surprising that the swift IL-12 production detected in this study may have come from the same source.

VII. Pathways of IL-12 Induction

A. IL-12 INDUCTION BY PATHOGENS Interleukin-12 production can be induced in three major pathways. The first pathway is represented by the ability of bacteria and their products to directly stimulate macrophages or DCs. This is a T-cell-independent mechanism of production of IL-12 in innate responses (D’Andrea et al., 1992; Kanangat et al., 1995). A wide range of intracellular bacteria, both gram-positive and -negative, as well as their products such as LPS, are potent inducers of IL-12 by phagocytes. Microbial lipoproteins such as those derived from Mycobacteria are strong stimulators of IL-12 production by human macrophages. The T-cell-independent mechanisms are important for the proinflammatory and immunoregulatory role of IL-12 at the interface of innate resistance and adaptive immunity. In addition to gram-negative bacteria via LPS, gram-positive bacteria can also


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stimulate macrophages to produce IL-12. For example, lipoteichoic acid (LTA), a predominant surface glycolipid of gram-positive bacteria, potently induced IL12 p40 gene expression (Cleveland et al., 1996). A competitive LPS antagonist, Rhodobacter sphaeroides LPS, inhibits LTA-induced IL-12 production, suggesting a common pathway for LPS and LTA in IL-12 activation. Pretreatment of cells with anti-CD14 monoclonal antibody blocks both LPS and LTA induction of IL-12 p40 expression. LTA also can induce Th1 development in na¨ıve CD4 T cells by an IL-12-dependent mechanism, indicating direct induction of physiologic levels of IL-12 (Cleveland et al., 1996). In this particular study, muramyl dipeptides as well as the major muramyl tetrapeptide component of Streptococcus pneumoniae were inactive for inducing IL-12. However, in another study, muramyl dipeptide conjugated to maleylated bovine serum albumin (BSA) was shown to be a potent inducer of macrophage-derived production of proinflammatory cytokines, especially for IL-12, through scavenger receptor-mediated endocytosis. Maleylated BSA alone did not induce any IL-12 secretion (Srividya et al., 2000). Bacterial cell wall components (e.g., LPS and peptidoglycans) are wellcharacterized pattern-recognition factors (PRFs) that are structurally conserved, invariant microbial constituents. Recognition of these factors by antigenpresenting cells activates the cells to exert various effector functions. Toll-like receptors (TLR) likely play a major role in the recognition of bacterial components and, through their ability to activate NFκB, induce IL-12 production. TLR4 is required for response to LPS (Poltorak et al., 1998), whereas TLR2, probably in cooperation with either TLR1 or TLR6, is involved in the recognition of different products of gram-positive bacteria (Brightbill et al., 1999; Ozinsky et al., 2000; Takeuchi et al., 1999). Over the last few years, bacterial DNA has also been recognized as a powerful stimulus of innate immune cells in vivo and in vitro (Wagner, 1999) and as a PRF. Its immune-stimulatory potential is based on the presence of unmethylated CpG-motifs, which often contain the nucleotide sequence purine– purine–cytidine–guanine–pyrimidine–pyrimidine. These sequences are present in bacterial DNA at almost the statistically expected frequency but are evolutionarily suppressed (CG suppression) and methylated in vertebrate DNA (Sved and Bird, 1990). The stimulatory principle contained in double-stranded DNA can be transferred to single-stranded oligonucleotides (ODNs) that contain the described CpG motif (CpG ODN) (Yamamoto et al., 1992). In macrophages and bone-marrow-derived DCs, CpG-DNA has been shown to induce release of the cytokines TNF-α, IL-6, and IL-12 (Sparwasser et al., 1997, 1998; Stacey et al., 1996). Additionally, CpG-DNA induces maturation of DCs, characterized by up-regulation of MHC class II and costimulatory molecules such as CD80 and CD86, which results in highly efficient antigen presentation (Sparwasser et al., 1998). Induction of this DC maturation accompanied by the production of large

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amounts of IL-12 may explain the exceptional potency of CpG-DNA as an adjuvant (Lipford et al., 1997). CpG-DNA is believed to enter the cell via a receptor complex, an essential component of which has been postulated to be TLR-9, based on the CpG-DNA-unresponsive phenotype of TLR-9 deficient mice (Krieg and Wagner, 2000). Hacker and coworkers (Hacker et al., 1999) analyzed the mitogen-activated protein kinase (MAPK) pathways triggered by CpG-DNA and their significance for cytokine production in macrophages and DCs and found that CpG-DNA induced extracellular signal-regulated kinase (ERK) activity in macrophages in a classic MAPK/ERK (MEK)-dependent way. This pathway up-regulated TNFα production but down-regulated IL-12 production. However, in DCs, CpGDNA and LPS failed to induce ERK activity. Consistent with a specific negative regulatory role for ERK in macrophages, chemical activation of this pathway in DCs suppressed CpG-DNA-induced IL-12 production. These results suggest that differential activation of MAP kinase pathways may be a basic mechanism by which distinct subsets of innate immune cells regulate their effector functions. A synthetic double-stranded RNA, polyriboinosinic polyribocytidylic acid (Poly I:C) is often used in models of viral infection. Poly I:C is a potent IFN inducer and can activate monocytes to produce CSF, IL-1β, prostaglandin E2 (PGE2) (Akiyama et al., 1985), and IL-12 (Manetti et al., 1995). Poly I:C has been described to bind to scavenger receptors of macrophages (Yoshida et al., 1992) and to induce stable maturation of functionally active human DCs producing high levels of IL-12 and low levels of IL-10 (Verdijk et al., 1999). B. IL-12 INDUCTION BY LOW-MOLECULAR-WEIGHT GLYCOSAMINOGLYCAN HYALURONAN The second pathway of IL-12 production is also T-cell-independent and involves the interaction of macrophages with components of the extracellular matrix (ECM) selectively expressed during inflammation. Components of the ECM can regulate leukocyte activation and function at inflammatory sites. Lowmolecular-weight fragments of the ECM glycosaminoglycan hyaluronan (LMWHA) that accumulate in inflammation, but not the ubiquitous high-molecularweight form of HA (HMW-HA), have been shown to induce cytokine and/or chemokine production by alveolar and bone-marrow-derived macrophages. Hodge-Dufour et al. (1997) compared the effects of HMW-HA and LMW-HA on resident and thioglycollate-elicited murine peritoneal macrophages and demonstrated that treatment of elicited macrophages with LMW-HA, but not with HMW-HA, stimulated production of the chemokines RANTES and macrophage inflammatory protein-1α and -1β. Furthermore, LMW-HA induced the production of biologically active IL-12, which was inhibited by an anti-CD44 mAb that blocks HA binding. In contrast to elicited macrophages, freshly explanted resident peritoneal macrophages did not respond to LMW-HA. However, preculture


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in vitro before stimulation led to adhesion-dependent priming for LMW-HAinduced cytokine and chemokine production by resident macrophages. In a more recent study, Termeer et al. (2000) reported that only small HA fragments of tetra- and hexasaccharide size (sHA), but not of intermediate size (m.w. 80,000– 200,000) or HMW-HA (m.w. 1,000,000–600,000) induced immunophenotypic maturation of human monocyte-derived DCs (up-regulation of HLA-DR, B7-1/2, CD83; down-regulation of CD115). Likewise, only sHA increased DC production of the cytokines IL-1β, TNF-α and IL-12, as well as their allostimulatory capacity. These effects were highly specific for sHA, because they were not induced by other glycosaminoglycans such as chondroitin sulfate or heparan sulfate or their fragmentation products. Interestingly, sHA-induced DC maturation does not involve the HA receptors CD44, because DC from CD44-deficient mice and wild-type mice both responded similarly to sHA stimulation. These results, although not entirely reconciled on the issue of CD44 involvement in macrophages vs. DC, provide further evidence of the potential importance of CD44/LMW-HA interactions in regulating the immune response at sites of inflammation and demonstrate that the state of differentiation of macrophages may determine their sensitivities to matrix components. C. IL-12 INDUCTION BY ACTIVATED T CELLS The third pathway of IL-12 induction involves macrophages or DCs and T cell engagement through CD40/CD40 ligand (CD40L) interaction, a T-celldependent productive pathway. Kennedy et al. (1996) first showed that the expression of CD40L by activated T cells is critical for T-cell-dependent IL-12 production by mouse macrophages. Snijders et al. (1998) subsequently demonstrated, using populations of na¨ıve and memory Th cells, recombinant CD40L, neutralizing and blocking antibodies, and by determining IFN-γ production and CD40L expression levels, that T-cell-induced IL-12 production by DCs results from the action of two signals, mediated by CD40L and IFN-γ and that the inability of na¨ıve Th cells to induce IL-12 production is due to their failure to produce IFN-γ . LPS could replace either signal for IL-12 production by DCs. The two-signal requirement is unique for the production of IL-12, since either CD40 engagement or LPS was sufficient for the efficient production of TNF-α, IL-8, and the p40 subunit of IL-12, suggesting that IFN-γ production from T cells is critical for activation of the IL-12 p35 gene expression. The two-signal hypothesis for IL-12 production (limited by the synthesis of the p35 subunit) in DC has been confirmed in vivo by a recent study which demonstrates that the ability of DC to produce IL-12 in response to CD40 ligation in vivo is nonreciprocally dependent on exposure to an innate signal from an appropriate type of infection (Schulz et al., 2000). The T-cell-dependent mechanisms of IL-12 induction play an important role in the T cell immunoregulatory role of IL-12 and in the maintenance of Th1 responses. The CD40/CD40L-induction of IL-12

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production is not the only pathway in which APC–T cell interaction could lead to IL-12 synthesis. Another member of the TNF receptor family, RANK (receptor activator of NFκB), when activated by its ligand (RANKL/TRANCE) expressed on activated T cells, also induces IL-12 production from DCs (Josien et al., 1999). Lymphocyte activation gene-3 (LAG-3) is an MHC class II ligand structurally and genetically related to CD4. Although its expression is restricted to activated T cells and NK cells, the functions of LAG-3 remain unknown. Avice et al. 1999) investigated the expression and function of LAG-3 on proinflammatory bystander T cells that are activated in the absence of TCR engagement. LAG-3 is expressed at high levels on human T cells cocultured with autologous monocytes and IL-2 and synergizes with the low levels of CD40L expressed on these cells to trigger TNF-α and IL-12 production by monocytes. Anti-LAG-3 mAb inhibits both IL-12 and IFN-γ production in IL-2-stimulated cocultures of T cells and autologous monocytes. Soluble LAG-3Ig fusion protein markedly enhances IL-12 production by monocytes stimulated with infra-optimal concentrations of sCD40L, whereas it directly stimulates monocyte-derived DCs for the production of TNF-α and IL-12, unraveling an enhanced responsiveness to MHC class II engagement in DCs. Thus similar to CD40L and RANKL, LAG-3 may be involved in the proinflammatory activity of cytokine-activated bystander T cells and most importantly it may directly activate DCs. These findings are consistent with a previous report that signaling through class II MHC in DC induces IL-12 (Koch et al., 1996). Although not completely demonstrated experimentally, it is believed that at the start of an immune response (i.e., during antigen uptake and presentation) DCs that have picked up antigens and have migrated to the T cell area of regional lymph nodes produce large amounts of IL-12 triggered by the CD40/CD40L interaction with a na¨ıve CD4+ T cell. The same mechanism may also apply later in an immune response; i.e., a differentiated Th1 cell could activate a pathogeninfected macrophage through the CD40/CD40L interaction to produce IL-12. The production of IL-12 by APC in response to activated T cells is likely to play a major role in the maintenance of the chronic pathogenic Th1 response in autoimmune diseases, but less so in the rapid Th1 response to acute infections. VIII. Molecular Regulation of the Expression of IL-12 Genes

The study of the regulation of IL-12 gene expression is compounded by the necessity to analyze the coordination of expression of the p40 and p35 genes, which are encoded on different chromosomes. Expression of the p40 gene is restricted to cells that produce IL-12 p70, compared with the more ubiquitously expressed p35 gene. Simultaneous analysis of nuclear transcription, steady-state mRNA, and secreted protein levels of IL-12 established that the human IL-12 p40 gene is primarily regulated at the transcriptional level by IFN-γ and LPS


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in monocytic cells (Ma et al., 1996), whereas the p35 gene has been reported in the mouse to be regulated at the level of transcription and translation in that there are multiple transcription start sites and translation initiation signals whose usages are determined by external stimuli (Babik et al., 1999). Comparative studies with cyclohex-mide (CHX) revealed other striking differences in mRNA regulation between the p40 and p35 genes; i.e., the Staphylococcus aureus (SAC)- or LPS-induced p40 mRNA was abrogated when cells were pretreated with CHX, suggesting that the regulation of IL-12 p40 gene requires the induction of a CHX-sensitive transcription factor(s). In contrast, IL-12 p35 mRNA was “superinduced” by CHX, indicating that the activation of the p35 mRNA requires only a presynthesized activator(s) that can be induced either by SAC or LPS at the post-translational level (Aste-Amezaga et al., 1998). A. CONTROL OF IL-12 p40 GENE EXPRESSION 1. Critical Promoter Elements in the IL-12 p40 Gene Both the mouse and human IL-12 p40 promoters have been cloned (Ma et al., 1996; Murphy et al., 1995). A detailed functional dissection of the human and mouse p40 promoters in RAW 264.7 and J774 mouse macrophage cell lines initially identified two critical cis-elements involved in the regulation of the p40 gene transcription by LPS and IFN-γ (Ma et al., 1996; Murphy et al., 1995): an ets site at –211/–206 (TTTCCT) and an “NFκB half site” at –117/–107 (TGAAATTCCCC). The ets site and its surrounding sequences (–292/–196) interacts in the murine RAW 264.7 cell line with a large complex named F1 which is induced by either LPS or IFN-γ and which is composed of ets-2, interferon regulatory factor-1 (IRF-1), interferon consensus binding protein (ICSBP), NFκB c-Rel, and a novel ets-2-related protein (Gri et al., 1998; Ma et al., 1996; Wang et al., 2000). In bone-marrow-derived mouse macrophages, the ets site binds a complex upon stimulation with IFN-γ and LPS that is composed of PU.1, ICSBP, and c-Rel (our unpublished data). The NFκB half site binds p50/p65 and p50/c-Rel heterodimers induced by LPS (Gri et al., 1998; Murphy et al., 1995; Plevy et al., 1997). The two heterodimers bind to this site with comparable affinities and exhibit equivalent transactivation activities in in vitro assays. However, p40 mRNA and protein concentrations were reduced dramatically in c-Rel–/– macrophages and only modestly in p65–/– macrophages, suggesting that c-Rel is physiologically more selective than p65 for IL-12 p40 gene activation (Sanjabi et al., 2000). This in vivo observation is well reflected also in an in vitro study in which c-Rel was found to be the most important of the three members of the NFκB family—p50, p65, and c-Rel—for the transcriptional activation of the human IL-12 p40 gene (Gri et al., 1998). Both the NFκB and ets elements are essential since deletion or mutation of specific nucleotides within these sites abolishes the human p40 promoter activity. However, the importance

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of the ets site is somewhat ambiguous, depending on the system used. In the mouse system, two groups that study the mouse IL-12 p40 promoter in J774 or RAW 264.7 cells found that the sequences upstream of the NFκB site appear to be insignificant because deletion of them does not affect the promoter activity stimulated with LPS (Murphy et al., 1995; Plevy et al., 1997). Studies of the human IL-12 p40 promoter in RAW264.7 cells, however, implicated the upstream region in response to IFN-γ and LPS (D’Ambrosio et al., 1998; Delgado and Ganea, 1999; Gri et al., 1998; Ma et al., 1996). The F1 complex is also present constitutively in RPMI-8866, the EBV-B cell line that spontaneously produces IL-12, with a subtle difference: the IRF-1 component is replaced with IRF-2 (Gri et al., 1998). Interestingly, when cotransfected in RAW264.7 cells, IRF-1 synergizes but IRF-2 antagonizes the activation of the IL-12 p40 promoter (Wang et al., 2000). The functional implication of such a difference is that the F1 complex variant in RPMI-8866 cells may serve to attenuate IL-12 p40 transcription, a notion consistent with the observation that IL-12 p40 promoter reporter constructs lacking the ets site but retaining the NFκB site downstream are functionally competent in RPMI-8866 but not in RAW264.7 cells (Gri et al., 1998). It is the authors’ view that the differences observed on the importance of the ets site may be principally attributable to the dissimilar differentiation states of the cells used in their respective studies. The J774 cell line used in the study of the mouse promoter is more differentiated than the RAW264.7 cell line used in the human promoter studies. J744 cells do not require IFN-γ priming for IL-12 p40 production, whereas RAW264.7 cells are completely dependent on it. The diverse activation states of the cells correspond to disparaging cellular environments, resulting in differential regulation of the IL-12 p40 gene by factors associated with their respective nuclear architectures. A third element, a CAAT element binding protein (C/EBP) site, has been identified downstream of the NFκB half site in the murine IL-12 p40 promoter. This site, located between -96 and -88 (GTGTTGCAA) interacts with C/EBP proteins and functionally cooperate with the NFκB half site upstream in response to stimulation by LPS or heat-killed Listeria monocytogenes (HKLM) (Plevy et al., 1997). 2. IFN-γ Priming Interleukin-12 production in primary monocytes is strongly dependent on the activation state of the cells. IFN-γ provides a powerful stimulation signal for monocytes to become activated macrophages and bactericidal with a much enhanced potential to produce IL-12, although it alone does not induce IL-12 p40 gene expression. This is defined as the “priming” effect of IFN-γ on monocytes or monocytic cell lines (Ma et al., 1996). IFN-γ , however, both induces IL-12 p35 transcription and strikingly primes cells for its induction by other stimuli, thereby strongly enhancing IL-12 p70 production (Hayes et al., 1995; Ma et al., 1996). The mediators of such an enhanced effect of IFN-γ are likely


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represented by members of the IRF family responsive to IFN-γ , particularly IRF-1 and ICSBP. The participation of IRF-1 in the regulation of p40 gene transcription is strongly supported by the deficiency in IL-12 production and impaired Th-1 responses in IRF-1-null mice (Taki et al., 1997). ICSBP, which is expressed exclusively in cells of the immune system, is highly inducible by IFN-γ . Mice with a disrupted ICSBP gene are selectively deficient in IL-12 p40 gene expression and highly susceptible to infection with intracellular pathogens such as L. monocytogenes and Toxoplasma gondii (Scharton-Kersten et al., 1997). ICSBP-transduced myeloid progenitor cells established from ICSBP–/– mice differentiated into mature macrophages with phagocytic activity coincided with growth arrest and the induction of macrophage-specific genes including IL-12 (Tamura et al., 2000). The ets site in the p40 promoter bears strong resemblance to the interferon stimulated response element (ISRE) with which ICSBP interacts. Functional analyses indicate that ICSBP can act as an obligatory factor through this site and synergize specifically with IRF-1 to activate the human p40 promoter in cells of the macrophage lineage by forming a complex on this site, although ICSBP itself does not appear to directly bind the IL-12 p40 promoter (Wang et al., 2000). This large complex may also interact with the NFκB complex approximately 100 bp downstream which primarily responds to LPS and other NFκB-inducing stimuli, resulting in a synergistic activation of the promoter in monocytic cells (Gri et al., 1998). The priming effect of IFN-γ for augmented IL-12 production may represent a mechanism by which IL-12-induced Th1 responses are maintained in vivo. 3. Role of the Transcriptional Factor PU.1 PU.1 belongs to the ets family of DNA binding proteins (Fisher and Scott, 1998; Pongubala et al., 1993). It is expressed predominantly in macrophages, B cells, and erythroid cells (Celada et al., 1996; Klemsz et al., 1990). PU.1 plays important roles in the development of hematopoietic cells. Genetic deletion of the PU.1 gene leads to a failure to produce mature B lymphocytes and macrophages (Scott et al., 1994). However, overexpression of PU.1 prevents terminal differentiation of hematopoietic cells and leads to the overproduction of erythroblasts (Schuetze et al., 1993). It was shown recently, using retroviral transduction of PU.1 complementary DNA into hematopoietic progenitors derived from PU.1deficient mice, that a graded expression of PU.1 can lead to distinct cell fates; i.e., a low concentration of PU.1 protein induces the B cell fate, whereas a high concentration promotes macrophage differentiation and blocks B cell development (DeKoter and Singh, 2000). In many instances, the transcriptional activation by PU.1 depends on the cooperation of PU.1 with other transcription factors. These factors can either become physically associated with PU.1 or they can bind to DNA adjacent to the ets site upon which PU.1 resides (Eisenbeis et al., 1995; Eklund et al., 1998; Himmelmann et al., 1997; Ortiz et al., 1999; Pongubala et al.,

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1992). PU.1 binds to the human IL-12 p40 promoter constitutively at two sites: immediately upstream of the NFκB half site, and at the ets site. Use of a dominant negative mutant of PU.1 (Fisher and Scott, 1998) cotransfected with the human IL-12 p40 promoter-luciferase gene into RAW264.7 cells abolished both the reporter activity as well as the endogenous IL-12 p40 protein secretion, suggesting that PU.1 is an obligatory factor for the transcriptional activation of IL-12 p40 (Cappiello et al., 2001). 4. Chromatin Structure of the IL-12 p40 Gene An analysis of nucleosome positioning, chromatin remodeling, and transcription factor binding in the regulation of the murine IL-12 p40 gene transcription (Weinmann et al., 1999) revealed that the p40 gene promoter in macrophages is normally configured in a nucleosome array. Upon activation of macrophages with HKLM or LPS, one of the nucleosomes (nucleosome 1) is selectively remodeled such that it allows accessibility to this region by transcription factor C/EBP. The remodeling at the IL-12 p40 promoter is a TLR4-dependent and Rel-independent event (Weinmann et al., 2001). The remodeling of nucleosome 1 is not sufficient, however, for p40 gene transcription, and additional inducible factor(s) or steps are clearly required (Weinmann et al., 1999). 5. Role of Mitogen-Activated Protein Kinases The p38 mitogen-activated protein kinase pathway, like the c-jun N-terminal kinase (JNK) MAPK pathway, is activated in response to cellular stress and inflammation and is involved in many fundamental biological processes. To study the role of the p38 MAPK pathway in vivo, Lu and coworkers (Lu et al., 1999) generated mice deficient in the Mkk3 gene, one of the two specific MAP kinases that activate p38 MAPK. Mkk3–/– mice were viable and fertile but were defective in IL-12 production by peritoneal macrophages stimulated with LPS or bone-marrow-derived DCs activated via CD40. IFN-γ production following immunization with protein antigens and in vitro differentiation of naive T cells is greatly reduced, suggesting an impaired type I cytokine immune response. The effect of the p38 MAPK pathway on IL-12 expression is at least partly transcriptional, since inhibition of this pathway blocks IL-12 p40 promoter activity in macrophage cell lines and IL-12 p40 mRNA is reduced in MKK3-deficient mice. An in vitro study by Feng et al. (1999) corroborated the above in vivo findings. These workers showed that the extracellular signal-related kinase and p38 MAPK play differential roles in the regulation of LPS-stimulated inducible NO synthase and IL-12 gene expression. In macrophages, LPS stimulates the simultaneous activation of all three classes of MAP kinases—ERK, c-jun N-terminal kinase, and p38—albeit with differential activation kinetics. Experiments using inhibitors selective for ERK (PD98059) and p38 (SB203580) indicated that, while p38 promotes induction of IL-12 (p40) mRNA, ERK activation suppresses


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LPS-mediated IL-12 transcription. The biological relevance of these regulatory signals is demonstrated by the finding that Leishmania lipophosphoglycans, which promote parasite survival, act by stimulating ERK MAP kinase to inhibit macrophage IL-12 production. 6. Polymorphisms within the Promoter Regions of IL-12 Genes Noteworthy, single-nucleotide polymorphisms within the promoter regions of the human IL-12 p35 and p40 genes have been described (Pravica et al., 2000). Transitions from C to T at position-916 from the transcription start site of the IL-12 p35 gene and G to T at position-1287 in the IL-12 p40 gene were observed. B. TRANSCRIPTIONAL AND POST-TRANSCRIPTIONAL CONTROL OF IL-12 p35 GENE EXPRESSION Analysis of IL-12 p35 gene expression was complicated not only by its ubiquity but also by its low expression, until the priming effect of IFN-γ on its expression was discovered (Hayes et al., 1995). The promoter regions of both the mouse and human p35 genes have been cloned (Hayes et al., 1998; Tone et al., 1996; Yoshimoto et al., 1996). A 1143-bp region (a BamHI-PstI genomic fragment) was sequenced. It contains some putative transcriptional motifs such as Sp1, IFN-γ -response element γ -IRE), PU.1, C/EBP, GM-CSF, and a T-cell-specific transcription factor TCF-1α The human p35 gene appears to initiate its transcription from at least two sites, one for B lymphoblastoid cells and one for monocytes (Hayes et al., 1998). The latter follows a “TATA” box-like sequence, which in the mouse gene has been suggested to be part of an ancestral IL-12 p35 promoter (Yoshimoto et al., 1996) and which would generate a shorter mRNA including only the second methionine at residue 35. The presence of multiple transcription initiation sites in the human and murine IL-12 p35 promoter raises an interesting prospect of different cell type usage of the promoter. The 1143bp p35 genomic fragment was linked to a luciferase reporter construct, transiently transfected into RPMI-8866 (an EBV-B cell line that constitutively produce IL-12), Jurkat (a T cell line that does not produce IL-12 but expresses the p35 gene constitutively), and RAW264.7 cells which produce IL-12 only after stimulation with IFN-γ and LPS. The p35 promoter is very active in B cells but not in T cells. TPA does not further activate the promoter. It is also inducible in RAW264.7 cells by a combination of IFN-γ and LPS treatment. Babik et al. (1999) characterized murine p35 expression in the B cell lymphoma line A20 and in bone-marrow-derived DCs. Multiple transcription start sites were identified in both cell types, resulting in four p35 mRNA isoforms (types I–IV) that differ in the number and position of upstream ATGs in their 5′ untranslated regions. In nonstimulated cells, the predominant forms of p35 message (types II and IV) contained an additional upstream ATG, whose presence

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was shown to inhibit the downstream translation of the p35 subunit. After LPS stimulation, however, transcription initiated from alternate positions, so that the proportion of transcripts not containing this upstream ATG (types I and III) was significantly increased in the population of p35 mRNA. These type I and type III transcripts readily supported translation of the p35 subunit and its incorporation into bioactive IL-12. Furthermore, p35 mRNA levels were substantially up-regulated after LPS stimulation in both cell types. These results show that p35 gene expression is regulated by both transcriptional and translational mechanisms (Babik et al., 1999). Murphy et al. (2000) examined the intracellular protein processing of both subunits of human IL-12 using rabbit reticulocyte-translated human IL-12 p40 and p35 and showed that the p40 and p35 subunits are processed by different mechanisms. Whereas processing of p40 conforms to the cotranslational model of signal peptide removal concomitant with translocation into the endoplasmic reticulum (ER), translocation of the p35 preprotein into the ER was not accompanied by cleavage of the signal peptide; rather, removal of the p35 signal peptide occurred via two sequential cleavages. The first cleavage took place within the ER, and the cleavage site localized to the middle of the hydrophobic region of the signal peptide. Although the preprotein was glycosylated upon entry into the ER, its glycosylation status did not affect primary cleavage. Subsequently, the remaining portion of the p35 signal peptide was removed by a second cleavage, possibly involving a metalloprotease, concomitant with additional glycosylation and secretion. Secretion could be inhibited by mutation of the second cleavage site or by inhibition of glycosylation with tunicamycin. In contrast, p40 secretion was not affected by inhibition of glycosylation. In a separate study, Carra et al. (2000) demonstrated that, whereas the IL-12 p40-chain showed similar pI pattern whether in the free form or associated in the heterodimer, either secreted or intracellular, the p35-chain in the secreted heterodimer was much more acidic than that present in the intracellular heterodimer. Deglycosylation experiments with neuraminidase and Endo-F combined with two-dimensional PAGE of single bands of the intracellular vs. extracellular IL-12 heterodimer revealed that the p35-chain was extensively modified with sialic acid adducts to N-linked oligosaccharides before secretion. Inhibition of N-glycosylation by tunicamycin did not alter the secretion of the free p40-chain while preventing the IL-12 heterodimer assembling and secretion, a finding similar to that by Murphy et al. (2000). Pulse-chase experiments indicated that IL-12 persists intracellularly as an immature heterodimer, and that glycosylation is the regulatory step that determines its secretion (Carra et al., 2000). Together, these findings suggest diverse mechanisms for regulation of IL-12 production with an important role for the IL-12 p35-chain in the control of the output of biologically active IL-12 p70.


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IX. Cytokine-Mediated Regulation of IL-12 Production

Many cytokines participate in cross talks that influence the function of their effector cells. IL-12 production during an immune response is tightly modulated by both positive and negative feedback signals. The positive feedback regulation is exemplified by IFN-γ , which is induced by IL-12 initially from NK and T cells and which in turn potently enhances the ability of monocytes and other cell types to produce IL-12 (Ma et al., 1996). Other cytokines that enhance the ability of the accessory cells to produce IL-12 indirectly are GM-CSF, IL-4, and IL-13. GM-CSF can prime monocytes for high levels of IL-12 p40 secretion; however, it is a rather poor primer for IL-12 p35 mRNA expression and IL-12 p70 secretion (Hayes et al., 1995). The physiological significance of GM-CSF priming is not clear. It might increase the possibility of forming either p40 homodimer or heterodimer of p40 with a secondary partner. Interleukin-4 and IL-13, together with IL-10 and TGF-β, are considered to be the most important macrophage-deactivating factors, with inhibitory effects on cytokine production. Unlike IL-10 and TGF-β, which appear to act as down-modulators of many phagocytic cell functions, the mode of action of IL-4 and IL-13 is more complex. Addition of IL-4 and IL-13 to peripheral blood mononuclear cell (PBMC) cultures inhibits production of IL-12, TNF-α, IL-10, and IL-1β induced by LPS or SAC added simultaneously with the cytokines. However, pretreatment of PBMC with IL-4 or IL-13 for ≥20 h enhances the production of IL-12 and TNF-α several-fold in response to LPS or SAC. IL-4 priming also enhanced the accumulation of IL-12 and TNF-α mRNA induced by LPS and SAC. The enhanced accumulation of transcripts for the IL-12 p35and p40-chains by IL-4 priming is reflected in enhanced secretion of both the IL-12 free p40-chain and the p70 heterodimer. These results suggest an unexpected complexity in the regulatory role of IL-4 and IL-13 in immune responses (D’Andrea et al., 1995). IL-4 and IL-13 enhance in human monocyets both mRNA accumulation and transcription of the IL-12 p35 gene much more efficiently than those of the IL-12 p40 gene, perhaps explaining the striking effect of these two cytokines on the secretion of the p70 heterodimer, compared with a relatively modest effect on p40 secretion (Marshall et al., 1997). Interestingly, IL-4 or IL-13 treatment of mononuclear cells from HIV-infected patients almost completely corrected their defective ability to produce IL-12 (Marshall et al., 1997). In a separate study, Takenaka et al. noted that, although IL-4, when added simultaneously with the inducing stimuli, inhibited LPS plus IFNγ -induced IL-12 production, it potentiated the production of IL-12 induced by CD40L (Takenaka et al., 1997). Kalinski and coworkers reported that human Th2 cells interacting with monocyte-derived DCs effectively induce bioactive IL-12p70 and revert to Th0/ Th1 phenotype (Kalinski et al., 2000). The induction

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of IL-12 p70 in Th2 cell–DC cocultures is prevented by IL-4-neutralizing mAb, indicating that IL-4 acts as a Th2-cell-specific cofactor of IL-12 p70 induction. Like IFN-γ , IL-4 strongly enhances the production of bioactive IL-12 p70 in CD40 ligand-stimulated DCs and macrophages and synergizes with IFN-γ at low concentrations of both cytokines. However, in contrast to IFN-γ , IL-4, when added simultaneously to the inducing stimulus, inhibited the CD40 ligandinduced production of IL-12 p40 and the production of either form of IL-12 induced by LPS, confirming the early studies by D’Andrea et al. (1995) and Takenaka et al. (1997). It was hence hypothesized that the ability of IL-4 to act as a cofactor of Th-cell-mediated IL-12 p70 induction may allow Th2 cells to support cell-mediated immunity in chronic inflammatory states, including cancer, autoimmunity, and atopic dermatitis (Kalinski et al., 2000). A study by Hochrein et al. (2000) also indicated that IL-4 is a major regulatory cytokine governing bioactive IL-12 production by mouse and human DCs by selectively and differentially regulating IL-12 p70 and p40. Although IL-12 is essential for host defense against infection, it is also toxic and even lethal at high doses (Cousens et al., 1999; Leonard et al., 1997); therefore, it must be strictly regulated. IL-10 is a major macrophage-deactivating and immunosuppressive cytokine. It is a critical component in the maintenance of the fine balance between swift and potent immune responses against invading pathogens, on the one hand, and the control of detrimental systemic inflammation on the other. IL-10 is a potent inhibitor of IL-12 production in accessory cells. Exogenously added IL-10 rapidly inhibited SAC- or LPS-induced cytokine mRNA expression in human PBMCs and monocytes, with a maximal effect observed when IL-10 was added from 20 h before until 1 h after the addition of the inducers (Aste-Amezaga et al., 1998). Nuclear run-on assays revealed that the inhibition of IL-12 p40, IL-12 p35, and TNF-α gene expression was at the transcriptional level and that the addition of IL-10 to SAC- or LPS-treated PBMC did not affect mRNA stability. The inhibitory activity of IL-10 was abrogated by CHX, suggesting the involvement of a newly synthesized protein(s). These results indicate that in human monocytes the mechanism(s) of IL-10 suppression of both IL-12 p40 and IL-12 p35 genes is primarily seen at the transcriptional level. A comparative study analyzing the endogenous mIL-12 p40 secretion in RAW264.7 cells and the activity of a sub-line with a stably integrated human IL-12 p40 promoter-luciferase reporter gene in response to IFN-γ /LPS stimulation and simultaneous IL-10 treatment led to the conclusion that the 3.3-kb human IL-12 p40 upstream promoter sequence does not contain an IL-10responsive element that mediates the suppressive effect of IL-10 (Aste-Amezaga et al., 1998). Alternatively, the way the human IL-12 p40 promoter-reporter was integrated may have generated a chromatin structure different from that of the endogenous gene, and the IL-10-mediated inhibitory mechanism that acts on the native gene may not be functional on the integrated gene.


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Interestingly, the immunosuppressive effects of IL-10 are not confined to proinflammatory cytokines. Recent in vivo studies of the pathogenesis of Schistosomiasis in IL-10/IL-4- and IL-10/IL-12 double-deficient mice that developed highly polarized type 1 and type 2 cytokine responses, respectively, suggest that IL-10 significantly suppresses both type 1 and type 2 cytokine development and reveal the central regulatory role of IL-10 in the pathogenesis of microbial infections (Hoffmann et al., 2000). Transforming growth factor (TGF)-β, a product of activated platelets, macrophages, malignant tumors, and other cell types, is also an inhibitor of IL-12 production. Unlike IL-10, which suppresses IL-12 production primarily at the level of transcription, TGF-β appears also to reduce the stability of the IL-12 p40 mRNA (Du and Sriram, 1998). Type I interferons (IFN-α/β) are members of a multigene family encoding for proteins that are potent antiviral and immunoregulatory cytokines. Although first noted for their ability to inhibit viral replication, type I interferons are also known to exert multiple immunoregulatory effects on NK and T cells (Biron et al., 1999), with some of the functions overlapping those of IL-12. Certain viral infections induce IL-12 to elicit NK cell IFN-γ production and antiviral mechanisms. However, high levels of IFN-α/β are dominant in the context of viral infections and act to regulate other innate responses, including induction of NK cell proliferation in vivo and overall negative regulation of IL-12 production (Cousens et al., 1997). Paradoxically, this property of IFN-α/β may explain in part their therapeutic effects on multiple sclerosis, for which the animal model, experimental allergic encephalomyelitis (EAE), is clearly attributable to the presence of IL-12 inducing autoimmunity in the brain (Karp et al., 2000). It may also explain to some degree why IL-12 synthesis during a viral infection goes through two phases: an initial induction by the virus and a subsequent prolonged suppression, since most viruses induce a type I interferon response, which may suppress IL-12 production induced by the primary infection. The mechanism of the IFN-mediated inhibition of IL-12 may be in part dependent on the down-regulation of the PU.1 transcription factor binding to the IL-12 p40 promoter at the ets site (Almeida et al., 2001). Tumor necrosis factor (TNF)-α and IL-12 are two major mediators of inflammatory responses in mammals, and they both play prominent roles in bridging the innate and adaptive phases of immunity. The interdependent and crossregulatory relations between them have key implications in influencing macrophage activation and the development of antigen-specific immune responses. Hodge-Dufour et al. (1997) showed that in thioglycollate-elicited mouse macrophages stimulated with IFN-γ plus either LPS or low-molecular-weight HA, IL-12 production could be suppressed by TNF-α. TNF-α-deficient mice showed little initial response and a late but vigorous and disorganized inflammatory response to heat-killed Corynebacterium parvum, leading to death with high

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levels of serum IL-12, unlike the prompt response (granuloma formation and hepatosplenomegaly) and subsequent resolution phase observed in C. parvuminjected wild-type mice (Hodge-Dufour et al., 1998; Marino et al., 1997). Ma et al. (2000) demonstrated that in human monocyte-derived macrophages TNF-α inhibits both LPS-induced and IFN-γ -enhanced IL-12 production at the level of transcription of the p40 gene, but not of the p35 gene. This inhibition appears to be independent of IL-10, a major IL-12 synthesis inhibitor, and it is not due to a down-regulation of either NFκB, a major IL-12 transcriptional activator in response to LPS, or IRF-1, a major IL-12 activator in response to IFN-γ . Thus, the nuclear mediators of TNF-α inhibition of IL-12 remain elusive. Contrary to these findings, Zhan and Cheers (1998) reported that mice lacking both receptors for TNF-α (TNFR1 and TNFR2) were severely deficient in IL-12 production in the serum and spleen upon infection with L. monocytogenes. The reason for this apparent contrast is not clear. Seemingly conflicting data exist in similar experimental settings. Endres et al. (1997) found that IL-12 mRNA expression measured by RT-PCR was unaffected in Listeriainfected mice lacking the p55 TNFR1. Flesch et al. (1995), using TNFR1deficient mice infected with Mycobacterium bovis BCG, found a marked depression of IL-12 p40 mRNA in vivo and an inability of macrophages in vitro to produce IL-12 p40 protein. These differences may have arisen either due to a yet undiscovered role of the two TNF receptors in the induction of IL-12 synthesis that is independent of its natural ligand TNF-α, or due to an unknown property of L. monocytogenes and M. bovis BCG that may induce different IL-12 responses in the presence or absence of the TNFR. TNF-α and IL-12 have multiple effects on macrophage function in innate immunity and on T and B cells in adaptive immunity. Their intricate interregulation is a reflection of their delicacy and importance in orchestrating immune responses against invading pathogens and keeping the reaction under control in order to clear the pathogen, as well as to resolve the inflammation. A significant tilt of the balance would have strong repercussions. The ability of TNF-α to inhibit IL-12 production by murine and human macrophages provides direct evidence that, in addition to the cascade of inflammatory and anti-inflammatory cytokines induced during inflammation, TNF-α signaling can selectively inhibit IL-12 gene expression upon macrophage activation as part of the scheme of cytokine feedback and self-limiting modulation. This may benefit the host in controlling inflammatory responses as exemplified in numerous murine models while at the same time providing a potential immune-evasion mechanism to intracellular pathogens whose infection is associated with chronic TNF-α expression such as in HIV-1 infection where sustained immune activation and expression of TNF-α are coupled with a decrease in IL-12 production and cellmediated responses. In this context, it is of interest to note that chronic exposure to TNF-α suppresses the response of both Th1 and Th2 subsets and attenuates


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T cell receptor signaling in vivo (Cope et al., 1997), highlighting important implications for our understanding of TNF-α and IL-12 in the pathogenesis of chronic inflammatory diseases and immune suppression. X. Role of Cell-Surface Receptors in the Regulation of IL-12 Production

Cellular receptors are “antennas” of the cell through which it interacts with the outside world. Their primary functions are ligand binding and signal transduction, resulting in cellular activation or deactivation. The control of IL-12 production is also mediated through some of these receptors, which interact with their respective ligands. A. CCR5-MEDIATED INDUCTION OF IL-12 PRODUCTION IN DENDRITIC CELLS The activation of DCs to produce IL-12 is thought to be a key step in the initiation of cell-mediated immunity to intracellular pathogens. Aliberti et al. (2000) showed that chemokines were rapidly induced in the spleen of mice injected with an extract (STAg) of tachyzoites of Toxoplasma gondii. Ligation of the C-C chemokine receptor 5 (CCR5) can provide a major signal for the induction of IL-12 synthesis by the CD8+ subset of DCs by T. gondii, and this pathway appears to be important in establishing interferon-dependent resistance to the parasite. These findings support the concept that the early induction of chemokines by invading pathogens is a critical step not only for the recruitment of DC but also for the determination of their subsequent immunologic function. B. CD11b/CD18-ASSISTED INDUCTION OF IL-12 PRODUCTION The CD11/CD18 β 2 integrins are a family of obligate heterodimeric glycoproteins expressed on leukocytes as a 95-kDa CD18 β subunit noncovalently associated with one of three subunits—CD11a (LFA-1), CD11b (Mac-1, CR3), or CD11c (CR4) (Todd and Petty, 1997). Although all three types of β 2 integrins are detectable on macrophages, macrophages express CD11b/CD18 heterodimers predominantly. The CD11/CD18 integrins were originally identified as LPS receptors in studies that demonstrated binding of gram-negative bacteria and LPS-coated erythrocytes to these receptors on human macrophages (Wright and Jong, 1986). Subsequent studies performed on monocytes and macrophages derived from CD18-deficient patients found normal levels of TNF- and IL-1β and led to the conclusion that CD18 is not essential for cellular responses to LPS (Wright et al., 1990). Perera et al. (2001) investigated the hypothesis that three membrane-associated proteins, CD14, CD11b/CD18, and TLR4, may act in concert as LPS recognition and/or signaling receptors in murine macrophages by using the respective knockout mice. Induction of IL-12 p35 and p40 mRNA expression and p70 production in response to low concentrations of LPS requires

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the participation of both CD14 and TLR4, whereas high concentrations of LPS elicit their expression in the absence of CD14. In contrast, for optimal induction of IL-12 p35, and p40 genes by low concentrations of LPS, CD11b/CD18 is also required. Mitigated induction of IL-12 p35 and IL-12 p40 gene expression by CD11b/CD18-deficient macrophages is correlated with a marked inhibition of NFκB nuclear translocation in response to LPS (Perera et al., 2001). C. COMPLEMENT RECEPTOR-MEDIATED INHIBITION OF IL-12 PRODUCTION Measles virus (MV) induces profound and prolonged abnormalities and depression in the cellular immune responses of infected hosts. Karp and collaborators (Karp et al., 1996) demonstrated that MV specifically ablates monocyte/macrophage and dendritic cell production of IL-12. Cross-linking of the cellular receptor for MV, the complement regulatory protein CD46, is sufficient to inhibit IL-12 production. The inhibition is seen with both IL-12 p40 and p70. It is primarily at the level of transcription; it is stimulus independent, occurring with either bacterial or CD40 stimulation of monocytes; it is highly specific, as infection of monocytes with MV does not suppress the stimulated production of IL-6, TNF-α and macrophage inflammatory protein 1; and it does not appear to show MV strain specificity (Karp et al., 1996). CD46-mediated down-regulation of IL-12 is one specific instance of a more general pattern of tight inhibitory control of IL-12 production effected by complement and phagocytic receptors on antigen-presenting cells, which might also be exploited by other intracellular pathogens to evade immune attacks. Subsequently, Marth and Kelsall reported that cross-linking of complement receptor (CR) 3 (also known as CD11b or Mac-1) with antibody or certain particulate ligands (including particles coated with iC3b) inhibits IL-12 production by both murine and human monocytes/macrophages with little if any down-regulation of the production of other proinflammatory cytokines or chemokines (Marth and Kelsall, 1997; Sutterwala et al., 1997). The ability of CR ligation to specifically inhibit IL-12 production suggests that complement activation products can directly regulate the type of immune response through interaction with APCs. D. FCγ RECEPTOR-MEDIATED INHIBITION OF IL-12 PRODUCTION IN MACROPHAGES Receptor-mediated inhibition of IL-12 production is not confined to CR and regulatory molecules. Indeed, ligating Fcγ receptors (that bind to the Fc portion of IgG often complexed with an immunogen) and scavenger receptors (that normally are involved in the phagocytosis of extarcellular pathogens) on macrophages results in profound and selective suppression of IL-12 production (Sutterwala et al., 1997). The inhibition of IL-12 p40 and p35 gene expression by Fcγ R cross-linking occurs at the level of transcription. The region responsive


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to this inhibition maps to the ets site of the p40 promoter. PU.1, ICSBP, and c-Rel form a complex on this element upon macrophage activation. Receptor ligation abolishes activation and/or nuclear translocation of PU.1, ICSBP, and IRF-1 but not cRel and, thus, prevents the formation and DNA binding of this PU.1-containing activation complex and abrogates p40 transcription without affecting the activation of NFκB. ICSBP and IRF-1 are known to be essential for IL-12 p40 gene expression in vivo (Scharton-Kersten et al., 1997; Taki et al., 1997). The role of PU.1 in the regulation of IL-12 p40 transcription is strongly suggested by the use of a dominant negative construct of PU.1, which diminishes IL-12 p40 promoter activity and endogenous IL-12 p40 protein secretion (Cappiello et al., 2001). These data provide insights into the cellular and molecular pathways in which pathogens and immune effector cells interact. Of interest, a separate study (Berger et al., 1997) showed that immune complexes (IC) formed of tetanus toxoid and polyclonal anti-tetanus toxoid antiserum as well as heat-aggregated human serum IgG completely abolished IL-12 p40 and p70 production by human monocytes stimulated with IFN-γ and LPS. However, neutralization of TNF-α by specific antibodies completely restored IL-12 secretion. Therefore, IC, which typically appear in the course of chronic inflammatory processes, may influence the balance between Th1 and Th2 responses. E. CD47-MEDIATED INHIBITION OF IL-12 PRODUCTION CD47 antigen, also named integrin-associated protein (IAP), regulates the function and the binding of vitronectin (Vn) to αVβ3, its associated integrin (Lindberg et al., 1993). However, Vn does not bind CD47, and the natural ligand of CD47 is reported to be thrombospondin (TSP) (Gao et al., 1996), which transiently accumulates at the inflammatory site. Engagement of CD47 by anti-CD47 monoclonal antibodies, by TSP, or by 4N1K, a peptide of the COOH-terminal domain of TSP selectively binding CD47, inhibits IL-12 release by monocytes (Armant et al., 1999). The suppression occurred after either T-cell-dependent or -independent stimulation of monocytes and was selective for IL-12 inasmuch as the production of TNF-α IL-1, IL-6, and GM-CSF was not inhibited. CD47 ligation also did not alter TGF-β and IL-10 production, and the suppressive effect on IL-12 was not due to autocrine secretion of TGF-β or IL-10. The IL-12 inhibition was not mediated by Fcγ R ligation, did not require extracellular Ca2+ influx, but was reversed by the phosphoinositide 3-kinase inhibitors wortmannin and Ly294002. Furthermore, CD47 ligation selectively inhibits the development of human na¨ıve T cells into Th1 effectors in the presence of exogenous IL-12, suggesting that it also interferes with IL-12 downstream signaling (Avice et al., 2000). Thus, engagement of CD47 on monocytes by TSP is a novel pathway to selectively down-regulate IL-12-mediated proinflammatory response.

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XI. Endotoxin-Tolerance-Mediated Inhibition of IL-12

Endotoxin tolerance, the deactivation of a subset of endotoxin-driven responses after an initial exposure to endotoxin, may provide protection from uncontrolled immunological activation of acute endotoxic shock. On the other hand, the inhibition of monocyte/macrophage functions associated with endotoxin tolerance can lead to an inability to respond appropriately to secondary infections in survivors of endotoxic shock, a phenomenon known as “immunological paralysis.” IL-12 plays an important role in pathological responses to endotoxin. Karp et al. (1998) examined the regulation of IL-12 during endotoxin tolerance and found that pre-exposure of human monocytes to small doses of LPS (priming) ablates IL-12 production induced by secondary challenge with LPS. This suppression of IL-12 production is primarily transcriptional. Unlike the down-regulation of TNF-α under such conditions, the mechanism of IL-12 suppression during endotoxin tolerance is not dependent upon IL-10, TGF-β, IL-4, PGE2, TNF-α, or nitric oxide (Wittmann et al., 1999). IL-12 production is not rescued by IFN-γ or GM-CSF, two potent enhancers of IL-12 production (Karp et al., 1998). Moreover, preincubation of monocytes with LPS did not downregulate CD14 expression (Wittmann et al., 1999), whereas it has been recently reported that endotoxin tolerance in mouse peritoneal macrophages correlates with down-regulation of surface toll-like receptor 4 expression (Nomura et al., 2000). Endotoxin tolerance is also observed in human DCs with potent downregulation of IL-12 production (Karp et al., 1998). Thus, endotoxin-tolerancemediated suppression of IL-12 production as a physiological negative-feedback control mechanism could lead to the anergy seen during the immunological paralysis, which follows septic shock. XII. Inhibition of IL-12 Production by Anti-inflammatory Hormones and Small Molecules

Many of the anti-inflammatory and immunosuppressive agents can, not surprisingly, inhibit IL-12 production by inflammatory macrophages. The first such agent identified is prostaglandin-E2 (PGE2) (van der Pouw Kraan et al., 1995). Subsequently, more agents were identified that share the ability to suppress IL-12 secretion, such as pentoxifyline (Moller et al., 1997b), β2-agonists (PaninaBordignon et al., 1997), thalidomide (Moller et al., 1997a), glucocorticoids (Visser et al., 1998), β-andrenoceptors (Hasko et al., 1998), corticosteroid, budesonide (Larsson and Linden, 1998), acetyl salicylic acid (Mazzeo et al., 1998), histamine (Elenkov et al., 1998; van der Pouw Kraan et al., 1998), and cholera toxin (Braun et al., 1999; Procopio et al., 1999). In a few cases where the mechanism of IL-12 inhibition by various agents has been explored, the target seems to involve NFκKB. For example, D’Ambrosio and colleagues (D’Ambrosio et al., 1998) analyzed the effect of 1,25-dihydroxy-


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vitamin D3 (VD3) on the human IL-12 p40 promoter activation by cotransfecting monocytic RAW264.7 cells with p40 promoter-reporter constructs and expression vectors for the VD3 receptor (VDR) and/or retinoid X receptor (RXRα). Transcriptional repression of the p40 gene by VD3 was observed, which required coexpression of VDR with RXR and an intact VDR DNA-binding domain. The repressive effect maps to NFκB half site of the p40 promoter. Another example is the vasoactive intestinal peptide (VIP) and the structurally related neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP), which act as “macrophage-deactivating factors.” Delgado and Ganea (1999) showed that VIP and PACAP inhibit the production of macrophagederived TNF-α, IL-6, nitric oxide, and IL-12. VIP and PACAP inhibit IL-12 p40 gene expression by affecting both NFκB binding and the composition of the ets-2 binding complex. Both neuropeptides prevent the activation-induced nuclear translocation of the NFκB components p65 and c-Rel by inhibiting the reduction in cytoplasmic IκBα. Moreover, VIP and PACAP inhibit synthesis of the IRF-1. The decrease in nuclear IRF-1 and c-Rel results in alterations of the ets-2-binding complex. Two transduction pathways, a cAMP-dependent and a cAMP-independent pathway, are involved in the inhibition of IL-12 gene expression and appear to differentially regulate the transcriptional factors involved (Delgado and Ganea, 1999). The underlying connection among most of these agents seems to lie, at least in part, in their common ability to elevate intracellular cyclic AMP (cAMP), rather than their general capacity to stimulate IL-10 production. How cAMP inhibits IL-12 production is not currently clear, although protein kinase A has been implicated (Link et al., 2000; Procopio et al., 1999). cAMP may act either to suppress a transcription factor(s) or to induce a transcription repressor(s). An intriguing observation was made (Rieser et al., 1997) that PGE2 can stimulate IL-12 production by human monocyte-derived DCS in the absence of LPS, whereas it inhibits IL-12 secretion in the presence of LPS. Moreover, PGE2 and TNF-α, another potent inhibitor of IL-12 synthesis, cooperate to activate IL-12 production, illustrating the complexity in the cross-regulation of immunologic modulators. Obviously, the study of the mechanisms of these agents by which they suppress IL-12 synthesis in the course of inflammatory responses have important implications in the design of better drugs that minimize the undesirable effects while exerting their therapeutic efficacy. XIII. Looking to the Future

In the last decade since the discovery of IL-12, large strides have been made in terms of exploring the biological functions of IL-12 and, to a lesser extent, the molecular regulation of IL-12 production. Important challenges still lie ahead. First, we are far from having a complete picture of how IL-12 is controlled in a cell-type-specific manner, although some of the past work has touched on the

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issue—for example, the synergy between ets-2 and c-Rel enabling IL-12 p40 transcription in cells that normally do not express the gene (Gri et al., 1998) and the demonstration of the role of ICSBP and PU.1, transcription factors predominantly expressed in myeloid cells, in the transcriptional activation of IL-12 genes (Wang et al., 2000). It is likely that the tissue specificity of IL-12 gene expression is achieved through interactions among multiple factors that form hierarchical complexes under appropriate stimulation. Complex formation and interaction along the IL-12 promoters awaits thorough investigation. Second, IL-12, although it was discovered 11 years ago, remains somewhat a mystery in the sense that its constituents could have multiple dimerization partners, which could perform agonistic, antagonistic, or yet-to-be-discovered functions, adding more complexity to the regulation of the biological equilibrium. Third, understanding how IL-12 production is controlled systemically and locally in vivo during an immune response presents a much greater challenge. It involves macrophages, DC, and B cells and is different at different stages of an immune reaction. It also involves many feedback pathways between different cell types and cytokines in the environment. Understanding the in vivo control of endogenous IL-12 production will aid in the design of therapeutic strategies alternative to the use of exogenous IL-12. Faced with a seemingly intractable undertaking, one cannot help but wonder what to do. As the Chinese proverb goes: “Drops of water become a river.” Let’s all roll up our sleeves and start collecting the drops. REFERENCES Airoldi, I., Gri, G., Marshall, J. D., Corcione, A., Facchetti, P., Guglielmino, R., Trinchieri, G., and Pistoia, V. (2000). Expression and function of IL-12 and IL-18 receptors on human tonsillar B cells. J. Immunol. 165, 6880. Akiyama, Y., Stevenson, G. W., Schlick, E., Matsushima, K., Miller, P. J., and Stevenson, H. C. (1985). Differential ability of human blood monocyte subsets to release various cytokines. J. Leukoc. Biol. 37, 519. Aliberti, J., Reis e Sousa, C., Schito, M., Hieny, S., Wells, T., Huffnagle, G. B., and Sher, A. (2000). CCR5 provides a signal for microbial induced production of IL-12 by CD8a+dendritic cells. Nature Immunol. 1, 83. Almeida, A., de, Ma, X., Cuomo, P., Wahl, L., Wolf, S., Zhou, H., Trinchieri, G., and Karp, C. L. (2001). Type I interferons, interleukin-12, and interferon-(γ ): convergence and cross-regulation among mediators of cellular immunity (submitted). Armant, M., Avice, M. N., Hermann, P., Rubio, M., Kiniwa, M., Delespesse, G., and Sarfati, M. (1999). CD47 ligation selectively downregulates human interleukin 12 production. J. Exp. Med. 190, 1175. Aste-Amezaga, M., Ma, X., Sartori, A., and Trinchieri, G. (1998). Molecular mechanisms of the induction of IL-12 and its inhibition by IL-10. J. Immunol. 160, 5936. Avice, M. N., Sarfati, M., Triebel, F., Delespesse, G., and Demeure, C. E. (1999). Lymphocyte activation gene-3, a MHC class II ligand expressed on activated T cells, stimulates TNF-α and IL-12 production by monocytes and dendritic cells. J. Immunol. 162, 2748. Avice, M. N., Rubio, M., Sergerie, M., Delespesse, G., and Sarfati, M. (2000). CD47 Ligation selectively inhibits the development of human native T cells into Th1 effectors. J. Immunol. 165, 4624.


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Mechanisms of Signaling by the Hematopoietic-Specific Adaptor Proteins, SLP-76 and LAT and Their B Cell Counterpart, BLNK/SLP-65 DEBORAH YABLONSKI∗ AND ARTHUR WEISS† ∗Department of Pharmacology, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Bat Galim, Haifa 31096, Israel; and †Departments of Medicine and of Microbiology and Immunology, and the Howard Hughes Medical Institute, University of California, San Francisco, California 94143

Adaptor proteins lack catalytic activity and contain only protein–protein interaction domains. They have been shown to interact with an ever-growing number of signaling proteins and to play essential roles in many signaling pathways. SLP-76 and LAT are cell-type-specific adaptor proteins expressed in T cells, NK cells, platelets, and mast cells. In these cell types, SLP-76 and LAT are required for signaling by immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors, including the T cell receptor (TCR), the pre-TCR, the high-affinity Fcε receptor, and the platelet GPVI collagen receptor. In B cells, an analogous adaptor, BLNK/SLP-65, is required for signaling by the ITAM-containing B cell receptor. This review summarizes recent research on SLP-76, LAT, and BLNK. A major challenge in understanding adaptor protein function has been to sort out the many interactions mediated by adaptor proteins and to define the mechanisms by which adaptors mediate critical signaling events. In the case of LAT, SLP-76, and BLNK, the availability of tractable genetic systems, deficient in expression of each of these adaptor proteins, has facilitated in-depth investigation of their signaling functions and mechanisms of action. The picture that has emerged is one in which multiple adaptor proteins cooperate to bring about the formation of a large signaling complex, localized to specialized lipid microdomains within the cell membrane and known as GEMs. Adaptors not only recruit signaling proteins, but also play an active role in regulating the conformation and activation of many of the proteins recruited to the complex. In particular, recent research has shed light on the mechanisms by which multiple adaptor proteins cooperate to bring about the recruitment and activation of phospholipase Cγ in response to the activation of ITAM-containing C 2001 Academic Press. receptors.

I. The Role of Adaptors in Signaling by ITAM-Coupled Receptors: An Overview

A. KEY EVENTS IN ITAM-COUPLED SIGNALING PATHWAYS The T cell antigen receptor (TCR) is one of a family of immune receptors that signal through characteristic tyrosine-containing motifs called ITAMs (Qian and 93 C 2001 by Academic Press. Copyright All rights of reproduction in any form reserved. 0065-2776/01 $35.00

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Weiss, 1997; van Leeuwen and Samelson, 1999). Other members of this family include the B cell antigen receptor (BCR), the high-affinity IgE receptor (FcεRI), the GPVI platelet collagen receptor, activating natural killer cell receptors, and a number of Fcγ receptors. The signal-transducing subunits of these multi-subunit receptors are characterized by a cytoplasmic domain containing one or more ITAM motifs. The signaling pathways initiated by ITAM-coupled receptors have been intensively studied and are relatively well understood. For details, the reader is referred to several excellent reviews (Qian and Weiss, 1997; van Leeuwen and Samelson, 1999). Here, we will briefly summarize only the best-characterized, key events of ITAM-coupled signaling pathways. ITAM-containing receptors have no intrinsic tyrosine kinase activity and are coupled to downstream signaling events by cytoplasmic tyrosine kinases. Genetic studies have confirmed that representatives of three families of tyrosine kinases (src-, Syk-, and Tec-family kinases) are required for signaling by ITAMcontaining receptors. Upon receptor ligation, the receptor ITAM motifs are phosphorylated by a src-family tyrosine kinase, triggering recruitment of a Sykfamily tyrosine kinase to the phosphorylated ITAMs. Recruitment and activation of src- and Syk-family tyrosine kinases, along with concomitant activation of Tecfamily tyrosine kinases, lead to tyrosine phosphorylation of a large number of signaling proteins and to activation of downstream signaling pathways. A central event in this process is the tyrosine phosphorylation and activation of phospholipase Cγ (PLC-γ ) (Weiss et al., 1991; Mustelin et al., 1990; Park et al., 1991; Secrist et al., 1991; Wang et al., 2000; Takata et al., 1995; Irvin et al., 2000). PLC-γ catalyzes the formation of two second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol, which trigger calcium flux and contribute to protein kinase C and Ras activation, respectively (Carpenter and Ji, 1999). The coordinate activation of Ras- and calcium-dependent signaling pathways is required for activation of NFAT (nuclear factor of activated T cells), a regulator of interleukin-2 (IL-2) transcription (Woodrow et al., 1993; Rao et al., 1997). In addition to the above-described events, ITAM-coupled receptors trigger signaling events dependent on Rho-family G proteins, leading to activation of the serine/threonine kinases: Pak1, JNK, and p38 (Yablonski et al., 1998a; Su et al., 1994; Wardenburg et al., 1998; Turner et al., 1998; Genot et al., 1996). Possibly connected to the activation of Rho-family-dependent signaling pathways, ITAM-coupled receptors also trigger cytoskeletal rearrangements, which facilitate such critical immune responses as the interaction of T cells with their target cells, the secretion of cytokines and cytotoxic granules in the direction of target cells, and the degranulation of platelets and mast cells (Serrador et al., 1999; Kupfer et al., 1991, 1994). In addition, cytoskeletal responses may facilitate optimal TCR-mediated transcriptional activation (Holsinger et al., 1998).

SLP-76 AND LAT AND THEIR B CELL COUNTERPART, BLNK/SLP-65

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A major theme that has emerged from the study of ITAM-coupled signaling pathways is the requirement for hematopoietic-specific adaptor proteins to couple tyrosine kinases to the activation of PLC-γ and to other downstream signaling events. This role is fulfilled by the adaptors SLP-76, LAT, and Gads in T cells and by BLNK/SLP-65 in B cells. In the absence of these adaptors, receptor-induced activation of the src- and Syk-family tyrosine kinases fails to trigger activation of the Ras pathway, calcium flux, and subsequent transcriptional events. This review will focus on recent studies that are beginning to uncover the mechanisms of signaling by the SLP-76 and LAT adaptor proteins and their B cell counterpart, BLNK/SLP-65. The domain structures of the adaptor proteins discussed in this review are depicted in Fig. 1. Notably, all of these adaptors are composed exclusively of four types of protein–protein interaction domains: SH2 domains, SH3 domains, and potential SH2 and SH3 domain-binding sites. SH2 domains bind to phosphotyrosyl residues in the context of a short consensus sequence, while SH3 domains bind to polyproline motifs. Differences in the consensus sequences preferred by different SH2 and SH3 domains create some specificity in these interactions. However, the specificity of SH2 and SH3 domains is not absolute, which can lead to confusion when many different signaling proteins can be shown to bind to a given adaptor. In this section, we will present the overall structure and expression pattern of each adaptor. In Section II we will describe the genetic systems, which have been developed for the study of these adaptors. These systems, most notably the availability of SLP-76- and LAT-deficient T cells and BLNK-deficient B cells, are enabling in-depth structure function studies, which should facilitate the identification of functionally important interactions while ruling out the less significant ones. In Section III we will summarize recent studies, which are beginning to shed light on the relative functional significance of the various protein–protein interactions mediated by these adaptors. Finally, in Section IV, we will present models, based on available evidence, for the mechanisms by which these adaptors may mediate signaling by ITAM-coupled receptors. B. INTRODUCTION TO THE CELL-TYPE-SPECIFIC ADAPTOR PROTEINS PARTICIPATING IN ITAM-COUPLED SIGNALING PATHWAYS 1. LAT: A Membrane-Anchored Adaptor Protein Linker for activation of T cells (LAT) was originally described as a prominent, membrane-bound tyrosine phosphorylated doublet of 36–38 kD, which was detected following TCR stimulation and which interacted with the SH2 domains of PLC-γ 1 and Grb2 (June et al., 1990; Gilliland et al., 1992; Buday et al., 1994; Sieh et al., 1994). It was postulated that the function of this

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FIG. 1. Domain structure of the adaptor proteins discussed in this review. (A) Domain structure of LAT. (B) SLP-76-family adaptor proteins, including SLP-76, BLNK, and CLNK. The number of amino acids comprising each protein is indicated at right. In cases where more than one splice form has been described, the long form is shown. (C) Domain structure of Gads.


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protein, known as pp36–38, was to recruit PLC-γ 1 and Grb2–SOS to the plasma membrane, leading to activation of the calcium and Ras pathways, respectively (Buday et al., 1994; Sieh et al., 1994; Motto et al., 1996a). Cloning of pp36–38 proved elusive. Recently, the gene encoding pp36–38, now designated LAT, was cloned based on mass spectrometry and sequencing of peptide fragments obtained from the purified protein (Zhang et al., 1998a; Weber et al., 1998). As expected, the LAT cDNA encodes a 36–38-kD doublet, which is rapidly and transiently tyrosine phosphorylated following TCR stimulation. LAT can be phosphorylated in a heterologous expression system by the Syk-family kinases Syk and ZAP-70, but not by src-family kinases (Zhang et al., 1998a). Furthermore, phosphorylated LAT associates with Grb2, PLCγ 1, and the p85 subunit of phosphatidylinositol-3-kinase (PI3K) (Zhang et al., 1998a). These characteristics confirm the identification of LAT as pp36–38. The sequence of LAT revealed it to be a type III transmembrane protein, with an extremely short extracellular domain of four amino acids and a long cytoplasmic tail that does not appear to contain any type of catalytic domain (Fig. 1A). The transmembrane domain is necessary and sufficient for localizing LAT to the plasma membrane. In addition, the juxtamembrane portion of the cytoplasmic domain contains two palmitoylation sites, responsible for targeting LAT to specialized membrane microdomains known as GEMs (Zhang et al., 1998b). The cytoplasmic tail includes a large number of potential tyrosine phosphorylation sites, eight of which are conserved between human, mouse, and rat LAT (Zhang et al., 1998a; Weber et al., 1998). The potential tyrosine phosphorylation sites include five consensus binding sites for the Grb2 SH2 domain (YXNX) and one consensus PLC-γ SH2 domain binding site (YLVV) (Songyang et al., 1993, 1994). Given that human and murine LAT are only 66% identical, the extensive conservation of the putative SH2-domain binding sites suggests that these motifs constitute the functional domains of LAT. Conceptually, the LAT adaptor protein resembles the cytoplasmic tails of receptor tyrosine kinases (RTKs) or the recently described FRS2 protein (Kouhara et al., 1997), whose autophosphorylation sites serve to recruit multiple signaling proteins to the membrane. LAT expression is restricted to a subset of hematopoietic cells. Expression has been detected in T cells, natural killer (NK) cells, mast cells, platelets, and megakaryocytes but not in B cells or in other myeloid- and monocytic-derived cell types (Zhang et al., 1998a; Weber et al., 1998; Facchetti et al., 1999). Further, LAT is expressed at all stages of thymocyte differentiation, suggesting that it plays a role in T cell development, in addition to its role in signaling by mature T cells (Facchetti et al., 1999), a notion that has been born out in mice made deficient in LAT expression (Zhang et al., 1999b).

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2. SLP-76: A Cytoplasmic Adaptor Protein The SH2-domain-containing leukocyte protein of 76 kD (SLP-76) is a 76-kD adaptor protein, inducibly tyrosine phosphorylated following stimulation by the TCR, which was purified and cloned by virtue of its ability to interact with the C-terminal SH3 domain of Grb2 (Jackman et al., 1995; Reif et al., 1994). Overexpression of SLP-76 augments TCR-induced transcriptional responses (Motto et al., 1996b; Wu et al., 1996; Wardenburg et al., 1996), and affects regulation of the actin cytoskeleton (Wardenburg et al., 1998). These findings were the first indication of the central role played by SLP-76 in the TCR pathway. The primary structure of SLP-76 includes three domains capable of mediating intermolecular interactions: an N-terminal acidic domain containing three tyrosine phosphorylation sites, a central proline rich region, and a C-terminal SH2 domain (Fig. 1B) (Jackman et al., 1995; Fang et al., 1996). These domains mediate an array of protein–protein interactions, discussed in Section III.B. Interestingly, at least three domains of SLP-76 are required for augmentation of TCR-induced transcriptional responses; these are the N-terminal tyrosine phosphorylation sites, the Gads binding region, and the SH2 domain (Musci et al., 1997b; Fang et al., 1996; Wardenburg et al., 1996). These results are consistent with the idea that multiple protein–protein interactions play a role in SLP-76 function. The expression pattern of SLP-76 is similar to LAT. Like LAT, SLP-76 is expressed in T cells, NK cells, platelets, and myeloid cells of the granulocyte and monocyte lineage, but not in B cells (Jackman et al., 1995; Robinson et al., 1996; Clements et al., 1998a; Peterson et al., 1999). However, SLP-76 is evolutionarily more conserved than LAT—84% identity of human and murine SLP-76 (Jackman et al., 1995) versus 66% identity of human and murine LAT (Zhang et al., 1998a)—suggesting a more complex function of SLP-76 which may depend on the precise structure of its protein–protein interaction domains. 3. Gads Mediates Association of SLP-76 with LAT Although SLP-76 was cloned by virtue of its ability to interact with Grb2 in vitro (Jackman et al., 1995), it is now known to bind with higher affinity to Gads (Liu et al., 1999; Asada et al., 1999), a hematopoietically expressed, Grb2related adaptor protein, also known as Grb40, GrpL, GRID, Mona, and Grap-2 (Liu et al., 1999; Asada et al., 1999; Law et al., 1999; Ellis et al., 2000; Qiu et al., 1998; Bourette et al., 1998). Like Grb2, Gads contains a central SH2 domain flanked by two SH3 domains (Fig. 1C). In addition, Gads contains a unique region, rich in proline and glutamine, located between the SH2 domain and the C-terminal SH3 domain. The SH2 domains of Gads and Grb2 exhibit similar binding specificity (Ellis et al., 2000; Liu and McGlade, 1998). As a result, both Grb2 and Gads associate via their SH2 domains with tyrosine-phosphorylated LAT following TCR


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stimulation (Liu et al., 1999; Asada et al., 1999). By contrast, the specificity of the SH3 domains differs. Gads interacts with higher affinity with SLP-76 than with SOS, whereas the reverse is true for Grb2 (Liu et al., 1999; Asada et al., 1999; Law et al., 1999). Competition studies show that in vivo SLP-76 binds preferentially to Gads, even in the presence of a large excess of Grb2 (Asada et al., 1999). Due to the constitutive interaction of the Gads SH3 domain with SLP-76, and the inducible interaction of its SH2 domain with LAT, Gads mediates TCRinducible recruitment of SLP-76 into the LAT complex. Consistent with this function, the expression pattern of Gads is similar to that of SLP-76 and LAT. Gads expression is restricted to hematopoietic tissues and primarily to T cells (Law et al., 1999; Ellis et al., 2000). In particular, Gads has been detected in T cells, megakaryocytes, mast cells, NK cells, and macrophages but not in the Raji B cells line (Liu et al., 1999). A further similarity in the expression pattern is that T cell activation has been shown to upregulate the expression of both SLP-76 and Gads (Clements et al., 1998a; Ellis et al., 2000). The overlapping expression pattern of Gads, SLP-76, and LAT, as well as their recruitment into the same signaling complex, suggest that these three adaptors may function in concert to mediate signaling through ITAM-coupled signaling pathways. Recent experimental results supporting this model will be described below, in Section IV.A. 4. BLNK: The B Cell Analog of SLP-76 Intriguingly, SLP-76, LAT, and Gads are not expressed in B cells, despite the many similarities between the BCR and TCR signaling pathways. Instead, a B-cell-specific adaptor protein is expressed, named B cell linker protein (BLNK), also known as SLP-65 and BASH (Fu et al., 1998; Wienands et al., 1998; Goitsuka et al., 1998). BLNK was independently identified by virtue of its BCR-induced tyrosine phosphorylation (Wienands et al., 1998), its ability to interact with the C-terminal SH2 domain of PLC-γ 1 (Fu et al., 1998), and its preferential expression in B cells (Goitsuka et al., 1998). The overall structure of BLNK is similar to SLP-76 and includes an N-terminal basic domain, followed by an acidic domain containing several potential tyrosine phosphorylation sites, an extensive proline-rich region, and a C-terminal SH2 domain (Fig. 1B). However, SLP-76 and BLNK are only 33% homologous overall and are therefore considered to be analogous, but not highly homologous, adaptors. Functionally, BLNK is often thought of as the B cell analog of both LAT and SLP-76, since it fulfills some of the roles ascribed to each of these adaptors in T cells. While SLP-76 has only three tyrosine phosphorylation sites, BLNK has six potential tyrosine phosphorylation sites, some of which may associate with proteins that in T cells are recruited to tyrosine-phosphorylated LAT (Fu et al., 1998). Further, BLNK is tyrosine phosphorylated by Syk and recruited to the

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membrane following B cell stimulation and may perform a role analogous to LAT in that it may recruit PLC-γ and other associated signaling proteins to the membrane (Fu et al., 1998; Ishiai et al., 1999b). 5. CLNK, A Cytokine-Induced Analog of SLP-76 Cytokine-dependent hematopoietic cell linker (CLNK), also known as MIST, is an analog of SLP-76, expressed in cytokine-stimulated hematopoietic cell lines (Cao et al., 1999; Goitsuka et al., 2000). CLNK shares its overall domain structure with SLP-76 (Fig. 1B), although the level of homology is quite low outside of the SH2 domain, which is 40 to 53% identical with those of SLP-76 and BLNK (Cao et al., 1999). Interestingly, CLNK expression was not detected in unstimulated bone marrow, lymph node, spleen, or thymus, but expression was detected following cytokine stimulation of hematopoietic cell lines. In addition, CLNK could be detected following cytokine stimulation of normal murine hematopoietic tissues, including anti-CD3+ IL-2-stimulated splenic T cells, IL-3-propagated bone-marrow-derived mast cells (BMMCs) and IL-2-activated NK cells (Cao et al., 1999). Furthermore, staining of serial skin sections with antiCLNK antibodies revealed its expression in the mast cells infiltrating the skin of NC/Nga mice, which spontaneously develop dermatitis-like lesions, suggesting that CLNK may be expressed in mast cells in vivo under certain conditions (Goitsuka et al., 2000). Consistent with the overall structural similarity between SLP-76 and CLNK, several functional similarities have been noted. First, cross-linking of the TCR or of the FcεR on CLNK-expressing T or mast cell lines, respectively, induces tyrosine phosphorylation of CLNK and its association with other tyrosine-phosphorylated molecules (Cao et al., 1999; Goitsuka et al., 2000). Second, overexpression of CLNK in Jurkat T cells augments basal and TCR-induced activation of NFAT, AP-1, and IL-2-luciferase reporter plasmids, to an extent similar to or greater than that induced by SLP-76 (Cao et al., 1999). Finally, overexpression of a mutant form of CLNK, in which six potential tyrosine phosphorylation sites have been mutated to phenylalanine, inhibits FcεR-mediated degranulation, calcium flux, and NFAT activation in stably transfected RBL-2H3 cells (Goitsuka et al., 2000). These results suggest that SLP-76 and CLNK may perform at least partially overlapping or redundant signaling functions, in the context of cytokine-stimulated hematopoietic cells. II. Genetic Models Reveal the Essential Roles of SLP-76, LAT, and BLNK in ITAM-Coupled Signaling Pathways

The development and characterization of LAT-, SLP-76-, and BLNK-deficient experimental systems have made an immense contribution to our understanding of the signaling functions mediated by these adaptor proteins. While some


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aspects of their function were inferred from the biochemical characterization of these adaptors and their interacting proteins (see Section III), the phenotypic analysis of adaptor-deficient cells, mice, and patients has enabled the precise identification of the signaling events that depend on each adaptor. Indeed, the existence of these experimental systems has established the TCR and BCR signaling pathways as important model systems for the investigation of adaptor protein function. A. LAT-DEFICIENT T CELL LINES: LAT COUPLES TCR STIMULATION TO THE ACTIVATION OF RAS AND PLC-γ 1 The initial biochemical characterization of LAT revealed it to be an inducibly, heavily tyrosine-phosphorylated, membrane-bound molecule that interacted inducibly with Grb2–SOS and with PLC-γ 1 following TCR stimulation (Fig. 2A). Based on these characteristics, it was hypothesized that LAT recruits a complex of Grb2–SOS to the membrane, leading to activation of the Ras pathway, and also mediates membrane recruitment and activation of PLC-γ 1, leading to production of IP3 and diacylglycerol, which trigger calcium flux and contribute to the activation of protein kinase C (PKC) and the Ras pathway, respectively (Buday et al., 1994; Sieh et al., 1994; Motto et al., 1996a). Characterization of LAT-deficient T cell lines has confirmed all major points of the above model, as well as revealing a few unexpected functions of LAT. Two independently isolated, LAT-deficient derivatives of the Jurkat T cell line have been described, and exhibit essentially identical signaling defects (Finco et al., 1998; Zhang et al., 1999a). One of these, J. CaM2, was isolated based on its failure to elevate intracellular calcium levels following TCR stimulation (Goldsmith et al., 1988). Further characterization revealed that J.CaM2 lacks expression of LAT (Finco et al., 1998). The LAT deficiency results in impaired transcriptional responses to TCR stimulation which are restored to approximately wild-type levels in J.CaM2 clones stably reconstituted with wild-type LAT. A comparison of TCR-induced signaling events in J.CaM2 and LAT reconstituted J.CaM2 revealed that LAT is required for TCR-induced tyrosine phosphorylation and activation of PLC-γ 1 and for TCR-induced calcium flux. These calcium pathway defects are consistent with a model in which the recruitment of PLC-γ 1 to tyrosine-phosphorylated LAT is required for its activation. In addition, LAT is required to mediate Ras-dependent signaling events, including TCR-induced activation of Erk, the AP-1 transcription factor, and surface expression of CD69. These defects are consistent with a model in which the recruitment of Grb2 and associated SOS to tyrosine-phosphorylated LAT is required for Ras activation. However, these results are also consistent with an alternative model, in which Ras activation is mediated by a PLC-γ 1- dependent signaling pathway, perhaps involving the diacylglycerol-regulated Ras exchange factor, RasGRP (Hashimoto et al., 1998; Ebinu et al., 2000).

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FIG. 2.


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In addition to the above signaling defects, which were largely expected, J.CaM2 cells exhibit several other intriguing signaling defects. Tyrosine phosphorylation of several signaling proteins is selectively affected. For instance, the inducible tyrosine phosphorylation of SLP-76, Vav, Itk, and PLC-γ 1 is reduced, whereas the inducible tyrosine phosphorylation of Cbl is augmented (Finco et al., 1998; Shan and Wange, 1999). By contrast, tyrosine phosphorylation of ZAP-70 and of the TCR zeta chain is unaffected (Finco et al., 1998). The mechanism whereby LAT selectively regulates the tyrosine phosphorylation of SLP-76, Vav, PLC-γ 1, and Cbl is not well understood but is likely related to the recruitment of these proteins into a LAT-nucleated signaling complex (Zhang et al., 1998a). In addition, we have noted that TCR-induced activation of the Rho GTPase family member, Rac1, is abrogated in J.CaM2 cells; nonetheless, activation of the Rac1/Cdc42 effector, Pak1, is relatively unaffected (Ku et al., 2001). These results suggest that LAT is required for a subset of TCR-induced, Rho-family-dependent signaling events. Again, this finding may be related to the recruitment of Vav into the LAT-nucleated signaling complex, since Vav functions as an exchange factor for the Rho-family GTPases Rac and Cdc42 (Crespo et al., 1997; Han et al., 1997). Finally, LAT is required for TCR-induced activation of HPK1, a serine-threonine kinase that has been implicated in negative regulation of TCR-mediated activation of Erk2 and AP1 (Liou et al., 2000). The mechanism underlying this requirement appears to involve the interaction of HPK1 with the adaptors Grb2 and the related Grap, which also interact with LAT. B. A SLP-76-DEFICIENT T CELL LINE: SLP-76 COUPLES TYROSINE KINASES TO THE ACTIVATION OF PLC-γ 1 AND OTHER KEY TCR-INDUCED SIGNALING EVENTS Phenotypic characterization of the SLP-76-deficient Jurkat-derived T cell line, J14, has revealed the TCR-induced signaling events that depend on SLP-76

FIG. 2. The role of SLP-76 in LAT-dependent signaling events. (A) In the absence of SLP-76, both PLC-γ 1 and Grb2-SOS are recruited to LAT, but downstream signaling events are not efficiently triggered. In particular, PLC-γ 1 is not efficiently tyrosine phosphorylated or activated. Evidence suggests that the Src Homology region of PLC-γ 1 forms a cap, which inhibits the basal activity of the enzyme (Carpenter and Ji, 1999). Based on this model, one may hypothesize that binding to LAT alone is insufficient to disrupt the structure of this inhibitory cap or to expose the tyrosine phosphorylation sites required for PLC-γ 1 activation. (B) SLP-76 is recruited to LAT by Gads and also binds to the SH3 domain of PLC-γ 1. Thus, each of the depicted molecules is stablized at the complex by a bivalent interaction (Gads and PLC-γ 1 each interact with both LAT and SLP-76). This web of interactions may exert a conformational strain on PLC-γ 1, disrupting the inhibitory conformation of its SH region and/or exposing the PLC-γ 1 tyrosine phosphorylation sites. (C) In addition to the above, SLP-76 may recruit a Tec-family kinase to the complex, thereby facilitating the phosphorylation and activation of PLC-γ 1.

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(Yablonski et al., 1998b). Strikingly, SLP-76 is required for optimal TCR-induced tyrosine phosphorylation and activation of PLC-γ 1 but is not required for tyrosine phosphorylation of any other proteins examined, including LAT, Vav, ZAP-70, and Itk. From this evidence, we concluded that SLP-76 is specifically required to couple TCR-activated cytoplasmic tyrosine kinases to the tyrosine phosphorylation and activation of PLC-γ 1. As a result of the reduced activation of PLC-γ 1, TCR-induced calcium flux is significantly reduced, to below the threshold required for activation of the NFAT transcription factor. In addition, activation of Ras-dependent signaling events is reduced, reflected in the reduced activation of Erk, the AP-1 transcription factor, and CD69 surface expression. As in the case of LAT, it is not known whether the Ras pathway defects are secondary to the PLC-γ 1 defect or whether they represent an independent function of SLP-76 in mediating signaling through the Grb2–SOS pathway. All of the above signaling defects are complemented by transfection with wildtype SLP-76, indicating that the SLP-76 deficiency is solely responsible for the phenotypes exhibited by J14 cells. Further, all of the signaling defects can be bypassed by treatment of the cells with PMA and ionomycin, pharmacological agents that trigger Ras activation and calcium flux, respectively, indicating that SLP-76 acts early in the TCR signaling pathway, upstream of the activation of Ras and calcium flux. The phenotype of J14 resembles that of the LAT-deficient cell line, J.CaM2, but is less severe. Like LAT, SLP-76 is required for optimal TCR-mediated activation of PLC-γ 1, and for subsequent PLC-γ 1-dependent and Ras-dependent signaling events. This similarity prompted us to examine whether LAT-mediated signaling events are affected in J14. Strikingly, we found that TCR-induced recruitment of Grb2, SOS, and PLC-γ 1 to LAT is independent of SLP-76 (Fig. 2A) (Yablonski et al., 1998b). We conclude that recruitment of PLC-γ 1 to LAT is necessary but insufficient for its tyrosine phosphorylation and activation; similarly, recruitment of Grb2–SOS to LAT is insufficient for optimal activation of the Ras pathway. Rather, a SLP-76-dependent signaling event is required for the optimal activation of PLC-γ 1 and for subsequent downstream signaling events. Since SLP-76 inducibly associates with Vav and Nck (see Section III. B), two proteins implicated in TCR-mediated activation of the Rac1/Cdc42-Pak1 signaling pathway (Yablonski et al., 1998a; Wardenburg et al., 1998), it was hypothesized that SLP-76 participates in TCR-mediated activation of this pathway (Wardenburg et al., 1998). Surprisingly, we found that TCR-mediated activation of Rac1 and of Pak1 is independent of SLP-76 (Ku et al., 2001). This finding underscores the power of adaptor protein-deficient experimental systems, such as J14, in that they allow us to rigorously test the signaling functions of adaptor proteins and to identify the functionally critical interactions from among the wide array of interactions potentially mediated by these proteins.


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C. LAT- AND SLP-76-DEFICIENT MICE REVEAL THEIR ESSENTIAL ROLES IN SIGNALING THROUGH MULTIPLE ITAM-COUPLED RECEPTORS The creation and characterization of SLP-76-deficient and LAT-deficient mice has revealed a wealth of information regarding the essential signaling functions fulfilled by these adaptor proteins in vivo. Indeed, in most of the cell types in which LAT and SLP-76 are expressed, they are required for signaling by ITAM-coupled receptors, including the pre-TCR, the high-affinity Fcε receptor, and the platelet GPVI collagen receptor. In addition, SLP-76 is required for hemostasis, possibly due to a requirement for SLP-76 for signaling through the αIIβ3 integrin of platelets. In this section, we will briefly describe the phenotypes of SLP-76-deficient and LAT-deficient mice, which reveal their essential roles in ITAM-coupled signaling pathways. The process of T cell development depends on many of the same signaling molecules that participate in the TCR signaling pathway (van Oers, 1999). The α and β chains of the TCR, which determine the specificity of antigen recognition, undergo gene rearrangement to generate a repertoire of clonotypically expressed TCRs. An intermediate step in this process is expression of the pre-TCR, consisting of a rearranged β chain paired with a surrogate α chain. Signaling by the pre-TCR is required for the progression of T cell precursors from the double negative (DN, or CD4−CD8−) to the double positive (DP, or CD4+CD8+) stage of development, and for allelic exclusion, a process that prevents productive rearrangement of more than one TCR β chain. Strikingly, mice made deficient in either SLP-76 or LAT exhibit a block in T cell development at the DN to DP transition and a complete lack of peripheral T cells (Clements et al., 1998b; Pivniouk et al., 1998; Zhang et al., 1999b). More specifically, thymocyte development is blocked at the CD25+CD44− stage within the DP compartment. This is the precise stage at which Rag1-, Rag2-, or CD3εdeficient mice arrest thymocyte development, indicating that development beyond this stage depends on the expression and function of a pre-TCR. Since the TCR β chain is rearranged and the pre-TCR is expressed on the surface of both LAT-deficient and SLP-76-deficient thymocytes (Zhang et al., 1999b; Clements et al., 1998b; Aifantis et al., 1999), it appears that their developmental defects result from a block in the signaling pathway initiated by the pre-TCR. Indeed, in vivo treatment with the anti-CD3ε antibody, 2C11, a treatment that drives Rag2-deficient DN thymocytes to develop into DPs, does not drive this transition in the SLP-76-deficient or LAT-deficient mice, confirming that the pre-TCR signaling pathway is impaired (Pivniouk et al., 1998; Zhang et al., 1999b). As a result of the failure of the pre-TCR to signal, SLP-76-deficient thymocytes do not exhibit allelic exclusion, as indicated by the finding that 31% of SLP-76-deficient CD25+CD44− DN thymocytes have two productively rearranged TCR β alleles, as compared to less than 5% in wild-type mice (Aifantis et al., 1999).

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In contrast to the T cell compartment, other hematopoietic compartments develop normally in the absence of SLP-76 or LAT. B cell development is unaffected, as expected, since B cells do not express either SLP-76 or LAT. Macrophages, NK cells, and mast cells develop in normal numbers in SLP-76deficient mice; furthermore, plateles develop normally, albeit in slightly reduced numbers (Clements et al., 1998b, 1999; Pivniouk et al., 1998, 1999). Similarly, NK cells, mast cells, and platelets develop normally in LAT-deficient mice (Zhang et al., 1999b; Saitoh et al., 2000; Pasquet et al., 1999). Thus, these mice provide a source of mature, SLP-76- or LAT-deficient hematopoietic cells, a fact that has facilitated the investigation of the signaling functions fulfilled by SLP-76 and LAT in vivo. The high-affinity Fcε receptor (FcεRI), expressed on mast cells, is an ITAMcoupled receptor, which signals by a pathway closely resembling the TCR signaling pathway. As in the TCR pathway, cross-linking of FcεRI triggers Sykdependent tyrosine phosphorylation of SLP-76 (Hendricks-Taylor et al., 1997) and LAT (Saitoh et al., 2000). Strikingly, both SLP-76 and LAT are required for physiologic responses to FcεRI ligation. SLP-76-deficient mice are resistant to IgE-mediated passive systemic anaphylaxis; furthermore, bone-marrow-derived mast cells (BMMCs) derived from these mice fail to degranulate or secrete IL-6 following FcεRI cross-linking (Pivniouk et al., 1999). These defects reflect reduced FcεRI-induced tyrosine phosphorylation of PLC-γ 1 and calcium flux, while tyrosine phosphorylation of Syk and Vav are normal (Pivniouk et al., 1999). Thus, as in T cells, SLP-76 is specifically required to couple receptor-induced tyrosine kinases to PLC-γ 1 and subsequent downstream signaling events. LATdeficient mice exhibit a similar mast cell phenotype, including resistance to IgE-mediated passive systemic anaphylaxis, reduced FcεRI-induced tyrosine phosphorylation of PLC-γ 1 and SLP-76, but normal phosphorylation of Syk and Vav, reduced calcium flux, reduced activation of MAPK and JNK, and impaired FcεRI-induced degranulation and cytokine production (Saitoh et al., 2000). In all, the signaling defects of LAT-deficient mast cells closely resemble those of J.CaM2 (LAT-deficient T cells), suggesting that the signaling function of LAT is conserved in ITAM-coupled signaling pathways in the cell types in which LAT is expressed. Like the FcεRI, the platelet GPVI collagen receptor is an ITAM-coupled receptor that signals through a pathway closely resembling the TCR signaling pathway (Watson and Gibbins, 1998), and requires both SLP-76 and LAT. Collagen can bind to two different receptors on platelets: the α 2β 1, integrin, which mediates adhesion to collagen, and the GPVI receptor, which mediates collagen-induced platelet activation (Watson and Gibbins, 1998). A collagenrelated peptide (CRP), which activates the GPVI receptor, but not α 2β 1, was used to demonstrate the role of SLP-76 and LAT in GPVI signaling. Significantly, CRP triggers Syk-dependent tyrosine phosphorylation of SLP-76 (Gross


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et al., 1999) and LAT (Pasquet et al., 1999). Whereas CRP-induced Syk activation was normal, downstream physiologic responses to CRP, including tyrosine phosphorylation of PLC-γ 2, calcium flux, platelet aggregation, and degranulation are absent in SLP-76-deficient platelets (Gross et al., 1999; Clements et al., 1999). By contrast, G-protein-coupled thrombin receptor-induced platelet activation is unaffected in SLP-76-deficient mice, demonstrating that the signaling defects are largely restricted to ITAM-coupled signaling pathways (Gross et al., 1999). Similarly, LAT-deficient platelets exhibit normal CRP-induced tyrosine phosphorylation of Syk, but reduced tyrosine phosphorylation of PLC-γ 2 and SLP-76 and reduced CRP-induced degranulation (Pasquet et al., 1999). Possibly related to the defect in platelet function, SLP-76-deficient mice exhibit a diffuse subcutaneous hemorrhage, in utero and at birth, which is associated with significant perinatal lethality (Clements et al., 1999; Pivniouk et al., 1998). By contrast, LAT-deficient mice, though impaired in platelet responses to CRP, exhibit no subcutaneous bleeding or perinatal lethality (Zhang et al., 1999b). This difference prompted an exploration of whether additional signaling pathways, required for platelet-dependent hemostasis, might be impaired in SLP-76-deficient mice. Indeed, a recent study shows that SLP-76-deficient platelets exhibit reduced tyrosine phosphorylation of cellular substrates in response to fibrinogen and reduced spreading on a fibrinogen matrix (Judd et al., 2000). These defects may underlie the bleeding phenotype of SLP-76-deficient mice. Nonetheless, since fibrinogen binds to integrins and is not known to activate ITAM-coupled receptors, the mechanistic basis of this phenotype is currently unclear. The previous examples suggest that signaling by ITAM-coupled receptors depends on SLP-76 and LAT as a rule; nonetheless, two apparent exceptions to this rule have been noted. First, no functional defects were observed in SLP-76- or LAT-deficient NK cells, despite the expression of SLP-76 and LAT in NK cells (Peterson et al., 1999; Zhang et al., 1999b). Most surprisingly, IL-2 stimulated SLP-76-deficient NK cells exhibited normal antibody-dependent cellular cytotoxicity (ADCC) (Peterson et al., 1999), a reaction that is mediated by the ITAM-coupled receptor, CD16 (Fcγ RIIIA). No BLNK expression was detected in NK cells, ruling out the possibility that NK cell function may be mediated by BLNK, rather than SLP-76 (Peterson et al., 1999). It is possible that CD16-induced ADCC is mediated by CLNK, a SLP-76-related protein that is expressed in cytokine-stimulated hematopoietic cell lines, including IL-2activated NK cells (Cao et al., 1999). In a second example, SLP-76 is not required for signaling by Fcγ receptors on bone-marrow-derived macrophages, probably due to the expression of both SLP-76 and BLNK in macrophages (Myung et al., 2000; Bonilla et al., 2000). Indeed, BLNK may be the dominant adaptor protein in macrophages, since this cell type is not known to express LAT. Consistent with the normal function of SLP-76-deficient NK cells and macrophages,

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SLP-76-deficient mice appear to exhibit unimpaired innate immune responses (Myung et al., 2000). D. A BLNK-DEFICIENT B CELL LINE, MOUSE, AND HUMAN PATIENT REVEAL THAT BLNK IS REQUIRED FOR ITAM-MEDIATED SIGNALING IN B CELLS As in the case of SLP-76 and LAT, the development and characterization of a BLNK-deficient B cell line and BLNK-deficient mice have facilitated the precise definition of signaling events that depend on BLNK, while the identification of a BLNK-deficient patient highlights the physiologic importance of this adaptor molecule. In general, the findings are consistent with the idea that BLNK is the B cell analog of SLP-76 and serves similar signaling functions, in the context of the B cell antigen receptor (BCR) signaling pathway. Nonetheless, the findings highlight remaining uncertainties concerning the signaling pathways activated by ITAM-coupled pathways, in particular, concerning the mechanism by which ITAM-coupled receptors induce Ras activation and the causal relationship between Ras and Erk activation. The major findings are briefly summarized below. Characterization of a BLNK-deficient derivative of the DT-40 chicken B cell line revealed that BLNK is required for optimal BCR-induced recruitment of PLC-γ 2 to the membrane and for tyrosine phosphorylation of PLC-γ 2 (Ishiai et al., 1999a). Not unexpectedly, BLNK is required for downstream, PLCγ 2-dependent events, including IP3 generation and calcium flux (Ishiai et al., 1999a). In addition, BLNK is required for optimal activation of the three MAP kinases, Erk2, JNK, and p38 (Ishiai et al., 1999a). The most intriguing aspect of the BLNK-deficient phenotype is its effect on the Ras pathway. The evidence suggests that Ras activation is unaffected; nonetheless, BCR-induced Erk activation is significantly reduced (Ishiai et al., 1999a). This finding is consistent with a previous study from the same laboratory, which suggested that two BCR-induced pathways independently contribute to Erk2 activation, a Ras-dependent pathway, and a PLC-γ 2-dependent pathway (Hashimoto et al., 1998). Consistent with this model, the residual Erk2 activation seen in BLNK-deficient DT-40 cells was eliminated upon transfection of the cells with a dominant negative form of Ras (Ishiai et al., 1999a), suggesting that the Ras-dependent pathway leading to Erk2 remains functional in BLNK-deficient cells. To further test this model, Ishiai and colleagues performed a transfection experiment designed to test whether the reduction in PLC-γ 2 activation is the cause of the reduced Erk2 activation (Ishiai et al., 1999a). PLC-γ 2 was expressed as a membrane-bound chimera, fused to the extracellular domain of Fcγ RIII and the transmembrane domain of the TCR ζ chain. Cross-linking of this chimera with the BCR rescued BCR-induced PLC-γ 2 tyrosine phosphorylation, calcium flux, and Erk2 activation, indicating that BCR-induced Erk2 activation correlates with the activation of PLC-γ 2 and is likely to result from a PLC-γ 2-dependent


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signaling pathway. Interestingly, the PLC-γ 2 chimera did not rescue activation of JNK or p38, suggesting that these defects represent independent signaling functions of BLNK. Together, the data suggest that BLNK is required to couple the BCR to the activation of PLC-γ 2 and for PLC-γ 2-dependent, but not Ras-dependent, activation of Erk2. In this respect, BLNK shows a strong functional resemblance to SLP-76, which is also required for the activation of PLC-γ and Erk. In light of the model suggested by Ishiai and colleagues, one wonders whether the reduced activation of Erk seen in SLP-76-deficient J14 cells may be secondary to the PLC-γ 1 defect and may not reflect a defect in Ras activation, as we have previously assumed. BLNK-deficient mice have been independently generated in four different laboratories (Xu et al., 2000; Pappu et al., 1999; Jumaa et al., 1999; Hayashi et al., 2000). Characterization of the BLNK-deficient mice revealed that they are viable and have normal numbers of T cells, but B cell development in the bone marrow is largely blocked at the pro- to pre-B cell transition. The number of IgM+ peripheral B cells is significantly reduced to less than 10% of wild type, whereas the number of mature IgMlo IgDhi cells is reduced to less than 1% of wild type (Pappu et al., 1999; Hayashi et al., 2000; Xu et al., 2000). The pro- to pre-B cell block suggests that BLNK is required for signaling by the pre-BCR, as mice deficient in components of the pre-BCR exhibit a similar developmental block. In addition, there is a complete absence of CD5-expressing peritoneal B1 B cells (Pappu et al., 1999; Jumaa et al., 1999; Hayashi et al., 2000). Consistent with the block in B cell development, the mice exhibit reduced levels of serum Ig, especially IgM and IgG3 (Pappu et al., 1999; Jumaa et al., 1999). Upon immunization, BLNK-deficient B cells fail to produce an IgM response to T-independent antigens and show a reduced primary IgM response to T-dependent antigens (Jumma et al., 1999; Xu et al., 2000). Surprisingly, the secondary IgM response is intact (Jumaa et al., 1999; Xu et al., 2000), suggesting that T cell help is able to bypass the requirement for BLNK, possibly by the induction of CLNK expression in B cells, by T-cell-secreted cytokines. Alternatively, memory B cells may have the intrinsic capacity to signal independently of BLNK. Overall, the phenotype of BLNK-deficient mice closely resembles the phenotype of Btk-deficient mice, suggesting that these two signaling proteins act in the same pathway (Fruman et al., 2000). Further supporting the functional connection between Btk and BLNK is the discovery of a patient carrying a homozygous splice defect in the gene encoding BLNK. The patient exhibited early-onset hypogammaglobulinemia and an absence of mature B cells, a phenotype characteristic of patients carrying homozygous mutations in Btk (known as X-linked agammaglobulinemia, or XLA). Thus, BLNK and Btk appear to function in concert, to mediate signaling by the pre-BCR, both in mice and in humans. Indeed, Btk associates with BLNK and is required to mediate BCR-induced tyrosine

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phosphorylation of PLC-γ 2 in DT-40 B cells (Hashimoto et al., 1999; Su et al., 1999; Takata and Kurosaki, 1996) (see Section IV.B). III. Interactions Mediated by LAT, SLP-76, and BLNK and Their Role in Antigen Receptor Signaling

Due to their structure, which consists of protein–protein interaction domains, adaptor proteins interact in vitro with a seemingly limitless variety of signaling proteins. A major challenge is not only to identify the interacting proteins, but also to determine their functional significance and to define the mechanisms by which adaptors mediate downstream signaling events. In ongoing studies, the availability of LAT- and SLP-76-deficient genetic backgrounds is facilitating a detailed analysis of the domains required for signaling by each adaptor. These studies, combined with biochemical analyses of the interactions mediated by each domain, should yield a coherent picture of how each adaptor functions. A. LAT RECRUITS SIGNALING PROTEINS TO THE GEMS LAT is a transmembrane protein with an extremely short extracellular domain and a long cytoplasmic tail, including nine potential tyrosine phosphorylation sites (Zhang et al., 1998a). Just adjacent to the transmembrane domain, on the cytoplasmic face of the protein, are two palmitoylation sites (Zhang et al., 1998b). While the transmembrane domain is sufficient to localize LAT to the plasma membrane, palmitoylation is required to localize LAT to GEMs (Zhang et al., 1998b), also known as DIGs or lipid rafts. These are specialized lipid domains within the plasma membrane, enriched in glycosphingolipids, cholesterol, and phosphatidylinositol bisphosphate (PIP2) (Xavier et al., 1998). Mutation of the palmitoylation sites eliminates the localization of LAT to GEMs, prevents TCR-induced tyrosine phosphorylation of LAT, and leads to its functional inactivation, as measured by an inability to rescue TCR signaling events in a LAT-deficient cell line (Zhang et al., 1999a; Lin et al., 1999). By contrast, LATdependent signaling functions are rescued by a chimeric construct, in which the N-terminus of Lck, including an alternative GEM targeting sequence, replaces the transmembrane domain and palmitoylation sites of LAT (Lin et al., 1999). These findings demonstrate that localization of LAT to GEMs is required for its signaling function and suggest that that a major function of LAT may be to recruit other signaling molecules to the GEMs. Indeed, a small amount of Vav, PLC-γ 1, Gads, and SLP-76 redistribute to the GEMs following TCR stimulation, possibly reflecting their recruitment into the LAT complex (Zhang et al., 1998b; Boerth et al., 2000). A number of signaling proteins co-immunoprecipitate with LAT, following TCR stimulation. Grb2, Gads, and the p85 subunit of PI3K appear to bind directly to LAT via their SH2 domains (Liu et al., 1999; Asada et al., 1999; Zhang


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et al., 1998a; Gibbins et al., 1998). Likewise, the association of PLC-γ 1 with LAT appears to be direct and to be mediated by the N-terminal SH2 domain of PLC-γ 1 (Stoica et al., 1998). Additionally, LAT associates with SLP-76, Vav, Cbl, and the Tec-family kinase Itk (Zhang et al., 1998a; Shan and Wange, 1999). Some of these interactions may be indirect. For example, the interaction of SLP-76 with LAT is likely mediated by Gads (Liu et al., 1999; Asada et al., 1999), and the association of Vav with LAT may be mediated by SLP-76. The importance of these associations is suggested by a dominant negative LAT construct, in which tyrosines 171 and 191 have been mutated to phenylalanine. The mutated tyrosines are both in VYNV motifs, consensus binding motifs for the Grb2 and Gads SH2 domains (Liu and McGlade, 1998). This mutant form of LAT is inducibly tyrosine phosphorylated but shows greatly reduced binding to Grb2, PLC-γ 1, the p85 subunit of PI3K, SLP-76, Vav, and Cbl (Zhang et al., 1998a). The mechanism by which mutation of only two sites affects the recruitment of so many signaling proteins is not yet clear. Nonetheless, overexpression of this mutant inhibits TCR-induced transcriptional responses (Zhang et al., 1998a). This finding is consistent with the hypothesis that recruitment of key TCR signaling proteins to LAT plays an essential role in the TCR signaling pathway and provided the first strong experimental confirmation of this hypothesis. While LAT contains nine potential tyrosine phosphorylation sites, the specific sites at which LAT is tyrosine phosphorylated upon TCR stimulation have not yet been mapped. Zhang and colleagues have attempted to determine the functional significance of specific phosphorylation sites, focusing on four sites that correspond to consensus binding motifs for PLC-γ 1 or Grb2/Gads (Zhang et al., 2000). To this end, mutant forms of LAT, bearing one or more tyrosine to phenylalanine substitutions, were stably transfected into the LAT-deficient T cell line, J.CaM2. The results of this structure–function analysis are not easy to interpret and hint at the complexity of the signaling events that occur at LAT. The results are inconsistent with a simple model, in which PLC-γ 1, Grb2, and Gads are independently recruited to LAT and execute their signaling functions independently of each other. Rather, the results suggest a web of complex, multivalent protein–protein interactions that occur at LAT, in which binding of any one signaling protein affects the binding and function of other signaling proteins. A few of the most significant findings of this study and some of the possible conclusions are summarized below; for additional details, the reader is referred to the original study (Zhang et al., 2000). The four potential phosphorylation sites that were examined include three Grb2-consensus binding motifs (YXNX, at tyrosines 171, 191, and 226) and one consensus PLC-γ 1-binding motif (YLVV, at tyrosine 132). In addition to individual mutations, combinations of tyrosines were mutated to create the following constructs; 2YF (Y171 and 191 mutated to F), 3YF (Y171, 191, and 226 mutated

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to F), and 4YF (all four tyrosines mutated). Significantly, all mutants, including 4YF, retained some degree of TCR-induced tyrosine phosphorylation, suggesting that additional TCR-induced tyrosine phosphorylation sites remain to be identified. Some of the results appear straightforward, and consistent with expectations. For example, LAT appears to bind to Grb2 at multiple sites, since mutation of all three consensus Grb2-binding sites was required to eliminate TCR-inducible binding of LAT to Grb2. By contrast, mutation of tyrosines 171 and 191 (2YF) was sufficient to eliminate binding of Gads, and in particular tyrosine 191 appears to be the major Gads-binding site. Mutation of Y132, the PLC-γ 1-consensus binding site, was sufficient to eliminate detectable binding of LAT to PLC-γ 1, as well as TCR-induced tyrosine phosphorylation of PLC-γ 1. Current models suggest that Grb2 recruits SOS to LAT, leading to Ras activation. In addition, Gads recruits SLP-76 to LAT, and PLC-γ 1 is recruited independently, leading to calcium flux. Many of the results of Zhang et al. are inconsistent with this simple model and hint at complex interactions between the signaling molecules recruited to LAT. The 3YF mutation, which eliminates the association of LAT with Grb2 and Gads, also eliminates its association with PLC-γ 1 and reduces the inducible tyrosine phosphorylation of PLC-γ 1 to nearly background levels. This result may indicate that tyrosine phosphorylation of the PLC- γ 1-binding site, Y132, is dependent on phosphorylation of the three Grb2binding sites. Alternatively, it may indicate that the recruitment and activation of PLC-γ 1 are complex events depending on the association of Grb2 or Gads with LAT. Similarly, Zhang et al. find that mutation of Y132, which disrupts the recruitment of PLC-γ 1, but not Gads, to LAT, markedly reduces the recruitment of SLP-76 to LAT. Again, this finding suggests a functional interaction between Grb2/Gads-dependent events and PLC-γ 1-dependent events. Possibly connected to these results, we have found that the proline-rich domain of SLP-76 binds to PLC-γ 1 as well as to Gads (Yablonski et al., 2001). Thus, the surprising results of Zhang et al. may be explained by a model in which PLC-γ 1 and Gads cooperatively recruit SLP-76 to the LAT complex, by virtue of their SH3 domains, which bind to SLP-76, and by their SH2 domains, which bind to LAT (Fig. 2B). According to this model, disruption of any one interaction could undermine the stability of the complex as a whole. Looking at downstream signaling events, the authors found that recruitment of Grb2 to the LAT complex does not correlate well with activation of Erk, suggesting that recruitment of SOS to LAT is not sufficient for activation of Ras and, subsequently, Erk. Similarly, we found that recruitment of Grb2-SOS to LAT is insufficient for optimal Erk activation, in the absence of SLP-76 (Yablonski et al., 1998b). By contrast a good correlation was observed between recruitment of PLC-γ 1 and Erk activation. Indeed, Zhang et al. found that all mutations or combinations of mutations that disrupt recruitment of PLC-γ 1 to the LAT


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complex are associated with significantly reduced TCR-induced calcium flux, impaired TCR-induced Erk activation, and an abrogation of TCR-induced activation of the NFAT transcription factor (Zhang et al., 2000). This correlation is consistent with the idea that the activation of PLC-γ 1 is a central signaling event in ITAM-coupled signaling pathways, not only responsible for calcium flux, but also contributing significantly to the activation of the Erk pathway (Hashimoto et al., 1998; Ishiai et al., 1999a; Ebinu et al., 2000). B. FOUR FUNCTIONAL DOMAINS AND MULTIPLE PROTEIN–PROTEIN INTERACTIONS COUPLE SLP-76 TO DOWNSTREAM SIGNALING EVENTS Many different signaling proteins are known to interact with SLP-76; these interactions are summarized in Table I. The three tyrosine phosphorylation sites in the N-terminal acidic domain of SLP-76 are found within YEXP motifs and preceded by three acidic amino acids (Jackman et al., 1995; Fang et al., 1996). These motifs are phosphorylated with high efficiency by the Syk-family tyrosine kinases Syk and ZAP-70 (Wardenburg et al., 1996; Gross et al., 1999; Raab et al., 1997) but may also be phosphorylated by the Tec-family tyrosine kinase Rlk (Schneider et al., 2000). Tyrosines 113 and 128 of SLP-76 mediate the TCRinducible association of SLP-76 with the Nck adaptor protein (Wardenburg et al., 1998; Wunderlich et al., 1999) and with Vav, a hematopoietic specific exchange factor for Rho-family GTPases (Wu et al., 1996; Tuosto et al., 1996; Fang and Koretzky, 1999; Raab et al., 1997). A short stretch (amino acids 224–265) within the proline-rich domain of SLP-76 was originally identified as a Grb2-binding site (Motto et al., 1996b) but is now known to bind with higher affinity to Gads (Liu et al., 1999; Asada et al., 1999). Interestingly, this interaction is mediated by an atypical SH3 domain-binding motif (PX3RX2KPX7PLD), which was only recently identified (Lock et al., 2000). We have defined an additional binding site within the proline-rich domain, designated the P-I domain. The P-I domain, encompassing residues 157–223 of SLP-76, associates with the SH3 domain of PLC-γ 1 (Yablonski et al., 2000) and may also associate with two kinases critical for TCR signaling: the src-family kinase Lck and the Tec-family kinase Itk, (Sanzenbacher et al., 1999; Bunnell et al., 2000). The SH2 domain of Itk has been further suggested to bind to N-terminal tyrosine phosphorylation sites in SLP-76 (Su et al., 1999). Finally, the C-terminal SH2 domain of SLP-76 interacts with an additional adaptor protein, SLAP-130/Fyb (Musci et al., 1997a; da Silva et al., 1997). We have recently found that the SH2 domain also binds to tyrosine phosphorylated HPK1 and facilitates its activation (Sauer et al., 2001). The functional importance of each of the protein–protein interactions mediated by SLP-76 is the subject of ongoing investigation. Recently we took advantage of the SLP-76-deficient background afforded by J14 cells to revisit the structure-function analysis of SLP-76, previously carried out on a wild-type background (Motto et al., 1996b; Wu et al., 1996; Wardenburg et al., 1996). Our

TABLE I SLP-76-INTERACTING PROTEINS Interacting protein

Description

Mode of interaction

Reference

Vav

Hematopoieticspecific exchange factor for rho-family G proteins

SH2 domain of Vav binds to N-terminal phosphotyrosyl residues of SLP-76

Wu et al., 1996; Tuosto et al., 1996; Raab et al., 1997; Fang and Koretzky, 1999

Nck

Adaptor protein

SH2 domain of Nck binds to N-terminal phosphotyrosyl residues of SLP-76

Wardenburg et al., 1998; Wunderlich et al., 1999

Grb2

Adaptor protein

C-terminal SH3 domain of Grb2 binds to short motif within the proline-rich domain of SLP-76 (residues 224–244)

Jackman et al., 1995; Motto et al., 1996b; Reif et al., 1994

Gads

Grb2-like adaptor protein

C-terminal SH3 domain binds to the same site as Grb/2, above, but with a higher affinity

Liu et al., 1999; Asada et al., 1999; Law et al., 1999

Lck

Src-family tyrosine kinase

SH3 domain of Lck binds to short motif within the proline-rich domain of SLP-76 (residues 185–194)

Sanzenbacher et al., 1999

Itk

Tec-family tyrosine kinase

SH3 domain of Itk binds to short motifs within the proline-rich domain of SLP-76 (residues 184–208), and SH2 domain binds to N-terminal phosphotyrosyl residues of SLP-76

Bunnell et al., 2000; Su et al., 1999

PLC-γ 1

Phospholipase C family member

SH3 domain of PLC-γ 1 binds to a region encompassing residues 157–223 within the proline-rich domain of SLP-76

Yablonski et al., 2000

SLAP/130Fyb

Adaptor protein

Phosphotyrosyl residues in SLAP/130Fyb bind to the SH2 domain of SLP-76

Musci et al., 1997a; da Silva et al., 1997; Raab et al., 1999; Geng et al., 1999

HPK1

Serine/threonine kinase

Tyrosine-phosphorylated HPK1 binds to the SH2 domain of SLP-76

Sauer et al., 2000


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studies revealed that three domains of SLP-76 are required for TCR-mediated activation of PLC-γ 1 and activation of the NFAT transcription factor—specifically, the N-terminal tyrosine phosphorylation sites, the Gads binding domain, and the P-I domain, a proline-rich region encompassing amino acids 157–223 of SLP-76 (Yablonski et al., 2000). The P-I domain associates with the SH3 domain of PLC-γ 1, a finding that may underlie the requirement for this domain to mediate activation of PLC-γ 1 (Yablonski et al., 2001). The SH2 domain of SLP-76 is not required for activation of PLC-γ 1, Erk, or NFAT (Yablonski et al., 2001) but is required for TCR-mediated activation of HPK1 (Sauer et al., 2000), a serine/threonine kinase that negatively regulates signaling through the TCR pathway (Yablonski et al., 2000). Thus, SLP-76 appears to mediate a number of independent signaling functions, which may depend on different domains and different protein–protein interactions. One approach, which has been productive in the study of SLP-76, is cooverexpression of putative interacting proteins, together with SLP-76. In many cases, this produces a synergistic augmentation of TCR-induced signaling events, suggesting that the interaction in question is part of the mechanism by which SLP-76 mediates TCR signaling. For example, overexpressed Gads synergizes with SLP-76 to augment activation of the NFAT transcription factor and the IL-2 promoter, suggesting that the signaling function of SLP-76 depends on Gads (Liu et al., 1999; Asada et al., 1999; Law et al., 1999). Indeed, recent studies support a model in which Gads and SLP-76 function in concert to recruit key signaling proteins to LAT (see Section IV.A). The functional significance of the interaction of SLP-76 with the Tec-family kinase Itk is more controversial. Notably, the signaling defects exhibited by Itkdeficient T cells resemble those of SLP-76 deficient T cells (Yablonski et al., 1998b; Liu et al., 1998; Schaeffer et al., 1999), suggesting that these signaling molecules may cooperate to fulfill a common function. Nonetheless, the interaction between SLP-76 and Itk was not detected between endogenous proteins in T cells, only between proteins overexpressed in a baculovirus system (Bunnell et al., 2000). Furthermore, overexpression of Itk inhibited SLP-76-induced augmentation of NFAT activity (Bunnell et al., 2000), suggesting that the interaction between Itk and SLP-76 may not contribute to the positive role of SLP-76 in the TCR signaling pathway. Possibly, another Tec-family kinase member, Rlk, may function in concert with SLP-76. A recent study demonstrated that Rlk can phosphorylate the N-terminal tyrosine phosphorylation sites of SLP-76; furthermore, overexpression of Rlk synergizes with SLP-76 to augment TCR-induced activation of NFAT (Schneider et al., 2000). To our knowledge, it is not known whether Rlk may interact with SLP-76 in a manner similar to the described interaction with Itk, nor has SLP-76 tyrosine phosphorylation been examined in Rlk-deficient mice. Two other controversial interactions involve the proteins, Vav and Nck. Cooverexpression of Vav and SLP-76 leads to a striking, synergistic augmentation

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of TCR-induced transcriptional responses, suggesting that the two proteins may function together and that each protein may be limiting, under conditions where the other is overexpressed (Wu et al., 1996). In addition, by binding to the N-terminus of SLP-76, Vav and Nck have been proposed to form a tri-molecular complex with SLP-76, which contributes to activation of the serine threonine kinase, Pak1, and to TCR-induced remodeling of the action cytoskeleton (Wardenburg et al., 1998). However, TCR signaling can occur in the absence of a detectable association between Vav and SLP-76 (Raab et al., 1997). Further, we have found that TCR-induced Pak1 activation is independent of both Nck and SLP-76 (Ku et al., 2001). Finally, a recent study has determined that the synergistic potentiation of TCR signaling by Vav and SLP-76 does not depend on their association (Fang and Koretzky, 1999). Thus, at present, the functional importance of these interactions remains uncertain. A final example of the uncertainty surrounding the mechanisms of signaling by SLP-76 is the adaptor protein, SLAP-130/Fyb, which binds to the SH2 domain of SLP-76. Overexpression of SLAP-130/Fyb was reported to inhibit TCR- and SLP-76-induced NFAT activation in Jurkat cells (Musci et al., 1997a) but was also found to augment TCR-induced IL-2 production in the DC27.10 T cell hybridoma (da Silva et al., 1997). These opposing results may reflect the different cell types used and, in particular, differences in the level of expression of Fyn-T, a kinase of the Src family. Fyn-T phosphorylates SLAP-130/Fyb on sites that mediate its association with the SH2 domain of SLP-76 (Raab et al., 1999; Geng et al., 1999). Indeed, strikingly synergistic activation of the NFAT transcription factor and the IL-2 promoter in Jurkat cells was obtained by coexpression of three proteins: SLAP-130/Fyb, Fyn-T, and SLP-76 (Raab et al., 1999; Veale et al., 1999). These results suggest that these three proteins function in concert to influence TCR signaling and further suggest that, in Jurkat cells, the phosphorylation of SLAP-130/Fyb by Fyn-T may be a rate-limiting event. C. BLNK-ASSOCIATING PROTEINS Interestingly, SLP-76, LAT, and Gads exhibit overlapping patterns of expression, including T cells, NK cells, mast cells, platelets, and megakaryocytes but excluding B cells. Instead, B cells express BLNK/SLP-65, an adaptor with an overall structure similar to SLP-76. No B cell analog of LAT has been identified; rather, BLNK is thought to fulfill the signaling functions of both SLP-76 and LAT. Consistent with this model, BLNK interacts with many signaling proteins, both those that interact with SLP-76 in T cells and those that interact with LAT. Nonetheless, it must be emphasized that transfection of BLNK alone fails to reconstitute TCR signaling events in SLP-76-deficient T cells (Wong et al., 2000). This finding suggests that BLNK may function in concert with a B cell analog of LAT, which remains to be identified.


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Among the proteins reported to interact with BLNK are Grb2, PLC-γ 1, Vav, Nck, Cbl, and Btk (Fu et al., 1998; Wienands et al., 1998; Yasuda et al., 2000; Su et al., 1999; Hashimoto et al., 1999). The first four (Grb2, PLC-γ 1, Vav, and Nck) are familiar from our previous discussion about LAT and SLP-76 and appear to fulfill similar signaling functions in T and B cells. Cb1 functions as a negative regulator of antigen receptor signaling by poorly understood mechanisms (van Leeuwen and Samelson, 1999), which may include the triggering of ubiquitin-mediated degradation of key signaling proteins (Levkowitz et al., 1999). Btk is a Tec-family kinase, and its involvement in BCR signaling appears analogous to the involvement of the Tec family members Itk and Rlk in TCR signaling. Specifically, Btk is required for BCR-induced activation of PLC-γ 2, IP3 generation, and calcium flux (Takata and Kurosaki, 1996). Thus far, all of the identified BLNK-interacting proteins appear to bind via interactions with the tyrosine phosphorylation sites of BLNK. Although the interaction of BLNK with Grb2 has a constitutive component, it is significantly augmented by BCR stimulation (Fu et al., 1998). Likewise, the interaction of BLNK with PLC-γ 1, Vav, Nck, Cbl, and Btk is dependent on BCR stimulation (Fu et al., 1998; Yasuda et al., 2000; Hashimoto et al., 1999), suggesting that the SH2 domains of the interaction proteins and the tyrosine phosphorylation sites of BLNK mediate these interactions. Indeed, the association of BLNK with PLC-γ 1 depends on the N-terminal SH2 domain of PLC-γ 1 (Ishiai et al., 1999b; DeBell et al., 1999), and the association with Cbl depends on the recently defined atypical SH2 domain of Cbl (Yasuda et al., 2000). The significance of the interaction of BLNK with most of the above proteins has not yet been analyzed in detail; however, evidence suggests that the interactions with PLC-γ 1 and Btk are functionally significant. In a striking experiment, co-expression of Syk and PLC-γ 1 in insect cells failed to result in efficient tyrosine phosphorylation of PLC-γ 1, whereas co-expression of BLNK, Syk, and PLC-γ 1 resulted in efficient tyrosine phosphorylation of PLC-γ 1 (Fu et al., 1998). This result suggests that the interaction of BLNK with PLC-γ 1 increases the accessibility of PLC-γ 1 to tyrosine phosphorylation. In a similar experiment, co-expression of Syk and Btk with PLC-γ 2 in 293 T cells produced only a low level of tyrosine phosphorylation of PLC-γ 2, whereas additional co-transfection of BLNK led to a significant increase in the tyrosine phosphorylation of PLC-γ 2 (Hashimoto et al., 1999). These studies suggest a model in which binding of PLC-γ 2 and Btk to Syk-phosphorylated BLNK significantly increases the efficiency of phosphorylation of PLC-γ 2 by Btk. In other words, BLNK appears to functionally mediate the interaction between Btk and PLC-γ 2. Intriguingly, in Cbl-deficient B cells, BCR-induced association of PLC-γ 1 with BLNK and BCR-induced activation of PLC-γ 1 are increased, suggesting that Cbl and PLC-γ 1 may compete for interaction with BLNK (Yasuda et al., 2000).

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IV. Mechanisms of Signaling by LAT and SLP-76

A. COOPERATIVE ACTIVATION OF PLC-γ MEDIATED BY A COMPLEX OF THREE CELL-TYPE-SPECIFIC ADAPTOR PROTEINS: SLP-76, LAT, AND GADS Despite the overall structural similarities between SLP-76 and BLNK, these adaptor proteins are not functionally interchangeable. BLNK fails to reconstitute the signaling defects of SLP-76-deficient Jurkat cells; correspondingly, SLP-76 does not reconstitute signaling in BLNK-deficient DT-40 cells (Wong et al., 2000; Ishiai et al., 2000). Evidence suggests that the failure of SLP-76 to function in B cells reflects a requirement for other T-cell-specific molecules, specifically LAT and Gads. Conversely, a B cell analog of LAT, though not yet identified, is likely to be required for BLNK to function. Indeed, recent studies provide compelling evidence for a model in which SLP-76, Gads, and LAT act in concert to bring about the activation of PLC-γ 1 and other downstream signaling events (Wong et al., 2000; Ishiai et al., 2000; Boerth et al., 2000). According to this model, LAT and Gads function primarily to recruit SLP-76 to specialized lipid microdomains within the plasma membrane, known as GEMs. Once recruited, SLP-76 is competent to activate PLC-γ 1 and other downstream signaling events, independently of LAT or Gads. The evidence supporting this model is briefly summarized below. Two groups have independently investigated which T-cell-specific molecules must be present, in addition to SLP-76, in order to reconstitute BCR-induced signaling events in a BLNK-deficient derivative of the DT-40 B cell line (Wong et al., 2000; Ishiai et al., 2000). Despite efficient BCR-induced tyrosine phosphorylation of SLP-76 (Ishiai et al., 2000), neither SLP-76 nor LAT alone can substitute for BLNK in mediating BCR-induced signaling events (Wong et al., 2000). Wong and colleagues found that cotransfection of SLP-76 together with LAT restored BCR-induced calcium flux, as well as activation of Erk, JNK, p38, and the NFAT transcription factor (Wong et al., 2000). Essentially, all of the signaling defects of the BLNK-deficient cells were corrected by the combination of SLP-76 and LAT. Nonetheless, the authors mention that BCR signaling events were further augmented by co-transfection of Gads, in addition to SLP-76 and LAT. To better simulate the TCR signaling pathway, Ishiai and colleagues investigated the reconstitution of BCR signaling, in BLNK- and Syk-deficient DT-40 B cells reconstituted with SLP-76 and ZAP-70 (Ishiai et al., 2000). On this background, cotransfection of both LAT and Gads was required to optimally reconstitute BCR-induced PLC-γ 1 tyrosine phosphorylation, IP3 generation, calcium flux, and C-jun N-terminal Kinase (JNK) activation (Ishiai et al., 2000). Furthermore, Gads was required to induce optimal association of SLP-76 with LAT and recruitment of SLP-76 to GEMs (Ishiai et al., 2000). Therefore, the function of SLP-76 and LAT appears to be strongly facilitated by Gads, which


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mediates the recruitment of SLP-76 to LAT. In this respect, BLNK differs from SLP-76, since BLNK appears to function well, even in B cells lacking the Grb2 family members Grb2 and Grap (Ishiai et al., 2000). Given the localization of LAT to the GEM fraction of the membrane (Zhang et al., 1998b), several groups have hypothesized that the major role of LAT and Gads is to recruit SLP-76 to the GEM fraction. Evidence for this hypothesis has been obtained, both in the B cell reconstitution system, described above, and in T cells (Boerth et al., 2000; Ishiai et al., 2000). TCR stimulation induces translocation of a fraction of SLP-76 to the GEM fraction, an event that depends on the Gads-binding site of SLP-76 (Boerth et al., 2000). To probe the functional importance of this event, Boerth and colleagues forced recruitment of SLP-76 to the GEM fraction by expressing it as a chimera, fused to the extracellular and transmembrane domain of LAT (Boerth et al., 2000). The chimera included the LAT palmitoylation sites, which target LAT to the GEM fraction, but lacked all of the LAT tyrosine phosphorylation sites. Incredibly, this LAT/SLP-76 chimera reconstituted TCR signaling events in the LAT-deficient Jurkat-derived cell line, J.CaM2 (Boerth et al., 2000). Furthermore, the Gads binding site, which is normally required for SLP-76 function (Yablonski et al., 2000), was not absolutely required for rescue of NFAT, PLC-γ 1 phosphorylation, or Erk activation in the context of the chimera (Boerth et al., 2000). These results demonstrate that LAT and Gads are dispensable for TCR signaling, if SLP-76 is recruited to the GEM fraction by other means. The above findings raise important mechanistic questions. An accepted paradigm of TCR signaling is that recruitment of PLC-γ 1 to LAT is required for its activation. Indeed, the N-terminal SH2 domain of PLC-γ 1, which mediates its association with LAT, is required for TCR-induced tyrosine phosphorylation of PLC-γ 1 (Stoica et al., 1998). Furthermore, mutational inactivation of the single, consensus PLC-γ 1 binding site in LAT leads to its functional inactivation (Zhang et al., 2000). Given the demonstrated importance of LAT in the TCR signaling pathway, how can GEM-targeted SLP-76 activate PLC-γ 1 in the absence of LAT? While the answer to this question is not certain, our recent findings, described in Section III.B, may be relevant. We identified a binding site for the SH3 domain of PLC-γ 1, within the proline-rich domain of SLP-76, and demonstrated that this site is required for TCR-induced activation of PLCγ 1 (Yablonski et al., 2001). Taken together with the results of Boerth and of Zhang (Zhang et al., 2000; Boerth et al., 2000), the results suggest a model (Fig. 2B) in which Gads recruits SLP-76 to LAT, thereby bringing SLP-76 into the GEMs. PLC-γ 1 is recruited primarily by its interaction with LAT; in addition, the association of SLP-76 with PLC-γ 1 may stabilize their recruitment to LAT (Zhang et al., 2000). Once recruited to the GEMs, the interaction of the PLC-γ 1 SH3 domain with SLP-76 brings about the activation of PLC-γ 1. Consistent with this idea, the SH3 domain of PLC-γ 1 is required for its activation

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by the BCR (DeBell et al., 1999). While the interaction of the SH2 domains of PLC-γ 1 with LAT likely contributes to its activation, this interaction is evidently not required in the context of the chimeric, GEM-targeted SLP-76. Nonetheless, the claim that the sole function of LAT is to recruit SLP-76 to GEMs is likely to be an oversimplification. Evidence suggests that LAT is likely to have other, SLP-76-independent functions. For example, Vav tyrosine phosphorylation depends on LAT (Finco et al., 1998), but not on SLP-76 (Yablonski et al., 1998b). Similarly TCR-induced Rac1 activation depends on LAT but not on SLP-76 (Ku et al., 2001). Thus, LAT is required for SLP-76-independent signaling functions, which include mediating the recruitment, tyrosine phosphorylation and activation of Vav, and the consequent activation of Rac1. In the special case in which overexpression of a LAT/SLP-76 chimera is used to rescue J.CaM2, the interaction of SLP-76 with Vav may substitute for LAT in mediating activation of Vav and Rac1. B. THE ROLE OF TEC-FAMILY TYROSINE KINASES IN SLP-76-MEDIATED SIGNALING EVENTS Increasingly, the evidence suggests that SLP-76-type adaptors (SLP-76 and BLNK) function in concert with Tec-family tyrosine kinases to mediate the activation of PLC-γ 1 and PLC-γ 2. The Tec-family kinases that may participate in activation of PLC-γ are Btk and Tec in B cells and Itk, Rlk (also known as Txk), and Tec in T cells. Phenotypic similarities between Tec-family-deficient and SLP-family-deficient experimental systems and patients provide evidence that these two types of signaling molecules function in concert. In the DT-40 B cell system, extensive similarities have been noted between the phenotypes of Btk-deficient and BLNK-deficient B cells (Takata and Kurosaki, 1996; Ishiai et al., 1999a). Similarly, Btk-deficient XLA patients were found to exhibit defects in GPVI collagen-receptor-induced platelet aggregation, similar to the defect seen in SLP-76-deficient mouse platelets (Quek et al., 1998). Even more striking, a patient with a XLA-like illness (usually caused by a mutation in Btk) was found to harbor a homozygous splice mutation in BLNK (Minegishi et al., 1999). Finally, T cells from Itk-deficient or Itk, Rlk double-knockout mice exhibit signaling defects reminiscent of those seen in SLP-76-deficient J14 cells, including a specific reduction in TCR-induced tyrosine phosphorylation of PLC-γ 1 and impaired TCR-induced IP3 production and Erk activation (Liu et al., 1998; Schaeffer et al., 1999). The mechanisms by which Tec-family kinases cooperate with SLP-type adaptors to mediate PLC-γ activation are still incompletely understood but appear to involve the N-terminal tyrosine phosphorylation sites of SLP-76, which are required for SLP-76-mediated activation of PLC-γ 1 (Boerth et al., 2000; Yablonski et al., 2000). The requirement for Tec-family kinases to mediate tyrosine phosphorylation of PLC-γ may indicate that Tec family kinases directly phosphorylate


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PLC-γ (Hashimoto et al., 1999); alternatively, it may indicate a requirement for Tec-family kinases to phosphorylate SLP-76, which in turn is required to activate PLC-γ (Schneider et al., 2000). Either way, increasing evidence suggests that Tec-family kinases inducibly associate with the N-terminal phosphorylated tyrosine residues of SLP-76 (Su et al., 1999; Bunnell et al., 2000) and BLNK (Su et al., 1999; Hashimoto et al., 1999). Given the association of SLP-76 with the SH3 domain of PLC-γ 1 (Yablonski et al., 2001), it appears plausible that SLP-76 may bridge the interaction between Tec-family kinases and their substrate, PLC-γ (Fig. 2C). Indeed, results from a heterologous reconstitution system support this model (Hashimoto et al., 1999). C. FUTURE PROSPECTS: ADAPTORS AS SIGNALING CHECKPOINTS As described in this review, genetic and biochemical studies demonstrate the essential role played by adaptor proteins in ITAM-coupled signaling pathways. SLP-76 and LAT function at critical regulatory junctures and, most significantly, cooperate to mediate the activation of PLC-γ isozymes, an event that is essential for the activation of immune cells (Wang et al., 2000; Takata et al., 1995; Irvin et al., 2000). Further, these adaptors are essential for medically important processes, including T cell activation, anaphylactic responses, and platelet aggregation. Given their cell-type-restricted expression pattern and essential signaling functions, SLP-76 and LAT represent potentially potent and cell-type-specific targets for therapeutic intervention. In fact, nature may have taken this approach, as some studies suggest that inhibitory receptors act by triggering the selective dephosphorylation of these adaptor proteins (Binstadt et al., 1998; Valiante et al., 1996). REFERENCES Aifantis, I., Pivniouk, V. I., Gartner, F., Feinberg, J., Swat, W., Alt, F. W., von Boehmer, H., and Geha, R. S. (1999). Allelic exclusion of the T cell receptor β locus requires the SH2 domain-containing leukocyte protein (SLP)-76 adpator protein. J. Exp. Med. 190, 1093–1102. Asada, H., Ishii, N., Sasaki, Y., Endo, K., Kasai, H., Tanaka, N., Takeshita, T., Tsuchiya, S., Konno, T., and Sugamura, K. (1999). Grf40, a novel Grb2 family member, is involved in T cell signaling through interactions with SLP-76 and LAT. J. Exp. Med. 189, 1383–1390. Binstadt, B. A., Billadeau, D. D., Jevremovic, D., Williams, B. L., Fang, N., Yi, T., Koretzky, G. A., Abraham, R. T., and Leibson, P. J. (1998). SLP-76 is a direct substrate of SHP-1 recruited to killer cell inhibitory receptors. J. Biol. Chem. 273, 27518–27523. Boerth, N. J., Sadler, J. J., Bauer, D. E., Clements, J. L., Gheith, S. M., and Koretzky, G. A. (2000). Recruitment of SLP-76 to the membrane and glycolipid-enriched membrane microdomains replaces the requirement for linker for activation of T cells in T cell receptor signaling. J. Exp. Med. 192, 1047–1058. Bonilla, F. A., Fujita, R. M., Pivniouk, V. I., Chan, A. C., and Geha, R. S. (2000). Adapter proteins SLP-76 and BLNK both are expressed by murine macrophages and are linked to signaling via Fcγ receptors I and II/III. Proc. Natl. Acad. Sci. USA 97, 1725–1730. Bourette, R. P., Arnaud, S., Myles, G. M., Blanchet, J.-P., and Rohrschneider, L. R. (1998). Mona, a

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Xenotransplantation DAVID H. SACHS, MEGAN SYKES, SIMON C. ROBSON, AND DAVID K. C. COOPER Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School, and Department of Medicine, Beth Israel Deaconess Medical Center/Harvard Medical School, Boston, Massachusetts 02129

I. Introduction

Two compelling reasons to expect that discordant xenotransplantation will indeed be possible are provided by experiments of nature, the nude mouse and the SCID (severe combined immunodeficiency) mouse. Each of these mouse strains bears a spontaneous mutation affecting the immune system. The nude mouse is born without a thymus, leading to defective T cell immunity (Manning et al., 1973). Part of the original description of this mutation involved skin grafting, and the nude mouse was found capable of accepting skin grafts across widely disparate barriers, including even the growth of chicken feathers! The SCID mouse is deficient in both T and B cell immunity due to mutation in a gene required for the rearrangements needed by both types of immune receptors (Bosma and Carroll, 1991). It has been demonstrated that highly disparate xenogeneic hematopoietic cells can repopulate SCID mice (Mosier et al., 1988; McCune et al., 1988; Kyoizumi et al., 1992), establishing that lymphohematopoietic tissue does not have an absolute species-specific requirement for engraftment. Since both results depend only on the immune defects caused by the respective mutations in these animals, they suggest that successful xenografting may be possible, at least for tissue grafts, through effective manipulation of the immune system even across discordant species barriers. In this paper, we shall concentrate on the most relevant aspects of both immunologic and nonimmunologic barriers to xenotransplantation and on the progress that has been made to date in overcoming these barriers. Special emphasis will be placed on efforts in our laboratory to explore the possibility of inducing immunologic tolerance across species barriers. II. The Need

The field of xenotransplantation provides remarkable validation of the old adage, “Necessity is the mother of invention.” The enormous success we have witnessed in clinical allotransplantation over the past two decades has paradoxically led to a new limitation to further progress in this field, by causing a shortage of available donor organs. Indeed, the insufficient supply of donor organs is one of the critical problems now facing the field of transplantation. The discrepancy 129 C 2001 by Academic Press Copyright All rights of reproduction in any form reserved. 0065-2776/01 $35.00

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between the number of patients waiting for an organ transplant and the number of organs that become available each year is steadily increasing (Cecka, 1998; Wolfe et al., 1999; Miranda and Matesanz, 1998; Keck et al., 1996; Belle et al., 1996). Thousands of people die each year while waiting for an organ. In addition, because of the severe shortage of organs, the criteria for receiving a transplant remain highly restrictive and many patients whose lives might be extended by a transplant are denied placement on the waiting list. Xenotransplantation provides a potential means for meeting this increasing need. III. History of Clinical Xenotransplantation

A. EARLY ATTEMPTS Clinical attempts at xenografting go back to the early 17th century when blood transfusions from animals to humans were carried out in England and France (Brent, 1997). In the 19th century, the list of animal tissues that were transplanted into human subjects was extensive, with skin being the most common (Gibson, 1955). Frog skin had the great advantage of not having hair, fur, or feathers. It was used extensively to cover leg ulcers and burns and possibly had some effect in allowing the natural skin to heal over the burned area while it was protected (Cooper, 1997; Cooper and Lanza, 2000). In the early part of the 20th century, several attempts were made to transplant animal kidneys into humans (Dubernard et al., 1974; Taniguchi and Cooper, 1997a,b; Cooper and Lanza, 2000), none with success, and it was not until the work of Reemtsma in the 1960s that xenotransplantation began to be developed in a more scientific manner (Reemtsma, 1991). B. KIDNEY XENOTRANSPLANTATION Reemtsma et al. (1964) transplanted a series of chimpanzee kidneys into humans at a time when dialysis was not commonly available and human organ donors were scarce. They demonstrated that acute cellular rejection of a chimpanzee kidney could be reversed by a course of increased steroid therapy. Survival of their patients ranged from 11 days to 2 months, except for one patient who survived for almost 9 months. The majority of deaths were related to rejection or infection, as was similarly the case with a series of baboon kidney xenografts carried out by Starzl et al. (1964) for which survival ranged from 19 to 60 days. C. HEART XENOTRANSPLANTATION The first heart transplant performed in a human by Hardy et al. in 1964 utilized a chimpanzee as the donor, but the heart proved too small to support the patient’s circulation. Further attempts using closely related donor species were made by Marion (1969) and Barnard et al. (1977) without significant success. After the

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introduction of cyclosporine, Bailey et al. (1985) performed a baboon heart transplant in a newborn infant in 1984, which functioned for 20 days. Attempts at pig or sheep heart transplantation in the late 1960s resulted in hyperacute rejection (HAR) before the end of the operation (Cooley et al., 1968; Ross, 1969). There has been one relatively recent and unsuccessful attempt at pig heart transplantation in a human (Czaplicki et al., 1992). D. LIVER TRANSPLANTATION Starzl and his colleagues (Starzl, 1969; Starzl et al., 1966, 1974; Giles et al., 1970) were again pioneers in the field of liver xenotransplantation, performing four chimpanzee liver transplants in humans between 1966 and 1974 with the grafts functioning from 99% and >97%, respectively (Watts et al., 2000), but, in order to maintain depletion, other therapeutic modalities need to be employed, such as inhibiting antibody production. This has proved much more difficult and is perhaps the major barrier to successful xenotransplantation facing us at the present time (see below). An alternative approach to the use of a synthetic oligosaccharide column is the intravenous (i.v.) infusion of large quantities of soluble Gal oligosaccharides. These are bound by the specific anti-Gal antibodies, thus preventing their binding to a transplanted pig organ (Good et al., 1992; Cooper, 1992; Cooper et al., 1993a, 1994; Ye et al., 1994a; Simon et al., 1998; Romano et al., 1999). However, results have been disappointing with delay of HAR, but not its prevention. The initial studies using this approach (the infusion of soluble sugars specific for the targeted antibody) were in relation to anti-A or anti-B blood group antibodies and were successful in allowing organ transplantation across the ABO barrier (Cooper et al., 1993c). The reasons why studies of discordant xenotransplantation have been so much less successful probably relate to such factors as the greater titer of anti-Gal NAbs in the circulation compared with anti-ABO antibodies and the absence of species-specific complement regulatory proteins in xenotransplantation, which provide some protection from antibody-mediated complement injury. A very recent advance appears to have overcome some of the deficiencies of the oligosaccharide haptens previously available. The infusion of albumin to which has been conjugated Gal sugar molecules provides a much more stable target to which anti-Gal NAbs can bind with more avidity. Metabolism (probably

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in the liver) and/or excretion of the albumin–Gal conjugate results in a reduction in the level of circulating antibody (Teranishi et al., manuscript in preparation). 2. Antiidiotypic Antibodies An alternative approach to using synthetic oligosaccharides is the use of antiidiotypic antibodies (AIAs) directed against idiotypes expressed on anti-Gal antibodies and also against the B lymphocyte subpopulations that bear the same idiotypes as surface receptors. Koren et al. (1996) produced AIAs to anti-Gal NAbs in mice, several of which, when incubated with human serum, had a major inhibitory effect on serum cytotoxicity towards pig PK15 cells in vitro. When a combination of two AIAs was infused i.v. into baboons, serum cytotoxicity to pig cells was reduced by approximately 90%. Recently, we have produced a polyclonal AIA by immunizing a pig with human anti-Gal antibodies (Buhler et al., 2000b). The purified final preparation contained 1 to 2% AIA. After repeated i.v. administration to a baboon (following repeated EIA of anti-Gal NAbs), a delayed return of anti-Gal antibody and reduced cytotoxicity to pig cells was observed. Furthermore, at this time point, the baboon serum was able to partially inhibit the cytotoxicity of other highly cytotoxic sera. These data suggest that the repeated administration of the AIA resulted in a change in the isotype of anti-Gal antibody that resulted in loss of cytotoxicity. Other approaches to this problem include the use of anti-IgM antibodies to inactivate circulating IgM. This has been reported to be effective for this purpose both in rodent (Soares et al., 1993) and nonhuman primate (Dehoux et al., 1999) models. Whether this approach might also deplete B or plasma cells that secrete IgM remains uncertain. 3. Concentrated Human Immunoglobulin The i.v. infusion of commercially available concentrated human immunoglobulin (IVIg) has been used successfully to reduce anti-HLA antibodies in highly sensitized patients awaiting organ transplantation. Our own observations indicate that, although repeated i.v. infusion of IVIg over several weeks was successful in reducing anti-HLA antibodies in sensitized patients, it had no effect on the level of anti-Gal NAbs (Buhler et at., 1999), presumably because the IVIg was not depleted of anti-Gal antibodies. Gautreau et al. (1995) have demonstrated that the i.v. infusion of IVIg into rats significantly delayed rejection of a guinea pig heart and that the addition of IVIg to human serum greatly reduced cytotoxicity to pig red blood cells. Magee et al. (1995) have demonstrated that the i.v. infusion of IVIg could prevent the HAR of pig organs transplanted into baboons. Different hypotheses have been proposed to explain the mechanism of action of IVIg in these experiments, including the presence of AIAs or complement inhibition.

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4. Accommodation Using plasma exchange, circulating anti-A and anti-B blood group antibodies have been depleted from potential kidney transplant patients, enabling ABOincompatible kidney allotransplantation to be performed successfully (Alexandre et al., 1987). HAR is avoided by transplantation of the organ during the period of antibody depletion. Accommodation, a state where return of antibody directed against antigens expressed on the graft vascular endothelium does not lead to rejection even in the presence of normal levels of complement, has been documented in these cases. Some in vitro studies (Dalmasso et al., 1996; Dorling et al., 1996), though not all (McKane et al., 2000), indicate that incubation of pig ECs with xenoreactive IgM or IgG prior to exposure to human complement can increase the resistance of the cells to lysis, inducing a state comparable to accommodation. In some rodent models of concordant xenotransplantation, xenografts are not rejected by AHXR and continue to function in the presence of xenoreactive antibodies and complement (Bach et al., 1997). However, accommodation has not been definitively documented in the pig-to-nonhuman primate model, and graft failure develops from AHXR. Potential mechanisms responsible for accommodation that have been suggested by Bach et al. (1991) include: (1) The antibodies that return to the circulation may be different in isotype, affinity, and/or specificity from the original and cannot initiate rejection; (2) the surface antigens on the graft ECs may change during the absence of antibody, and recognition by the returning antibodies is prevented; and (3) ECs may adapt during the return of antibody and react differently to these antibodies. During accommodation, the expression of “protective genes” has been demonstrated in the graft ECs in concordant rodents, and these are believed to induce resistance of the ECs to antibody-mediated destruction (Bach et al., 1997). If accommodation could be achieved after discordant xenotransplantation in the pig-to-baboon model, maintenance immunosuppressive treatment or, alternatively, the induction of T cell tolerance might be sufficient to prevent the cellular response and prevent rejection. If accommodation cannot be achieved, it will prove necessary to suppress anti-Gal Nab production indefinitely, induce a state of B cell tolerance, or use a Gal-deficient pig as the organ donor (if one becomes available). 5. Suppression of Production of Anti-Gal Antibodies Antibodies are produced by B lymphocytes and plasma cells. If it were possible to suppress selectively the cells responsible for the production of anti-Gal, a major step would be taken in achieving long-term xenograft acceptance. As this is not yet possible, recent and current studies are directed towards the nonspecific

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temporary killing or suppressing all B cells and/or plasma cells using various methodologies. After total body irradiation (TBI) of 300 cGy, B cell numbers recover within 2 to 3 weeks and anti-Gal antibody levels remain close to baseline (Alwayn et al., 1999). TBI, therefore, has only a minimal and transient effect on antibody production. Splenectomy has been demonstrated to prolong survival of guinea pig organs in rats (Reding et al., 1989) and of pig organs in nonhuman primates (Schmoeckel et al., 1999). White and coworkers have used numerous pharmacologic agents in their hDAF transgenic pig-to-primate heart and kidney transplant models (Schmoeckel et al., 1998, 1999; Zaidi et al., 1998; Vial et al., 2000); also, pharmacologic immunosuppressive therapy to suppress antiGal NAb production following antibody depletion by EIA was investigated extensively by Lambrigts et al. (1998b). In summary, pharmacologic agents, even in combination in relatively high dosage, have proved capable of only partially suppressing anti-Gal NAb production. AHXR has been significantly delayed, but not prevented. Furthermore, observations in patients receiving long-term pharmacologic immunosuppressive therapy after organ allotransplantation indicate that no combination of the drugs currently used leads to a significant reduction in the level of anti-Gal NAbs, suggesting little or no effect on NAb production (Gojo et al., 2000). B cells can be targeted with mAbs directed against B-cell-specific surface antigens. Several such mAbs have proven successful in reducing the mass of B cell lymphomas in in vitro, preclinical, and clinical studies. Following coating by mAb, the lymphoma cells are cleared mainly through ADCC. We have investigated whether these mAbs are effective in depleting normal (non-neoplastic) B cells and plasma cells in baboons (Basker et al., 2000; Alwayn et al., 1999). After four doses of the chimeric murine-human anti-CD20 mAb (IDEC-C2B8, Rituximab, Rituxan, IDEC Pharmaceuticals, San Diego, CA; Genentech, San Francisco, CA) at weekly intervals, no B cells could be detected by flow cytometry in the blood or BM, and there was an 80% depletion in the lymph nodes. With the addition of low-dose TBI (150 cGy) after the course of mAb treatment, B cells were depleted even in the lymph nodes for up to 3 months. However, after a course of extracorporeal immunoadsorption (EIA) carried out during this period of B cell depletion, anti-Gal IgG remained at approximately 50% of pretreatment level but anti-Gal IgM returned almost to baseline within 2 weeks. This result suggested to us that the major source of anti-Gal NAb is plasma cells rather than B cells, a conclusion supported by in vitro data from our laboratory (Alwayn et al., 2001). 6. Depletion or Inhibition of Complement Complement is another important target for therapies aimed at preventing HAR. Genetic engineering approaches to increase the protection of the pig cells

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provided by complement regulatory proteins are discussed below. We shall here review approaches for depleting or inhibiting exogenous complement. Because cobra venom factor is approximately five times more stable than C3bBb and is resistant to decay acceleration and proteolytic inactivation, its administration causes continuous activation of complement, resulting in its rapid depletion. Cobra venom factor therapy can, therefore, prolong discordant xenograft survival significantly (Moberg et al., 1971; Leventhal et al., 1993; Kobayashi et al., 1997). The addition of concomitant pharmacologic immunosuppressive therapy to suppress function of B and T cells delays graft rejection further, but eventually proves inadequate and AHXR occurs (Kobayashi et al., 1997). Human complement receptor I (CR1, C3b/C4b receptor) is a single-chain, cell-surface glycoprotein found on erythrocytes, most white blood cells, and some other cells. It is also found circulating in a soluble form in plasma at low concentrations. The interaction of CR1 with C3b and C4b can regulate complement activation through its convertase decay-accelerating factor activity and its factor I cofactor activity. A soluble form of complement receptor 1 (sCR1), which lacks the transmembrane and cytoplasmic protein domains, has been demonstrated to be a potent and selective inhibitor of both the classical and alternative complement pathways. The administration of sCR1 has prolonged cardiac graft survival in guinea-pig-to-rat and pig-to-primate models. sCR1 therapy combined with pharmacologic immunosuppression has prolonged pig cardiac xenograft survival for up to 6 weeks (Davis et al., 1996) and has been demonstrated to be of benefit even when the transplanted organ is from an hDAF pig (Soin et al., 2000). FUT-175, which is a synthetic inhibitor of serine proteases, has been demonstrated to inhibit C3a and C5a anaphylatoxin generation, and K76COOH, which is a monocarboxylic acid derivative of a fungal product, inhibits complement activity at the C5 stage. Kobayashi et al. (1996) demonstrated that neither agent proved as effective as cobra venom factor or sCR1. Evidence was provided in 1999 by Fiane et al. (1999a) that compstatin, a C3-binding peptide that inhibits complement activation by blocking C3 convertase-mediated cleavage of C3, attenuates HAR of pig organs perfused ex vivo with human blood. The same group reported a similar result with a C-1 inhibitor (Fiane et al., 1999b). Whether these agents will prove more efficient than current agents remains uncertain. 7. Prevention of the Induced Antibody Response It has been suggested that induced high-affinity anti-Gal IgG and possibly antibodies directed against new porcine (non-Gal) antigenic determinants play a major role in AHXR (Galili, 1997). It is believed that these newly synthesized antibodies are produced by a T-cell-dependent mechanism. The costimulatory pathway of CD40 and the T cell ligand CD154 (CD40L) can play a role in activation of T cells to antigen and in the activation of resting B cells by helper T cells.

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In our laboratory, the i.v. infusion of large numbers (3 × 1010/kg) of mobilized porcine peripheral blood progenitor cells into baboons that had undergone a regimen aimed to inducing tolerance led to sensitization to Gal, with a 100-fold increase in anti-Gal IgG and the development of antibody to new porcine (nonGal) determinants (Alwayn et al., 1999; Buhler et al., 2000a). The addition to the immunosuppressive regimen of an anti-CD154 mAb prevented sensitization to all pig antigens (Alwayn et al., 1999; Buhler et al., 2000a). Although, after EIA, return of anti-Gal IgM and IgG to baseline levels was observed, there was no increase in either immunoglobulin above baseline. This indicated that blockade of the CD40-CD154 pathway prevented the T-cell-dependent (induced) antibody response, but had no effect on the production of T-cell-independent NAbs. We have subsequently confirmed that anti-CD154 mAb therapy prevents the induced antibody response to a transplanted pig organ in a baboon (Buhler et al., 2001). Anti-CD154 mAb therapy may therefore have considerable potential in facilitating the development of mixed chimerism after porcine hematopoietic cell transplantation and the induction of immunological tolerance to pig antigens. Even if tolerance is not induced, anti-CD154 mAb may prolong pig organ survival in primates without the need for excessive dosages of pharmacologic immunosuppressive agents. Our studies indicate, however, that AHXR can occur in the absence of induced antibody to both Gal and non-Gal porcine epitopes (Buhler et al., 2001). Anti-Gal IgM alone may initiate either AHXR or EC activation sufficient to cause a state of disseminated intravascular coagulation, necessitating urgent graft removal (Buhler et al., 2000d). Both AHXR and disseminated intravascular coagulation occurred in the absence of evidence of IgG or complement deposition on the grafted tissues (although mild IgM deposition was documented), and with no measurable complement (by CH50 assay) and minimal levels of anti-Gal IgG in the blood. 8. Suppression of the Cellular Response Numerous immunosuppressive agents have been developed to suppress cellular immunity to allografts, and some of these are likely to have a suppressive effect on the cellular response to a discordant xenograft. However, it seems likely that the intensity of immunosuppression that will be required to avoid rejection of a discordant xenograft will be greater than that required for control of allograft rejection. To date, it has proved impossible in the pig-to-nonhuman primate model to assess the full severity of the cellular response as irreversible AHXR has prevented a clear assessment of subsequent cellular events. The fact that a cellular response that can be differentiated histopathologically from AHXR (Pino-Chavez, 2001) has been documented despite profound pharmacologic immunosuppression adds support to in vitro data indicating that the

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cellular response to a pig organ is strong. It also suggests that the currently available agents may be insufficient to suppress this response and supports the notion that successful xenotransplantation may require the induction of T cell tolerance to at least the most potent xenogeneic antigens. Until T cell tolerance can be induced reliably, new agents that profoundly deplete T cells will continue to be examined. C. GENETIC ENGINEERING Recent approaches to xenotransplantation have involved attempts to modify either the recipient or the donor, or both, through genetic engineering. Modification of the recipient has been directed toward induction of tolerance, either at the humoral or cellular level, and has utilized retroviral transduction of autologous recipient BM cells with genes encoding porcine antigens toward which tolerance is desired. Modification of the donor has involved production of transgenic pigs into which human genes have been introduced in an attempt to decrease differences relevant to the prospective primate recipient’s immune response. 1. Approaches Involving Modification of the Recipient As described below (see Section VI.E), successful engraftment of allogeneic or xenogeneic BM cells carries with it the induction of tolerance to any other tissues or organs from the same donor. However, the potential also exists to induce tolerance to the products of the most relevant genes determining histocompatibility by transferring those genes into autologous BM cells and then reconstituting with these modified cells rather than with allogeneic or xenogeneic cells (Sachs et al., 1993). This approach was demonstrated to induce prolonged acceptance of skin grafts in mice across a strain combination differing only by a single class I locus (Sykes et al., 1993; Fraser et al., 1995) and has recently been demonstrated to permit the induction of tolerance across a full MHC barrier in pigs following transfer of the allogeneic class II genes (Emery et al., 1997; Sonntag et al., 2000). In the latter case, reconstitution of recipients with autologous BM transduced with an allogeneic class II c-DNA permitted successful renal transplantation following a short course of cyclosporine treatment in fully mismatched, partially inbred miniature swine (Sonntag et al., 2000). This same immunosuppressive treatment had previously been shown to lead to tolerance across a class I mismatch but not across a full MHC mismatch, indicating that differences at class II loci were of overwhelming importance in determining the fate of such vascularized allografts (Pescovitz et al., 1984; Rosengard et al., 1992). a. Transfer of MHC Genes. Following up on these results, Ierino et al. have demonstrated that similar transfer of porcine class II genes into BM of nonhuman primates was capable of modifying the response of those primates to subsequent porcine renal xenografts (Ierino et al., 1999). Following reconstitution of these

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primates with transduced autologous BM cells, the transgene was detected by polymerase chain reaction (PCR) in mononuclear cells for more than 12 weeks in five of the six animals. Unlike the allogeneic studies, in which kidneys expressing the same class II were accepted longterm, renal xenografts from pigs of the same genotype as the class II transgenes succumbed to antibody-mediated rejection. However, in contrast to control animals, in which rejection of porcine kidneys led to the production of induced, T-cell-dependent antibodies to new porcine antigenic determinants in addition to Gal, two of these animals developed no such new antibodies (Ierino et al., 1999). These results suggest that the gene transfer led to diminution or possibly tolerance of the T cell response to at least some of the most important porcine xenoantigens. b. Transfer of the Gene for Gal. While retroviral transfer of MHC genes into autologous BM appears to be capable of inducing tolerance at the T cell level, it is possible that a similar approach may be able to inhibit the anti-Gal antibody response at the B cell level. Thus, since it is presumably the absence of the GalT gene in Old World primates and humans that leads to their production of anti-Gal NAbs (Galili et al., 1999), introduction of the gene encoding the GalT enzyme into the BM of these animals might inhibit their production (Bracy et al., 1998). To test this hypothesis, Iacomini and colleagues utilized the Galknockout mouse, a strain of mice in which the GalT gene has been disrupted by homologous recombination (Cooper and Thall, 1997) and which, therefore, like Old World monkeys, makes anti-Gal NAbs (LaTemple and Galili, 1998). They introduced the GalT c-DNA gene through retroviral transduction of Galknockout BM and then reconstituted irradiated Gal-knockout mice with the transduced BM. Unlike unmodified Gal-knockout animals and control animals, in which the BM was transduced with a control gene, the GalT-reconstituted mice lost their anti-Gal NAbs and showed no return of these antibodies after recovery from the conditioning (Bracy et al., 1998). In addition, they made no detectable anti-Gal antibodies in response to a subsequent challenge with pig cells (Bracy and Iacomini, 2000). This gene transfer approach is now being attempted in baboons, conditioned similarly to those studied previously for porcine class II gene transfer (Ierino et al., 1999). Although initial attempts have not been successful (Iacomini et al., unpublished), it is likely that this is because baboons have much higher titers of anti-Gal NAbs than do Gal-knockout mice. Such antibodies undoubtedly coated the injected, transduced BM cells and may have led to their destruction before they had a chance to engraft and affect the B cell response. Current research is aimed at obtaining lower levels of anti-Gal NAbs in the post-reconstitution period through repeated EIA, as well as better levels of gene transduction, resulting in a longer period of GalT expression, all with the aim of improving chances of engraftment of GalT-transduced BM cells.

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2. Approaches Involving Modification of the Donor As mentioned above, the breeding characteristics of swine make it possible to produce genetically modified animals in which human genes can be introduced or swine genes deleted in order to diminish the primate immune response to xenografts. a. Modification of Complement-Regulatory Proteins. Among the genes that have so far been introduced into pigs for this purpose are several speciesrestricted, membrane-bound, complement-inhibitory proteins, including human decay accelerating factor (hDAF, CD55), human membrane cofactor protein (hMCP, CD46), and human CD59. Each of these proteins, when expressed at the level of the EC surface, is thought to have the capacity to decrease the activation of human complement by antibodies of the same (but not discordant) species (Hansch et al., 1981). Thus, in a nonhuman primate, these genes are thought to down-regulate complement activation following the binding of primate antibodies (especially NAbs) to the endothelium of the vessels of a transplanted pig organ. Pigs expressing the hDAF gene, so-called hDAF pigs, have been studied most extensively as xenograft organ donors and have been shown to provide a means of avoiding HAR (reviewed by Lambrigts et al., 1998a; Cozzi et al., 1997; Schmoeckel et al., 1998; Zaidi et al., 1998). However, prolongation of organ survival by this genetic manipulation is far from permanent and, on its own, hDAF appears to protect the pig organs for only a few days (Cozzi et al., 1997; Lambrigts et al., 1998a). This result is consistent with the fact that the effect of hDAF and the other complement-regulatory proteins must be only relative, since even in allogeneic systems, high levels of human serum antibody can activate human complement, causing HAR, such as may occur in ABO-incompatible allografts (Cooper, 1990; Cooper et al., 1993). Recent reports of transgenic pigs expressing more than one of the complement-regulatory proteins have likewise prolonged survival of the modified organs by a few days (Lambrigts et al., 1998a). The addition of intensive pharmacologic immunosuppressive therapy to the recipient delays rejection further, sometimes for as long as 2 to 3 months, though median graft survival is approximately one month (Lambrigts et al., 1998a). Recently, data have been published indicating that an overexpression of porcine complement-regulatory protein may be as efficient in protecting the organ as the introduction of a human complement-regulatory protein (which results, of course, in co-expression of pig and human proteins). Morgan’s group in Cardiff has carried out extensive in vitro studies that indicate that the introduction of the porcine analog of CD59 protects cells equally to the expression of human CD59 (van den Berg and Morgan, 1994; Hinchcliffe et al., 1998). Fisicaro et al. (2000) have confirmed this to be protective in vivo in the wild-type-to-Galknockout mouse model. It may therefore be that protection is related to the quantity of complement-regulatory protein expressed on the ECs rather than

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FIG. 1. Natural biosynthetic pathway for synthesis of the Gal epitope (Galα1-3Gal), and methods by which this can be modified by transgenic techniques: (A) Galactose is added to the N-acetyllactosamine (Galβ1-4GlcNAc) substrate by the α1,3galactosyltransferase enzyme to form Galα1-3Gal. Both (B) gene inactivation by homologous recombination and (C) transgenic approaches to inhibit/breakdown RNA could prevent production of α1,3galactosyltransferase (GalT) and eliminate Galα1-3Gal. (D) Potentially specific α1,3galactosyltransferase inhibitors could act upon the enzyme. (E) Galβ1-4GlcNAc can also form the substrate for the H(O) histo-blood group epitope when the gene for the α1,2 fucosyltransferase enzyme is transgenically introduced. (F) Furthermore, cleavage of Galα1-3Gal occurs when the gene for the α-galactosidase enzyme is introduced. Modification of the natural pathway has been demonstrated in cells in culture and in Gal-knockout (Gal-negative) mice by transgenic techniques but has not yet been successfully achieved in pigs. (Modified from Sandrin et al., 1997.)

to the “quality.” Species-restriction of the proteins may be less important than previously suspected. b. Modification of Antigen Expression. (Competitive Glycosylation). Genetic engineering approaches are also being attempted to decrease the Gal antigen expression on pig ECs. One approach involves the introduction of a gene for an enzyme that will compete with GalT for the underlying substrate, N-acetyllactosamine (Table II, Fig. 1). Suggested candidates for such genes include those for a sialic acid (α2,3 or α2,6 neuraminic acid) or that for the H (O) histo-blood group antigen (α1,2 fucosyltransferase or H-transferase) (Cooper et al., 1993). As both sialic acid and the H histo-blood group antigen are, or can be, expressed on the human vascular endothelium (Table II), no NAbs are present in humans against these epitopes. Indeed, the presence of the H antigen constitutes the human universal allograft donor. The theoretical basis for this approach is that because, for example, a fucosyl residue is found as a terminal sugar on the vascular endothelium in primates, the resulting cell surface molecules should not be targets of primate NAbs. Pigs do, in fact, have the gene

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for H-transferase and express H oligosaccharide epitopes, not on the vascular endothelium, but in certain other tissues (Oriol et al., 1993). It is essential, therefore, that any H-transferase gene introduced should function at the desired site. In mice, although this genetic manipulation has decreased the level of Gal on EC surfaces by as much as 90%, this has not proved sufficient to prevent antibodymediated rejection (McKenzie et al., 2000). The few attempts to introduce the H-transferase gene into pigs have largely been unsuccessful, and expression of the H antigen has been low (Koike et al., 1996; Cowan et al., 2000). It seems clear, therefore, that, unless H epitopes replace Gal epitopes completely, the number of Gal epitopes remaining still makes the transplanted pig organ susceptible to HAR. To try to resolve this problem, it has been suggested that a further gene should be introduced, namely that for the enzyme αgalactosidase (Cooper et al., 1996; Sandrin et al., 1997). αGalactosidase has the opposite effect of GalT in that it removes the terminal Gal molecule rather than adding it. Cell culture studies by Sandrin et al. (1997) indicate that the combined presence of αgalactosidase and H-transferase results in a great reduction in Gal expression. It would appear that, if H-transferase is not successful in competing for substrate with GalT, some Gal epitopes that remain are removed by the αgalactosidase. It will prove more difficult, however, to replace or remove Gal epitopes in a pig than from cells in culture. c. Gal-Knockout Technology. An exciting possibility for eliminating Gal altogether from the cell surfaces of pigs has been proposed through the use of “knockout” technology. Until recently, this approach was thought to be feasible only in the mouse, since mice are the only species in which true embryonic stem cell lines are available. Unlike transgenic animals, in which a new gene is introduced into the genome, specific elimination of a gene requires targeted homologous recombination. This is a process by which a piece of DNA similar enough to recombine with the targeted gene is introduced into proliferating cells. The introduced DNA carries a mutation, however, that makes the resulting gene nonfunctional, in essence “knocking out” the targeted gene. While integration of a transgene somewhere in the genome is a relatively frequent event after DNA is injected into a single fertilized egg, homologous recombination is very infrequent. Therefore, knockout technology requires introduction of the DNA into large numbers of proliferating cells, along with a selectable marker that can help to select those rare cells in which the desired homologous recombination event has occurred. Embryonic stem cells provide a source of such proliferating cells and have enabled knockout technology to produce numerous valuable knockout mice (Bronson and Smithies, 1994), such as the Gal-knockout mouse. In species such as the pig, however, for which no true embryonic stem cells are available, it is not feasible to inject enough fertilized eggs to ever produce a knockout by the same technology.

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d. Nuclear Transfer. What has changed the outlook for knockout technology in large animals has been the report of successful cloning of sheep (Wilmut et al., 1998; Kuhholzer and Prather, 2000) and, more recently, of pigs (Betthauser et al., 2000; Polejaeva et al., 2000; Onishi et al., 2000) through a process called nuclear transfer. This procedure involves replacing the nucleus of a fertilized egg with a nucleus taken from the cell of an embryo or adult animal. Since these cells can be grown in tissue culture prior to removing the nucleus for this transfer, the cultured cells can potentially be subjected to the same kind of selective technology that has been used to effect homologous recombination in mouse embryonic stem cells. Thus, it is theoretically possible that a knockout of the GalT gene in a pig could be produced by this procedure. However, to date, no reports of cloning of large animals have utilized adult cells that have been extensively cultured in vitro prior to transfer. It therefore remains to be seen whether or not the number of generations in culture necessary to produce a knockout cell line will be consistent with production of a viable pig after nuclear transfer. The end product, however, would be so valuable to the field of xenotransplantation that it is likely to be achieved if it is at all possible. On the other hand, Galili’s group (Tanemura et al., 2000a,b) has drawn attention to the fact that Gal expression in the pig is up to 500 times higher than in the mouse and may therefore play an essential physiologic role. They have questioned, therefore, whether a Gal-negative pig will be viable. However, humans and Old World primates have survived after having lost expression of Gal. Furthermore, there are human subjects who do not express the “corresponding” ABH antigens either (Table II), the so-called “Bombay” histo-blood type, who appear to be clinically well in all respects. D. APPROACHES TO PREVENT DISORDERED ACTIVATION OF COAGULATION AND PLATELETS IN XENOGRAFTS Combinations of select anti-platelet modalities with parallel approaches to control thrombin generation (e.g., with heparin and more novel therapies) would be an optimal first step to regulating the thrombotic components of AHXR. The challenge remains to either specifically target these interventions to the xenograft or develop well-tolerated systemic pharmacological approaches. Thrombin inhibition in an ex vivo pig-to-human xenoperfusion model prolonged survival (Robson et al., 1996b). Thrombin inhibition following decomplementation in normal and intrinsically C6-deficient PVG recipient rats significantly improved graft survival in HAR, but not AHXR (Lesnikoski et al., 1997). Overexpression of thrombomodulin and tissue factor pathway inhibitor by retroviral and/or adenoviral vectors (Kopp et al., 1998a,b) may have therapeutic utility in the genetic modification of both porcine xenografts and BM-derived cells (Robson et al., unpublished).

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1. Platelet Inhibitors and Thromboregulatory Factors Inhibition of platelet aggregation by treatment of xenograft recipients with antagonists to the platelet fibrinogen receptor, GPIIbIIIa (Candinas et al., 1996b; Robson et al., 1996a), by the use of P-selectin or platelet activating factor (PAF) antagonists (Coughlan et al., 1993; Makowka et al., 1990; Ohair et al., 1993) or by administration of a soluble ATPDase (Koyamada et al., 1996) has been generally shown to prolong graft survival in several discordant xenotransplantation models. 2. Inhibitors of vWF-GPIb Prostacyclin and nitroprusside, vasodilator compounds that enhance cellular and platelet cAMP (cyclic adenosine monophosphate) and cGMP (cyclic guanosine monophosphate) concentrations, respectively, can cause a drastic inhibition of vWF-induced platelet responses (Francesconi et al., 1996). vWF-deficient pigs have been used as renal donors to xenoantibody-depleted baboons without effect on graft survival or on the microvascular thrombosis associated with AHXR (Meyer et al., 1999). However, the effect on platelet counts was not studied and circulating C activity was not inhibited. 3. Inhibitors of Fibrinogen-GPIIbIIIa The integrin GPIIbIIIa is known to be crucial to the formation of platelet aggregates and potentiates adhesion to subendothelial matrices via fibrin(ogen), vWF, and vitronectin. The combination of the GPIIbIIIa antagonist and cobra venom factor results in a significant decrease in intragraft platelet aggregation, P-selectin expression, and leukocyte infiltration (Candinas et al., 1996b). a. Thromboregulatory Vascular ATPDases. CD39 is an integral membrane protein of ECs that degrades adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to AMP with ultimate generation of adenosine. These nucleotides are released from damaged EC and promote platelet and EC activation. Adenosine has potent anti-platelet aggregatory and anti-inflammatory actions. This use of CD39 as an anti-platelet agent acting at an earlier phase of platelet activation would have certain benefits over the GPIIbIIIa antagonists that operate at a final common pathway and do not preclude release of platelet mediators (Robson et al., 2000). Additional anti-inflammatory effects and the maintenance of vascular integrity by CD39, a natural vascular-associated thromboregulatory factor, are other putative theoretical benefits. Finally, targeted local expression of CD39 may have advantages to systemic administration of soluble NTPDase derivative. However, both options remain to be explored. In summary, while thrombosis may be a consequence of the immunological response to the xenograft and validly viewed as a component of rejection, it may also arise as a consequence of molecular incompatibilities of nonimmunological origin. These molecular barriers may persist even if the xenogeneic immune

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response is completely inhibited and xenoantibodies fully controlled. The presence of an incompatible thrombophilic vasculature within the xenograft may compromise long-term survival and function. Hence, additional genetic engineering of donor animals with expression of human anticoagulant- (e.g., tissue factor pathway inhibitor and thrombomodulin) and platelet-regulatory factors (e.g., CD39) may be required. E. TOLERANCE In light of the powerful immune responses to xenografts discussed above, it is felt by many investigators that the induction of immunological tolerance will ultimately be essential to the success of xenotransplantation in humans (Dorling and Lechler, 1998; Auchincloss, 1995). Considering the many cell types of the innate and acquired immune systems that can participate in xenograft rejection, it may be necessary to induce tolerance for all of these pathways if xenotransplantation is to be successful. These pathways include those mediated by T cells, antibody-producing B/plasma cells, NK cells, and macrophages. Alternatively, a tolerance approach for some of these pathways may be combined with selective immunosuppression for others. In addition, activation of some of these pathways may depend on activation of others, in which case it would only be necessary to tolerize the cell type responsible for initiating the rejection pathway involved. Thus far, three approaches to inducing T cell tolerance across xenograft barriers have been attempted. These included induction of mixed chimerism, gene therapy of autologous hematopoietic stem cells, and thymic transplantation. In addition, donor antigen infusion with NK-cell-depletion and B-cell-suppressive therapy is an approach to tolerizing the non-T-cell innate immune system. Genetic engineering of autologous BM has been discussed above and will therefore not be discussed further here. The others are discussed below in the context of the pathways of rejection that they are capable of controlling. 1. Induction of Mixed Hematopoietic Cell Chimerism The word “chimerism” is used in this review to describe the existence in a recipient of hematopoietic elements from a donor that is allogeneic or xenogeneic to the recipient. In our terminology, “microchimerism” refers to chimrism that is not measurable by flow cytometry (which usually has a detection limit in the range of 0.1 to 1%) and requires sensitive techniques, such as PCR, for its detection. “Mixed chimerism” refers to a state in which donor and host hematopoietic elements of multiple lineages coexist at levels detectable by flow cytometry. “Full chimerism”, on the other hand, is a state in which essentially all hematopoietic elements are derived from a donor stem cell inoculum. The pioneering work of Owen, Medawar, and others, beginning 50 years ago, led to the observation that hematopoietic chimerism can be associated with a state of donor-specific tolerance (reviewed in Charlton et al., 1994).

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When pre-existing peripheral T cells are adequately eliminated and allogeneic or xenogeneic BM engraftment is achieved, tolerance to the most immunogenic allografts, such as fully MHC-mismatched skin grafts and small bowel grafts, is reliably attained (Sharabi and Sachs, 1989; Orloff et al., 1994). The challenge in bringing hematopoietic cell transplantation to the clinic for the purpose of tolerance induction has been to develop a conditioning regimen that permits allogeneic BM engraftment in immunocompetent recipients without producing inordinate toxicity. In recent years, several advances have brought the mixed chimerism approach closer to clinical application for this purppse. An advance in this direction was the demonstration that fully MHC-mismatched BM engraftment and specific tolerance could be achieved by pretreating recipients with depleting doses of anti-CD4 and anti-CD8 mAbs along with a sublethal dose (6 Gy) of total body irradiation (TBI) (Cobbold et al., 1986). Similar outcomes could be achieved even with a minimally myelosuppressive (Tomita et al., 1994a) dose of TBI (3 Gy), if additional selective irradiation was given to the thymic area (referred to as thymic irradiation, TI) (Sharabi and Sachs, 1989). More recently, a number of modifications have led to the development of even less toxic conditioning regimens. TI can be replaced by a second injection of depleting anti-T-cell mAbs (Tomita et al., 1996a,b), TBI can be omitted if very high BM doses are given (Sykes et al., 1997), both TI and host T-cell-depleting mAbs can be excluded if single injections of each of two costimulatory blockers are given (Wekerle et al., 1998), and TI can be replaced by a single injection of one co-stimulatory blocker (Wekerle et al., 1999). Lasting mixed chimerism and donor-specific tolerance have been achieved with a regimen that requires no host pre-conditioning, by giving a high dose of fully MHC-mismatched donor BM followed by a single injection of each of two costimulatory blockers (Wekerle et al., 2000). When allogeneic stem cells engraft, they provide a life-long source of progenitor cells that seed the thymus, giving rise to T cells and to cells that mediate clonal deletion of developing T cells that recognize their antigens (Ardavin et al., 1993). In the thymus, self-reactive T cells are clonally deleted during their maturation when they recognize antigens expressed on cells of hematopoietic origin (Brocker et al., 1997; Inaba et al., 1991). In mixed chimeras, hematopoietic cells from both the recipient and the donor locate to the thymus and hence delete both host-reactive and donor-reactive T cells, resulting in a peripheral T cell repertoire that is tolerant towards the donor and the host (Tomita et al., 1994b, 1996a; Wekerle et al., 1998, 1999; Manilay et al., 1998a). Deletional tolerance remains as long as chimerism persists and requires the continuous presence of donor antigen in the thymus (Khan et al., 1996). Allogeneic chimerism has generally been more difficult to induce in large animals than in rodents, perhaps, in large part, due to differences in the types and amounts of irradiation and reagents used in the different species. For example,

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T-cell-depleting antibodies have not been used in primates that achieve the level of T cell depletion that has been possible in mouse models of mixed chimerism. Stable mixed chimerism has recently been achieved in dog leukocyte antigen (DLA; i.e., MHC)-identical dogs using a nonmyeloablative protocol involving a limited course of pharmacological immunosuppression after BM transplantation (Storb et al., 1997). However, success with this approach has yet to be demonstrated across MHC barriers. Recent results in a porcine model have applied approaches developed in mice (Sharabi and Sachs, 1989; Sykes et al., 1997) to achieve mixed chimerism. In MHC-identical pigs receiving a depleting anti-CD3 immunotoxin, 3-Gy TBI and 7-Gy TI with conventional stem cell doses (Huang et al., 2000) and in fully MHC-mismatched recipients of high-dose stem cell transplants (Fuchimoto et al., 2000), durable mixed chimerism has been achieved. In a non-human primate model, cynomolgus monkeys were conditioned with anti-thymocyte globulin (ATG), fractionated TBI (3 Gy), local TI (7 Gy), and splenectomy before transplantation of MHC-mismatched BM and kidney. Cyclosporine (CyA) was given for four weeks after BM transplatation (Kawai et al., 1995; Kimikawa et al., 1997) with no further immunosuppression. Transient multilineage chimerism has been achieved in association with long-term stable graft function for over 6 years (T. Kawai, personal communication). The goal of using nonmyeloablative BM transplantation to induce allograft tolerance has been recently realized in a multiple myeloma patient who received a combined kidney and BM transplant from the same HLA-identical donor (Spitzer et al., 1999). The patient has now accepted her donor kidney without immunosuppressive therapy for more than 2 years. Mixed chimerism has also been shown to effectively induce xenograft tolerance. Mixed chimerism was originally shown to successfully tolerize xenoreactive T cells in lethally irradiated mice reconstituted with a relatively high dose of T-cell-depleted rat BM cells in combination with a much lower dose of T-celldepleted host-type BM (Ildstad and Sachs, 1984; Ildstad et al., 1985a,b; Ildstad and Wren, 1991). Analysis of deletion of Vβ that could only recognize endogenous superantigens presented by donor rat and not recipient MHC suggested that tolerance occurred through an intrathymic deletion mechanism (Ildstad et al., 1992). More recently, a less toxic, more clinically relevant, nonmyeloablative approach to inducing mixed chimerism in the rat-to-mouse species combination was developed (Sharabi et al., 1990). This regimen was based on a similar approach that was successful in an allogeneic combination, but some additional host manipulations were required in the xenogeneic combination. Whereas BM engraftment in the allogeneic model requires anti-CD4 and anti-CD8 mAb treatment of the host, along with thymic irradiation and 300-cGy TBI, host pretreatment with mAbs against NK1.1 and Thy1 must be added in order to reliably

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FIG. 2. Method for producing mixed xenogeneic (rat-to-mouse) chimeras using a nonmyeloablative conditioning regimen. Anti-mouse CD4 mAb is GK1.5 {453}, anti-mouse CD8 mAb is 2.43 {455}, anti-Thy1.2 mAb is 30H-12 {202}, and anti-NK1.1 mAb is PK136 {232}. Rat marrow was T cell depleted as described {839}.

achieve engraftment and tolerance in the xenogeneic combination (Sharabi et al., 1990). This treatment protocol is outlined in Fig. 2. Thymic irradiation can be omitted from the protocol if two injections of the mAb cocktail are administered prior to BM transplantation (Tomita et al., 1994c). T cell depletion of the donor rat is essential in order to avoid graft-vs.-host disease (GVHD) in this model. Central deletion of donor-reactive thymocytes is a major mechanism of host-vs.graft tolerance in these animals (Tomita et al., 1994c), and this correlates with the presence in the thymus of donor class IIhigh cells with a dendritic cell or macrophage-like morphology (Nikolic et al., 1998). T cell tolerance is systemic, as evidenced by specific prolongation of donor skin and primarily vascularized heart grafts (Sharabi et al., 1990; Ohdan et al., 2000b), donor-specific unresponsiveness in mixed lymphocyte reactions and cell-mediated lympholysis assays (Nikolic et al., 1998), and acceptance of repeat donor BM grafts given to chimeras treated with only a low dose of TBI (Lee et al., 1995). Although chimerism is not stable in these mixed xenogeneic chimeras (Sharabi et al., 1990), as it is in mixed allogeneic chimeras (Sharabi and Sachs, 1989), the gradual decline in donor chimerism is due to a competitive advantage of host hematopoiesis over xenogeneic donor hematopoiesis, not to incomplete tolerance (Lee et al., 1995; Gritsch and Sykes, 1996b). The basis for this xenogeneic disadvantage is discussed further below. A similar approach has been used successfully to induce mixed chimerism and tolerance in a concordant non-human primate species combination (Bartholemew et al., 1997; Ko et al., 1998). We have attempted to achieve similar outcomes in a discordant pig-to-primate model and have encountered a number of obstacles, both immunologic and nonimmunologic, as described below.

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Mixed chimerism also has the potential to tolerize natural antibody-producing cells. Preformed NAb constitute a significant barrier to xenogeneic BM engraftment in the concordant rat-to-mouse BM transplantation model (Aksentijevich et al., 1991) and they are likely to do so in discordant xenogeneic systems, as well (Yang et al., 1998). Mixed chimerism was shown in the nonmyeloablative rat-to-mouse BM transplantation model (Fig. 2) to be associated with tolerance of cells that produce anti-rat natural antibody (Aksentijevich et al., 1992; Lee et al., 1994a), in addition to T cell tolerance. Importantly, it has recently been demonstrated that B cell tolerance for the important Gal antigen recognized by human natural antibody on porcine donors occurs following the induction of mixed chimerism in αGT-knockout hosts receiving Gal-expressing BM from wild-type allogeneic donors (Yang et al., 1998; Ohdan et al., 1999). This tolerization applies to both pre-existing B cells (Ohdan et al., 1999) that are not eliminated by the conditioning regimen (Ohdan et al., 2000a), and to B cells developing after the BM transplant (Yang et al., 1998; Ohdan et al., 1999). Antibody-mediated rejection of Gal-positive cardiac allografts is prevented, as is cellular rejection, in these mixed chimeras (Ohdan et al., 1999). More recently, it has been shown that the anti-Gal antibody barrier to xenogeneic BM engraftment can be overcome by administering higher than usual rat BM doses to αGT-knockout mice and that the induction of mixed xenogeneic chimerism in this manner simultaneously prevents HAR, AHXR, and cell-mediated rejection of primarily vascularized cardiac xenografts (Ohdan et al., 2001). Using a fluorochrome-labeled Gal polymer combined with ELISPOT assays, it has been possible to identify the phenotype of anti-Gal-producing cell populations in Gal-knockout mice and to assess whether or not they are present in tolerant mice. These studies show that anti-Gal IgM natural antibody is produced primarily by a CD5-negative and Mac1-negative, but otherwise B1b-like, B cell population in the spleen. Although anti-Gal surface Ig-bearing cells are present in the peritoneal cavity in larger numbers than in the spleen, the peritoneal cavity B cells do not produce antibody unless stimulated with LPS for several days in vitro (Ohdan et al., 2000b). Mixed allogeneic and xenogeneic chimeras produced in Gal-knockout mice show an absence of anti-Gal surface Ig-bearing cells in the spleen, along with tolerance in ELISPOT assays (Ohdan et al., 1999, 2001). However, under certain conditions, anti-Gal surface Ig-bearing cells may persist in the peritoneal cavity (Ohdan et al., 1999). Together, these data suggest that mixed chimerism leads to tolerization of newly developing NAb-producing cells in the BM, either by receptor editing or clonal deletion, but that pre-existing anti-Gal-producing cells may be tolerized by other mechanisms. We are now actively investigating the mechanism of tolerization of pre-existing anti-Gal-producing cells, as well as the interrelationship between the non-antibody-producing peritoneal cavity anti-Gal surface

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Ig-bearing cells, which have a more typical B1 cell (Mac-1+ ) phenotype, and the Mac1– actively NAb-producing cell population in the spleen. It remains to be determined whether or not primate anti-Gal-producing cells have characteristics similar to those in mice. Because macrochimerism of more than a few days’ duration has not yet been achieved in a pig-to-primate species combination (see below), it has not been possible to assess the induction of B cell tolerance with the mixed chimerism approach in this model. It is not yet known whether or not tolerance of donor-reactive recipient NK cells develops in mixed xenogeneic chimeras. However, chronic host NK depletion did not significantly delay the gradual loss of rat chimerism in mixed chimeras prepared with the regimen in Fig. 2 (Lee et al., 1995), suggesting that a failure of host NK cell tolerance to the donor is not the predominant reason for the loss of chimerism that occurs in animals that are tolerant of their donors at the T and B cell levels (Lee et al., 1995; Aksentijevich et al., 1992; Nikolic et al., 1998). Somewhat conflicting data on the issue of NK cell tolerance induced by hematopoietic chimerism are beginning to emerge in allogeneic BM transplantation models. It seemed possible that regulation of inhibitory receptors, such as the Ly-49 receptors, which recognize specific MHC class I ligands, could play a role in inducing tolerance of NK cells in chimeric mice. Individual NK cells in mixed chimeras might be expected to be tolerant of both donor and host antigens, and this might be associated with alterations in the expression of Ly-49 receptors. When this question was examined, the presence of the MHC ligand for a given Ly-49 receptor was shown to reduce the expression levels of that receptor in mixed chimeras on cells of the mouse strain that lacked the ligand (Sykes et al., 1993; Manilay et al., 1998b, 1999). For example, B6 NK cells, regardless of whether they were of donor (B6 → BALB/c mixed chimeras) or recipient (BALB/c → B6) origin, showed reduced levels of expression of Ly49A and Ly49G2 compared to control B6 NK cells whenever BALB/c cells, which express Dd and Dd/Ld ligands for Ly-49A and Ly-49G2, respectively, were present (Sykes et al., 1993; Manilay et al., 1998b). Furthermore, in mixed chimeras with varying levels of chimerism, a quantitative relationship between the level of down-regulation of Ly-49A, Ly-49C, and Ly-49G2 and the number of hematopoietic cells expressing their MHC ligands was observed (Manilay et al., 1999). Surprisingly, however, in vitro functional assays to test whether host and donor NK cells were tolerant to each other showed that donor NK cells from mixed chimeras efficiently lysed host targets, and host NK cells were not tolerant to the BALB/c donor (Manilay et al., 1998b). In all of these studies, however, NK cells from mixed chimeras did not kill syngeneic targets to any greater extent than was observed for NK cells from nontransplanted controls or normal mice of the same strain. Thus, tolerance to “self”, as defined by the MHC antigens expressed by the

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NK cell itself, appears to develop normally in the mixed chimeric environment. The lack of tolerance to non-self MHC suggests that an inhibitory receptor for allogeneic MHC may not be expressed on all NK cells in mixed chimeras. In contrast to these in vitro findings, in vivo studies from several groups have demonstrated NK cell tolerance in irradiation chimera models (Wu and Raulet, 1997) and in mice with mosaic expression of an MHC transgene (Tg) (Johansson et al., 1997; Rosenzweig et al., 1996), and these have not always been consistent with in vitro data showing a lack of such tolerance (Chadwick and Miller, 1992; Johansson et al., 1997). In these and our own in vitro assays, NK cells are activated by culture in IL-2, whereas NK cells were not intentionally activated in any of the in vivo assays. A tolerance mechanism that operates at the level of regulation of NK cell activation in vivo might explain the in vivo tolerance observed in models in which tolerance is not preserved in in vitro assays, when exogenous IL-2 is added. We have initiated in vivo studies to determine whether or not this mechanism of tolerance is operative in our mixed chimera model. Clear evidence of in vivo tolerance to hematopoietic cells of the B6 recipient and BALB/c donor strains was obtained in BALB/c → B6 mixed chimeras (Y. Zhao et al., manuscript in preparation). Following lethal irradiation, these chimeras did not resist engraftment of fresh B6 or BALB/c BM but showed normal resistance to the engraftment of β2m–/– BM cells. These data, which are consistent with the observation that chimerism tends to remain stable over the life of these mixed chimeras, suggest that donor and host NK cells, while not tolerant to one another by the same mechanisms as that involved in tolerance to their own MHC antigens, are nevertheless functionally tolerant of one another in vivo. Similar studies have yet to be performed in a xenogeneic BM transplantation model. Given the lack of understanding of the mechanisms of NK cell tolerance and the molecular interactions involved, the outcome of studies to address this issue cannot be predicted. 2. Physiologic Barriers to Allogeneic and Xenogeneic Hematopoietic Cell Engraftment The concept that “space” must be created in the hematopoietic compartment in order to allow donor stem cells to engraft has long been widely accepted. In a syngeneic BM transplantation system in which donors and hosts differed only by nonimmunogenic alleles of the leukocyte common antigen Ly-5, a low dose (150 to 300 cGy) of TBl was required to make physiologic “space” for engraftment of syngeneic BM cells given in numbers similar to those which could be obtained from BM of living human allogeneic donors (Tomita et al., 1994a). However, it is now clear that this requirement can be overcome by the administration of very high doses of syngeneic BM (Ramshaw et al., 1995; Sykes et al., 1998). Thus, the dose of TBI required to achieve allogeneic BM engraftment in mice can be reduced by administering increasing numbers of donor BM cells (Bachar-Lustig

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et al., 1995). Engraftment of high doses of allogeneic BM can be achieved without myelosuppressive treatment in mice that receive T-cell-depleting mAbs (Sykes et al., 1997), and similar results have now been obtained in a porcine model (Fuchimoto et al., 2000). However, at least in the murine model, it is helpful to create “space” in the thymus and to achieve high levels of early donor T cell repopulation in order to achieve permanent skin graft tolerance (Sykes et al., 1997; Tomita et al., 1996a,b; Nikolic et al., 2000b). Apparently, thymic “space” and peripheral hematopoietic “space” are regulated independently, and the stem cell pools contributing to thymopoiesis and hematopoiesis of all other lineages (including B cells) are nonidentical (Sykes et al., 1998). Costimulatory blockade has been used successfully to avoid the requirement for thymic irradiation and for recipient T-cell-depletion to achieve lasting mixed chimerism using high doses of donor BM (see above) (Wekerle et al., 2000). The successful engraftment of large numbers of T-celldepleted human leukocyte antigen (HLA)-mismatched mobilized peripheral blood and BM stem cells supports the concept that alloengraftment can also be enhanced in humans by increasing the number of hematopoietic progenitors administered (Aversa et al., 1994). Using peripheral blood stem cells collected after cytokine mobilization or by expanding progenitor cells in vitro (Petzer et al., 1996; Emerson, 1996; Brandt et al., 1998; Brugger et al., 1995), it may be possible to transplant large numbers of xenogeneic hematopoietic cells to humans. Inbred porcine donors could provide a virtually limitless supply of genetically identical hematopoietic progenitors to be used for this purpose. The administration of large numbers of hematopoietic cells is currently being explored as an approach to overcoming the competitive disadvantage of xenogeneic BM compared to host BM (Gritsch and Sykes, 1996b; Sharabi et al., 1990; Gritsch et al., 1994; Buhler et al., 2000a) (see below). The mechanisms by which myelosuppression promotes BM engraftment are not fully understood and could include both the creation of physical niches due to the destruction of host hematopoietic cells and the upregulation of cytokines that promote hematopoiesis. Homing to the BM environment depends on interactions between adhesion molecules and their ligands (Papayannopoulou et al., 1995; Simon et al., 1999), and active hematopoiesis also depends on specific molecular interactions, including cytokines, chemokines, and adhesion molecules, between the stroma and hematopoietic cells (Levesque et al., 1995, 1996; Verfaillie et al., 1991; Voura et al., 1997; Schick et al., 1998; Goltry and Patel, 1997; Kovach et al., 1995; Peled et al., 1999, 2000; van der Loo et al., 1998; Aiuti et al., 1997). The species specificity of some of these interactions accounts, at least partially, for the competitive advantage enjoyed by recipient BM over xenogeneic donor BM (see below). As is discussed above, a mAb-based nonmyeloablative approach to host conditioning has been used to achieve tolerance in a xenogeneic combination,

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rat-to-mouse (Fig. 2). However, in the xenogeneic model, large numbers of donor BM cells are needed to achieve engraftment, and the level of rat hematopoietic reconstitution gradually declines over time, despite persistent tolerance (Sharabi et al., 1990; Lee et al., 1995). This decline, which can be averted by the late administration of repeated BM injections (Lee et al., 1995), is due to a competitive advantage enjoyed by host hematopoietic cells over xenogeneic cells that becomes increasingly evident as recovery of the host from low-dose TBI occurs (Gritsch and Sykes, 1996a). Achievement of xenogeneic hematopoietic repopulation has proved to be an even more formidable challenge in more disparate species combinations. Human and pig progenitor cells have been clearly shown to be capable of repopulating murine recipients at low levels (Pallavicini et al., 1991; Lapidot et al., 1992; Gritsch et al., 1994), but the species specificity of critical regulatory molecules may limit the level of donor repopulation. Administration of exogenous donor-species-specific cytokines can partially overcome this barrier (Lapidot et al., 1992; Yang et al., 1996), and studies with porcine cytokine transgenic mice demonstrate the powerful capacity of high levels of donor cytokines to enhance donor stem cell engaftment and hematopoiesis, and even to permit the spontaneous appearance, for at least 20 weeks, of class-II-positive dendritic cells or macrophages in the murine host thymus (Yang et al., 2000; Chen et al., 2000). These cell types are of importance in inducing central tolerance of developing thymocytes (Inaba et al., 1991; Matzinger and Guerder, 1989; Brocker et al., 1997), and the presence of such donor cells in the thymus across such a disparate species barrier is highly encouraging for the potential of this approach to induce deletional tolerance. Evidence implicates clonal deletion in the longterm tolerance induced in mixed chimeras prepared in the rat–mouse species combination (Ildstad et al., 1992; Tomita et al., 1994c; Nikolic et al., 1998). In these animals, donor class IIhigh cells with a dendritic cell or macrophage morphology that are detectable in the recipient thymi are probably responsible for this deletion (Tomita et al., 1994c; Nikolic et al., 1998). Among the adhesion molecules that are expressed on hematopoietic progenitor cells, and that may therefore play a role in hematopoietic cell homing and function, are LFA-1, ICAM-1, VLA-4, VLA-5, and CD44 (reviewed in Simon et al., 1999). Porcine VLA-4 has been shown to interact well with human VCAM-1, but in vitro studies suggest that this interaction does not appear to have the same critical role in porcine hematopoiesis as it does for human cells (Warrens et al., 1998). Likewise, VLA-5 blockade has an inhibitory effect on human, but not porcine, hematopoiesis in a long-term BM culture system (M. Giovino et al., manuscript in preparation). Blocking ICAM-1-mediated interactions did not inhibit human or porcine hematopoiesis in vitro (Warrens et al., 2000), and anti-CD44 mAb has been shown to inhibit human and porcine hematopoiesis to a similar extent (Warrens et al., 1998). Another important observation has been

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that porcine hematopoietic progenitors, including long-term culture-initiating cells, appear to be more sensitive to the inhibitory effect of species cross-reactive cytokines, TGF-β and TNF- α (M. Giovinno et al., manuscript submitted). These observations of differing physiologies of hematopoiesis between the species suggest several approaches to improving the capacity of porcine hematopoietic cells to function in a human microenvironment. Clearly, a better understanding of the physiology of porcine hematopoiesis, and its differences and similarities with human hematopoiesis, is needed. Studies in SCID mice receiving porcine BM cells have provided evidence that, even in the absence of antibody, complement can cause the destruction of xenogeneic BM cells and reduce their engraftment (Yang et al., 1999). Thus, the expression of human complement regulatory factors by transgenic pigs might enhance the survival of porcine BM in primate recipients. 3. Attempts to Apply the Mixed Xenogeneic Chimerism Approach in a Pig-to-Primate Model In view of the immunologic and physiologic obstacles discussed above, it is not surprising that achievement of lasting chimerism at sufficient levels to induce T and B cell tolerance in a pig-to-primate species combination has, thus far, been elusive. As described above with respect to prevention of sensitized humoral responses, a conditioning regimen has been developed that is based on the rat-to-mouse nonmyeloablative BM transplantation model (Sharabi et al., 1990). TBI (300 cGy) and TI (700 cGy) are used as in the murine model. Since mAbs that completely deplete non-human primate T cells and NK cells have not been available, we have made use of a combination of anti-thymocyte globulin, cyclosporine, anti-CD154 mAb, and mycophenolate mofetil in order to overcome these barriers. In an effort to prevent antibody-mediated rejection of the porcine hematopoietic cells and kidneys that have been transplanted with them, EIA of anti-Gal antibodies as well as cobra venom factor and host splenectomy have been used. In addition, recipients are given recombinant porcine cytokines. This protocol is presented schematically in Fig. 3. In initial studies using porcine BM as the source of hematopoietic cells (2–30 × 108/kg), pig cells could not be detected, even initially, by flow cytometry but were detectable intermittently for months by the more sensitive PCR technique (Kozlowski et al., 1998, 1999). Most of the infused BM was rapidly destroyed, probably by host macrophages. This possibility is supported by the observation that treatment with liposomes containing a macrophage-depleting diphosphonate agent, medronate, markedly prolonged the survival of porcine hematopoietic cells. (This, unfortunately, was offset by the appearance of an induced anti-pig antibody response in these animals despite therapy with anti-CD154 mAb, which normally prevents this response (Buhler et al., 2000c).

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FIG. 3. Schematic representation of nonmyeloablative protocol for attempts to induce transplantation tolerance across the discordant xenogeneic barrier pig → baboon through establishment of mixed chimerism.

In more recent studies, infusion of large numbers of cytokine-mobilized miniature swine peripheral blood progenitor cells (3 × 1010/kg) resulted in the survival of sufficient porcine cells that chimerism was detectable by flow cytometry for 4 to 6 days and continuously for at least one month by PCR (Buhler et al., 2000a,b,e). In a few animals, chimerism that is measurable by flow cytometry has reappeared at 10 to 20 days, suggesting successful engraftment. Nevertheless, the return of anti-Gal antibodies has not yet been successfully avoided, and it is clear that means of overcoming macrophage-mediated destruction and the physiologic barriers described above must be more successfully overcome if this approach is to be used as an effective means of inducing T and B cell tolerance. 4. Donor Antigen Infusion with NK-Cell-Depletion and B-Cell-Suppressive Pharmacotherapy It has been demonstrated in nude rats that several components of T-cellindependent innate immunity can be tolerized by xenogeneic antigens under certain conditions. Such animals, when pre-treated with an infusion of donor antigen along with NK-cell-depleting antibody and leflunomide, a B cell immunosuppressive drug, accept hamster cardiac xenografts and show long-term specific tolerance to the donor species at the level of T-cell-independent antibodies and NK cells (Ji et al., 1999; Lin et al.,1998a,b). The mechanisms involved in this tolerance induction have not been fully defined. Recently, the use of donorspecific blood transfusion has been shown to enhance the induction of tolerance of the T-cell-independent NK cell and antibody-mediated rejection pathways in

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this model (Xia et al., 2000). In animals not receiving donor-specific transfusion, graft survival was prolonged, but they underwent chronic rejection with obliterative vasculopathy associated with infiltration of NK cells and macrophages (Xia et al., 2000), suggesting that this can occur in the absence of T and B cell responses to the donor in this concordant xenogeneic combination. These results suggest that these cells of the innate immune system can reject xenografts even in the absence of responses of acquired immunity, and that it is possible to tolerize the above components of the innate immune system. 5. Thymic Transplantation Because of the difficulties encountered in inducing xenogeneic hematopoietic cells to migrate to a recipient thymus and induce central tolerance, an alternative approach might involve replacement of the recipient thymus with a xenogeneic donor thymus after host T cell depletion and thymectomy. Immunocompetent mice treated in this way demonstrate recovery of CD4 T cells in xenogeneic porcine thymic grafts (Lee et al., 1994b). These cells repopulate the periphery, are competent to resist infection (Zhao et al., 1997a), and are tolerant of donor antigens, even by the stringent measure of discordant xenogeneic skin grafting (Zhao et al., 1996). Tolerance to both donor and host develops, at least in part, by intrathymic deletional mechanisms in these animals, and this reflects the presence of class IIhigh cells from both species within the graft (Zhao et al., 1996, 1997b; Y. Zhao and M. Sykes, manuscript in preparation). Since MHC restriction is believed by most to be determined by the MHC of the thymus, it was surprising that T cells that differentiated in a xenogeneic thymus were able to respond to peptide antigens presented by host MHC and to clear opportunistic infections (Zhao et al., 1997a). However, the excellent immune function achieved in humans receiving HLA-mismatched allogeneic thymic transplantation for the treatment of congenital thymic aplasia (DiGeorge syndrome) suggests that this “restriction incompatibility” may not be a major obstacle to the achievement of adequate immune function (Markert et al., 1997). It has been clearly demonstrated that porcine MHC mediates positive selection of murine CD4+ cells in grafted porcine thymi, as high levels of CD4 reconstitution are achieved when such grafts are placed into class-II-deficient mice (Zhao et al., 1997b). Furthermore, the use of thymectomized, T-cell-depleted TCR transgenic mice on selecting and nonselecting MHC backgrounds as recipients of porcine thymic grafts demonstrated no role for murine MHC in positive selection in these grafts. Equally efficient selection of the TCR transgenic murine T cells was observed in porcine thymic grafts of mice with both the selecting and nonselecting MHCs (Zhao et al., 1998b). Perhaps this high level of TCR cross-reactivity with MHC, even between species, reflects the fact that MHC reactivity is inherent in unselected TCR sequences (Zerrahn et al., 1997).

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In view of recent data indicating that MHC class II/peptide complexes similar to those mediating positive selection in the thymus are required to maintain CD4 cell survival in the periphery (Kirberg et al., 1997; Brocker, 1997; Maroto et al., 1999), we were somewhat surprised to observe no difference in the decay rates of murine CD4 cells developing in procine vs. murine thymus grafts following removal of the grafts (I. Rodriguez-Barbosa and M. Sykes, manuscript in preparation). These and other recently published results (Takeda et al., 1997; Clarke and Rudensky, 2000) challenge the above conclusions regarding the need for the same MHC in the periphery and the thymus to maintain CD4 cell survival. In thymectomized, T-cell-depleted mice grafted with porcine thymic tissue, only the CD4 subset and not CD8 T cells repopulate the periphery, despite the fact that functional, mature CD8 single-positive cells are detectable in the thymus grafts (Lee et al., 1994b; Zhao et al., 1998a). However, porcine thymus grafts can also support human T cell maturation, and human T cells developing in these grafts are specifically tolerant of the MHC of the porcine thymus donor, with normal reactivity to human alloantigens and non-donor porcine MHC. In addition to CD4 repopulation in the periphery, human CD8 cells are also detected in the periphery of the immunodeficient mice in which these porcine thymus grafts are implanted with human hematopoietic progenitor cells (Nikolic et al., 1999). Thus, in the most relevant pig-to-human species combination, generation of fully functional, tolerant CD4 and CD8 cells seems to be possible. In addition to the potential to achieve donor-specific T cell tolerance, porcine thymic xenografts, which appear to be resistant to the effects of HIV infection (B. Nikolic, S. Stanley, and M. Sykes, unpublished data), might also have a role in the achievement of immune restoration in the presence of human immunodeficiency virus (HIV) infection, and this possibility is currently being explored in the mouse model. While porcine thymic grafts present a promising approach to the induction of T cell tolerance to xenografts, the approach has several limitations. One is that the grafts do not appear to tolerize xenoreactive NAb-producing B cells (Lee et al., 1994b); (I. Rodriguez-Barbosa and M. Sykes, manuscript submitted). Thus, an additional approach would be required to overcome the NAb barrier to xenografts. A second concern is that a CD4 cell-mediated autoimmune syndrome has been described in nude mice receiving thymic xenografts from several different species (Taguchi et al., 1986; Nishigaki-Maki et al., 1999). We have also observed this phenomenon in nude mice receiving porcine thymic grafts, but its frequency is markedly reduced ( mouse species combination. J. 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Regulation of Antibacterial and Antifungal Innate Immunity in Fruitflies and Humans MICHAEL J. WILLIAMS Ume˚a Centre for Molecular Pathogenesis, Ume˚a University, S-901 87 Ume˚a, Sweden

Insects have been very successful in adapting to their environment, and the ability of the insect immune system to detect and elicit the appropriate response against various invading pathogens has helped in this success. Unlike the vertebrate immune system, which consists of both innate and adaptive components, insect immunity probably consists entirely of an innate immune response, as no evidence of an adaptive response has been found. The innate immune response is described as either a reaction against “lack of self,” or the interaction between host germlineencoded receptors and molecules unique to a particular class of invading organisms. Once the invading organism is recognized, the host immune response can be activated via signaling pathways that lead to the appropriate reaction. This review endeavors to put forth how through genetic, molecular, and biochemical studies of the fruit fly Drosophila melanogaster, as well as other insects, it is now understood that aspects of the insect and vertebrate innate immune system are very C 2001 Academic Press. similar.

I. Introduction

The insect immune response has been very successful; evidence of this is the ability of insects to invade and survive various hostile environments where they are in close contact with potential pathogens. Yet, interactions between insects and potentially pathogenic microorganisms rarely result in an infection with any long-term deleterious effects. The insect immune response was first recognized at the end of the 19th century. As early as 1884, Metchnikoff published a paper describing the phagocytic ability of blood cells from Daphnia pulex, a crustacean (Metchnikoff, 1884). It was not until the early part of the 20th century that any descriptions of the insect humoral response were published (For a review of this early work, see Brey, 1998). These studies continued throughout the century but real advances in humoral activation were not made until the late 1970s and the early 1980s, when the first antimicrobial effector molecules were identified (Hultmark et al., 1980; Steiner et al., 1981), and the 1990s when transcriptional control of the immune response started to be investigated (Sun and Faye, 1992a; Ip, 1993; Reichhart, 1993). Advances in the molecular biology of blood cells occurred in the 1990s with the discovery of signaling pathways necessary for 225 C 2001 by Academic Press Copyright All rights of reproduction in any form reserved. 0065-2776/01 $35.00

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hemocyte development in both embryos and larvae (Tepass et al., 1994; Harrison et al., 1995; Luo et al., 1995; Qiu et al., 1998). How the insect immune response is activated is best understood in the fruit fly Drosophila melanogaster. In Drosophila, the combination of a well-defined genetic system together with molecular biology and biochemistry has led to the discovery of proteins and pathways involved in the activation and regulation of this response. Now, with the completion of the Drosophila genome project, another powerful tool has been added to help in the advancement of this field (Adams, 2000; Khush and Lemaitre, 2000). Using Drosophila as a model, and with evidence gathered from other insects, it is now evident that the insect immune response closely resembles the innate immune system of vertebrates. The vertebrate immune system has both adaptive and innate components (Medzhitov and Janeway, 2000). The adaptive immune response consists of T cells and B cells that express receptors generated by somatic recombination during their development. This developmental process allows each T cell and B cell to express a receptor that is structurally unique, leading to the creation of a large repertoire of receptors able to recognize infinite numbers of antigens. This system of randomly created antigenic receptors could potentially lead to deleterious effects, so energy must be expended to protect the host from its own immune system. Among all of these randomly generated receptors only a subset are useful, and cells expressing these receptors are selected for clonal expansion. Other cells expressing receptors that are not useful, or that may even recognize “self”-expressed antigens, are selected for destruction. Since T cell and B cell receptors are created somatically, and thus cannot be passed on to the next generation, the energy expended to create this potent response is not conserved. However, the evolutionarily older innate immune response consists of germlineencoded receptors that can be passed on from generation to generation. This means that the innate immune response does not have to re-invent itself with each new generation and has evolved together with the epitopes its receptors recognize. Since the innate immune response receptors are “hardwired” and vary little from cell to cell or generation to generation, they may not recognize every possible antigen, but rather only a few, well-defined, pathogenic epitopes that can be found on many different invading organisms. Once the innate receptors recognize their epitopes, peptides that further activate the innate immune response, along with antimicrobial peptides, are produced. In vertebrates, proteins involved in activating the adaptive portion of the immune response are also produced (Medzhitov and Janeway, 2000). Insects lack T and B cells and other components of adaptive immunity. However, like in the vertebrates, the innate immune system allows a rapid response to an invading organism, by both humoral and cellular defenses. In Drosophila, the fat body is the major tissue involved in the humoral response. When an infection is recognized by the immune system, antimicrobial proteins are produced by the

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fat body and released into the hemolymph. Antimicrobial proteins consist of antibacterial proteins, bacteriostatic proteins, and antifungal proteins (Hultmark, 1993; Cociancich et al., 1994; Engstrom, ¨ 1999; Imler and Hoffmann, 2000). Until recently it was thought that the genes encoding these proteins were all induced upon infection with no specificity to the invading pathogen. It is now understood that the Drosophila immune response is to some extent able to differentiate between different microorganisms and produce the appropriate response. Expression of these genes is mainly controlled at the level of transcription. Cells circulating in the hemolymph, called hemocytes, propagate the cellular response. In larvae these hemocytes develop in lymph glands that are located behind the brain in five pairs of lobes closely associated with the dorsal vessel. When the morphology of circulating blood cells is compared, three basic types of cells can be identified (Shrestha and Gateff, 1982; Rizki and Rizki, 1984). The most abundant cells are the plasmatocytes; these are able to produce antimicrobial peptides and are also able to phagocytize invading organisms. The largest and least abundant cells are the lamellocytes. These cells along with the crystal cells are involved in the encapsulation of invading pathogens. Crystal cells secrete components of the phenoloxidase cascade that is involved in sclerotization of the cuticle and in the formation of melanin in the capsules formed around invading organisms, and in wound repair. During development Drosophila come into contact with various pathogens. Drosophila larvae spend their entire life burrowing in decaying fruit containing different bacteria and fungi. Drosophila can also be parasitized by different wasp species during larval development. The first line of defense against invading pathogens is the cuticle, or exoskeleton, and the peritrophic membrane lining the gut epithelium. If an invading organism can get past these defenses, then various reactions occur. When the infection is local and does not actually involve the hemolymph, antimicrobials are produced at the site of infection (Brey, 1998; Ferrandon et al., 1998). If the infection reaches the hemolymph a more systemic reaction occurs, and antimicrobials produced by the fat body, and hemocytes, are released into the hemolymph. At the same time circulating plasmatocytes can, by phagocytosis, engulf the invading organism(s) (Engstrom, ¨ 1999; Imler and Hoffmann, 2000). If these reactions are not sufficient to sequester or defeat the pathogen, then the lamellocytes can encapsulate the organism, and the crystal cells can melanize it. This forms a melanized nodule that effectively removes the invading organism from the system (Kraaijeveld et al., 1998). The mechanisms that regulate local versus systemic reactions are still under intense investigation. A model for the sequence of events that may take place during the Drosophila immune response is diagrammed in Fig. 1. This is a very simplified model of how an invading organism is recognized and defeated by the immune response. This review, though not exhaustive, is an attempt to explain this model, by integrating the most recent discoveries in the activation and regulation of the immune

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FIG. 1. A model of the Drosophila immune response. A pathogen or parasite that succeeds in penetrating the cuticle or peritrophic membrane is recognized as foreign. The interaction of pathogen-associated molecular patterns (PAMPS) with pattern-recognition receptors (PRRs) may activate a serine protease cascade or trigger cell-surface receptors directly. These extracellular signaling events induce intracellular signal transduction pathways. Signal transduction events in hemocytes may lead to the production of antimicrobial peptides or to phagocytosis, encapsulation, and melanization of the invading organism. In the fat body, these signaling events lead to the production of antimicrobial peptides.

response in Drosophila and other insects, and to compare it with what is known from vertebrates. II. Recognition of Foreign Molecules

Whereas immunoglobulins and T cell receptors provide an intellectually pleasing explanation for the accurate discrimination between “self ” and “non-self ” of the acquired immune reactions, it is less clear how the innate immune system can make this discrimination without prior experience of the invading microorganism or parasite. Two models have been proposed to explain this phenomenon. According to one hypothesis, particularly well formulated by Janeway (1989), large groups of microorganisms share common epitopes, or patterns. These microbial epitopes include the lipopolysaccharides from Gram-negative bacteria, peptidoglycan and lipoteichoic acids from Gram-positive bacteria, the unmethylated dinucleotide CpG in DNA from bacteria, and β-1,3-glucans and


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mannans from fungi. It is proposed that specific receptors, or recognition molecules, of the innate immune system recognize these patterns. Janeway has dubbed the microbial epitopes PAMPs, for “pathogen-associated molecular patterns,” and the recognition molecules PRRs, for “pattern-recognition receptors ” (Medzhitov and Janeway, 2000). We prefer to use the two terms “microbial patterns” and “recognition molecules,” since we think that the recognition of nonpathogens is also very important, and may indeed be the reason why they are nonpathogens. This will also somewhat reduce the otherwise heavy use of three-and four-letter acronyms. A second model proposes that recognition could be mediated by a large number of low-specificity receptors that can be triggered by any encountered surface. Attack against “self” is avoided by the presence of specific markers on the organism’s own cells. These markers are recognized by specific receptors, leading to an inhibitory signal. Thus, the specific discrimination of foreignness is based on the absence of such markers, and this model has been dubbed the “missing self” hypothesis. The missing self hypothesis has been proposed in various contexts for different insect systems but there is so far only good evidence for this mechanism from vertebrates, where natural killer cells use molecules of the major histocompatibility complex as markers of self (Moretta et al., 1992). However, good candidates have been found for the specific receptors predicted by the pattern recognition hypothesis and we will review some of them here. A. PEPTIDOGLYCAN RECOGNITION PROTEINS Peptidoglycan is the major cell wall component in most bacteria. In Grampositive bacteria, the peptidoglycan layer is generally very thick, whereas Gramnegative bacteria have a thinner wall covered by a lipopolysaccharide-containing outer membrane. In mammals, peptidoglycan can potentiate the immune system, induce fever, and activate macrophages. In insects, it was discovered that injecting peptidoglycan into the hemolymph can activate the phenoloxidase system and induce the production of antimicrobial peptides (Ashida et al., 1983; Dunn et al., 1985; Kanost et al., 1988). In the silkworm, Bombyx mori, Ashida and collaborators showed that the activation of prophenoloxidase is mediated by a specific peptidoglycan recognition protein, PGRP (Yoshida et al., 1986, 1996; Ochiai and Ashida, 1999). This work provided the first evidence for a specific pattern-recognition molecule mediating an immune response in any organism. Independently, a PGRP homolog from Trichoplusia ni was cloned in a search for genes activated in the immune response. They could show that closely related genes are present in several organisms including man (Kang et al., 1998). In the Drosophila genome, there are twelve genes that encode PGRP-like proteins (Werner et al., 2000). They fall into two major classes, the short-form

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PGRPs, and the long-form PGRPs. The short PGRPs are small soluble peptides containing one typical PGRP domain, similar to those found in other insects. These short-form PGRPs contain a signal peptide, and like the lepidopteran PGRPs it is presumed that they are secreted into the hemolymph. There are seven genes that encode short-form PGRPs, five of which are inducible upon infection. Of these five inducible PGRPs, PGRP-SA and PGRP-SC1b are known to be able to bind peptidoglycan. Surprisingly, unlike most of the proteins that are induced in the immune response, PGRP-SA is not transcriptionally induced in the fat body or hemocytes, but upon infection is highly expressed at other sites, probably the epithelia. Thus, PGRP-SA may be involved at the point of wounding, or in wound repair, in the earliest part of the immune response. Since PGRP-SC1b is mainly upregulated in the gut, it may also function in the earliest part of the immune response protecting the fly from pathogens trying to invade through this tissue. The other three short forms induced upon infection, PGRPSB1, -SC2, and -SD, are all mainly induced in the fat body where they are likely to contribute to PGRP levels in the hemolymph. The long-form PGRPs can be divided further into smaller families. Three of the long form PGRPs, PGRP-LA, -LC, and -LD, have no obvious signal peptide, but do have a putative transmembrane region. Since these PGRPs are specifically expressed in hemocytes and a cell line derived from hemocytes, it is speculated that they encode transmembrane receptors expressed on the hemocyte plasma membrane (Werner et al., 2000), though this has yet to be shown. These receptors have typical PGRP domains extracellularly and unique intracellular domains. Similar to other known pattern recognition receptors, PGRP-LA has two splice forms, and PGRP-LC may have as many as five alternative forms, thus allowing for the possibility that they are able to recognize many different types of petidoglycan. Two other long forms, PGRP-LB and -LE, have no signal peptide or transmembrane domain and thus may remain cytoplasmic. It is postulated that these PGRPs may be involved in the recognition of bacteria that can invade the cytoplasm. This may be a viable explanation considering that, unlike the other long-form PGRPs, these two genes are expressed in many tissues, and PGRP-LB is inducible upon infection (Werner et al., 2000). B. β-1,3-GLUCAN RECOGNITION/GRAM-NEGATIVE BINDING PROTEINS Gram-negative bacteria represent a major group of invading pathogens known to cause diseases from invertebrates to humans. These bacteria have their cell wall hidden under an outer membrane, the principal component of which is a glycolipid called lipopolysaccharide (LPS). Bacterial lipopolysaccharides are therefore primary targets for the innate immune system. Although the chemical structure of lipopolysaccharide has been known for a long time, the molecules that recognize this structure and elicit a response have only been recently discovered. The first lipopolysaccharide binding protein to be discovered was recovered


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from rabbit serum and named lipopolysaccharide-binding protein (LBP) (Tobias et al., 1986). More recently, CD14, the first leucine-rich repeat protein reported to be involved in the innate immune response, was discovered to bind lipopolysaccharide (Wright et al., 1990). The role of LBP and CD14 in lipopolysaccharide recognition is not entirely clear and will be further discussed below in the context of the Toll-like receptors. No LBP or CD14-like proteins have been discovered in Drosophila or in any other insect, but a class of β-1,3-glucan- and lipopolysaccharide-specific proteins has been found (Ochiai and Ashida, 1988). A β-1,3-glucan recognition protein (βGRP) was isolated from Bombyx and shown to mediate the activation of prophenoloxidase (Yoshida et al., 1986; Ochiai and Ashida, 1988). A Gramnegative binding protein (GNBP) was later purified from Bombyx mori and the corresponding cDNA cloned (Lee et al., 1996). Cloning and sequencing of βGRP showed that the two proteins are related with 38% overall sequence identity (Ochiai and Ashida, 2000). They are also homologous to a β-1,3-glucanase from a sea urchin, Strongylocentrotus purpuratus (Bachman and McClay, 1996) and to bacterial glucanases. This could explain the affinity for β-1,3-glucans, whereas LPS binding is more surprising. However, the dual specificity of this class of proteins is independently confirmed for a related lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) which was recently isolated from a crayfish (Lee et al., 2000). Homologous genes were later isolated from Hyphantria cunea, Manduca sexta, Anopheles gambiae, and Drosophila melanogaster (Shin et al., 1998; Richman et al., 1997; Kim et al., 2000; Ma and Kanost, 2000). Although these genes have been called GNBP, they are in fact most closely related to βGRP. In this review, we will retain the term βGRP to designate the entire family of LPS-β-1,3glucan-binding proteins. In Bombyx, Hyphantria, and Anopheles it was shown that the βGRPs are transcriptionally induced after infection with Gram-negative bacteria (Lee et al., 1996; Richman et al., 1997; Ochiai and Ashida, 2000). In Drosophila, three cDNAs encoding βGRP-like proteins have been recovered. Unlike βGRPs in other insects, none of the Drosophila βGRPs is inducible upon infection (Kim et al., 2000a). The only Drosophila βGRP to be extensively studied is DGNBP-1. Similar to mammalian CD14, DGNBP-1 can be found in two forms, one soluble and the other GPI-linked to the plasma membrane in cell culture. It is not known whether these two forms actually interact or act independently of each another. It was also found that DGNBP-1 had a high affinity for both lipopolysaccharide and β-1,3-glucan but would not bind peptidoglycan, β-1,4-glucan, or chitin. Furthermore, overexpression of DGNBP-1 in immunocompetent Drosophila Schneider cells enhanced lipopolysaccharide and β-1,3-glucan induced antimicrobial gene expression. This showed that not only can Drosophila GNBP1 interact with lipopolysaccharide, but this interaction can also lead to the induction of the immune response. The authors went

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on to show that the effect of DGNBP-1 overexpression could be blocked by a specific antibody (Kim et al., 2000a). Although βGRP was previously shown to mediate the activation of phenoloxidase, this was the first evidence of a pattern-recognition receptor being involved in the induction of antibacterial peptides. Since DGNBP1 has no cytoplasmic domain of its own, it is interesting to speculate that, similar to CD14, DGNBP1 may interact with a transmembrane receptor to signal in the immune response. It may also be possible that the soluble form can interact first with lipopolysaccharide and then with the membrane-bound DGNBP1 or with a transmembrane receptor. In addition to the three βGRPs already reported, we found at least three other genes that encode possible βGRPs in the Drosophila genome. It has yet to be determined if any of these other possible βGRPs function in the Drosophila immune response. The reason for so many possible βGRPs may be redundancy in the system, or they may have different affinities for various microbial patterns. C. C-TYPE LECTINS Protein–carbohydrate interactions serve multiple functions in the immune system. The structure of prokaryotic carbohydrates is different, and distinct, from those found on eukaryotic membranes. These carbohydrate determinants are probably used extensively by the immune response to determine self from non-self. In mammals, the natural killer cells express a family of carbohydratebinding receptors known as lectins (sugar-binding proteins) that mediate both pathogen recognition and cell–cell interactions using structurally related Ca2+dependent carbohydrate-recognition domains (Lanier, 1998; Drickamer, 1999). The collectins, a sub-class of C-type lectins, also have a collagen-like tail. Soluble collectins, such as serum mannose-binding protein and pulmonary surfactant proteins, as well as the macrophage cell-surface mannose receptor, recognize pathogens by binding to terminal monosaccharide residues characteristic of bacterial and fungal cell surfaces. The broad selectivity of the monosaccharidebinding site and the geometrical arrangement of multiple, related, Ca2+dependent, carbohydrate-recognition domains in the intact lectins explains the ability of the proteins to mediate discrimination between self and non-self (Weis et al., 1998). Many lectins have been cloned from invertebrates, and, though the biological role of many of these lectins has yet to be determined, some C-type lectins are involved in the insect immune response. An inducible lipopolysaccharidebinding lectin was found in a cockroach, and a constitutively expressed C-type lectin that binds to lipopolysaccharide was recovered from immunized Bombyx mori (Jomori and Natori, 1991; Koizumi et al., 1999). Two different inducible lipopolysaccharide-binding C-type lectins, involved in phenoloxidase activation, have been recovered from Manduca sexta (Yu et al., 1999; Yu and Kanost, 2000).


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A galactose-specific C-type lectin, recovered from the flesh fly Sarcophaga peregrina, was one of the first insect lectins to be studied in detail (Komano et al., 1980). This lectin, undetectable in uninfected third instar larvae, was seen to accumulate in the hemolymph of immunized third instar larvae (Komano et al., 1980, 1981). Later a similar lectin, named lectin-galC1, was recovered from Drosophila melanogaster (Haq et al., 1996) Similar to the Sarcophaga lectin, lectin-galC1 binds galactose and is transcriptionally upregulated when third instar larvae are infected. Like so many other defense molecules, lectingalC1 is also upregulated when the larvae nears pupation (Haq et al., 1996). Since lectin-galC1 is also expressed in imaginal tissue, this may be a developmental effect as well as a protective one. There is at least one more C-type lectin mapping near, and having similarity to, lectin-galC1. Besides these two genes there are at least 35 more C-type lectins in the Drosophila genome, some of which may be involved as pattern-recognition receptors for the immune response (Theopold et al., 1999). D. SCAVENGER RECEPTORS The scavenger receptors constitute a heterogenous class of membrane proteins, defined by their ability to bind oxidized or acetylated low-density lipoproteins and therefore implicated in arteriosclerosis. They were first isolated from the cell membranes of mammalian monocytes and macrophages but similar proteins were later also found on phagocytic Drosophila cells (Pearson et al., 1995). There are three groups of scavenger receptors. The first group includes the collagenous trimeric receptors and consists of class A type I and type II macrophage scavenger receptors (MSR-A) and a macrophage receptor with collagenous structure (MARCO) that mediate the uptake of chemically modified low-density lipoproteins (Kodama et al., 1996; Ito et al., 1999). The second group of scavenger receptors consists of CD36-like proteins involved in the uptake of oxidized low-density lipoproteins and apoptotic bodies (Endemann et al., 1993; Kodama et al., 1996). The third group consists of the class C scavenger receptors that have a high affinity for fucoidan, lipopolysaccharide, lipotechoic acid, and nonopsonized matter (Hughes et al., 1995). The scavenger receptors are predicted to have broad ligand-binding specificities for different microbial patterns. In mammals, a macrophage-specific scavenger receptor (SR-A) has been characterized as having broad polyanionic ligand specificity and may serve as a pattern-recognition receptor for the innate immune response (Krieger et al., 1993). There is mounting evidence that this receptor can recognize and bind to surface determinants of Gram-negative and Gram-positive bacteria, such as lipopolysaccharide and lipoteichoic acid (Krieger et al., 1993). CD36 is another mammalian protein that has been characterized as a scavenger receptor (Endermann et al., 1993). Though CD36 was initially characterized as a scavenger receptor involved in the innate immune response, its main role may

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be in the recognition and removal of apoptotic cells (Ren et al., 1995; Navazo et al., 1996). A different kind of scavenger receptor was described from Drosophila, defining the class C of scavenger receptors (Pearson et al., 1995). Though there is no significant sequence homology between the two proteins, Drosophila SR-CI has many of the same properties as mammalian SR-A. Similar to mammalian SR-A, SR-CI is expressed on hemocytes, the Drosophila version of macrophages. SR-CI also has broad polyanionic ligand-binding abilities and can bind to microbial β-glucans. Strinkingly, SR-CI has two N-terminal complement control protein (CCP) domains (Pearson et al., 1995). CCP domains have been identified in over 30 different mammalian and invertebrate proteins, including many complement proteins, as well as a Limulus coagulation factor (Blom et al., 2000; Bevilacqua and Nelson, 1993; Iwanaga et al., 1992). The exact function of the CCP domain is unknown, but in many cases they have been shown to interact with other proteins. This leads to the possibility that the CCP domains of SR-CI are involved in ligand binding. There are at least two more genes having similarity to SR-CI described in the Drosophila genome. As yet none of these Drosophila scavenger receptors has been described as being involved in immune response activation. In Drosophila, two homologs of mammalian CD36 have been described: epithelial membrane protein (emp) and croquemort (CRQ) (Hart and Wilcox, 1993; Franc et al., 1996). Both emp and CRQ may have developmental functions, but as yet there has been no report of either protein functioning in the immune response (Har and Wilcox, 1993; Franc et al., 1999). During embryogenesis, emp is expressed in epithelial cells, and in larvae may function in imaginal tissues to help in the formation of adult structures. CRQ is specifically expressed in embryonic hemocytes that are actively involved in the phagocytosis of apoptotic cells. CRQ is also expressed on larval hemocytes and, though its function there is unknown, it has been determined that it is not involved in the recognition and phagocytosis of bacteria (Franc et al., 1999). There appears to be three additional emp-like and four additional CRQ-like molecules in the Drosophila genome; possibly some of these may be scavenger receptors involved in the immune response. The Drosophila scavenger receptors described so far are all expressed on circulating hemocytes. They appear to be involved in the phagocytic activity of these cells during metamorphosis and embryonic development, but there is so far no evidence that they serve as specific pattern-recognition molecules of the immune defense. E. TOLL-LIKE RECEPTORS The first leucine-rich repeat protein to be identified in the mammalian innate immune response was CD14 (Wright et al., 1990). Later, another leucine-rich


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repeat protein, Toll, was identified as being involved in the Drosophila immune response (Rosetto et al., 1995; Lemaitre et al., 1996). Toll encodes a transmembrance protein that is a leucine-rich repeat extracellularly and has high homology to the interleukin-1 type 1 receptor intracellularly. More recently, in mammals, a family of Toll-like receptors has been cloned and studied. Studies using mammalian cell lines and knockout mice have shown that at least two–three of these Tolls can function as pattern-recognition receptors. Toll-like receptor 4 (TLR4) appears to bind lipopolysaccharide directly, while TLR2 interacts with peptidoglycan (Takeuchi et al., 1999), and TLR9 with unmethylated CpG dinucotides in DNA (Hemmi et al., 2000). In Drosophila there are nine TLR genes (Tauszig et al., 2000); although at least three of them, Toll, 18- wheeler, and Toll5, are known to function in activating the Drosophila immune response, none has been shown to directly bind any microbial patterns. Since the Drosophila Toll receptor requires the activated Spätzle ligand for its signaling during the immune response, it does not seem likely that Toll could function directly as a pattern recognition receptor (Levashina et al., 1999). This does not exclude the possibility that one, or more, of the other Toll-like proteins is able to bind directly to a microbial pattern. Williams et al. (1997) reported that in third instar larvae the Toll-like receptor 18-Wheeler expressed one insoluble and four soluble protein forms. It is possible that one of these soluble forms acts in a fashion similar to CD14, binding to lipopolysaccharide or some other microbial pattern. It will be interesting to see if any of the Drosophila Toll-like receptors can function directly as a recognition molecule, or if all of them are downstream of activated endogenous ligands. III. Serine Protease Cascades and Thioester-Containing Proteins

Interaction of microbial patterns with their respective recognition receptor in the insect leads to the activation of a series of serine proteases in the hemolymph that act in a cascade similar to that which activates complement in mammals (Johansson and Soderh¨ ¨ all, 1996; Ashida and Brey, 1998). One result of these serine protease cascades is the activation of the enzyme phenoloxidase by proteolytic cleavage of an inactive proenzyme. The hemolymph also contains members of an extended family known as the thioester proteins (Armstrong and Quigley, 1999; Kanost, 1999; Lagueux et al., 2000). Proteins of this family are abundant components of mammalian blood plasma and arthropod hemolymph (Sottrup-Jensen, 1989; Armstrong et al., 1996). In humans, these can account for as much as 3% of the proteins found in plasma (Ganrot and Schersten, 1967). Endogenous as well as exogenous proteases are active during an infection and if left uncontrolled can lead to serious tissue damage. In mammalian immune response α2-macroglobulin and complement C3, C4, and C5 are all active as protease inhibitors. It has also been shown that a fragment of C3, known as C3a,

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is an inducer of the inflammation response (Sottrup-Jensen, 1989; Armstrong and Quigley, 1999). Whether or not any of the Drosophila thioester proteins act as protease inhibitors or activators in the immune response is not yet known. A. PHENOLOXIDASE In Drosophila, prophenoloxidase is found in the hemolymph and in crystal form in specialized cells circulating in the hemolymph called crystal cells (Rizki et al., 1985). It is well known that activated prophenoloxidase plays an important role in cuticular melanization and sclerotization. In addition, studies dealing with immune response of various insects, suggest that phenoloxidase is also critical in the defense reactions against invading pathogens. Prophenoloxidase is activated when various microbial patterns, such as peptidoglycan, β-1,3-glucan, and lipopolysaccharide, are recognized by recognition proteins (Ochiai and Ashida, 1988; Yoshida et al., 1986; Tsuchiya et al., 1996; Ratcliffe et al., 1991). This leads to the activation of a serine protease cascade involved in the processing of prophenoloxidase into active phenoloxidase (Johansson and Soderh¨ ¨ all, 1996). Once prophenoloxidase is activated, quinones are produced from tyrosine derivatives, highly reactive quinoids may contribute to the production of superoxide anions involved in a cytotoxic role in defense. The end products of this cascade are melanin and sclerotin. In many insects, melanin is deposited around cuticular wounds, is involved in parasitic encapsulation, and is active at sites of fungal infection. Melanin has been shown to be involved in the Drosophila immune defense during encapsulation of invading parasites, such as parasitic wasps, and in wound healing (Marmaras et al., 1996). Phenoloxidase is a copper-containing enzyme. Genes encoding prophenoloxidase have been recovered from many different insects, including Bombyx mori, Manduca sexta, and Drosophila melanogaster (Yasuhara et al., 1995; Hall et al., 1995; Fujimoto et al., 1993). At the amino acid level all of the insect prophenoloxidases share 40–45% identity and belong to a superfamily that includes the arthropod hemocyanins, which also contain copper, and the hexameric hemolymph proteins that serve as storage proteins. It has been suggested that the phenoloxidases may themselves be able to form thioester cross-links to other proteins, like the thioester proteins described below (Hall et al., 1995). Although this would nicely explain the stickiness, which is a typical property of the active phenoloxidase, this proposal is based on rather weak sequence similarity to the thioester motif and so far it has no experimental support. B. THIOESTER PROTEINS As stated previously, in mammals the thioester family includes the complement proteins C3, C4, and C5 and the protease inhibitor α2-macroglobulin (Armstrong and Quigley, 1999). When these proteins are activated, they form


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covalent bonds with other proteins via their thioester domains. The Drosophila thioester proteins, known as TEPs, are most similar to mammalian complement C3 and α2-macroglobulin. In mammals, there are three ways to activate the complement cascade system, and thus complement C3 (Medzhitov and Janeway, 2000; Haeney, 1998). The first way is via the classical system involving activation downstream of an antibody–antigen interaction, eventually leading to the activation of complement protein C3. Since Drosophila lack antibodies, this cannot be the method of activation for their thioester complement-like proteins. The second pathway is the lectin pathway, involving the activation by C-type mannose-binding lectin of mannan-associated serine proteases 1 and 2 (MASP1 and MASP2) eventually leading to the activation of complement C3. Since Drosophila have many C-type lectins, some of which show similarity to mannose-binding lectins, and Drosophila also have genes that encode proteins similar to MASP1 and MASP2, the lectin pathway may function in the activation of the thioester proteins. The final method of activation is the alternative pathway where activated C3 can autoactivate more C3. In Drosophila, there are four genes for proteins having similarity to mammalian complement C3 and α2-macroglobulin, TEP1, -2, -3, and -4 (Lagueux et al., 2000). There is also a fifth gene that encodes a TEP, but no transcript for this gene has been detected, leading the authors to believe that it may encode a pseudogene. Of the four TEPS that express detectable transcripts, three are inducible upon infection (TEP1, -2, and, -4). The inducibility of TEP 1 and 2 was significantly higher in larvae than in adults; the reason for this is unknown. Similar to recognition receptors, TEP2 has five alternative transcripts, each one varying in what is known as the hypervariable region. Since α2-macroglobulin and complement C3 are both known to act as protease inhibitors, the variability of Drosophila TEP2 proteins may allow for the recognition of many different proteins or microbial patterns. TEP1 is the only Drosophila TEP studied in any detail (Lagueux et al., 2000). TEP1 is mainly transcribed in the fat body, and its inducibility is dependent on the Jak kinase pathway. This differs from the antimicrobials, whose induction seems to rely mainly on either the Toll pathway, or the Imd–Relish pathway. Since gain-of-function alleles of the Drosophila Jak kinase, Hopscotch, mainly affect hemocytes (Harrison et al., 1995; Luo et al., 1997), the authors speculate that somehow hemocytes are necessary for the induction of TEP1 during the immune response. Larvae may have more hemocytes than adult flies; if this is true and TEP1 requires hemocytes for its induction upon infection, this may be the reason TEP1 is more inducible in larvae than adults. As yet, no function for the TEPS in the Drosophila immune response has been described. However, by analogy to mammalian complement C3 and α2-macroglobulin, it is possible that they act as protease inhibitors or as regulators of a serine protease cascade in the immune response.

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IV. Induction and Regulation of the Antimicrobial Genes

Recognition of the microbial patterns by their respective recognition molecules activates the prophenoloxidase system, soluble signaling molecules, and the intracellular signaling pathways needed to regulate the immune response. One aspect of this regulation is the induction of antimicrobial peptides that are rapidly produced by the fat body and hemocytes and then secreted into the hemolymph. Many signaling molecules involved in the activation and regulation of the Drosophila immune response are similar to those found in vertebrate and plant innate immunity (Medzhitov and Janeway, 1998). This conservation suggests an ancient origin for the innate immune response and has allowed for the discovery of important proteins involved in its regulation. Both vertebrates and Drosophila use Toll-like receptor/NF-κB (Rel) pathways to regulate their innate immune response (Fig. 2). These pathways involve the activation of kinases that lead to the translocation of NF-κB-like transcription factor from the cytoplasm into the nucleus. Mammalian NF-κB is a homo- or heterodimer of Rel proteins. All Rel proteins share a similar N-terminal Rel homology domain that functions in dimerization and DNA binding. They also recognize a similar DNA sequence sharing the consensus 5′ -GGGRNNYYCC- 3′ (Grimm and Baeuerle, 1993). The mammalian family of Rel proteins includes p50, p52, p65, RelA, Rel-B, and c-Rel. Classical NF-κB consists of a p50–p65 heterodimer. Two other nonclassical Rel factors, p105 and p100, were also found. p105 and p100 differ from other Rel proteins in their C-terminal region, which consists of an IκB-like domain that acts as an inhibitor to Rel translocation (Hatada et al., 1992, 1993; Liou et al., 1992). It was also discovered that p105 is the precursor for p50 and p100 is the precursor for p52 (Ghosh et al., 1990; Bours et al., 1990). The IκB domain of p105 acts to hold the Rel inhibitor of transcription homodimer p50–p50 in the cytoplasm (Henkel et al., 1992). Different combinations of these Rel factors in mammals are known to act as either activators or inhibitors of gene expression (Baeuerle and Henkel, 1994). In the insect immune response, the emerging paradigm implicates a Rel/NF-κB cascade, analogous to that used in mammalian innate immunity and in dorsal–ventral patterning in the Drosophila embryo (Hultmark, 1993). The first Rel activity in response to infection detected in insects was discovered in the Cecropia moth. Sun and Faye (1992) describe a Cecropia immuno-responsive factor (CIF) recovered from immune-challenged insects. In Drosophila, three Rel-domain proteins, Dorsal (DI), Dorsal-like immunity factor (Dif), and Relish (Rel), are present in the fat body of larvae and adults and are induced in response to infection (Dushay et al., 1996; Lemaitre et al., 1996; Petersen et al., 1995; Engstrom, ¨ 1999; Govind, 1999). DI and Dif are most similar to the mammalian p65 Rel protein, while Relish is more similar to the mammalian proteins p100 and p105, containing both a Rel homology domain and an IκB domain (Steward, 1987; Ip et al., 1993; Dushay et al., 1996).


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FIG. 2. The Toll pathway in the immune response. In Drosophila, recognition of microbial epitopes activates a serine protease pathway, normally inhibited by the serpin necrotic. Activation of this serine protease cascade leads to the processing of the Spätzle ligand and thus to the activation of the Toll receptor. Signaling downstream of the Toll receptor requires an interaction between the death domains (DD) of the kinase Pelle and the novel protein Tube. These signaling events lead to Cactus phosphorylation and degradation and to the phosphorylation and translocation of Dif into the nucleus. In the human innate immune response, binding of a microbial epitope by a Toll-like receptor leads to an interaction between the cytoplasmic (Toll/interleukin-1 receptor, TIR) domain of the Toll receptor and the TIR domain of the adaptor protein MyD88. MyD88 then interacts with the Pelle-like kinase IRAK via their death domains. In turn, IRAK interacts with TRAF6 and the MAP3K TAK1. This interaction leads to the phosphorylation and activation of the IκB kinase complex and thus to the phosphorylation and degradation of IκB. Degradation of the inhibitor protein IκB allows the transcription factor NF-κB to translocate into the nucleus. In this figure, in the mammalian pathway, all proteins marked in gray have a Drosophila homolog.

There are multiple κB-sites in the regulatory regions of the antimicrobial peptide genes (Engstrom ¨ et al., 1993; Georgel et al., 1993, Roos et al., 1998). The sequence of these sites differs slightly from the mammalian sequence, having the consensus sequence 5′ -GGGRAYYYY-3′ (Hultmark, 1993). Some of the differential expression of the antimicrobial genes could be due to combinations of Rel proteins binding at these sites. Evidence of this is the fact that the Rel homology domains of Dl and Dif have differing affinity for the κB-sites in the regulatory regions of CecropinA1 and Diptericin (Diptericin A) (Gross et al.,

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1996), although in vivo neither D1 nor Dif appears to be necessary for the induction of these genes (Manfruelli et al., 1999; Meng et al., 1999). In vertebrates these pathways act to ensure an efficient innate immune response and thus activation of the acquired immune response. In Drosophila, this activation leads to the production and secretion of the antimicrobial peptides into the hemolymph. The best characterized of these pathways in the Drosophila immune response are the Toll/Dif and Imd/Relish pathways. A. THE TOLL PATHWAY As stated previously, in mammals and Drosophila, the Toll-like receptors are major regulators of the innate immune response (Wasserman, 2000; Anderson, 2000). All Toll-like receptors have very similar domains. The extracellular regions contain two types of domains, one contains multiple leucine-rich repeats and the other is a cysteine-rich domain. Following a single-membrane spanning region, they share approximately 200 amino acids of sequence similarity with the cytoplasmic domain of the IL-1R, which is now known as the Toll/IL-1R (TIR) domain (Rock et al., 1998; Tauszig et al., 2000). From the C-terminal to the TIR domain, sequence conservation is lost. There are nine genes encoding Toll-like receptors in Drosophila (Tausig et al., 2000) The original Toll protein was reported as a receptor involved in Drosophila embryonic dorsal–ventral patterning (Belvin and Anderson, 1996). In embryonic development, this pathway is activated when a serine protease cascade involving gastrulation-defective, Snake, and Easter processes the Toll-ligand Spätzle. Processing of Spätzle leads to the activation of the Toll receptor on the ventral side of the embryo. The activated Toll receptor then signals via the kinase Pelle and the novel adapter protein Tube to a complex composed of the Drosophila IκB homolog Cactus and a protein dimer consisting of the Rel protein Dorsal. This signaling event leads to the phosphorylation and degradation of Cactus and to the phosphorylation and nuclear translocation of the Dorsal homodimers (Belvin and Anderson, 1996; Drier et al., 1999). Later this same pathway was discovered to be an integral part of the Drosophila immune response (Rosetto et al., 1995; Lemaitre et al., 1996). As in embryos, during the immune response proteolytic cleavage of Spätzle by a serine protease cascade leads to activation of Toll (Fig. 2). Recently, it was reported that a loss-of-function mutation in a gene encoding for a hemolymph serpin, or serine protease inhibitor, necrotic leads to constitutive processing of Spätzle and thus to continuous activation of the Toll pathway (Levashina et al., 1999). In Toll or ¨ Spatzle loss-of-function mutants this constitutive signaling is lost, but not when necrotic was crossed in with gastrulation-defective or snake. This shows that the serpin necrotic required to block constitutive activation of a serine protease(s) involved in Spätzle processing is different from those involved in embryonic signaling. Since an activated endogenous ligand is necessary, and sufficient, for Toll


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to signal in the immune response, it seems unlikely that Toll itself is a patternrecognition receptor. This differs from mammalian TLR4, TLR2, and TLR9, which have been shown to directly interact with lipopolysaccharide, peptidoglycan, and CpG DNA, respectively (Rhee and Hwang, 2000; Tapping et al., 2000; Yoshimura et al., 1999; Takeuchi et al., 1999; Hemmi et al., 2000). Since Toll receptors have no intrinsic kinase activity of their own, they require downstream kinases to transmit their cytoplasmic signal. In Drosophila, the first such kinase described downstream of the Toll receptor was Pelle (Fig. 2). Pelle is a serine/threonine kinase homologous to mammalian interleukin-1 receptor-associated kinase (IRAK) (Belvin and Anderson, 1996). IRAK is a kinase that rapidly associates with the intracellular domain of IL-1 receptor upon binding of IL-1 (Croston et al., 1995; Cao et al., 1996a). Exactly how Pelle interacts, and functions, downstream of Toll is still unclear. In mammals, the story became a little more clear with the discovery of the adaptor protein MyD88 being necessary for Toll-like receptor signaling (Medzhitov et al., 1998) (Fig. 2). Knockout mice deficient for MyD88 are unable to respond to endotoxin, and the signaling pathways of both TLR2 and TLR4 are affected (Kawai et al., 1999; Rhee and Hwang, 2000: Takeuchi et al., 2000). MyD88 contains three functional domains: an amino-terminal death domain, an intermediate domain, and a carboxyterminal Toll/interleukin-1 receptor domain (Hultmark, 1994; Hardiman et al., 1996). It was discovered that MyD88 interacts directly, via its Toll/interleukin-1 receptor domain, with the Toll/interleukin-1 receptor domain of all known mammalian Toll receptors (Bowie et al., 2000; Xu et al., 2000). It has also been shown that the death domains of MyD88 and IRAK directly interact (Wesche et al., 1997). In this way the mammalian Toll receptors are connected to the kinase IRAK (Fig. 2). Recently, the sequence from the Drosophila genome project was reported and a gene encoding for a MyD88 homolog was predicted. This leads to the possibility that, similar to mammals, Toll is connected to Pelle via the Drosophila MyD88. IRAK has also been shown to interact directly with TRAF6 (Cao et al., 1996b) (Fig. 2). TRAFs were originally characterized as being downstream of the TNF receptor pathway, thus their name TNF-receptor-associated factors (TRAF). The interaction of IRAK with TRAF6 leads to TRAF6 phosphorylation and interaction with another protein called evolutionarily conserved signaling intermediate in Toll (ECSIT) pathways (Cao et al., 1996a; Kopp et al., 1999). TRAF6 and ECSIT are known to be necessary for NF-κB activation downstream of the Toll and interleukin-1 receptor pathways (Kopp et al., 1999; Yang et al., 1999; Cao et al., 1996a). Recently a Drosophila TRAF homolog and a homolog of ESCIT were cloned (Zapata et al., 2000; Kopp et al., 1999). Drosophila TRAF1, a homolog of mammalian TRAF4, was shown to directly interact with Pelle kinase, and it was determined by coimmunoprecipitation assays that TRAF1 and Drosophila ECSIT also directly associate. Furthermore, overexpression studies

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using ECSIT in a Drosophila immunocompetent cell culture induced the transcription of the antimicrobial genes in a manner similar to a gain-of-function Toll construct (Tl10B) or by infection with lipopolysaccharide (Kopp et al., 1999). These data suggest that Drosophila TRAF1 and ECSIT may be involved in the immune response downstream of Pelle kinase, and thus the Toll receptor. A note of interest may be the fact that there is also a Drosophila homolog of mammalian TRAF6 in the genome, called TRAF2. It will be interesting to see if, similar to mammals, this TRAF is also involved in regulating the Drosophila immune response. Activation of the Toll receptor also causes the adapter protein Tube to be recruited to the membrane (Belvin and Anderson, 1996). The exact function of Tube is unknown, and no mammalian homolog has been identified, but Tube may be involved in the regulation of the Rel proteins. In development, Tube is associated with Pelle via their respective death domains (Schiffmann et al., 1999; Xiao et al., 1999) (Fig. 2). The interaction of Tube with Pelle leads to the phosphorylation of Tube (Shen and Manley, 1998). Tube was also shown to directly interact with Dorsal, and Tube and Pelle oligomerization is necessary for the translocation of the Dorsal to the nucleus (Yang and Steward, 1997; Grosshans et al., 1999) (Fig. 2). Whether or not these interactions are important for Toll signaling during the immune response has yet to be determined, but the fact that Tube is required downstream of Toll for activation of the antifungal gene Drosomycin points to the importance of Tube in immunity (Lemaitre et al., 1996). In the mammalian immune response, once TRAF6 is activated by IRAK it can then interact with a MAP kinase kinase kinase (MAP3K) (Burns et al., 1998) (Fig. 2). This interaction may be important for the activation of the MAP3K. Many MAP3Ks require autophosphorylation for their activation, and this interaction with TRAF6 may be a method for bringing the MAP3Ks into contact for their activation. In mammals, different MAP3Ks have been shown to be involved in the activation of NF-κB. The best studied of these are NF-κB- inducing Kinase (NIK), MAP kinase kinase kinase 1 (MEKK1), and TGFβ-activated kinase 1 (TAK1) (Hatada et al., 2000; Takaesu et al., 2000). Hematopoietic progenitor kinase 1 (HPK1) and mixed lineage kinase 3 (MLK3) have also been implicated in the activation of NF-κB (Hu et al., 1999; Hehner et al., 2000). There are no good homologs for NIK or MEKK1 in Drosophila. On the other hand, there is a TAK1 homolog in Drosophila, which has been shown to function during embryonic development. By expressing a dominant negative form of Drosophila TAK1 during embryonic development, it was evident that functional TAK1 is necessary during dorsal closure (Takatsu et al., 2000). Though it has yet to be shown if Drosophila TAK1 signals in any of the immune pathways, in mammals TAK1 is known to signal downstream of TNF, IL-1, and the Toll receptors TLR4 and TLR2 (Ninomiya-Tsuji et al., 1999; Irie et al., 2000). In mammalian


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cell lines, recent experiments using an inactive mutant of TAK1 suppressed NFκB activation downstream of IL-1R, TLR4, and TLR2 (Irie et al., 2000). It was also shown recently that TAK1 interacts with TRAF6 in an IL-1-dependent fashion (Takaesu et al., 2000). The other two MAP3K possibilities, HPK1 and MLK3, have not yet been studied in Drosophila, though cDNAs for both genes do exist showing that they are expressed in Drosophila. To date, there is no evidence for HPK1 or MLK3 signaling in the mammalian TNF, IL-1, or Toll pathways, but this does not mean they are not involved in Drosophila immunity. In mammals, activation of the Toll pathway leads to the phosphorylation and degradation of IκB (Hatada et al., 2000). This phosphorylation is achieved by the IκB kinase (IKK) complex (Fig. 2) consisting of two serine threonine kinases (IKKα/IKKβ) and a regulatory subunit known as IKKγ (Karin and Delhase, 2000). Another IκB kinase, IKKǫ, is also able to phosphorylate IκB, leading to its ubiquitination and then degradation by the proteasome (Peters et al., 2000). The IKKs are themselves activated when phosphorylated by the various MAP3Ks. Two homologs to the human IκB kinases can be found in Drosophila. One was called IKKβ or DLAK and is closely related to human IKKα and IKKβ (Kim et al., 2000b; Silverman et al., 2000), and the presence of a second IKK homolog similar to human IKKǫ can be deduced from the sequence of the Drosophila genome. A homolog to the regulatory subunit IKKγ has also been reported (Silverman et al., 2000; Rutschmann et al., 2000b). Mutants have so far been isolated for Drosophila IKKβ and IKKγ (Lu et al., 2001; Rutschmann et al., 2000b). Cactus is the Drosophila IκB homolog known to be downstream of Toll both in development and in immunity (Belvin and Anderson, 1996; Lemaitre et al., 1996). Similar to the mammalian IκBs, Cactus is phosphorylated on its Nterminal regulatory region. This phosphorylation leads to ubiquitination by the Slimb-containing ubiquitin–ligase complex and is then degraded by the 26S proteasome (Spencer et al., 1999). The identity of the kinase phosphorylating Cactus is still unknown. Although IKKβ can phosphorylate Cactus in vitro (Kim et al., 2000b), Drosophila IKKβ and IKKγ are dispensable downstream of Toll signaling to Cactus in the immune response (Silverman et al., 2000) and therefore are probably not involved in this pathway. This is supported by the observation that loss of Drosophila IKKβ or IKKγ has little effect on the inducibility of the Toll-dependent gene Drosomycin (Lu et al., 2001; Rutschmann et al., 2000b). It is possible that the Drosophila IKKǫ kinase homolog is involved in the phosphorylation of Cactus in a Toll-dependent fashion. Degradation of Cactus leads to the translocation of the Rel factors Dorsal or Dif to the nucleus. In larvae, the translocation of Dif or Dorsal into the nucleus is sufficient to activate Drosomycin induction (Manfruelli et al., 1999; Meng et al., 1999). A partial redundancy can also be seen in embryonic development. Normally, Dorsal is the Rel factor downstream of Toll signaling in embryonic

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dorsal–ventral patterning, whereas Dif is not normally expressed in embryos. However, it has been shown that the expression of Dif in embryos deficient for Dorsal partially rescues the Dorsal developmental phenotype (Stein et al., 1998). In adults, Dif is absolutely required for the induction of Drosomycin downstream of the Toll receptor pathway. Recently, one more level of regulation downstream of Toll signaling was described; members only encodes a Drosophila nucleporin protein necessary for the translocation of Dif and Dorsal, but not Relish, into the nucleus (Uv et al., 2000). All macromolecular trafficking in and out of the nucleus occurs via the nuclear pore complex. Transport through these pores requires soluble receptors. These receptors bind to the cargo that will transport into the nucleus, and at the pore bind to specific docking proteins. This docking allows for the translocation of the cargo into the nucleus. In a members only mutant, Dif and Dorsal cannot enter the nucleus and Drosomycin induction is inhibited. This is the first evidence that a specific nucleoporin regulates the translocation of Rel proteins during immunity. Since more than one million molecules pass through the nuclear envelope every minute, nucleoporin specificity may be required to allow for the rapid transport of the Rel proteins into the nucleus during the immune response. B. THE IMD/RELISH PATHWAY The other pathway known to be involved in the Drosophila immune response that has been studied extensively leads to the activation and translocation of another Rel factor called Relish (Fig. 3). Relish activity is required for the induction of all the antimicrobials (Hedengren et al., 1999; 2000). As stated previously, Relish is a homolog of the mammalian p105 protein. Similar to mammalian p105, Relish consists of both an N-terminal Rel homology domain and a C-terminal ankyrin-repeat IκB- like domain (Dushay et al., 1996). The signaling pathway leading from infection to Relish activation is still poorly understood. The most recent data point to Relish being regulated by a pathway most similar to mammalian TNF receptor signaling (Fig. 3), though there is no TNF receptor homolog in Drosophila. The cell-surface receptor needed to activate the Relish pathway has to date not been discovered. Since Relish seems to be most active in inducing the transcription of the antibacterial genes, it might be expected that the cell-surface receptor is sensitive to lipopolysaccharide or peptidoglycan signaling. In mammals, TLR4 and TLR2 are involved in signaling downstream of lipopolysaccharide and peptidoglycan, respectively. Besides Toll, there are eight other Toll-like receptors in Drosophila, and it is possible that one or more of these receptors is involved in signaling to Relish. One possibility could be the Toll-like receptor 18-Wheeler. In an 18-wheeler mutant, the induction of the antimicrobial gene Attacin A is severely reduced, and the induction of another antimicrobial gene, CecropinA1,


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FIG. 3. The Imd/Relish pathway. Activation of the Drosophila immune response leads to two pathways responsible for regulating Relish cleavage in the immune response; One pathway consists of the IκB kinase complex that is necessary for Relish phosphorylation, and the other consists of the Drosophila caspase Dredd upstream of Relish cleavage. The identity of imd is still unknown, but in an imd mutant Relish cleavage and translocation into the nucleus are inhibited. The regulation of Relish in the Drosophila immune response is reminiscent of the mammalian TNF receptor pathway that bifurcates at the membrane immediately after the receptor. One pathway leads to activation of the IκB complex and thus to NF-κB translocation, while the other pathway leads to the activation of Caspase-8. Many of the proteins involved downstream of TNF signaling (those marked in gray) have Drosophila counterparts that may also be involved in the regulation of Relish processing and translocation during the immune response.

is also reduced (Williams et al., 1997). The idea that 18-Wheeler would be the main regulator of Relish processing is unlikely, since in a Relish mutant the induction of the antibacterial gene Diptericin is completely abolished, and in an 18- wheeler mutant Diptericin induction is normal (Hedengren et al., 1999; Williams et al., 1997). Other possibilities are that another of the Drosophila Toll-like receptors regulates Relish, more than one Toll-like receptor is able to activate Relish, or a pathway other than the Toll receptors is involved in Relish activation. The immune deficiency (imd) locus is mostly involved in activating the antibacterial response and does not affect the Toll responsive gene Drosomycin

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(Lemaitre et al., 1995; Corbo and Levine, 1996). The identity of imd is still unknown, but in an imd mutant Relish cleavage and translocation into the nucleus are inhibited (S. Stoven, ¨ personal communication). This points to imd being upstream of Relish in the immune response (Fig. 3). Surprisingly, the processing of Relish is very different from that of mammalian p105. Similar to IκB, when mammalian p105 is phosphorylated the ankyrin domain is then ubiquitinated and targeted for degradation by the proteasome. Recently, it was found that Relish is activated by endoproteolytic cleavage in response to bacterial infection (Stoven ¨ et al., 2000). This cleavage leads to the production of a Rel homology N-terminal domain, which translocates to the nucleus, and an ankyrin C-terminal domain, which, unlike other IκB-like factors, is stable and remains in the cytoplasm. Inhibitors that block proteasome activity had no effect on Relish activation and translocation, indicating that another protease must mediate the signal-dependent processing of Relish (Stoven ¨ et al., 2000). For Relish to be cleaved it must first be phosphorylated. Silverman et al. (2000) have identified the Drosophila IκB kinase complex, consisting of Drosophila IKKβ and IKKγ , as being required for the activation of Relish cleavage (Fig. 3). They show that active IKKβ is able to phosphorylate Relish, and that this phosphorylation is necessary for Relish cleavage. Normally, Relish is phosphorylated and cleaved in response to lipopolysaccharide, but when a dominant negative form of Drosophila IKKβ was expressed in cell culture Relish cleavage was abolished. This shows that activated IKKβ is necessary upstream of Relish processing in the immune response. Silverman et al. (2000) showed by RNA interference in cell culture that IKKβ and IKKγ are necessary to induce the antibacterial genes (Attacin A, Diptericin A, and CecropinA1) in response to lipopolysaccharide. The loss of IKKβ and IKKγ was not sufficient to block the induction of Drosomycin by dominant active Pelle (simulating an active Toll pathway) (Silverman et al., 2000). This is further evidence of both IKKβ and IKKγ being involved in the Relish and not the Cactus pathway. Phosphorylation of Relish is not enough to induce its translocation to the nucleus and thus not sufficient for Relish activity. Once Relish has been phosphorylated it must be cleaved for it to function as a transcription factor in the immune response. In genetic screens for mutants that affect the immune response, Elrod-Erickson et al. (2000) and Leulier et al. (2000) found that loss of function mutations in the Dredd gene inhibited antibacterial gene induction but did not affect Drosomycin. Dredd is the Drosophila homolog of caspase-8 (Chen et al., 1998). A caspase being involved in the regulation of the Drosophila immune response was a surprise, considering that caspases are usually described as inducers of apoptosis. However, recently it was reported that in the mammalian immune response caspase-8 was active downstream of the TNF receptor and was necessary for NF-κB translocation into the nucleus (Chaudhary et al., 2000).


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It is now evident that functional Dredd protein is necessary upstream of Relish for Relish cleavage to occur in response to lipopolysaccharide (Fig. 3). These data lead to the possibility that two pathways are responsible for regulating Relish cleavage in the immune response: One pathway consists of the IκB kinase complex that is necessary for Relish phosphorylation, and the other consists of the Drosophila caspase Dredd upstream of Relish cleavage. This situation is very reminiscent of the mammalian TNF receptor pathway that bifurcates at the membrane immediately after the receptor. In mammals, caspase-8 requires the adaptor protein FADD for its activity (Hu et al., 2000) (Fig. 3). Recently, a Drosophila homolog of FADD, dFADD, was cloned and shown to be required for Dredd’s activity during apoptosis (Hu et al., 2000). It is not yet known if dFADD is active upstream of Dredd, and thus upstream of Relish, in the immune response. Since different TRAFs are known to function in the TNF receptor pathway, and it seems as if this pathway is active in the regulation of Relish, it will also be interesting to see if any of the Drosophila TRAFs are involved in Relish activation. Also, it is not yet known if MAP3Ks are active upstream of the Drosophila IKK complex, although, since TAK1 is active in the TNF receptor pathway upstream of the mammalian IKK complex, it is a plausible candidate to be involved in regulating Relish during the Drosophila immune response. C. GATA FACTORS Another transcription factor involved in Drosophila development and larval immunity is the GATA factor Serpent. Serpent is necessary during embryonic development for the proper differentiation of hemocytes and fat body (Rehorn et al., 1996; Riechmann et al., 1998). In addition to the κB sites found in the regulatory regions of antimicrobial genes, GATA and GAAA sites have also been described (Kadalayil et al., 1997; Georgel et al., 1995; Engstrom, ¨ 1999). Unlike Rel proteins, Serpent is constitutively found in the fat body nucleus bound to its target sites. Antibodies against Serpent have found it bound to the GATA site in the regulatory region of the antimicrobial gene CecropinA1 prior to infection. In her review, Engstrom ¨ (1999) suggests that it is probably the interaction of Rel proteins with Serpent that induces the antimicrobial genes during infection. Though this may be true for the larval immune response, it was shown that Serpent is completely dispensable during the adult immune response. Evidence for Serpent involvement in larval immunity was provided by Roos et al., (1998), who reported that both κB and GATA regulatory elements were required for the full induction of the CecropinA1 gene. Constructs containing either the GATA or the κB elements can induce CecropinA1 to low levels, but both the κB sites and the GATA sites were required for full induction (Roos et al., 1998). These elements have also been reported in the regulatory regions of other antimicrobial genes (Engstrom, ¨ 1999). These data lead to the model

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that when a Drosophila larva is challenged the Rel factors translocate to the nucleus and once there interact with GATA factors as well as other factors to regulate the immune response. D. DIFFERENTIAL EXPRESSION OF ANTIMICROBIAL GENES The first inducible antibacterial peptide recovered from an insect was Cecropin, recovered from a bacterially challenged diapausing Hyalophora cecropia pupae (Hultmark et al., 1980; Steiner et al., 1981). Since that time more than 170 antimicrobial peptides and proteins have been discovered from various insects. When infected, Drosophila produce a battery of antimicrobial peptides that are secreted into the hemolymph. The regulatory regions of a few of these antimicrobial genes have been characterized molecularly. Differences in their sequence and organization suggest that they each have the capability of being differentially regulated (Engstrom, ¨ 1999). The signaling pathways that control the induction of the various antimicrobial genes during the immune response are just now beginning to be elucidated. As described earlier, one pathway known to control antimicrobial gene expression goes from the Toll receptor to the Rel protein Dif. The Toll pathway seems chiefly to control the expression of the antifungal gene Drosomycin. In Toll mutants, the induction of Drosomycin is severely reduced, while the induction of the antibacterial genes is only slightly affected. In toll gain-of-function or cactus loss-of-function mutant flies, Drosomycin is constitutively induced (Lemaitre et al., 1996). The Toll pathway also seems to be involved in the regulation of two other antimicrobials, Defensin and Cecropin (Lemaitre et al., 1996; Manfruelli et al., 1999; Rutschmann et al., 2000a). While a Toll gain-of-function mutation is not sufficient to induce either of these genes, Defensin and Cecropin induction is reduced in loss-of-function Toll pathway mutants, including Dif (Lemaitre et al., 1996; Rutschmann et al., 2000a). Most recently, Toll was also reported to be involved in the induction of new attacin genes (Attacin C and Attacin D) and a new diptericin gene (Diptericin B) (Hedengren et al., 2000). Another Drosophila Toll-like receptor, 18-wheeler, is involved in controlling the expression of the antimicrobial genes Attacin A and Cecropin A1 but has no affect on Diptericin A or Drosomycin (Williams et al., 1997). Hedengren et al. (2000) also report that 18-wheeler is necessary for wild-type induction of the Attacin C gene. Since the effect of loss-of-function 18-wheeler is strongest on the attacins, this may be a special pathway just for attacin induction. The antibacterial genes are also known to be under the control of the Imd/ Relish pathway. When filies, mutant for genes involved in this pathway, are infected all of the antimicrobial genes fail to induce at wild-type levels (Lemaitre et al., 1995, 1996; Corbo and Levine, 1996; Hedengren et al., 1999; Leulier et al., 2000; Silverman et al., 2000). The most severely reduced antimicrobials are


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Diptericin A, Cecropin A1, and Defensin, while the antifungal gene Drosomycin and the antimicrobial gene Metchnikowin are still inducible. There are some discrepancies between different reports of antimicrobial gene regulation, and some of these discrepancies could be due to varying methods of inducing the immune response. While some groups prefer to infect with only Gram-negative or Gram-positive bacteria, others use a mixture. Still, a review of the research of antimicrobial induction may reveal the existence of classes of genes that are regulated in a similar fashion. When flies are infected with fungi or Grampositive bacteria, the antifungal gene Drosomycin requires the Toll-Dif pathway for its induction (Lemaitre et al., 1996; Rutschmann et al., 2000a). Yet, when flies are infected with Gram-negative bacteria, then Drosomycin requires the Imd/Relish pathway for its induction (Hedengren et al., 1999). CecropinA1 seems to be regulated in a similar fashion. When Relish mutants are infected with Enterobacter cloacae, a Gram-negative bacteria, Cecropin A1 fails to induce, but when Relish mutants are infected with Micrococcus luteus (a Grampositive bacteria) Cecropin A1 is still somewhat inducible (Hedengren et al., 1999; Leulier et al., 2000). The antibacterial genes Attacin A and Diptericin A seem to be mainly under the control of the Imd/Relish pathway, no matter what infectious agent is used. Defensin seems to require both the TI/Dif pathway and the Imd/Relish pathways for its induction, no matter what is used for infection (Rutschmann et al., 2000a; Leulier et al., 2000). Another antimicrobial with both antibacterial and antifungal properties is the proline-rich peptide Metchnikowin. Metchnikowin is still inducible in Toll pathway mutants and in imd mutants but is not inducible in Tl/imd double mutants (Levashina et al., 1999). By being under the control of both pathways, Metchnikowin may be equally inducible by bacteria or fungi. Even here, there is some controversy; Hedengren et al. (1999) report that Metchnikowin is severely reduced in a Relish loss-of-function mutant when infected with Gram-negative bacteria alone. Again, this controversy is probably due to the different methods used to infect flies. V. Concluding Remarks

Though this review is not exhaustive, I have tried to describe the immune proteins and pathways involved in regulating the Drosophila immune response. There are known components of the immune response I have not included in this review, such as hematopoiesis and cellular immune reactions. The reactions of the hemolymph, hemocytes, and fat body are complex and presumed to have a close connection with each other. This leads to many interesting problems that need to be addressed to determine how the Drosophila immune response is regulated, and points to the possibility of finding novel factors and pathways as yet unknown in innate immunity. It is known that the regulation of the antimicrobials is complex, too complex to be explained by the two major immune

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pathways presented here. Also, how an invading organism is recognized, either by recognition of lack of self or by the interaction of recognition molecules with microbial epitopes, is still nuclear. Besides lack of self, or microbial epitopes, another means of activation or recognition could be the proteases secreted by some pathogenic organisms that could themselves activate a protease cascade or signaling molecules leading to an immune response. It is also known that parasitic wasps use virus-like particles to try to avoid the immune system. The mechanisms used to defend against these potential problems are still unknown and lead to many interesting questions. It is also becoming clear that regulation of the larval and adult immune responses is different. Using the power of Drosophila genetics and the molecular techniques now available, it is clear that the fruit fly is an essential model organism in the study of innate immunity. ACKNOWLEDGMENT I would like to thank Dr. Dan Hultmark for his assistance in preparing this manuscript.

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Functional Heavy-Chain Antibodies in Camelidae VIET KHONG NGUYEN, ALINE DESMYTER, AND SERGE MUYLDERMANS Vlaams Interuniversitair Instituut voor Biotechnolgie, Vrije Universiteit Brussel, Sint Genesius Rode, 1050 Bruxelles, Belgium

I. Introduction

The molecular biodiversity within vertebrates is enormously large. However, the discovery of bona fide antibodies devoid of light chains occurring exclusively in Camelidae was one of the major surprises of molecular immunology in the past decade. These so-called heavy-chain antibodies (HCAbs) bind antigen solely with one single variable domain, referred to as VHH. Obviously, the appearance of HCAbs requires the acquisition of multiple events to allow their generation and maturation into functional molecules and opens up new perspectives in antibody engineering. Here, the composition of these curious antibodies is described, and the gene adaptations that occurred in the Camelidae and allowed the generation of such HCAbs are discussed. We also speculate on the emergence of these dedicated genes within the common ancestor of camel, dromedary, and llama and how these genes evolved thereafter. We focus on the steps that are involved in the ontogeny of a HCAb, starting from distinct genes. Finally, the current methods that are employed to isolate antigen-specific VHHs, either polyclonal or monoclonal, are reviewed. The primary and tertiary structure of the antigen-specific VHHs is used to explain their biochemical properties. Their soluble behavior, stability, diverse structural repertoire of the antigen-binding site, and antigen-binding capacity are emphasized. Finally, the biotechnological fields where VHHs might have competitive advantages over scFvs or any other antigen-binding fragment derived from conventional antibodies are briefly summarized. II. Natural Occurrence of Heavy-Chain Antibodies in Camelidae

A heavy-chain antibody (HCAb) has been defined as an immunoglobulin devoid of light (L) chains (Franklin et al., 1964). The presence of HCAbs in human serum has been reported as a pathological disorder. It seems that, besides the absence of a L chain, also the heavy (H) chain of the HCAb is truncated. It was found, largely from mouse studies, that the smaller H chain is caused by a somatic deletion involving large and variable parts of the VH and the CH1 (Seligmann et al., 1979). Obviously, in absence of the L chain and various parts of the VH, it is safe to conclude that such antibodies will fail to recognize an antigen (Cogne et al., 1989). The human HCAbs are therefore supposedly nonfunctional in antigen binding. 261 C 2001 by Academic Press. Copyright All rights of reproduction in any form reserved. 0065-2776/01 $35.00

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An accidental observation led to the discovery of large amounts of HCAbs in camel serum (Hamers-Casterman et al., 1993). Differential protein A and protein G affinity chromatography could separate at least three different antibody fractions. One fraction contained conventional antibodies composed of two H and two L chains. But two fractions contained antibodies that clearly missed the L chains. Moreover, the H chain within these fractions had a reduced MW compared to that of the conventional antibody, conform the H chain of human or mouse HCAbs. It was immediately noticed that the serum of camels (Camelus bactrianus and Camelus dromedarius) and llamas (Lama glama, Lama pacos, Lama guanoco, and Lama vicugna) shared the presence of high titers of functional HCAbs (Hamers-Casterman et al., 1993). These animals belong to the family of Camelidae, the only surviving family within the suborder of Tylopoda that is taxonomically placed with Ruminantia and Suiformes in the order of Artiodactylas. However, to the best of our knowledge, the animals belonging to the Ruminantia (bovines, gazelles, giraffes, deer) and Suiformes (Suidae [pigs] and Hyppopotamidae) do not possess large amounts of HCAbs in their serum. Quantification of the different fractions indicated that, in general, the conventional antibodies present within a dromedary or llama is around 3 mg/ml serum, and the HCAb fractions total up to 3 mg/ml as well (Table I) (Hamers and Muyldermans, 1998; van der Linden et al., 2000). Of course, the exact ratio differs among animals and time of sampling the serum even from the same animal. However, the equivalent amount of HCAb and conventional antibodies present in the serum suggests that the humoral immune system of camels and llamas relies (equally) on both types of antibodies. All the conventional IgG antibodies of llama and camel bind to protein A and protein G. In contrast, the HCAbs can be divided into two fractions. Both fractions bind protein A but only one adsorbs on protein G (Table I). The apparent MW of the proteins in the former fraction in llamas and dromedaries TABLE I IgG ISOTYPES IN CAMELIDAE A G IgG1a IgG1b IgG2a IgG2b IgG2c IgG3

+ + + + + +

MW MW Camel Llama

+ 3 mg/ml 55,000 55,000 + 55,000 55,000 − 1 mg/ml 45,000 42,000 − 42,000 − 45,000 42,000 + 2 mg/ml 43,000 43,000

Hinge Sequence ELKTPQPQSQPE - - - - - - - EPHGG- - - - - - - - - - - EPKIPQPQPKPQPQPQPQPKPQPKPEPE EPKIPKPQPQPQPQPQPNPTAE - - AHHSEDPS - - - - - - - - - GTNEV- - - - - - - - - - - -

-

-CRCPKCP -CTCPQCP -CTCPKCP -SKCPKCP -SKCPKCP -CKCPKCP

19 aa 12 aa 35 aa 29 aa 15 aa 12 aa

Note. A = adsorbs on protein A; G = adsorbs on protein G; MW Camel and MW Llama denote the apparent MW of the H chain of each. Last column gives number of amino acids (aa) in the hinge.

FUNCTIONAL HEAVY-CHAIN ANTIBODIES IN CAMELIDAE

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FIG. 1. Schematic representation of conventional antibody and naturally occurring heavy-chain antibody in Camelidae. The folded domains of the polypeptide chains are shown by cylinders. ∼S∼ denotes the interchain disulfide bonds.

is about 43,000 (Hamers-Casterman et al., 1993; van der Linden et al., 2000). In contrast, the MW of the proteins in the latter fraction differs in dromedaries and llamas: In llamas, the MW is less than 43,000, whereas in dromedaries it is larger (∼45,000). Different llama and dromedary clones carrying the cDNA of γ -isotypes have been sequenced. These sequences proved that the HCAbs contained an intact variable domain, but the entire CH1 domain was deleted. Consequently, the variable domain was immediately followed by the hinge sequences in the H chains of the HCAbs in Camelidae (Fig. 1). Combining the data of the MW of the nonreduced HCAbs and the cDNA sequence analysis indicated that the HCAbs will be bivalent and will bind the antigen by one single domain only. To distinguish the variable domain of the heavy chain in conventional antibodies with that of HCAbs, we refer to the latter as VHH instead of VH. The H chains of conventional antibodies of Camelidae contained the expected VH–CH1–hinge–CH2–CH3 organization (Fig. 1). Taking the hinge sequence—the most diverse part—to distinguish the isotypes, we found that the llama contains six isotypes, and the dromedary seems to have five expressed γ -genes. In both species, two conventional H chains belonging to two isotypes (γ 1a, γ 1b) were identified. Originally, the dromedary IgG1, IgG2, and IgG3 were named according to the decreasing MW of the dromedary immunoglobulins (Hamers-Casterman et al., 1993). The first available cDNA sequences of dromedary IgG hinges of HCAbs showed a hinge of 35 or 12 codons. It was therefore hypothesized that the shorter hinge of 12 amino acids would constitute the IgG3 fraction, and the

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longer 35-amino-acid hinge would be present in the serum IgG2 fraction. The same hinge sequences were found in the llama cDNAs, and the attribution to IgG2 and IgG3 was maintained. Additional sequences revealed the presence of an immunoglobulin with a 29-amino-acid-long hinge in the llama (Vu et al., 1997). Due to the high sequence homology with IgG2 we named the antibodies with 35- and the 29-amino-acid-long hinges IgG2a and IgG2b, respectively. However, the llama immunoglobulin fraction that does not bind to protein G has an apparent MW of 42,000, definitely smaller than the protein-G-binding HCAb isotype of dromedary. Therefore, it might be that the attribution of the long hinge to the protein G minus fraction requires revision. The presence of an isotype with a 15-amino-acid-long hinge was found afterwards both in the llama and dromedary (van der Linden et al., 2000; Woolven et al., 1999). Due to sequences within the CH2 domain we are sure that it belongs to the HCAb fraction that is not retained on protein G columns, and it is therefore named IgG2c. The Pro–Xaa (Xaa = Gln, Lys, or Glu) repetition in the hinge sequences of llama IgG2a and IgG2b and dromedary IgG2a suggests that it might adopt an extended rigid-rod conformation. The TolB and procyclin sequences were found to have a similar repeat, and structural data of these peptides revealed an extended conformation (Evans et al., 1986; Roditi et al., 1989). The gene for the 35-amino-acid hinge isotype was also extracted from a genomic library of dromedary liver (Nguyen et al., 1999). The length of the dromedary γ 2a genetic hinge (105 nc) is comparable in size to the human γ 3 that is encoded in a tandem array of four separated exons (Huck et al., 1986). In contrast, the dromedary γ 2a is contained within one single exon with an eightfold repeated sequence of the hexameric motif CAACCA. This region of 48 nc is embedded in a stretch of 68 bp that has 87% sequence identity to the coding region of the Ac9 maize transposable element (Nguyen et al., 1999). Therefore, the immunoglobulin hinge sequences were hypothesized to originate from an exon with inserted transposon that was later excised to different extents. This would explain the large variability in sizes of the hinges among both the γ isotypes and animals. The most important difference between the HCAb of Camelidae and human HCAb or mouse myeloma mutants is that only the former is functional in antigen binding. This was proven on multiple occasions. Analyzing the serum immunoglobulins of a dromedary infected with trypanosomes (Trypanosoma evansi) showed a very clear and diverse response against these parasites in the HCAb classes (Hamers-Casterman et al., 1993). In addition, the possibility of immunizing dromedaries and llamas to obtain antigen-specific immune responses in their HCAb classes is now well established (Hamers and Muyldermans, 1998; Lauwereys et al., 1998; Ghahroudi et al., 1997; van der Linden et al., 2000).


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III. Adaptations in the H Chain

Two major differences between the H chain of a conventional antibody and that of a HCAb were noticed: The CH1 domain is missing in the HCAbs and the N-terminal variable domain contains a few amino acid substitutions not found in the H-chain variable domains of conventional antibodies (Hamers-Casterman et al., 1993). These adaptations are crucial to convert the H chain from a fourchain (heterotetrameric) into a two-chain (homodimeric) antibody. A. REMOVAL OF THE CH1 DOMAIN Normally, the translation product of the immunoglobulin H-chain mRNA is directed to the endoplasmic reticulum. The H-polypeptide chain is bound by chaperon proteins, which need to be replaced by the antibody L chain before secretion of the antibody occurs. The chaperon proteins anchor the H chain at the VH and the CH1 (Henderschot, 1987; Knarr et al., 1995) at regions that overlap with the contact sites of the two domains of the L chain (Padlan, 1994). Of these, the CH1 domain plays an essential role because it harbors the most conserved and important interaction sites for the CL. Apparently, CH1-deleted H chains are deprived of the primordial binding site for the chaperon proteins and the L chain; such truncated H chains will escape form the endoplasmic reticulum because they cannot be retained by the chaperon system and they do not need the L chain for secretion. All H chains of HCAbs of the dromedary and llama lack the entire CH1 domain, but their variable domain is intact and the hinge is present, as well. This is substantially different from the situation of pathogenic human/mouse HCAbs where various parts of the VH, CH1, and hinge are deleted. The consistent absence of the entire CH1 domain in camelid HCAbs could suggest that an alternative splicing of a conventional γ -gene, as described for the isoforms of duck IgY (Magor et al., 1994), generates these HCAbs. Alternatively, Camelidae could use a special set of genes that lack the CH1 region at the genomic level or from which the CH1 exon is specifically removed after transcription. The last explanation turns out to be correct. Clones containing the dromedary γ 2a or the llama γ 2c gene have been retrieved from genomic libraries (Nguyen et al., 1999; Woolven et al., 1999). The sequences revealed the presence of a point mutation that modifies the consensus splicing signal at the 3′ end of the CH1 exon. Consequently, the splicing machinery will no longer recognize the signal, and this will remove the CH1 coding part by splicing the 3′ end of the exon coding for the variable domain with the 5′ end of the hinge exon. This genotypic point mutation is conserved in dromedary γ 2a, γ 3, and γ 2c genes and the llama γ 2c gene, suggesting that the mutation occurred in the common ancestor of Lama and Camelus. Recent expression studies with a reconstituted gene containing a rearranged VHH and the dromedary γ 2a in mouse myeloma cells

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confirmed the proper removal of the CH1 exon in this heterologous system. Therefore, there are no camel-specific factors required in this splicing event. The CH1 coding part of the dromedary γ 2a gene also contained a point mutation that would substitute the Cys208 by a Ser. This Cys is normally involved in the conserved intradomain disulfide bridge of the immunoglobulin fold. The llama CH1 exon of the identified genomic γ 2c gene contained a frame shift mutation (Woolven et al., 1999). Therefore, we can eliminate the possibility that these genes might form a typical CH1-containing H chain after an unlikely alternative mRNA processing. The presence of a CH1 domain in an immunoglobulin spaces the two antigen◦ binding sites by some 80 to 160 A within one antibody molecule (Padlan, 1994; Harris et al., 2000). This spacing is important for optimal recognition and crosslinking of repeated epitopes. The absence of the CH1 domain in a HCAb would bring the two antigen-binding sites closer together so that steric hindrance might occur during antigen binding. However, the γ 2a (and γ 2b in llama) isotypes of camelids with their rod-like long hinge could separate the two antigen-binding sites sufficiently far apart to allow the simultaneous interaction with two antigens and thus provide improved antigen capturing. The CH2 and CH3 domains harbor the effector functions. Sequence inspection of the Fc part of the HCAbs shows the preservation of Asn297–Ser–Thr, which acts as a glycosylation site. In addition, the Fc parts also contain the signals for FcR, Clq, and protein A binding sites. It seems, therefore, that the HCAbs will exert the conventional effector functions (Attachouch et al., 1997; Nguyen et al., 1999). Hence, in this respect, as well, the dromedary and llama HCAbs appear to be fully functional and an integral part of their immune system. The immunity of a newborn llama or camel has to be acquired via the colostrum since the six-layer epitheliochorial placentation of Camelidae prevents passage of the immunoglobulins to the fetus. The maternal colostrum IgG concentration of camelids can reach levels of 250 mg/ml. The presence in colostrum of IgG, IgM, and low amount of IgA has been reported (Azwai et al., 1996). The published figures suggest the presence of all IgG classes in the camel colostrum including the HCAbs. The HCAbs were also predicted to be transported through the mammary gland because of the presence of the mammary gland transport signal (Jackson et al., 1992) at the beginning of the CH2 domain of γ 2a and γ 2c and γ 3 genes. Thus, the HCAb subclasses contribute to the immunity of the newborns. This again emphasizes the importance of the HCAbs in the Camelidae. B. ADAPTATIONS IN THE VARIABLE DOMAIN The first sequencing results of VHHs immediately revealed the presence of a number of amino acid substitutions located in the part that normally interacts with the VL (Fig. 2) (Muyldermans et al., 1994). The association of the VH and


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FIG. 2. Schematic representation of the differences between VHs and VHHs. The hallmark amino acids of VH and VHH sequences are given, and the positions of the CDRs, SDRs, and H loops are indicated. The regions originating from the VH, VHH, D, and J genes are shown by arrows.

VL domain is mediated by hydrophobic interactions (Horne et al., 1982), and the amino acids that participate in the VH-VL interaction have been very well conserved throughout evolution (Chothia et al., 1985). It was, therefore, striking to find that Leu45, conserved throughout the VHs of all species (Kabat et al., 1991), was substituted by Arg45 in all the VHHs. Additional mutations between VHs and VHHs of conserved hydrophobic amino acids that are found frequently in this region are Val37Phe, Gly44Glu, and Trp47Gly (Fig. 2). It is clear that these substitutions will abolish the interaction of a VHH with a VL domain. In addition, the conserved amino acids in this region of the VH form the major interaction site of the chaperon proteins (Knarr et al., 1995). An algorithmic calculation (BlondElguindi et al., 1993) has predicted that this region of the VHH will fail to attach to the chaperon proteins (Vu et al., 1997). In conclusion, since the CH1 domain is missing and the VHH lacks the chaperon binding site, the H chain of a HCAb can be readily secreted from the endoplasmic reticulum without L-chain association. The VHH also contains additional amino acid substitutions (Fig. 2). The conserved Leu11 that in VHs normally forms a hydrophobic ball-and-stick joint with amino acids of the CH1 (Lesk and Chothia, 1988; Padlan, 1994) is replaced by Ser in dromedary VHHs. This substitution of an extended, hydrophobic sidechain by a short hydrophilic side-chain is logical considering the absence of the CH1 domain in HCAbs and is expected to increase the solubility of the molecule (Nieba et al., 1997). Nevertheless, many llama VHHs do not exhibit this mutation, suggesting that this amino acid substitution is not crucial. Other mutations in this region of the folded domain involve a frequent switch of Pro14 and Ala84 in VH sequences to Ala14 and Pro84 in VHH sequences. However, the importance of these substitutions is currently less evident. Finally, most of the dromedary VHHs contain four Cys, of which two are conserved between

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VH and VHHs and form the interdomain disulfide bond. The additional two Cys are located in the CDR1 and CDR3, respectively, and form an interloop disulfide bond. Occasionally, a Cys in found at position 45 in the dromedary or at position 50 in the CDR2 in the llama, but in these VHHs the CDR1 Cys was missing (Muyldermans et al., 1994; Vu et al., 1997). A hypervariability plot of the dromedary and llama VHH (as obtained from cDNA sequences) revealed three remarkable differences compared to VH hypervariability plots. First, the overall variability in VHHs is larger throughout the entire sequence, probably reflecting an active somatic mutation mechanism (Nguyen et al., 2000). Second, the first hypervariable region (known as CDR1) of the VHHs is extended toward its N-terminal end so that it also includes amino acids 27 to 30 (Fig. 2) (Vu et al., 1997). This suggests that these amino acids (27–30) in VHHs are also involved in antigen recognition. Third, the VHH bears a longer hypervariable region 3 or CDR3 loop (Muyldermans et al., 1994). In mouse and human VHs, this loop is on average 9 and 12 amino acids long (Wu et al., 1993). In the VHH of Camelidae we found an average CDR3 length of 16 to 17, although in llamas a considerable fraction has short CDR3 loops (Vu et al., 1997). A longer CDR3 loop suggests an increase in the actual antigenbinding surface area, and this could compensate for the absence of antigen interactions provided by the VL partner in a Fv. From the dromedary and llama VH sequences we obtained, it became evident that the CDR3 of VHHs is longer (nearly twice as long) compared to those in VHs of Camelidae. However, bovines that utilize apparently a very limited repertoire of VH genes also possess a long CDR3 (17 or more residues) (Saini et al., 1997, 1999). Remarkably, the long CDR3 of bovine VHs and that of dromedary VHHs are made rigid by a disulfide bond. Thus, among Artiodactyla, the CDR3 size and Cys content are shared by bovine VHs and camel VHHs, whereas shorter CDR3 without Cys is inherited by conventional VHs in pig and camel. The VHH hallmarks could be encoded in the germline or could be introduced by a directed somatic hypermutation process operating in conventional VH genes. To differentiate between these possibilities, we screened a genomic bank of dromedary liver DNA and this resulted in the identification of two putative functional V genes that could be used in the V–D–J recombination (Nguyen et al., 1998). The V-coding part possesses the codons for either the VH or the VHH specific hallmarks. Both germline V genes contained the regulatory octamer, Ig promoter sequences (Parslow et al., 1984), a leader exon that could function as secretion signal at their 5′ ends, and the consensus recombination signal sequence (RSS) (Ramsden et al., 1994) at their 3′ end for recombination with the D–J segments. This observation proves that distinct VH and VHH genes are imprinted in the dromedary genome, and the presence of the RSS ensures that the VHH can be rearranged to a recombined DJ segment. It further eliminates the possibility that the VHH domains are shaped by a directed somatic hypermutation mechanism.


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Subsequent PCR experiments revealed the presence of ∼40 different VHH germline genes and ∼50 VH germline genes in the dromedary (Nguyen et al., 2000). Here, again, the ratio of VH/ VHH being close to 1 supports the equal importance of HCAbs and conventional antibodies in the dromedary immune response. The study further indicated that the vast majority of the VHH germline genes contain a Cys residue in the CDR1 or at position 45. From cDNA sequences of dromedary VHHs it is known that an additional Cys within the CDR3 will allow the formation of an interloop disulfide bond with the CDR1 or with the Cys45. Although all the VHH germline genes belonged to VH family III (Schroeder et al., 1989), they could be classified in seven distinct subfamilies. Each subfamily coincides with a particular CDR2 length and the actual presence of Cys in the CDR1 or at position 45. At this moment it is uncertain whether all VHH germline genes cloned from dromedary can be used in the primary recombination event. Indeed, only five out of the seven VHH subfamilies were found after RT-PCR on blood peripheral lymphocytes (Nguyen et al., 2000). Nevertheless, for the two VHH subfamilies that have not yet been cloned as rearranged products, at least some of the their members contain the proper promotor sequence and the recombination signal sequence (conserved heptamer 23-bp spacer and nonamer sequences). Hence, it is assumed that they might be used to produce functional HCAbs. IV. The L Chains

Apparently, the HCAbs are bona fide antibodies within the camelids. The role and necessity of the L chain in the conventional antibodies of these species can therefore be questioned. To address this issue, the L chains of the dromedaries were analyzed (Legssyer et al., 1995). The SDS-PAGE of the L chains revealed two distinct bands of different MW (∼27,000 and ∼30,000). The purification of the L chains from SDS-PAGE and the N-terminal amino acid sequencing suggested the presence of both λ and κ chains in the fastest migrating band, whereas the band with reduced electrophoretic mobility seems to contain only the λ type. The lower mobility is attributed to the presence of carbohydrates on this L chain since it binds to lectins. Clones containing λ and κ sequences were obtained after RT-PCR on mRNA extracted from peripheral blood lymphocytes. All these sequences encoded polypeptides that are compatible with a normal L chain fold. However, some of the Cλ sequences contained a glycosylation signal sequence due to an Asn143– Val–Thr mutation of a Ser143–Val–Thr sequence. Also, the mouse Cλ1 and Cλ2 genes and the rabbit Cκ2 gene of b9 allotype present glycosylation sites at codon 138, 138 and 151, and 161, respectively, but it is not known whether these light chains are effectively glycosylated (Kabat et al., 1991). The glycosylation site of the dromedary Cλ is located at the top of the CL domain, where the

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carbohydrate could possibly interact with the VL or VH domain. If such interactions would occur, then the dromedary conventional antibody molecule containing the glycosylated L chain could have some interesting architectures. Much can be speculated about the importance of the glycosylated L chain in the conventional antibodies or possibly in the HCAb ontogeny; however, a direct role or function still needs to be discovered. The absence of a glycosylated L chain in the llama indicates that it might be a curiosity of the dromedary antibodies rather than a necessity. V. Heavy-Chain Antibody Generation

The exact generation mechanism of the HCAb in Camelidae remains obscure at several points. However, as mentioned above, dedicated VHH germline genes and γ genes used to form a HCAb are imprinted in the dromedary and llama genome. A separate set of VH and γ genes is employed to produce conventional antibodies. In this section, we focus on the emergence of these special genes to support this dichotomic humoral immune system, and on the ontogenic process of these unique HCAbs in Camelidae. A. EMERGENCE AND PHYLOGENY OF VHH GENES IN CAMELIDAE Like the horse, the camel has its origin in North America. Fossil records indicate that Tylopoda emerged in the Middle Eocene (∼50 million years ago) and were developed by the Upper Eocene (∼40 million years ago) (Webb, 1972; Harrison, 1985). The two Old World camels (C. dromedarius and C. bactrianus) and four New World llamas (L. vicugna, L. guanoco, L. pacos, and L. glama) have 37 pairs of chromosomes (Bianchi, 1986; Taylor et al., 1968; Koulisher et al., 1971) and are the only extant species of the taxonomic family Camelidae (suborder: Tylopoda; order: Artiodactyla). Major vertebrate classifications placed the Tylopoda in between the Ruminantia and Suiformes within the Artiodactyla (Fowler, 1997). Although the Camelidae ruminate, it is not appropriate to classify them together with Ruminantia because this suborder of the Artiodactyla have four-chambered stomachs, whereas the Tylopada have a three-chamber form and Suiformes have simple stomachs. Apparently, the capacity to ruminate has been acquired independently by the two suborders and similarities may be due to convergent evolution. The dental pattern, lack of horns, and fusion of bones of tarsus and carpus are additional anatomical differences that support a separate grouping of Ruminantia and Tylopoda. The emergence of the HCAbs in Camelidae involved the selection and fixation of multiple changes in both the variable and the constant regions of the heavy chain in a co-evolutionary process. Apparently, this happened only in the Tylopoda, since no HCAbs have been reported for other mammals, including the closely related taxonomic species such as pigs (Suiformes) and the sheep


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and bovines (Ruminantia). However, it cannot formally be excluded that the antigen-binding receptor of HCAbs of Camelidae might have evolved from a primitive HCAb type since two cartilaginous fishes (nurse shark and spotted ratfish) have been reported to possess remote HCAbs, called NAR and CH1-type 2 antibody, respectively (Greenberg et al., 1995; Rast et al., 1998). To verify the evolutionary pathway of the VHHs, a phylogenetic analysis focusing on the relationship of the camelid VHH and VH with the entire group of known VHs of other vertebrates was initiated. Among 95 vertebrate VH families, all five available phylogenetic inference methods revealed a monoplyletic cluster of the camelid VHs and VHHs. Furthermore, the bifurcation of the VHs and VHHs within this cluster indicates clearly that the VHH and VH existed in the common ancestor of the Camelus and Lama prior to their divergence. Our molecular dating for the VHH emergence was estimated to arise 82–37 M years ago, while the Lama–Camelus divergence happened ∼45–11 million years ago, according to the fossil records (Webb, 1972). Consequently, we conclude that the VHH originated from the VH after the divergence of the Tylopoda from other Artiodactyla and before the divergence of Camelus and Lama. An orthologous relationship of the VH and VHH sequences of the Camelus and Lama was also observed, confirming the above conclusion and providing evidence that the VHH evolution followed the birth-and-death model (Nei et al., 1997). Note that the camel and llama γ genes of the HCAb isotypes also have a close relationship and co-existed in their ancestors. This type of co-evolutionary process seems to be the main reason for the camelid HCAbs being apomorphic among the Artiodactylas. There is no indication for a close relationship between VHH of Camelidae and the variable domains of new antigen receptor (NAR) or the CH1 type 2 antibodies. Nevertheless, their common phenotype of homodimeric antibodies suggests that these remote events might be the outcome of an evolutionary convergence perhaps due to equivalent evolutionary constraints (Roux et al., 1998). If this is true, and taking into account the huge time gap between the emergence of cartilaginous fishes (∼550 million years ago) (Litman et al., 1999) and Tylopoda (∼45 million years ago), we might expect that HCAbs that still remain to be discovered were also acquired by other species. The molecular evolutionary analysis further revealed a higher evolutionary rate for the VHHs, because the VHH cluster exhibited a more diverse branching than that of the VHs of Camelidae. The nearly equal number of VH and VHH genes in the dromedary genome reflects a rapid expansion of the latter gene family. The cause for the generation of these diverse VHH genes can be traced. An unusually high ratio of nonsynonymous to synonymous nucleotide substitutions at framework 2 indicates a positive (Darwinian) selection (Ota and Nei, 1995) operating in this region, while the sequence in this region is highly conserved in all conventional VHs (Hsu and Steiner, 1992). This

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could concur with these VH residues requiring to interact with the VL in the conventional four-chain antibodies and the absence of such interactions for VHHs in the HCAbs. However, the higher VHH germline divergence could also be explained by the acquisition of numerous DNA recombination hotspots within the VHH genes that are less abundant in the VH genes (Nguyen et al., 1998). B. ONTOGENY OF HCABS IN CAMELIDAE 1. VHH-D-J Rearrangements and Diversity Generation The spreading of the VH and VHH genes among or within the chromosomes remains uncertain. However, there are good reasons to accept that at least some of them reside within the same locus since we could identify a VH and a VHH minigene that recombined to the same D minigene and that used the same J gene (Nguyen et al., 2000). However, this observation does not reveal the number of VH and VHH genes residing in the locus or whether they are interspersed or clustered. Interestingly, data for the llama suggest that some VHH subfamilies and γ genes might group on discrete loci because a VHH subfamily characterized by Cys50 was always associated with the γ 2b, whereas the VHH subfamilies with Cys32 or Cys33 were confined to IgG3 or G2a (Vu et al., 1997). A clustered-type gene linkage is typical of primitive fish genomes in which multiple Ig loci are present consisting of VH-D clusters or VH-D-J genes in front of a μ gene (Litman et al., 1999). Also, humans have VH and D orphan genes on chromosomes 15 and 16, while functional VH, D, J, and all heavy-chain genes reside on chromosome 14 (Tomlinson et al., 1994). The final differentiation between one or multiple IgH loci residing on one or several of the 37 chromosomes in Camelidae necessitates in situ hybridization and/or YAC cloning. After immunizing a dromedary with ovalbumin, we could identify a VHH domain recognizing the antigen with nM affinity. The VHH domain was derived from a VHH gene rearranged to a D–J, where the part encoded by the VHH germline gene did not contain any somatic mutations. This indicates that VHH germline genes can recombine with D–J and that a VHH–D–J rearrangement product can be immediately functional in antigen binding. However, it remains an intriguing question as to how HCAbs can build up an immense antigen-binding repertoire in the absence of a VH–VL combinatorial diversity. Obviously, it should rely on the innate number of divergent sequences of the VHH germline segments, on the V–D and D–J junctional diversity, and on somatic mutations introduced after the initial V–D–J gene rearrangement (Tonegawa, 1983). Indeed, species with a limited V-gene family rely heavily on important somatic mutation processes to diversify their antigen-binding repertoire (Knight, 1992; Reynaud et al., 1989). In contrast, species with an elaborate


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V-gene pool use the somatic mutations largely for antigen affinity maturation (Foote and Milstein, 1991). At first sight, the size of the VHH gene pool in the dromedary is limited because all the VHH sequences that were amplified or were found in cDNAs belong to one single family, the equivalent of family III. However, this is not a drawback since this family is also the only one used in rabbits and chickens (Knight, 1992; Reynaud et al., 1989) and has the most widespread usage and genome complexity in humans (Kirkham et al., 1992; Tomlinson et al., 1992). Moreover, after combination with a VL it can be used to target all types of antigens: haptens, oligopeptides, and large proteins (Vargas-Madrazo et al., 1995). Thus, the members of this family can create all possible paratope architectures. In addition, an impressive VHH gene duplication followed by subsequent germline diversification yielded seven VH subfamilies (Nguyen et al., 2000), of which five are certainly rearranged to D–J recombined segments. The length and variability of the CDR3 reflects the V–D–J junctional diversification processes. A long CDR3 like that of a VHH is encountered in various organisms with low VH diversity such as sharks and rays (Litman et al., 1999) and bovines (Lopez et al., 1998). Likewise, the presence of an additional disulfide bridge is also shared between VHHs and VHs of several species with low VH segment diversity—for example, shark NAR (Diaz et al., 1998; Greenberg et al., 1995), rabbit (Mage, 1987), and bovine (Saini et al., 1999). Thus, other nonrelated species have these characteristics in common with VHHs but not VHs of Camelidae. The cause of the longer CDR3 loop, often with one Cys residue, in VHH versus a shorter CDR3 loop, without Cys residue, in VHs remains enigmatic, especially considering that VH and VHH use a common set of D and J genes. The longer CDR3 loop could be explained by the usage of two D genes or, more likely, a more active terminal deoxynucleotidyl transferase activity during the V–D–J recombination or during the antigen-binding site editing (Nemazee, 2000). However, it could just reflect the selective advantage of a larger antigen-binding surface area provided by the longer CDR3 loop during the antigen selection. Obviously, a combination of these explanations remains possible as well. The presence of a Cys in the CDR3 of VHH, but not in VH, indicates that it is somatically introduced and not encoded by the D or 5′ end of the J gene. It remains surprising, at least in our dromedary VHH cDNA sequences, that the majority of the clones contain a Cys in the CDR3 region and a second Cys in the CDR1 loop (or in framework 2 at position 45). A minority of the clones lack a Cys in their CDR3 and, strikingly, in all these clones the second Cys that was encoded in the germline gene is mutated out. This specific removal of Cys codons in the CDR1 or framework 2 region in VHH–D–J recombination products without Cys in the CDR3 is an additional argument for the presence of an antigen-independent somatic mutation mechanism to expand the VHH repertoire.

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Finally, we have several reasons to assume that the antigen-binding repertoire of VHHs is strongly reshaped. The retrieval of a high percentage of funcational VHHs of known antigen specificity, with a length that could not be recovered from the PCR clones of the VHH germline genes, suggests that a geneconversion mechanism might be very active in the antibody-generation mechanism of Camelidae. We then suppose that a gene conversion often leads to improper DNA rearrangements that result in a codon deletion or insertion (Wagner and Neuberger, 1996). However, the observation of the codon insertion/deletion close to a palindromic DNA could also indicate an aberrant DNA repair mechanism active during the somatic hypermutation (de Wildt et al., 1999). Whatever the cause, of all cDNA encoding VHH genes, some 30% are off-sized, whereas this percentage is marginal in cDNAs encoding VHs. This suggests that an “off-sized” VHH is more likely to be functional than an “off-sized” VH, which makes sense since the absence of a VL association should facilitate deviation of the conserved V sequence or length. The off-sized lengths are expected to enlarge the VHH diversity, and an enlarged VHH repertoire could be beneficial for the antigen-binding capacity. The enhanced presence of the palindromic sequences that might provoke the off-sized lengths in the VHH germline genes further reveals how this beneficial signal became imprinted in the VHH germline gene. The comparison of the hypervariability plots of dromedary germline and rearranged VHH sequences demonstrated the usage of somatic hypermutation mechanism around the antigen-binding loops (Nguyen et al., 2000). In human and mouse VH sequences, these loops contain a number of conserved amino acids at key positions to maintain the loop conformation. In the dromedary VHH, however, these amino acids are substituted by homologous amino acids encoded by sequences that are known to be hotspots for somatic mutation. These key amino acids can then be easily modified so that other loop conformations become possible. The most illustrative example is located in the solvent-exposed part of the loop around the CDR1. The side chains of Ala24, Phe27, Phe29, and Met34, which form a hydrophobic cluster, and Gly26, which forms a sharp turn, fix the structure of this loop. In the VHH germline genes, these sequences are conserved except for the homologous substitution of Phe27Tyr. Probably the presence of these Tyr residues already allows the loop to adopt a different shape, but the concomitant codon mutation from TTY (Phe) to TAY (Tyr) creates two somatic mutation hotspots (Milstein et al., 1998). Consequently, these regions become more susceptible to mutation and result in an enlargement of the structural repertoire of the paratope. Whether the somatic mutations of the rearranged VHH gene occur as part of the antigen affinity maturation (Foote and Milstein, 1991), or whether they occur as an antigen-independent process remains to be seen (Reynaud et al., 1995). We speculate that an antigen-independent mechanism would be most efficient for enlargement of the repertoire.


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2. µ–γ Switch We have a good understanding of what happens during VHH–D–J recombination and co-expression with a γ chain into a membrane-bound or secreted HCAb. However, we are limited to speculations about the IgM status of the VHH recombination products and the possibility of having a rearranged Vλ or Vκ gene in the HCAb producing B cells. PCRs on cDNA from peripheral blood lymphocytes with VHH- and μ-specific primers were unable to yield clonable amplification products. Similar PCRs on cDNA of spleen cells yielded a very low number of clones where apparently a VHH was attached to a μ chain. The clones of both VH and VHH contained the same μ gene and the intact CH1 exon (V. K. Nguyen, unpublished results). However, the number of VHH-μcontaining clones remains so disappointingly low that we cannot exclude that they were only obtained after a PCR cross-over. Consequently, we have no formal proof that VHHs ever occur as part of an expressed IgM. In contrast, if we accept a short-lived, transient existence of IgM with VHHs, then the presence of the CH1 domain in the μ chain would prevent its secretion from the endoplasmic reticulum unless the attached chaperon proteins are replaced by λ5 surrogate L chain in pre-B cells or by a mature L chain in B cells (Melchers et al., 1994). Possibly, the CH1 domain of the μ chain might interact with the λ5 surrogate light chain so that VHH-μ could be exposed on the pre-B-cell membrane. It is then possible that these pre-B cells expressing a VHH-μ chain with λ5 follow a different route compared to the cell lines expressing VH-μ chains with VpreB and λ5. The latter could start to rearrange the L locus (Rajewsky, 1996), whereas the former immediately switches the μ gene with the HCAbspecific γ genes, without rearranging the L locus. This immediate μ–γ switch might be an antigen-independent process and perhaps a cytokine-independent process, as well. In any case, the switch of the VHH from the μ genes to the γ genes with the CH1 splicing defect should be a highly specific process because we could barely amplify a VHH gene linked to a conventional γ or a true VH with a γ isotype used to produce HCAb. We realize that such HCAb ontogony is highly speculative, and several other alternative schemes might be proposed. VI. Isolation of Antigen-Specific Heavy-Chain Antibodies, or VHHs

A. IMMUNIZATION OF LLAMAS OR DROMEDARIES Dromedaries and llamas are successfully vaccinated against camelpox and tetanus, respectively (Wernery et al., 1997; Murphy et al., 1989). We routinely obtain a high titer of antigen-specific HCAb in the dromedary by giving four to six injections of 50 to 100 μg antigen (Lauwereys et al., 1998). The amount of antigen required for this immunization is similar to what is used to immunize,

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for example, a rabbit. Moreover, immunization with a cocktail of antigens or even a whole cell lysate raised an immune response to each antigen separately (Lauwereys et al., 1998; van der Linden et al., 2000). This makes the whole procedure more cost effective and applicable for proteomic research. The titer and diversity of the response in conventional or HCAbs seem to depend on the actual antigen. Proteinaceaous antigens yield a higher titer than haptens; the small hydrophobic haptens especially tend to give weak HCAb responses. However, a llama immunized with dyes coupled to BSA carrier resulted in a higher relative proportion of HCAb against the hapten than to the carrier (van der Linden et al., 2000). The same authors also reported that the proteins with a MW between 60,000 and 80,000 of a S. mutants lysate were bound preferentially by llama HCAb, although overall the HCAbs recognized a smaller number of proteins than the conventional antibodies. However, these findings need confirmation with other haptens, purified antigens, or whole-cell lysates and will probably also depend on the immunization protocol (carrier, adjuvant, vaccination scheme). Unexpectedly, the immunization of a dromedary with enzymes generates large quantities of HCAbs that react with the enzymatic active site. Therefore, a large fraction of the dromedary HCAbs directed against enzymes behaves as potent competitive inhibitors (Lauwereys et al., 1998). Two complementary experiments lead to this conclusion. First, it was shown for α-amylase and carbonic anhydrase that approximately half of the HCAb raised against these antigens could be displaced by adding a large excess of small competitive inhibitors such as acarbose and dorzolamide, respectively. In contrast, the conventional antibodies of the same animal failed to be displaced by these drugs. Second, it was demonstrated that the monomeric VHH prepared from the HCAb of a dromedary immunized with α-amylase could inhibit the cleavage of 2-chloro-4nitrophenyl-maltotriose, a substrate for α-amylase. B. OBTAINING ANTIGEN-SPECIFIC VHHS The antigen-specific VHHs can be obtained using one of the following methods: (1) protease digestion of HCAb of an immunized camelid; (2) direct cloning of the VHH genes from B cells of an immunized camel, dromedary, or llama; and (3) large na¨ıve or synthetic VHH libraries. 1. Polyclonal VHH from Proteolyzed HCAbs Methods to prepare Fab or Fab′ 2 fragments from conventional antibodies by pepsin or papain digestions are well established (Porter, 1959; Nisonoff et al., 1975). The same strategy can be applied to obtain VHH or VHH′ 2 fragments by using proteases cleaving the HCAb hinges. As a proof of principle, we prepared polyclonal VHHs—of which measurable amounts recognize α-amylase—


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from the IgG3 fraction of an immunized dromedary (Lauwereys et al., 1998). Endo-Glu V8 protease was used to cleave the short hinge region between the VHH and the CH2 of this isotype (R. Hamers, personal communication). The VHH purification was performed by protein A chromatography of the proteolyzed sample retaining the Fc-containing fragments, while the flow-through contains the majority of the VHHs. This method might be less suitable to prepare llama VHHs because the IgG3 titer is lower in llamas compared to dromedaries (van der Linden et al., 2000). In addition, a large fraction of the llama VHH, but not the dromedary VHH, seems to bind protein A, so different purification schemes should be devised. It is also feasible to obtain the homodimeric VHH′ 2 from the dromedary HCAb IgG3 isotype. In this case, pepsin, trypsin, or papain should substitute the endo-Glu V8 protease (Hamers and Muyldermans, 1998). These proteases attack the hinge region at various positions, but mainly after the first disulfide bond that links the two γ 3 chains. It should also be possible to prepare VHH or VHH′ 2 fragments from the other dromedary or llama HCAb isotypes, but once again at a lower yield due to the reduced titer in serum. However, in most cases, it will be far more appropriate to obtain antigen-specific VHHs employing genetic engineering techniques. Most of these techniques were developed for the generation of scFv or Fab fragments (reviewed in Hoogenboom and Chames, 2000; Hoogenboom et al., 1998; Winter et al., 1994). 2. Monoclonal VHH from Cloned VHH Banks a. Cloning of the VHH Repertoire of Immunized Camelidae. Cloning the repertoire of antigen-binding fragments of HCAbs from an immunized dromedary or llama is extremely simple (Fig. 3). Once there is evidence that antigenspecific HCAbs are present in the immunized animal, anticoagulated blood is collected to isolate the peripheral blood lymphocytes. These cells (107 isolated from a few milliliters of blood) are used as a source for mRNA isolation and cDNA synthesis. The gene fragments encoding the VHHs are specifically amplified by PCR. All the VHHs belong to one single family; therefore, all VHHs are encoded by an exon with homologous border sequences so that one set of primers should amplify the entire VHH repertoire present within the blood sample (Ghahroudi et al., 1997). However, amplification and cloning of VHs originating from the conventional antibodies should be avoided because the sticky behavior of VHs might interfere during subsequent selection of antigen-specific fragments (Davies and Riechmann, 1995). Two methods—based (1) on the observation that VH do not occur within a HCAb, and (2) on the inherent isotypic differences between conventional antibodies and HCAb—were employed to eliminate the VH gene fragments

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FIG. 3. Overview of the methods to clone the VHHs from an immunized dromedary and the selection procedures to retrieve antigen-specific VHHs from the “immune” library.

from the VHH pool. In the first method, primers were designed that anneal selectively on the hinge of the HCAb (van der Linden et al., 2000). The hinge region of the HCAbs was chosen because it harbors the largest sequence differences between the different isotypes. Such primers in combination with a primer annealing at the 5′ end of the VHH gene will amplify the VHH genes only (Fig. 3,


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procedure A). In an alternative approach (Fig. 3, procedure B), a primer that binds to a conserved region of all constant γ genes in combination with a back primer that anneals at the leader signal sequence of the VH and VHH amplifies all γ isotypes towards their VH and VHH end. As a result, two types of primary PCR products that differ by ∼300 bp in length are obtained: One type (the longest) contains the CH1 exon and is derived from the conventional antibody; the other type of fragment (the shorter) lacks the CH1 exon and is derived from HCAbs. These different types of PCR fragments are well separated by agarose gel electrophoresis, and the isolation of the shorter fragment eliminates the VH sequences efficiently. A second PCR with nested primers annealing at either end of the VHH is then performed to make sufficient material and to include restriction enzyme sites for cloning purposes (Fig. 3, procedure B). b. Cloning Na¨ıve and Synthetic VHH Banks. Besides the possibility of using “immune” VHH libraries, two “non-immune” VHH libraries can be generated to retrieve subsequently antigen-specific VHHs. The na¨ıve library tries to cover a maximum number of different VHHs as obtained from healthy, nonimmunized dromedaries or llamas. The synthetic VHH library uses a particular VHH or VHH-like scaffold onto which one or several antigen binding loops are randomized by synthetic oligonucleotides to create the diversity. The use of “non-immune” libraries has the advantage that the immunization is avoided and thus offers a solution for antigens that are very toxic, non-immunogenic, or not available at all. The generation of large na¨ıve VHH libraries (each ∼109 individual clones) was reported for the first time and simultaneously by two groups at the antibody engineering meeting (IBC’s 11th Annual Int. Conf. La Jolla, CA, 3–6, 2000). One group cloned the VHH from llamas (Tanha et al., Ottawa, Canada), the other group (Rousch et al., Maastricht, The Netherlands) used dromedaries. The authors claimed in their poster presentations to be successful in identifying antigen-specific VHHs from their libraries. Several formats of synthetic single-domain antibody libraries have already been constructed. The libraries contain a “camelized” human VH framework or a mouse VH scaffold into which a synthetic CDR3 is introduced (Davies and Riechmann, 1995; Reiter et al., 1999). The mouse VH used to generate the single-domain library is, obviously, not a bona fide VHH. However, the presence of a nonconventional Lys44 probably reshaped the framework 2 region so that the VHs expressed as inclusion bodies are easily refolded in soluble, monomeric domains (Reiter et al., 1999). Interestingly, this mouse VH belongs to VH family I, indicating that single-domain antibodies can also be formed by VH scaffolds outside VH family III. Tendamistat, fibronectin, and CTLA4 having a fold and monomeric appearance that resembles the VHH was also used as a scaffold to construct synthetic, single-domain libraries (McConnell and Hoess, 1995; Koide et al., 1998; Hufton, 2000).

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c. Selecting Antigen-Specific Binders from VHH Libraries. In each case, the VHHs are cloned in expression vectors in frame with a secretion signal to direct the VHH into the periplasm. The VHH libraries can be screened for the presence of antigen-specific binders, either by direct colony screening or by panning. Indeed, it was shown that ∼1% of the VHH clones within the “immune” library contained VHHs that recognized the antigen used for immunization. This is reasonable since the B cells expressing antigen-specific VHHs were probably proliferated during the immunization. Thus, the direct testing of the 103 to 106 individual clones of the VHH library by colony screening with labeled antigen as performed in the late 1980s (Huse et al., 1989) is expected to yield several antigen binders. Alternatively, testing the individual supernatants of 103 induced cultures in an ELISA with immobilized antigen are likely to identify clones carrying antigen-specific VHHs (Fig. 3, procedure C). The latter strategy was in fact used by Ward et al. (1989) to clone only the VHs from spleen cells in the absence of any VL after immunization of a mouse with lysozyme or KLH. Although the mouse VHs were reported to be sticky, the authors found 21/2000 and 14/2000 antigen-specific VHs, respectively. The retrieval of antigen-specific VHHs by panning (Fig. 3, procedure D) is obviously preferred over the screening of individual colonies (procedure C), because the panning conditions also select for binders with the highest affinities for the antigen and those that express better in bacteria. To this end, the VHH gene is inserted in front of the minor coat gene 3 protein of filamentous phages. This allows the production of phage particles that possess the VHH-g3p fusion protein at their tip and the corresponding VHH gene encoded in the encapsulated phage or phagemid genome. The antigen-specific VHH can therefore be selected—by several sequential rounds if needed—through panning of phage particles on immobilized antigen. Such pannings became a routine method in the past decade to identify antigen-specific molecules (Winter et al., 1994; Hoogenboom and Chames, 2000; Hoogenboom et al., 1998). d. “Immune” versus “Non-immune” VHH Libraries. The use of synthetic VHH libraries (camelid VHH, camelized human VH or mouse VH) is the only solution when immunizations are impossible. However, the success rate of isolating single-domain binders from synthetic libraries is currently difficult to infer from the literature. In addition, the binders currently retrieved from the “non-immune” or synthetic libraries have a good antigen specificity, but their affinity is very often in the μM range (Davies and Riechmann, 1995; Martin et al., 1997; Reiter et al., 1999). Thus, for many applications, subsequent and time-consuming in vitro affinity maturation steps should be included to improve the antigen-binding capacity of the primary VHHs (Davies and Riechmann, 1996a,b). Therefore, whenever antigens are available, even in a crude form,


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it might be advisable to exploit the dromedary immune system and to use a short immunization step to generate a small “immune” VHH library to retrieve antigen-specific binders that were matured in vivo to high affinity (Riechmann and Muyldermans, 1999). In addition, for the “immune” VHH libraries, we are working with relatively small libraries so that artefacts and pitfalls of large libraries (over-representation of phages/virions with incomplete scFv genes or carrying additional stopcodons or reading frame shifts) are avoided (de Bruin et al., 1999). C. CHARACTERISTICS OF THE ANTIGEN-SPECIFIC VHHS FROM “IMMUNE” LIBRARIES By panning the VHH libraries from immunized dromedaries we have currently isolated over 100 different binders. These binders are well produced in bacterial expression systems, have a soluble and stable behavior in aqueous solutions, seem to be antigen-specific, bind the antigen with high affinity, and are directed to unique epitopes that are less well targeted by conventional antibodies. 1. Production, Solubility, and Stability of Recombinant VHH The expression levels of recombinant VHHs in the periplasm of Escherichia coli is very satisfactory. A yield of 5 to 10 mg VHH per liter bacterial culture is routinely obtained from culture flasks, even before expression optimizations (Ghahroudi et al., 1997). However, higher yields were reported for recombinant llama VHHs. Production levels of 9.3 mg/1/OD660 or ∼250 mg secreted protein per liter of Saccharomyces yeast culture in shake flasks have been observed (Frenken et al., 2000). Therefore, it is expected that production levels of grams per liter should be achievable after high-cell-density fermentation. It is well established that VHs of family III adsorb on protein A (Potter et al., 1996); thus, such an affinity chromatography on protein A columns has been utilized to purify the camelized human VHs and the llama VHHs that belong to family III (Davies and Riechmann, 1996a; Frenken et al., 2000; van der Linden etal., 1999). Protein A affinity chromatography has the advantage that it also selects for the properly folded domains. Unfortunately, in our hands only a few dromedary VHHs have been purified by protein A chromatography, although they are properly folded (Ghahroudi et al., 1997). The purification of recombinant VHHs is facilitated by cloning six His codons at the 3′ end of the genes, so that the VHH can immediately be purified from periplasmic extracts by metal affinity chromatography on a Ni–NTA column followed by gel filtration chromatography on Superdex-75. The elution profile of this gel filtration always yields a single symmetric peak at the expected molecular weight for a VHH (15 kDa). Therefore, the recombinant VHH proteins are

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monomeric and show no tendency to form aggregates even at a concentration of 10 mg/ml. ScFv often have a tendency to dimerize or aggregate through their linker tethering the VH and VL domains (Whitlow et al., 1993). Evidently, due to the absence of such a linker, the recombinant single-domain VHH does not suffer from this linker-induced aggregation. However, it was noticed that the expression of recombinant VH in the absence of a VL partner leads to formation of inclusion bodies or to sticky, single-domain VHs (Ward et al., 1989). This is explained ◦ by exposure to the aqueous environment of a large hydrophobic area of ∼700 A2 of the VH that is normally involved in the VL association in conventional antibodies (Davies and Riechmann, 1994). In contrast, the recombinant VHHs harboring the hallmark amino acid substitutions in their framework 2 region are naturally soluble. It was experimentally proven that the framework 2 amino acid substitutions make the difference (Davies and Riechmann, 1994). The mutation of a human VH to mimic the camel VHH specific hallmarks rendered the human VH sufficiently soluble and monomeric at high concentration so that its structure could be solved by NMR (Riechmann, 1996). Although it can be modulated, the half-life of an Fv is relatively low (Glockshuber et al., 1990). In contrast, purified recombinant VHHs retained their full antigen-binding capacity after incubation at 37◦ C in mouse serum for 24 hours (Conrath et al., 2001). The VHHs also have a much longer shelflife. When tested under physiological conditions at 37◦ C, the antigen-binding activity was retained or dropped maximally by 20% even after a 1-week incubation (Ghahroudi et al., 1997). Interestingly, the VHH with the lower shelflife lacked a cystine between its CDRs, underlining the stabilizing effect of this disulfide bond. The stabilizing effect of an interloop disulfide bond was also confirmed by thermal stability measurements. The introduction of such an interloop disulfide bond in the camelized human VH increased the Tm from 60◦ C to almost 80◦ C (Davies and Riechmann, 1995). However, most striking is the fact that the llama VHHs remained associated with their hapten “Reactive Red” azo-dye (RR6) at temperatures as high as 90◦ C, a temperature where all mouse monoclonal antibodies against the same antigen lost their functionality (van der Linden et al., 1999). Furthermore, in the absence of antigen, the VHHs denature at 90◦ C; however, it is a reversible thermodenaturation since the VHHs regain their original antigen-binding capacity after cooling. VHHs are also chemically robust, as full denaturation with chaotropic agents such as urea is not always possible. Moreover, the VHH fully denatured with guanidinium HCl refolded immediately into antigen-binding units upon dilution with water. This indicates that the VHH should easily refold from inclusion bodies by a denaturation–renaturation step. VHHs could also be functionally immobilized on biosensor layers, and the sensing layer could be regenerated multiple times with acid or alkaline treatments. All of these findings support

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the potential of VHHs as immuno-adsorbents (Berry and Davies, 1992) or in biosensor applications. 2. Antigen Specificity and Affinity The affinities of the VHHs isolated from “immune” libraries for their respective antigens as determined by biosensor or ELISA are similar to the affinity of monomeric Fv fragments derived from monoclonal antibodies (Lauwereys et al., 1998; Riechmann and Muyldermans, 1999). Kinetics rate constants, kon and koff, ranged from 104 to 106 M−1 s−1 and 10−2 to 10−4 s−1, respectively (Table II). Even for the llama VHH binder to the azo-dye hapten (RR6), rate constants in the same range were measured (Spinelli et al., 2000). Consequently, the equilibrium dissociation constant, Kd, of the VHH–antigen complex is in the 1- to 100-nM range for both protein and hapten binders. Highly specific binders for the hapten phenyloxazolon and, proteinaceous antigens were also retrieved from synthetic single-domain libraries. Although, the antigen affinities of the VHH from the immunized libraries are usually an order of magnitude better compared to the camelized human VHH against the same proteinaceous model antigens (Riechmann and Muyldermans, 1999). However, it is expected that the larger libraries yield consistently higher affinity

TABLE II VHHS WITH KNOWN STRUCTURE Antigen specificity HEL TEL HEL RNase A Carb. anh. Carb. anh. Ch gonad. h. α-Amylase α-Amylase α-Amylase Antat 1.1 RR6

VHH/VH Inhibitory kon(M−1s−1)

cAb-Lys3 cAb-Lys3 D3L11-8 cAb-RN05 cAb-CA05 cAb-CA05 hCG-H14 cAb-AM7 cAb-AM9 cAb-AM10 cAb-AN33 R2 VH-P8 NS3 protease cVHE2 Pot-VH

Yes Yes Yes 80% No No — No Yes No — — — Yes —

4.1 × 104 4.4 × 104 NA 2.3 × 106 6.4 × 104 — — 1.5 × 105 2.4 × 105 — NA 1.5 × 105 — NA —

koff (s−1) × 10−3 × 10−3 × 10−3 × 10−2 × 10−3 — — 3.5 × 10−2 8 × 10−4 — NA 3.3 × 10−3 — NA —

2.7 1.8 4.6 8 5

Kd ASA Crystal of ◦ (nM) CDR3 ( A2) pdb-file VHH with 65 40 NA 35 72 — 300 230 3 24 NA 22 — NA —

24 24 18 12 19 19 8 19 14 14 10 16 8 11 12

850 770 NA 570 620 — — NA NA NA — 300 — — —

1mel — — 1bzq 1g6v 1f2x 1hcv — — — — 1qdo 1vhp — 1igm

HEL TEL HEL RNase A Carb. anh. None None Amylase Amylase Amylase None RR6 None — None

Note: ASA = accessible surface area. HEL = hen egg-white lysozyme; TEL = turkey egg-white lysozyme; Carb. anh = carbonic anhydrase; Ch. gonad. h. = human chorionic gonadotropin hormone; RR6 = Reactive Red azo-dye hapten; NS3 protease = hepatitis C virus NS3 protease that was used to select an inhibitor from a synthetic camelized human VH library. The VH-p8 is the camelized human VH with known NMR structure. Pot-VH is the VH of a human 1gM. NA = not available. — is used to denote that the information is not relevant.

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binders (Nutall et al., 2000) and that binders from na¨ıve scFv libraries perform better than those from synthetic libraries (Vaughan et al., 1996; Hoogenboom and Chames, 2000). Therefore, the binders that will be identified from the large, na¨ıve VHH libraries of the camel or llama might possess affinities comparable to the binders from the “immune” library. The VHHs from the “immune” libraries are highly specific for their target antigen and do not cross-react with other nonrelated antigens (Ghahroudi et al., 1997). For example, VHHs were obtained that discriminate the surface antigens of closely related hepatitis serotypes (M. Lauwereys, personal communication). However, the synthetic library of the camelized human VH was shown to yield binders that cross-reacted with nonrelated antigens. This was attributed to the presence of the Ile47, since its substitution by Gly47 to mimic more closely the most frequent dromedary VHH amino acid at that position solved the problem of retrieving nonspecific binders (Davies and Riechmann, 1995). 3. Recognition of Unique Epitopes The immunization of dromedaries with enzymes α-amylase, hen egg-white lysozyme, β-lactamases, and carbonic anhydrase) revealed that a significant proportion of the HCAbs behaves as an inhibitor that interacts with the activesite cavity on the surface of the antigen (Lauwereys et al., 1998). This enzyme inhibitory activity is only present in the HCAbs and not in the conventional immunoglobulins of the immunized dromedaries. This indicates that the enzyme inhibition of the HCAbs results from an inherent structural property of the single-domain antigen-binding domain. Also, the conventional monoclonal antibodies that are competitive enzyme inhibitors are, by far, more the exception than the rule. It is, therefore, clear that camelid HCAbs are capable of recognizing unique epitopes that are less immunogenic for conventional antibodies. Similarly, several of the dromedary VHHs from the “immune” libraries with specificity for enzymes turned out to be potent competitive inhibitors (Lauwereys et al., 1998; Transue et al., 1998). However, as could be expected, the targeting of the VHHs to the substrate cleft of the enzyme depends largely on the actual antigen. All (nine out of nine) hen egg-white lysozyme binders interacted with the groove on the antigen surface, whereas only one out of five binders inhibited the β-lactamase (K. Conrath, personal communication). The VHH capacity to associate preferentially with cavities on the antigenic surface and to block the enzymatic activity might be a property shared with the single-domain binders extracted from synthetic libraries, as well. Indeed, the affinity selection of the synthetic, camelized VH library yielded an inhibitor of hepatitis C virus NS3 protease (Martin et al., 1997).


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VII. VHH Structure and VHH–Antigen Interaction

A. OVERALL VHH STRUCTURE Over 100 different, antigen-specific VHHs have already been isolated from the “immune” libraries; of these, about a dozen have been crystallized in the presence or absence of antigen to solve their structure. The coordinates of six of these VHH structures (two llama VHHs) are available as pdb files (Table II). The NMR structure of the camelized, human VH is also available in the protein databank (pdb). For all these VHHs, it is clear that the immunoglobulin fold is well maintained (Fig. 4, see color insert). The VHHs have nine β-strands arranged in two sheets, typical for the variable domain. The three hypervariable loops cluster at one end of the domain. Furthermore, a conserved disulfide bond formed between Cys22 and Cys92 links the two sheets. The backbone structure of any of the VHHs superimposes very well on that of human Pot-VH, a family III member (Fan et al., 1992). Calculation of the root mean square (rms) deviations for the main-chain atoms of the framework residues (residues 2–24, 32–52, 55–72, 77–92, and 103–112) of the currently available VHH structures, the camelized human VH antibody fragment, and a human VH reference structure gave values ◦ between 0.58 and 0.88 A . These low rms values confirm the good overlap of the protein core main-chain atoms not only among the VHHs but also with the human Pot VH structure. Larger rms deviations were observed between the camelized VH structure (pdb code 1vhp) (Davies and Riechmann, 1994) and the other structures. This is largely due to the fact that the former structure was determined by NMR and the occurrence of significant scaffold distortions around Val37, Arg38, Glu39, Arg45, Ile47, and Trp103 in the camelized human VH (Riechmann, 1996). B. “VL-SIDE” CONFORMATION OF THE VHH Despite the overall good overlap, some structural adaptations are expected to occur in the VHH domain at the side that corresponds to the VL-interacting site of a VH. Both, framework and CDR residues of the VH contact the VL domain. The VH residues Val37, Gly44, Leu45, Trp47, Tyr91, and Trp103 are all conserved in sequence and structural position and are crucial in the VH– VL packing (Kabat et al., 1991; Chothia et al., 1985; Padlan, 1994). Four of these residues are nearly constitutively substituted in the VHH by more hydrophilic residues (Phe37, Glu44, Arg45, Gly47) (Muyldermans et al., 1994; Vu et al., 1997). The crystal structures illustrated how the Val37Phe mutation fills a hydrophobic pocket created by Gly47 (or Phe47 in llama RR2) and the side chains of Trp103, Tyr91, and the CDR3 where the conserved Tyr (Phe in llama RR2)—three amino acids upstream of Trp103—plays a central role (Fig. 4, see color insert). The structural analysis further revealed that the Trp103 side-chain

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rotates approximately 180◦ over its Cβ–Cγ bond in the VHH to expose its most polar part, the Nε, to the aqueous solvent to make this region even more hydrophilic (Desmyter et al., 1996). The Trp103, encoded by the JH, is absolutely conserved in VHs (98.8% occurrence) and is also the most frequently occurring amino acid at that position in VHHs since this amino acid is encoded by the J minigene and the VH and VHHs use probably the same J segments. However, due to the D–J junctional variation mechanisms and the absence of VL contacts, other amino acids are found in VHHs at this position; for example, Arg occupies position 103 in cAb-CA05, a VHH with specificity for carbonic anhydrase. This Trp103Arg substitution occurs repeatedly in VHH domains. According to the Kabat database (http://immuno.bme.nwu.edu/), 6 out of 70 camelid VHH sequences—or 8%—have an Arg residue at position 103. From the crystal structure of the cAb-CA05 it was shown that this Trp103Arg mutation changes the nature of the surface even more drastically without disturbing the main-chain conformation. The hydrophobic part of the Arg side-chain integrates well with the neighboring hydrophobic side-chains, whereas the guanido group extends into the solvent. Furthermore, the presence of Arg103 apparently induces a longrange shift of the Phe37 side-chain towards Try91 and a concomitant joining of the CDR3 loop. All these framework 2 and framework 4 modifications do not distort the framework Cα’s. In contrast, the partial camelization of a human VH in this area by Leu45Arg and Trp47Ile substitutions makes the isolated domain more soluble, but induces backbone deformations at positions 37–38 and 45–47 (Riechmann, 1996). Moreover, the side-chain of Trp103 takes a completely new position. These results illustrate how subtle side-chain shifts reshape the chemical nature of the VHH surface. C. ANTIGEN-BINDING LOOP STRUCTURES The antigen-combining site of conventional antibodies is located at the tip of the Fv portion and is composed of six hypervariable loops (Padlan, 1994). It was confirmed by crystallographic data of antibody fragments that the hypervariable loops or CDRs as defined by Kabat were located in this area. However, the CDR and solvent exposed part (in antigen-free antibody fragments) of the loops (in VH, referred to as H loops) were not always overlapping (Fig. 2) (Chothia et al., 1989, 1992; Chothia and Lesk, 1987), The crystal structure of antigen– antibody complexes further revealed the actual amino acids that participate in the antigen recognition (Padlan, 1994, 1996). These antigen-contacting regions are referred to as SDRs (specificity-determining regions) (Abergel et al., 1999; Padlan et al., 1995). In Fig. 2 we compare the locations of CDRs, H loops, and SDRs within VHs and VHHs. It is clear that the CDR1, SDR1, and H1 loops are superimposable for the VHH. Until now, the amino acids 27–29 of VHH only were reported to be hypervariable and to contact directly the antigen. This enlarged SDR1 in VHHs probably compensates for the absence of


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antigen-binding contacts provided by the VL loops. In contrast, for the VH the CDR1 and SDR1 overlap significantly but are offset of the solvent-exposed H1 loop. The N-terminal part of the H1 loop does not belong to the SDR1 and is not used for antigen recognition in VHs. The pioneering work of Chothia revealed that the H loops can only adopt a limited number of main-chain conformations, known as canonical structures (Chothia et al., 1989, 1992; Chothia and Lesk, 1987). This was observed after comparing the positions taken by the Cα atoms in the loop structures of many VHs. The actual canonical loop structure is determined by the length of the loop and by the presence of conserved amino acid residues. The canonical structures were refined, later, by taking into account the peptide backbone conformation (and not only the Cα positions), and a number of subtypes were introduced for some of the canonical structures (Al-Lazikani et al., 1997). Importantly, analysis of the loop lengths and occurrence of key amino acids of the antigen-binding site of cartilaginous fish antibodies revealed the same canonical structures in this species most distantly related to humans with a known immune system. This observation indicates that the canonical loop structures arose early in the evolution of the immune system and were conserved thereafter. Surprisingly, the relationship between the VHH loop structures and the canonical structures appears to be much less strict (Decanniere et al., 2000). Although true canonical structures were noticed in a number of VHHs, many more deviations were observed, for several reasons. First, the length of the loop is much more variable in VHHs; for example, a cDNA bank of dromedary VHH revealed that some 30% of the clones had “unnatural” loop lengths. It is evident that VHHs with “unnatural” loop lengths are functional since most of them were obtained from “immune” libraries and have known antigen-binding specificity. Second, although the residues supporting the loop conformation are unmutated or undergo conservative mutation in VH genes (Cook and Tomlinson, 1995), in the dromedary VHH germline genes we have observed that mutational hotspots encode some of these key residues of the VHH-CDR1 (Nguyen et al., 2000). These mutation hotspots lead to frequent modification of those residues and consequently to a reshaped H1 loop that no longer fits the canonical structures. Third, the CDR3 in VHHs is longer; nevertheless, its flexibility is constrained at its loop “entry” and “leaving” ends by the β-strand scaffold, by its Cys that is disulfide-bonded to a Cys in the CDR1 or CDR2 or at position 45 in framework 2, and by the Tyr or Phe three residues upstream of Trp103, where the side-chain clusters with aromatic residues of the “VL-side” of the VHH domain. Consequently, the CDR3 provides a large solvent-accessible area available for antigen recognition, and is folded over the “VL-side” of the domain, a position that cannot be taken in a paired VH–VL. The canonical structures occur in various combination in mouse or human antibody molecules (Almagro et al., 1997; Vargas-Madrazo et al., 1995). This creates an enormous complexity of antigen-binding sites. However, a very small

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number of all possible combinations are preferentially used to construct the main part of the antigen-combining site repertoire. Nearly 90% of the structural repertoire belongs to only 10 canonical structure classes. These canonical structure classes determine to a large extent the antigen preference (Vargas-Madrazo et al., 1995). The canonical structure combinations form an antigen-combining site with either a planar conformation or a groove or cavity (Webster et al., 1994; Padlan, 1996). The planar architecture is chosen for interactions with large antigens, while cavities are more likely to bind haptens. In VHHs, the loop structures can also form a cavity; however, here the cavity is not formed between VH and VL but rather in between the CDR loops. The crystal structure of an anti-hapten llama VHH shows how a noncanonical conformation of the CDR1 loop separates sufficiently from the CDR2 to provide a pocket into which a dimerized azodye RR6 subsides (Spinelli et al., 2000). A planar paratope conformation can also be created by VHHs as seen in cAb-RN05 and cAb-CA05, two VHHs binding to RNAse A and carbonic anhydrase, respectively (Decanniere et al., 1999) (A. Desmyter et al., J. Biol. Chem., in press). Remarkably, the antigen-binding site of VHH can form a large protruding loop (Fig. 4, see color insert). The threedimensional structure determination of the dromedary VHH with specificity for lysozyme revealed how the first 10 amino acids of the 24-amino-acid-long CDR3 extends from the remaining antigen-binding site and inserts into the active site of the lysozyme (Desmyter et al., 1996). This was the first example of an antigenbinding site with a large convex paratope. The residues of the protruding loop of cAb-Lys3 interact with the catalytic residues of the enzyme, and seven consecutive amino acids forming the bulge of the loop mimic the natural substrate of the enzyme (Transue et al., 1998). Because of the presence of a long CDR3, seen in most of the VHHs, and the large protruding CDR3, as observed in the cAb-Lys3, it is tempting to speculate about a common mechanism of camel VHHs to produce enzyme inhibitors. It was even anticipated that the long CDR3, of the VHH could lead to new drugs against various enzymes (Sheriff and Constantine, 1996). However, several VHHs with enzyme-inhibiting capacity but with much shorter CDR3 loops have been indentified (Table II). The inhibitor from the camelized human VH synthetic library that interacts with the hepatitis protease has a CDR3 of only 11 amino acids (Martin et al., 1997). The amylase inhibitor, cab-AMD9, has a CDR3 of only nine amino acids and its CDR1 is also shortened (Lauwereys et al., 1998). In addition, all nine VHHs with specificity for hen egg-white lysozyme seem to interact with the substrate site of the antigen, and one of them has a CDR3 of only 12 amino acids (K. Silence, personal communication). Combined, these observations argue against the necessity of a long, protruding CDR3 loop in the VHH for enzyme inhibition but support the notion that VHHs are better suited to interact with cavities on the antigen surface that are large, such as in lysozyme. We think that the smaller size of the antigen-binding domain (one


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domain instead of two domains in Fv) creates a greater access for its three CDR loops to interact with grooves or cavities on the antigenic surface. This is of importance because grooves and cavities play a critical role in multiple biological activities as they often form the specific interaction site between two molecules (e.g., receptor–ligand or enzyme–substrate) (Laskowski et al., 1996). Antibodies that specifically bind into such active sites therefore represent powerful tools to modulate biological activities. The VHHs might be better in doing this than Fvs having mostly flat antigen-binding surfaces (Webster et al., 1994; Padlan, 1996). Based on a structural analysis on VHH-antigen complexes, it is clear that there is no strict correlation between the antigen-binding surface and the antibody– antigen affinity (Table II). Indeed, whereas the cAb-lys3 has a large interacting ◦ ◦ surface of 850 A2 due to its protruding loop, that of cAb-RN05 is only 570 A2. Both binders bind their respective antigen with a comparable affinity. These antigen-interacting surface areas are equivalent to those formed by the Fv ◦ paratope to recognize proteinaceous antigens (630–850 A2) (Lo Conte et al., 1999). VIII. Biotechnological Importance of VHHs

Antibodies are widely used as a research tool, but more importantly are their applications for diagnostic and therapeutic purposes (Borrebaeck, 2000; Glennie and Johnson, 2000). It is expected that the VHH will also be applicable in these areas, especially when an economic production, small, size, and robust entity are required. The high expression yields of VHHs in yeast will be beneficial in applications where extremely large quantities of antigen binders are a necessity (Frenken et al., 2000). The single-domain nature of the VHH, having a good resistance against temperature and chemicals, and its easy surface immobilization (either chemically through the lysines scattered over its surface but mostly absent around the paratope, or through nonspecific adsorption on polystyreen or nitrocellulose) with retention of antigen-binding capacity opens up new perspectives in the development of antibody-based immunodiagnostics, such as antibodybased microarrays (Borrebaeck, 2000; de Wildt et al., 2000). It is expected that the impact of biosensors will grow steadily in the near future. Moreover, the robust property of VHHs will allow them to find their antigen under harsh conditions when antigen-binding fragments from conventional antibodies would not survive or would fail to complex the antigen. The absence of linker, often leading to nonspecific aggregation of scFv (Whitlow et al., 1993), makes the VHH a perfect modular binding unit in more complex entities, such as bispecific constructs or in immunofusions with enzymes or toxic substances (Hoogenboom, 1997). With VHHs it is in essence simple to

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engineer bispecific constructs. We tethered the C-terminus of one VHH to the N-terminus of the second VHH by the natural upper structural hinge of the llama γ 2a (Conrath et al., 2001). In our expression vector, we added a polyhistidine tail to facilitate purification by metal-affinity chromatography. The expression levels in E. coli were comparable to the levels obtained for the individual VHHs, and the constructs behaved in a perfectly monomeric manner. Importantly, each of the VHH fragments retained the original antigen-binding properties, and evidence for simultaneous binding to the bispecific antibodies of both antigens was provided by ELISA, biosensor, and gel filtration experiments. Moreover, we have experimental evidence that these molecules are stable, as no loss of activity or bispecificity is detected after incubation for 24 hours in plasma (Conrath et al., 2001). Finally, the observation of VHHs recognizing epitopes that are less immunogenic for conventional Fv fragments (e.g, active site of enzymes) might also lead to a number of applications. The enzyme-inhibiting capacity of VHH has already been explained, and this property could be exploited for immunomodulation studies to produce knockout phenotypes (Visintin et al., 1999; Rondon and Marasco, 1997). In addition, the combination of small size, good immobilization possibilities, and targeting of epitopes not recognized by human antibodies creates the possibility of using the VHH in diagnostic protocols to determine an antigen titer directly in human serum samples. ACKNOWLEDGMENTS The authors thank their colleagues at the VUB department Ultrastructure and the group of Prof. C. Cambillau at AFMB, CNRS, Marseille, France, for discussions and help with crystallography. Much of this work would not have been possible without the help of the research groups of CVRLDubai, NRCG-Iran, and I.A.V. Hassan II Rabat-Morocco. This work was supported by VIB, IWT, FWO, OZR-VUB, and EEC grants.

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Uterine Natural Killer Cells in the Pregnant Uterus CHAU-CHING LIU∗ AND JOHN DING-E YOUNG† ∗Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213; and †Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, New York 10021

I. Immunobiology of Pregnancy: An Overview

How a fetus, expressing paternal as well as embryonic antigens, escapes the immunological response and survives in the uterus of the genetically alien mother is a fascinating problem yet to be resolved. At first glance, the immunobiology of pregnancy appears to resemble that which occurs following transplantation of allogeneic organs (Medawar, 1953; Billingham, 1964), since a fetus is to its mother as a transplant is to its host. Pregnant females are able to mount systemic immune responses to microbial pathogens and even to paternal and fetal antigens (Ahrons, 1971; Weber and Nelson, 1986; Tafuri et al., 1995; Zhou and Mellor, 1998), indicating that maternal immunity remains functionally intact during pregnancy. Thus, the maintenance of the fetus must rely on a balance achieved by subtle immunoregulation at the maternal–fetal interface. The mechanisms that regulate the maternal–fetal balance have been postulated from at least three different aspects: (1) segregation of the fetus from the maternal immune system and/or “neglect” of the “fetus allograft” by the maternal immune cells, (2) active involvement of maternal secretory or cellular suppressive factors in tolerating the “fetus allograft,” and (3) interference of the potentially hazardous immunological attack by factors derived from the fetus. Several lines of evidence, outlined briefly below, have been reported to support these three mechanisms, each of which seems to be partially responsible for this immunologic paradox. It is now generally accepted that trophoblast cells (villous syncytiotrophoblasts in humans and labyrinthine trophoblasts in mice), which are in direct contact with maternal blood in the placenta, do not express classical class I and class II major histocompatibility complex molecules (MHC, in mice) or leukocyte antigens (HLA, in humans) (Head et al., 1987; Loke, 1989; Redline and Lu, 1989; Torry et al., 1997; Hutter et al., 1998). These “MHC-null” cells may serve as a mechanical barrier to physically protect the fetus, since they cannot be recognized by maternal T lymphocytes and are thus incapable of eliciting allogeneic immune reactions (Jenkinson and Billington, 1974; Smith, 1983). The trophoblast cells that invade decidualized endometrium (i.e., extravillous cytotrophoblasts in humans and spongiotrophoblasts in mice) nevertheless do express certain MHC class I– like molecules (Kovats et al., 1990; King et al., 1996a; Le Bouteiller and Blaschitz, 1999; Le Bouteiller et al., 1999). On the one hand, these molecules (e.g., HLA-G 297 C 2001 by Academic Press Copyright All rights of reproduction in any form reserved. 0065-2776/01 $35.00

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in humans), characterized by limited polymorphism, may not be sufficient to stimulate cytotoxic T-lymphocyte (CTL)-mediated reactions as would the classical MHC molecules. On the other hand, the presence of such nonpolymorphic class I molecules on extravillous trophoblasts confers on them the identity as “self” cells, recognizable by the inhibitory receptors expressed on maternal natural killer (NK) cells (Verma et al., 1997; Ponte et al., 1999; Rajagopalan and Long, 1999), allowing them to escape the attack by NK cells (Moretta et al., 1992; Karre, 1995; Pazmany et al., 1996; Rouas-Freiss et al., 1997; Yokoyama, 1997). With regard to immunosuppressive factors, hormonal changes on the maternal side may induce their production. For example, high concentrations of estrogen and progesterone in the pregnant uterus have been shown to cause suppression of phagocytosis, B cell functions, and T-cell-mediated cytotoxicity (Simmons et al., 1968). These effects may be mediated in part through a 34-kD protein named progesterone-induced blocking factor (PIBF) produced by human lymphocytes (Faust et al., 1999; Laskarin et al., 1999). High concentrations of estrogen and progesterone may also play a role in recruiting the so-called “suppressor/regulatory” lymphocytes into the uterus (Clark et al., 1983; Olding et al., 1997). Moreover, recent studies have demonstrated that the systemic and local (within the pregnant uterus) cytokine production in pregnant women is often skewed toward a T helper 2 (TH2) type of dominant response (Lin et al., 1993; Wegmann et al., 1993; Raghupathy, 1997; Iwatani and Watanabe, 1998). The TH2-type cytokines, such as IL-4, IL-6, IL-10, and IL-13, are known to play important roles in the generation of humoral immunity but exhibit immunosuppressive activities in some situations (Mosmann et al., 1991; Romagnani, 1996). Such an “immunosuppressive” TH2 type of dominant response has been demonstrated to be crucial for the success of pregnancy (Wegmann et al., 1993; Raghupathy, 1997). In contrast, a TH1-type response (e.g., IFNγ and IL-2) has been associated with failure of pregnancy (Piccinni et al., 1998; Raghupathy et al., 1999). On the fetal side, α-fetoprotein has been reported to be a potent inhibitor of humoral antibody response and of mitogen/alloantigen-induced lymphocyte proliferation (Toder et al., 1979; Olding et al., 1997). Some poorly characterized factors produced by trophoblasts cultured in vitro, which appear to be capable of inhibiting the growth of monocytes, have also been postulated to suppress both lymphocyte-mediated and antibody-dependent cell-mediated cytotoxicity (McIntyre and Faulk, 1982; Saji et al., 1987; Sanyal et al., 1989; Silver et al., 1990; Muller et al., 1999). These observations, taken together, suggest that the fetus may actively participate in the local suppression of the maternal immune activity during gestation. During pregnancy, the uterus of viviparous mammals undergoes distinctive changes in order to accommodate the fetus (De Feo, 1967; Bell, 1983).

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These histophysiological and morphological changes are referred to as “decidualization.” The remarkably heterogeneous and complex cell populations within the decidualized uterine tissue are likely to participate in mediating and modulating the above-mentioned immunological events occurring at the maternal–fetal interface (Stewart and Peel, 1977; Gambel et al., 1985). Among these cells, the uterine natural killer (uNK) cells merit special attention and are the focus of this review. II. Uterine Natural Killer Cells of Rodents: Granulated Metrial Gland Cells

The most remarkable population of cells in the decidualized uterine tissue of rodents is the granulated metrial gland (GMG) cells (Peel, 1989; Stewart, 1991; Croy and Kiso, 1993; Liu et al., 1994; Head, 1996; Whitelaw and Croy, 1996). GMG cells owe their name to both their striking morphology and their unique localization in the pregnant uterus. The metrial gland (MG), a term first introduced by Selye and McKeown (1935), is a region of maternal uterine tissue that differentiates in the myometrium at the conceptus implantation sites in pregnant rodents. It extends from the attenuated muscle layer adjacent to the decidua basalis out to the mesometrium and serosal surface of the uterus, an area sometimes referred to as the mesometrial triangle (Bridgman, 1948) (Fig. 1). This pregnancy-associated tissue has been distinguished in mice (Smith, 1966;

FIG. 1. (A) Schematic representation of the decidualized mouse uterus. The decidualized tissue is made of actively proliferating cells which outgrow into the uterine lumen (L). Three regions can be delineated: antimesometrial area (AMA), mesometrial area (MA), and mesometrial triangle (MT) which eventually develops into the metrial gland during normal pregnancy. (B) Schematic representation of the mouse uterus during mid-gestation. The embryo-derived portion of the placenta, labyrinthine, and basal regions joins the embryo to the decidua basalis (DB). Both DB and the adjacent tissue, the metrial gland (MG), are derived from maternal cells.

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Stewart and Peel, 1977), rats (Wislocki et al., 1957; Larkin and Cardell, 1971; Peel and Bulmer, 1977), hamsters (Bulmer and Sunderland, 1983), guinea pigs (Asplund and Holmegren, 1940), and rabbits (Asplund and Holmegren, 1940). The metrial gland consists of three major components: fibroblast-like stromal cells, vascular cells, and GMG cells, all of which are believed to be maternal in origin. Although the mesometrial triangles begin to develop following the complete differentiation of decidual stromal cells, prominent metrial glands with abundant GMG cells only appear in the pregnant uterus or in a pseudopregnant uterus that has received physical stimuli for deciduomata formation. It is generally believed, although this is still controversial, that the maintenance of the metrial gland depends on gestational hormones, particularly progesterone (Sharma et al., 1986; Stewart, 1987, 1988a; Hunt et al., 1997a). The transient existence of metrial glands in the uterus, specifically accompanying pregnancy, and the strategic location of metrial glands at the maternal–fetal interface imply that they may play a critical role in the establishment of pregnancy. The postulation of metrial glands being the immune barriers that protect the fetus has mainly evolved from the studies on mouse and rat GMG cells. Some distinct features of rodent GMG cells will be discussed in the following sections. A. ORIGIN OF GMG CELLS Granulated metrial gland cells were originally described in the mouse as “maternal glycogenic cells” by Jenkinson in 1902 (Jenkinson, 1902). In recent years, extensive studies on the origin, distribution, morphology, and functions of GMG cells have been carried out by Stewart and Peel (Peel, 1989; Stewart, 1991), Croy (1990), and others (Ritson and Bulmer, 1989; Linnmeyer and Pollack, 1991; Liu et al., 1994; Head, 1996). As previously mentioned, GMG cells reside mainly at the metrial gland in the pregnant uterus. These cells, however, are also found in smaller numbers in the decidua basalis and sometimes in the labyrinthine portion of the placenta (Stewart and Peel, 1978; Jbara and Stewart, 1982; Stewart, 1984; Parr et al., 1990b). Occasionally, they can also be found in the maternal blood vessels of the metrial gland and decidua basalis (Stewart and Peel, 1978; Parr et al., 1987). Similar to the other components of the metrial gland, GMG cells are of maternal origin. That GMG cells may be derived from blood cell precursors was first speculated in the 1960s but received little attention (Smith, 1966). Subsequently, Peel and Bulmer (Peel and Bulmer, 1977) and Stewart and Peel (1977) performed a series of studies to investigate the structure/morphology of GMG cells in the developing decidua basalis and metrial glands in mice and rats. The results led these investigators to propose a lymphoid origin for GMG cells. In the early 1980s, a bone marrow origin for murine decidual leukocytes was proposed by Kearns and Lala (Kearns and Lala, 1982). The definitive evidence supporting this


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hypothesis, however, was not available until Peel and his colleagues performed bone marrow chimera studies using lethally irradiated mice reconstituted with either mouse or rat bone marrow cells (Peel et al., 1983; Peel and Stewart, 1984, 1986). The distinct morphological features of rat and mouse GMG cells allowed these investigators to demonstrate that mice reconstituted with rat bone marrow contained only GMG cells with the rat phenotype, whereas those reconstituted with mouse bone marrow possessed only mouse GMG cells (Peel et al., 1983). This study therefore clearly demonstrated that GMG cells are derived from bone marrow precursors that already reside in the uterus and differentiate in situ during pregnancy (Peel and Stewart, 1984; Parr et al., 1990b; Kiso et al., 1992). Results obtained in a later study, however, suggested that GMG precursors could also enter the uterus during decidualization initiated by the implantation of blastocysts (Peel and Stewart, 1988; Parr et al., 1992; Head, 1996). During the gestation period of a pregnant mouse, GMG cells are found to differentiate and appear in the uterus in the following sequence (Peel and Stewart, 1979; Stewart and Peel, 1980, 1981). Prior to implantation of the conceptus (on day 4.5), a few GMG cells seem to randomly distribute in the endometrium. By day 5, GMG cells become conspicuous and reside throughout the developing decidua and uterus. The number of GMG cells increases dramatically by day 6 of gestation, with a preferential localization in the decidua basalis and mesometrial triangle at the implantation sites. Concomitantly, GMG cells disappear from other regions of the decidua and uterus, probably due to local degeneration of this special cell type. The disappearance of GMG cells from the uterus outside the implantation sites argues against an important role for progesterone in maintaining GMG cells, since the entire uterus is supposed to be under the influence of progesterone. In the mesometrial triangles, GMG cells continue to proliferate and become a prominent cellular population in those areas now termed metrial glands. GMG cells proliferation appears to reach the peak by days 11 to 13 of gestation. During mid-gestation, GMG cells remain nonproliferating but are probably functionally active (Croy, 1990). Toward the end of gestation, GMG cells again degenerate and only a few of them remain by parturition (Croy, 1990; Delgado et al., 1996). Most GMG cells appear to die with morphological and biochemical characteristics indicative of apoptosis (Kusakabe et al., 1999b). Based on the studies described above, it has become clear that GMG cells are derived from hematopoietic precursors originating in bone marrow and differentiate/proliferate transiently in situ in the pregnant uterus. The next obvious question therefore is to which lineage of hematopoietic cells do GMG cells belong? Several studies demonstrated that mice with severe combined immunodeficiency (SCID) mutation and nude (nu/nu) mice both have normal development of metrial gland and GMG cells (Bosma et al., 1983; Croy and Kiso, 1993). These findings indicate that GMG cells are unlikely to be derived from either T or B lymphocyte lineage (Croy et al., 1991a). Beige (bg/bg) mice which are

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TABLE I GMG CELLS IN MUTANT OR TRANSGENIC MICE Mouse Strain

Features

GMG Cells

nu/nu bg/bg scid/scid op/op Tgǫ26 transgenic P561ck/IL-2R double knockout IL-2/β 2m double knockout IL-2Rγ (γ c) knockout RAG-2/γ c double knockout

T cells– , B cells+, NK cells+, macrophages+ T cells+, B cells+, NK cells+, macrophages+ T cells– , B cells–, NK cells+, macrophages+ T cells+, B cells+, NK cells+, macrophages– T cells–, B cells+, NK cells–, macrophages+ T cells–, B cells+, NK cells– , macrophages+ T cells–, B cells+, NK cells+, macrophages+ T cells–, B cells+, NK cells– , macrophages+ T cells–, B cells–, NK cells– , macrophages+

Present Present Present Present Absent Absent Present Absent Absent

defective in NK cell functional activity have also been shown to possess normal GMG cells and breed normally (Yamashiro et al., 1989; Croy et al., 1991a,b). The finding of GMG cells in bg/bg mice, however, does not preclude the NK lineage of GMG cells, since these mice still have nonfunctional NK cells which contain giant cytoplasmic granules (Roder and Duwe, 1979). The lineage of GMG cells was eventually deciphered by a series of studies conducted by Croy and colleagues (Guimond et al., 1996, 1997; Croy et al., 1996, 1997a). These investigators examined the development of GMG cells using a panel of transgenic or gene-knockout mice whose NK cell development and/or functioning had been altered (Table I). It was found that GMG cells were essentially absent in four lines of mice: Tgǫ26transgenic (deficient in T and NK cells) (Guimond et al., 1996, 1997), P56lck/ IL-2Rβ double knockout (deficient in T and NK cells) (Croy et al., 1997a), IL-2Rγ (γ c) knockout (deficient in T and NK cells) (Croy et al., 1997a), and RAG-2/γ c double knockout (deficient in B, T, and NK cells) (Ashkar et al., 2000). Furthermore, the GMG cell deficiency and reproductive defects in Tgǫ26 mice and RAG-2/γ c double knockout mice were shown to be reversed by reconstitution using the bone marrow derived from SCID mice (deficient in T and B lymphocytes, but not in NK cells) (Guimond et al., 1998; Ashkar et al., 2000). Taken together, these results reveal unambiguously the NK lineage of GMG cells. B. MORPHOLOGY AND PHENOTYPES OF RODENT GMG CELLS Mouse GMG cells are large round cells, ranging from 20 to 50 μm in diameter (Stewart et al., 1977). They possess abundant cytoplasm, eccentric nucleus (sometimes binucleate and occasionally multinucleate; see Stewart and Peel, 1977; Parr et al., 1990b), and acidophilic cytoplasmic granules. The granules have an average diameter of 2 to 3 μm but occasionally may be as large as 5 μm (Stewart and Peel, 1977). These large granules contain glycoproteins and can be stained by periodic acid Schiff’s (PAS) reagent (Fig. 2). In addition to the


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glycoprotein-containing granules, glycogen deposits can also be found in the cytoplasm of GMG cells. Cells analogous to mouse GMG cells have been found in other rodents, such as rats (Wislocki et al., 1957; Larkin and Cardell, 1971; Peel and Bulmer, 1977), hamsters (Bulmer et al., 1983), and field voles (Stewart and Clark, 1990b). Phenotyping studies have been performed to delineate the lineage relationship between GMG cells and various hematopoietic cells. Both mouse and rat GMG cells were found to express the leukocyte common antigen (CD45) (Mitchell and Peel, 1984; Mukhtar et al., 1989) and the Thy-1 antigen (a surface protein present on all T lineage cells, some NK cells, and occasionally other cell types) (Bernard et al., 1978; Parr et al., 1987; Mukhtar et al., 1989; Redline and Lu, 1989), confirming a lymphoid lineage for GMG cells. The morphological similarity characterized by abundant cytoplasmic granules led several investigators to propose earlier that GMG cells may be uterine NK cells. Further studies by several investigators demonstrated that mouse GMG cells expressed Thy-1 and two NK cell markers, asialo-GM1 and LGL-1 (Mukhtar et al., 1989; Parr et al., 1990b; Linnmeyer and Pollack, 1991). Interestingly, the expression of LGL-1 and asialo-GM1 appeared to be inversely correlated; i.e., LGL- 1bright cells displayed little or no staining of asialo-GM1, whereas LGL- 1dim cells showed more intense staining of asialo-GM1 (Mukhtar et al., 1989; Parr et al., 1990b). This latter finding suggests that LGL-1+ NK cells may differentiate in situ to GMG cells. During the differentiation process, precursor cells may lose LGL-1 surface antigens and gain a higher concentration of asialo-GM1 antigens (Mukhtar et al., 1989; Parr et al., 1990b). In rats, GMG cells do not express Thy-1 antigen and several other T-cell-associated antigens, but they do express asialo-GM1 and the 3.2.3 antigen (also known as NKR-P1), a triggering molecule expressed on rat NK cells (Mitchell and Peel, 1984; O’Shea et al., 1988; Head, 1990; Head et al., 1994). Taken together, these results provide strong evidence supporting the NK lineage of GMG cells. The relationship between GMG cells and NK cells is further suggested by the finding that mouse GMG cells also express several of the granule mediators of NK cells, lymphokine-activated killer (LAK) cells, and cytolytic T lymphocytes (Masson and Tschopp, 1985; Podack et al., 1985; Young et al., 1986a; Masson and Tschopp, 1987; Liu et al., 1995). By employing immunofluorescence techniques, our laboratory showed that a population of cells localized at the implantation sites on cryostat sections of pregnant uterus stained intensively with several polyclonal antisera (Parr et al., 1987, 1990b) and a monoclonal antibody (Joag et al., 1991) against mouse perforin, a potent cytolytic mediator (Fig. 3). These perforin+ cells also expressed the Thy-1 antigen and the GMG cell marker, asialo-GM1, and contained typical PAS+ granules in the cytoplasm. The numbers of perforinexpressing cells and the level of perforin production appeared to correlate with the time course of the appearance of GMG cells at the implantation sites (Zheng et al., 1991b). The positively stained cells became detectable by day 5 of gestation

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FIG. 2. Morphology of mouse GMG cells. Mouse GMG cells in the metrial gland of day 14 pregnant uterus. (A) A section of the metrial gland was examined under light microscope. Some GMG cells are present in the blood vessel (top to the panel), but most of the GMG cells are found in the metrial tissue. (B) Another section of the metrial gland was stained with periodic acid Schiff (PAS) reagent to reveal the characteristic glycoprotein-containing cytoplasmic granules. (Magnification ×300.) (C) A portion of the tissue obtained from a mouse under similar conditions was processed for immuno-electron microscopy and used here to show the ultrastructure of GMG cells. The presence of abundant cytoplasmic granules is evident. The inset shows the structure of a granule that characteristically contains an amorphous central matrix bounded by a membrane with a vesicular “cap.” (Reproduced with permission from L. M. Zheng, D. M. Ojcius, C.-C. Liu, M. D. Kramer, M. M. Simon, E. L. Parr, and J. D.-E. Young. FASEB Journal 1991;5:79–85.)


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FIG. 3. Expression of perforin by mouse GMG cells. Mouse GMG cells of day 14 pregnant uterus were stained with a polyclonal anti-mouse perforin antiserum and visualized by either immunofluorescence microscopy (A and B) or immuno-electron microscopy (C). Intensive staining of perforin in the cytoplasmic granules can be seen under fluorescence microscope. (Part A magnification, ×90; part B magnification, ×350.) The subcellular localization of perforin and serine esterase is further demonstrated by immunogold double labeling. Arrows points to the protein A-conjugated gold particles in the cytoplasmic granules. Small particles demonstrate anti-perforin staining, while large particles represent anti-serine esterase staining. (Reproduced with permission from E. L. Parr, M. B. Parr, and J. D.-E. Young. Biology of Reproduction 1987;37:1327–1336. Copyright C 1987, the Society for the Study of Reproduction, Inc.)

in a sparser and dimmer pattern and increased in both number and brightness as gestation proceeded. These cells also began to accumulate at implantation sites, particularly in the metrial glands. The peak of perforin expression in pregnant uterus occurred around days 12 to 15 of gestation, comparable with the time at which proliferation and activities of GMG cells reach the maximum (Parr et al., 1992). By the time of parturition, perforin-expressing cells were almost absent, as were most GMG cells. The expression of perforin in GMG cells was further validated by in situ hybridization and northern blotting analysis using specific nucleotide probes (Zheng et al., 1991b). Similarly, the accumulation of perforin messages in metrial gland showed a good correlation with the time course of GMG cell differentiation/proliferation. The perforin message, however, appeared and then diminished 1 to 2 days earlier than the protein products (Zheng et al., 1991b). In addition to perforin, two members of the serine esterase family associated with killer lymphocytes (Young et al., 1986b; Masson and Tschopp, 1987), granzyme A and granzyme B, were also found to be expressed by mouse GMG cells (Parr et al., 1990b; Zheng et al., 1991c). At the ultrastructural level, perforin and granzymes were co-localized to the cytoplasmic granules (Zheng et al., 1991c). Similarly, rat GMG cells were shown to express perforin (Head et al., 1994). The identification of NK cell-associated proteins in GMG cells not only provides important information regarding their origin but may also throw light on their functions, as we shall see later.

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C. REGULATION OF GMG CELL DIFFERENTIATION AND FUNCTIONALITY Since the expression of perforin and granzymes coincides with the development of GMG cells in the metrial gland, it is likely that these two processes are controlled by the same gestational endocrine and/or paracrine factors. The origin of these factors is believed to be maternal, as we have observed that perforinexpressing GMG cells were also present in deciduomata of pseudopregnant mice (where no fetus was present) (Zheng et al., 1991a). The observations that GMG cells vary in number and maturation during pregnancy suggest that hormones, particularly progesterone and estrogen, play a crucial role in controlling the differentiation of these cells (Sharma et al., 1986; Stewart, 1987, 1988a; Hunt et al., 1997a). Nevertheless, nonhormonal factors (e.g., cytokines) may also be important. Several cytokines, including interleukin (IL)-2, IL-6, IL-7, IL-12, and IL-15, have been shown to be capable of inducing the differentiation and activation of NK cells. They are also able to induce and upregulate the expression of perforin and granzymes in these cells (Liu et al., 1989, 1990; Smyth et al., 1990a,b; Salcedo et al., 1993; Gamero et al., 1995; Ye et al., 1996a). Among them, IL-15 belongs to the four-helix bundle cytokine family and exhibits some biological activities previously identified for IL-2 (Grabstein et al., 1994). The receptor complexes for IL-15 and IL-2 are known to share two common subunits, the β chain and the γ c chain (Giri et al., 1994; Grabstein et al., 1994). In spite of the similarities to IL-2, IL-15 is unique in its cell and tissue distribution and probably involves distinctive functions. While IL-2 is produced predominantly by activated T cells (Smith, 1988), IL-15 appears to be produced by a variety of cell types and tissues (Grabstein et al., 1994). The mRNA coding for IL-15 has been detected in organs including spleen, skeletal muscle, and placenta (Grabstein et al., 1994). The abundant expression of IL-15 in placenta suggests that it may be involved in regulating the differentiation of GMG cells during pregnancy. Indeed, we have found that IL-15, rather than IL-2, may play a critical role in the differentiation of GMG cells (Ye et al., 1996b). Our results showed that IL-15, but not IL-2, was capable of inducing the bone-marrow-derived precursors of GMG cells present within the uterus to differentiate in situ into mature cells expressing NK surface markers and effector molecules. Using reverse transcription–polymerase chain reaction analysis, we have demonstrated that IL-15 and the α chain of its cognate receptor (but not IL-2 and the IL-2 receptor α chain) were expressed in the mouse pregnant uterus during the early/mid-gestational stages (Ye et al., 1996b), concomitant with the expression of perforin and granzymes (NK cytolytic mediators) in differentiating GMG cells in vivo. Moreover, IL-15, but not IL-2, was capable of inducing the abundant expression of perforin and granzymes in GMG cells explanted in vitro. Preliminary in situ hybridization studies have revealed that macrophages present in the pregnant uterus may contribute to the production of IL-15 (Ye et al.,


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1996b), which in turn may drive the NK progenitor cells to differentiate in this unique microenvironment. We have also observed that IL-15-expressing cells were absent in the labrynthine placenta, excluding the possibility of trophoblasts (fetally derived cells) being an important source of IL-15. This latter finding is in accord with the notion derived from our previous study using pseudopregnant mice that maternal, but not fetal, factor(s) are crucial for the differentiation of GMG cells (Zheng et al., 1991a). However, it still cannot be ruled out that other types of stromal cells within the maternal decidual tissue may also be capable of producing IL-15. Similar observations regarding the expression of IL-15 and its effect on GMG cell differentiation/maturation have recently been reported by Allen and Nilsen-Hamilton (1998) and by Head (1996). These authors have also detected the expression of IL-2 in the late gestational stage when GMG cells are already fully differentiated/matured. Thus, IL-2 may play an ancillary role in the further activation of mature GMG cells (Croy et al., 1997b; Allen and Nilsen-Hamilton, 1998). III. Uterine Natural Killer Cells of Human and Other Mammalian Species

Although non-rodent mammals lack an organized tissue analogous to the metrial gland, cells with a bone marrow origin that are similar to GMG cells have been found in many other species. In humans and other primates, a population of granulated lymphoid cells known as endometrial granulocytes (EGs) (Cardell et al., 1969; van Bogaert, 1975; Bulmer and Sunderland, 1983) have been observed in the endometrium during early pregnancy or certain phases of the menstrual cycle (King et al., 1989b). These cells are alternatively termed Kornchenzellen (“K”) cells, endometrial stromal granulocytes (Hamperl, 1955; Hamperl and Hellweg, 1958), decidual large granulocytes (Bulmer et al., 1987), or uNK cells (the term to be used below) (King et al., 1996b). Human uNK cells are smaller than rodent GMG cells, with a maximum diameter of about 12 μm. Just like the rodent GMG cells, they also contain eosinophilic, glycoproteincontaining granules. The proliferation and differentiation of human uNK cells, in parallel to those of rodent GMG cells, appear to be under the influence of ovarian hormones. In human nonpregnant endometrium, the number of uNK cells varies throughout the course of the menstrual cycle. The uNK cells become prominent in the late secretory phase, a status appropriate for implantation, provided conception occurs (King et al., 1989b). If pregnancy does not occur, these cells die by apoptosis a few days prior to the onset of menstruation. When pregnancy occurs, these cells will persist and then increase remarkably in number in the decidua as gestation advances into the first trimester (Starkey et al., 1988; Pace et al., 1989). At this stage, uNK cells may account for up to 70% of the decidual leukocytes and localize most densely at the implantation site where fetal trophoblasts infiltrate into maternal tissues (Starkey et al., 1988; King et al., 1996b). uNK cells will begin to disappear afterwards, become less conspicuous by 20 weeks of gestation, and

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TABLE II PHENOTYPIC AND FUNCTIONAL CHARACTERISTICS OF MOUSE AND RAT GMG CELLS AND HUMAN UNK CELLS

Surface markers

Cytolytic mediators (mRNA or protein) Cytokines (mRNA or protein) a

Mouse GMG Cells

Rat GMG Cells

Human uNK Cells

Thy-1+, CD45+, CD3–, CD4– , CD8–, asialo-GM-1+, LG1-1+, a NK1.1+ Perforin, granzymes, TNF-α, CSF-1, EGF, IL-1, LIF, IFNγ

CD45+, CD2+/–,a CD8+/–,a asialo-GM-1+, NKR-P1+/–a Perforin, TNF-α

CD2+/– ,a CD7+/– ,a CD3–, CD4– , CD8–, CD16–, CD57–, CD56+bright Perforin, TNF-α, TIA-1 GM-CSF, CSF-1, IFNγ

IFNγ

Levels of expression decrease in mature GMG or uNK cells.

virtually vanish by parturition (Kazzaz, 1972). Unlike rodent GMG cells, human uNK cells do not accumulate in specific regions of the pregnant uterus. These cells distribute throughout the decidual tissue but are more noticeable around the spiral arteries and near the degenerative endometrial glands (Bulmer and Sunderland 1983). Interestingly, they are absent in chorionic villi (Lin et al., 1991). Detailed characterization of human uNK cells by immunohistocytochemistry and flow cytometry has been carried out in several laboratories. The results obtained indicate that human uNK cells display a distinct surface phenotype. These cells express a number of surface molecules, including CD45 (common leukocyte antigen), CD2 (early T cell marker), and CD7 (early T cell marker) (King et al., 1989b, 1991a; King and Loke, 1991c). They lack, however, the 55-kD interleukin-2 receptor α chain (IL-2Rα; CD25) (Bulmer and Johnson, 1986) and other T cell markers such as CD3, CD4, and CD8 (Bulmer and Sunderland, 1984; Bulmer and Johnson, 1985; Christmas et al., 1990; King et al., 1991b; King and Loke, 1991c). Notably, most human uNK cells have been shown to possess little or no CD16 (Fcγ RIII) and CD57, but they strongly express CD56 (Ritson and Bulmer, 1987b; Starkey et al., 1988; Geiselhart et al., 1995), surface molecules previously associated with the NK cell phenotype (Table II). CD56, which was originally identified as the NKH1 antigen and later shown to be identical to the neural cell adhesion molecule (NCAM), is present on virtually all NK cells (Lanier et al., 1989). This unique phenotype of uNK cells, CD45+/CD2+/CD7 +/CD3–/CD16–/CD56+bright/CD57–, is reminiscent of, and yet somewhat distinct from, that of the NK cells circulating in peripheral blood. Most peripheral-blood NK cells express high levels of CD16 and CD57, but a much lower level of CD56 (CD16+/CD57+/CD56+dim). A small subset of CD56+bright NK cells circulating in peripheral blood lacks the cytoplasmic granules (Lanier et al., 1986), in contrast to the high granularity of CD56+bright uNK


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cells. These results, taken together, strongly suggest that human uNK cells are related to peripheral blood NK cells and yet may represent a special subtype. It should, however, be pointed out that the expression of CD16 on human uNK cells increases upon IL-2 stimulation in vitro (King et al., 1992). Flow cytometry analysis of single-cell suspensions prepared from first-trimester decidua, however, revealed a small group of cells expressing a significant level of CD16 but a low level of CD56 (Starkey et al., 1988). Whether this minor population belongs to the uNK family (possibly as precursors) or are in fact NK cells deriving from contaminating blood remains unclear. Another line of evidence indicating the lineage of uNK cells came from the identification of several cytolytic mediators, including perforin, granzymes, and TIA-1, in human uNK cells (Lin et al., 1991; King et al., 1993; Rukavina et al., 1995; Gulan et al., 1997) (Fig. 4). Through immunohistochemical analysis, these

FIG. 4. Expression of perforin by human uNK cells. A frozen section of human 6-week gestational endometirum was stained with a polyclonal anti-mouse perforin antiserum that crossreacts with human perforin (a) or anti-NKH1 antibody (b) (magnification ×400). The similar distribution of NKH-1+ and perforin+ cells suggests that they are the same population of uNK cells.

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molecules were shown to be expressed in uNK cells in the decidualized endometrial stroma and decidual tissue of first-trimester gestational endometrium (Lin et al., 1991; King et al., 1993), as in the case of rodent GMG cells. The expression of these cytolytic effector molecules suggests that uNK cells are potentially capable of functioning as effector cells to lyse target cells. With regard to the proliferation and differentiation of human uNK cells, both hormonal factors (e.g., progesterone) and nonhormonal factors (e.g., cytokines) derived from the maternal side are thought to be involved (Booker et al., 1994; King et al., 1996b). A recent study by Verma et al. (2000) has shown that, while IL-2 was absent from the decidua and placenta, both IL-15 mRNA and proteins were detected in decidual macrophages and other decidual cell types. Moreover, IL-15 was shown to induce proliferation of uNK cells and activation of their cytolytic activity toward JEG-3 choriocarcinoma cells in vitro. These results, consistent with those observed for mouse GMG cells (Ye et al., 1996b; Allen and Nilsen-Hamilton, 1998), suggest an important role for IL-15 in the proliferation and differentiation of human uNK cells. GMG-like cells have also been found in nonprimate mammals. In pigs, a population of NK-like cells, referred to as intraepithelial lymphocytes, has been identified (Croy et al., 1988; King, 1988; Engelhardt et al., 1997). Some CD45+ CD5– MHC class II– lymphocytes containing one to three granules in their cytoplasm have also been observed as the dominant cell type in the uterine and endometrial glandular epithelia in sheep (Lee et al., 1988). As in the case of uNK cells of rodents and humans, these cells are thought to play a role in the immunobiology of pregnancy. IV. Biological Functions of Uterine Natural Killer Cells during Pregnancy

In order to investigate their functional and biochemical properties, it is necessary to obtain a homogeneous population of uNK cells. uNK cells and other decidual cell types are often prepared through either mechanical dissociation or enzymatic digestion (Ritson and Bulmer, 1987a; Stewart and Mukhtar, 1988b; Ritson and Bulmer, 1989; Parr et al., 1990a). These methods, although convenient, appear to result in preferential damage of uNK cells, which are large cells having no strong cytoskeletal structures (Peel, 1989). A unique functional characteristics of mouse uNK cells, however, has been taken advantage of for their isolation (Dickson, 1980; Mukhtar and Stewart, 1988). Mouse uNK cells appear to acquire a migratory ability after differentiation and can enter the lumina of blood vessels located in metrial glands, decidua basalis, and labyrinthine placenta (Stewart and Peel, 1978, 1980; Dickson, 1980; Stewart and Jbara, 1980; Jbara and Stewart, 1982; Parr et al., 1987) (Fig. 2). These cells were also found, in pregnant mice and pseudopregnant mice with deciduomata, at certain extrauterine sites including the capillaries in the lung and kidney (Stewart,


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1985). These observations suggest that mouse uNK cells can migrate effectively in vivo. Under in vitro conditions, mouse uNK cells could also migrate out of the cultured metrial gland explants more quickly than would other decidual cell populations (Mukhtar and Stewart, 1988; Ye et al., 1996b; Croy et al., 1997c). Such a migratory ability of uNK cells thus provides investigators an alternative method for preparing homogeneous uNK cell populations (Croy et al., 1991b). Mouse uNK cell lines have also been generated by fusing these migrating cells with SP2/0 mouse myeloma cells (van den Heuvel et al., 1994, 1996). Due to the technical impediments associated with isolation and characterization of uNK cells, the functional roles of uNK cells currently understood are largely inferential (Zheng et al., 1991d; Croy, 1994; Head, 1996; King et al., 1996b). Some insight, however, is being obtained using transgenic or geneknockout mice (Guimond et al., 1996, 1997, 1998; Stallmach et al., 1995; Croy et al., 1997a, 1996; Ashkar et al., 2000). Several proposals pointing toward important roles of uNK cells in maintenance of placenta and the immunobiology of pregnancy have been put forward, but they have yet to be confirmed. A. IMMUNOREGULATORY FUNCTION: CYTOKINE PRODUCTION Cytokines are believed to play important roles throughout pregnancy in regulating the growth and differentiation of various maternal and embryonic cells in the uteroplacental unit. The candidate cytokine producers located in pregnant uterus include glandular uterine epithelial cells, decidual stromal cells, and decidual leukocytes. Among the possible functions of mouse uNK cells, the production of cytokines has been investigated (Croy et al., 1991b). Croy et al. analyzed the cytokine activities present in the culture supernatants which had been conditioned by either metrial gland explants or isolated migratory uNK cells (98–100% homogeneous population). It was shown that at least four cytokine activities were detectable in the conditioned supernatants: colony stimulating factor-1 (CSF-1), IL-1, leukemia inhibitory factor (LIF), and certain unidentified factor(s) cytotoxic against the macrophage cell line 5/10.14 as well as early embryonic tissues. More recently, Kusakabe et al. (1999a) demonstrated, by immunohistochemical techniques, that mouse uNK cells were capable of producing and releasing epidermal growth factor (EGF) during early gestation (days 6 to 9) and late gestation (day 15). By employing the more sensitive reverse transcription–polymerase chain reaction (RT–PCR) techniques, several laboratories including ours have examined the presence of transcripts for a number of cytokines in the mRNAs of migratory uNK cells or metrial gland explants (Croy et al., 1991b; Ye et al., 1996b; Allen and Nilsen-Hamilton, 1998). Transcripts for several cytokines known to be expressed by cells of T or NK lineage were not detected. These undetectable cytokines included IL-2, IL-3, IL-4, IL-6, IL-7, granulocyte-CSF (G-CSF),

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granulocyte-macrophage-CSF (GM-CSF), interferon-γ (IFNγ ), tumor necrosis factor-α (TNF-α), TNF-β (lymphotoxin), and erythropoietin (Croy, 1990; Croy et al., 1991b; Ye et al., 1996b). Transcripts of LIF, CSF-1, and IL-15, on the other hand, were readily detectable, in accordance with the presence of these cytokines in the mouse uNK cell-conditioned culture supernatant. Similarly, the expression of cytokine mRNA and proteins in human CD16–CD56+bright uNK cells which had been purified were investigated in several laboratories (Saito et al., 1993; Jokhi et al., 1994b,c; Deniz et al., 1996). mRNAs for G-CSF, GM-CSF, M-CSF, TNF-α, IFNγ and LIF were detected by RT–PCR and the encoded proteins were shown to be present in the culture supernatants of these cells (Saito et al., 1993; Jokhi et al., 1994 b,c; Deniz et al., 1996). Although mRNA for TNF-α was initially not detected in mouse uNK cells (Croy et al., 1991b), it was later found to be expressed and produced in the decidua of pregnant uterus of several mammalian species including mouse, rat, and human (Casey et al., 1989; Yelavarthi et al., 1991; De et al., 1993; Parr et al., 1995). Although decidual macrophages would seem to be the best candidate as producers of TNF-α, in situ hybridization studies failed to verify this assumption (Yelavarthi et al., 1991). Instead, uNK cells were found to be responsible, at least partially, for the expression of TNF-α in the uteroplacental unit (Yelavarthi et al., 1991; Hunt et al., 1997a). In mice, TNF-α was shown to be expressed in uNK cells between day 10 and day 14 of gestation (Parr et al., 1995). Moreover, isolated uNK cells were shown to release TNF-α in in vitro culture (Markert et al., 1997). The finding of CSF-1 and EGF production by mouse uNK cells (Croy et al., 1991b; Kusakabe et al., 1999a) and GM-CSF by human uNK cells (Saito et al., 1993; Jokhi et al., 1994b,c; Deniz et al., 1996) is of interest since it lends support to the hypothetical function of CSF-1, EGF, and GM-CSF in regulating the differentiation of trophoblasts and other placental components (Pollard et al., 1987; Arceci et al., 1989; Evain-Brion et al., 1995). Because a close association of mouse uNK cells with trophoblasts has been observed in histological studies (Stewart, 1990a), high concentrations of CSF-1, EGF, and GM-CSF locally might be delivered to trophoblasts in a paracrine fashion. It is worthwhile mentioning that receptors for CSF-1, EGF, or GM-CSF have been found on fetal trophoblasts (Pollard et al., 1987; Jokhi et al., 1993, 1994a; Evain-Brion et al., 1995). Interleukin-1 is a pleiotropic cytokine that acts as a central mediator in host defense mechanisms during inflammation, infection, and tissue injury (Grey et al., 1984). This monocyte-produced cytokine has a broad spectrum of biological effects including the activation of lymphocytes, regulation of the growth and differentiation of lymphocytes and fibroblasts, and modulation of the production of acute phase proteins. Since mouse uNK cells seem to interact closely in vivo with at least three kinds of cells—trophoblasts, small lymphocytes, and endothelial


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cells (Stewart, 1990)—these latter cells might represent the physiological targets of IL-1 secreted by uNK cells. How IL-1 affects these cells, from the viewpoint of physiology during pregnancy, is still unknown. Leukemia inhibitory factor (LIF), a pleiotropic cytokine in the IL-6 family, may exert different biological effects on a variety of cells/tissues/organs (Gearing et al., 1987; Gough and Williams, 1989; Gadient and Patterson, 1999). The linkage of LIF with pregnancy is obvious in view of its ability to regulate the differentiation of embryonic stem cells derived from blastocysts (Williams et al., 1988), its widespread distribution in the uterine tissue and placenta (Kojima et al., 1995; Yang et al., 1995; Senturk and Arici, 1998; Vogiagis and Salamonsen, 1999), and its importance in blastocyst implantation (Vogiagis et al., 1999). The low-affinity LIF receptor (LIFR) has been shown to be present on murine and human decidual macrophages, fetal trophoblasts, and certain embryonic tissues (Gough and Williams, 1989; Croy et al., 1991b; Hilton et al., 1991; Kojima et al., 1995; Yang et al., 1995; Sharkey et al., 1999). Abundant expression of LIF and LIFR in the mouse uterus has been shown to occur on days 4 to 5 of pregnancy when embryo implantation takes place (Bhatt et al., 1991; Yang et al., 1995). Likewise, Croy et al. (1991b) have demonstrated LIF production to be both a peri- and post-implantation event in mouse. For humans, LIF has been found to be expressed in uterine endometrium and decidua (Kojima et al., 1995; Sharkey et al., 1999). High levels of LIF expression were detected during the secretory phase of the menstrual cycle and early pregnancy, coinciding with the time for implantation and placenta development (Senturk and Arici, 1998). Moreover, LIFR was detected in villous and extravillous trophoblasts, suggesting the possibility that LIF regulates the growth and differentiation of trophoblasts. It is therefore conceivable that LIF may be involved in the initiation of blastocyst implantation and the subsequent differentiation as well as maturation of placental tissues (Senturk and Arici, 1998; Smith et al., 1998; Vogiagis and Salamonsen, 1999). Such a role for LIF in reproduction has been validated by the finding that blastocyst implantation and placenta development failed to take place in mice lacking the expression of LIFR in uterine endometrium (Ware et al., 1995). A defective production of LIF has also been implicated in infertility and recurrent abortions in women (Hambartsoumian, 1998; Piccinni et al., 1998). The biological function of LIF in reproduction may in fact extend beyond its involvement in implantation. Considering that LIF has the capacity to regulate the differentiation of leukocytes, it may also participate in modulating the local immune activities at the maternal–fetal interface. This latter role, nevertheless, awaits further investigation. Interferon-γ produced by NK cells has been shown to be an important component of the early host response to microbial infections by activating macrophages and other effector cells including NK cells themselves (Trinchieri, 1989; Biron et al., 1998, 1999). IFNγ , a TH1 cytokine, may also facilitate host defence by

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inducing or upregulating the expression of MHC and intercellular adhesion molecules on vascular endothelial cells, parenchymal cells, and immune cells (Boehm et al., 1997). The detection of IFNγ mRNA and protein in mouse uteroplacental units and uNK cells (Platt and Hunt, 1998) and human uNK cells (Guilbert et al., 1993; Jokhi et al., 1994a–c)as well as IFNγ receptors on mouse uNK cells (Chen et al., 1994), therefore, is of noteworthy interest. IFNγ secreted by uNK cells may exert activating effects on decidual macrophages or stromal cells in a paracrine fashion and/or on themselves in an autocrine fashion, thereby contributing to the local immunoregulatory mechanisms. A series of elegant studies by Croy and colleagues using transgenic or mutant mice has demonstrated clearly that mouse uNK cells are a major source of IFNγ in the pregnant uterus, since the concentrations of IFNγ at the implantation sites were considerably lower in the uNK cell-deficient (Tgǫ26 transgenic or RAG-2/γ c double knockout) mice than in the wild-type mice (Ashkar and Croy, 1999; Ashkar et al., 2000; Croy et al., 2000). Moreover, these studies have unraveled a novel function of uNK cells and their secreted cytokines such as IFNγ concerning the normal development of decidua and placenta tissues (Ashkar and Croy, 1999; Croy et al., 2000), because decidua of the pregnancy uterus in female uNK-deficient mice exhibited necrosis, hypocellularity, and abnormal vasculature (Ashkar and Croy, 1999; Ashkar et al., 2000; Croy et al., 2000) (see further discussion in Section IV.C below). The deficiency of uNK cells and the decidual abnormalities could be reverted by either transplanting bone marrow (BM) cells derived from wild-type, but not IFNγ -deficient, mice into the uNKdeficient female mice (Ashkar et al., 2000; Croy et al., 2000), or injecting these mice with IFNγ (Ashkar et al., 2000). These results strongly support the notion that IFNγ is required for normal pregnancy. IFNγ is also involved, via an autocrine pathway, in regulating the maturation of uNK cells, since uNK cells present in the pregnant uterus of RAG-2/γ c double knockout mice reconstituted with BM cells derived from IFNγ receptor-α-deficient mice displayed an immature phenotype characterized by fewer, smaller cytoplasmic granules (Ashkar et al., 2000). uNK cells in these BM-reconstituted mice were capable of producing IFNγ , but could not respond to IFNγ due to their deficiency of IFNγ receptor-α (Ashkar et al., 2000). Although the above-mentioned data indicate undoubtedly the importance of IFNγ during normal pregnancy, it should be pointed out that a TH1-dominant cytokine profile in the pregnant uterus has been implicated in the failure of pregnancy (Lin et al., 1993; Wegmann et al., 1993; Raghupathy et al., 1999). It is plausible that a cytokine network with an intricate balance between IFNγ and other cytokines and their crucial timely expression, by either uNK cells or other decidual and placental cells, are essential for maintaining a local milieu that tolerates the “allogeneic” placenta and fetus (Guilbert et al., 1993; Jokhi et al., 1997; Piccinni et al., 2000).


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B. IMMUNE-EFFECTOR FUNCTION: CYTOLYTIC ACTIVITY A cytolytic function for uNK cells has been proposed based on their expression of perforin and granzymes (Parr et al., 1987, 1990b; Zheng et al., 1991c; King et al., 1993) and the morphological resemblance of rodent GMG cells and human uNK cells to peripheral blood NK cells and LAK cells (Ginsburg et al., 1989). Previous studies in our laboratory and by others have shown that perforin, following the release from cytolytic lymphocytes, is capable of binding to the plasma membrane of the target cell and polymerizing to form transmembrane pores (Masson and Tschopp, 1985; Podack et al., 1985; Young et al., 1986a; Liu et al., 1995). The presence of these water-/salt-permeable transmembrane pores eventually leads to colloid osmotic lysis of the target cell (Liu et al., 1995). The lytic activity of perforin is dependent on calcium ions and exhibits no apparent specificity for target membranes; perforin has been shown to lyse a wide variety of targets ranging from liposomes to erythrocytes to nucleated tumor cells. Expression of perforin has also been associated with the activation of cytolytic cells, since resting CTLs do not express this molecule, and reagents that activate lymphocytes also enhance perforin expression (Liu et al., 1989, 1990; Joag et al., 1990; Smyth et al., 1990b). Similar to other activated cytolytic effector cells, rodent and human uNK cells have also been shown to express TNF-α (Chen et al., 1991; Yelavarth et al., 1991; Parr et al., 1995) and TIA-1 (King et al., 1993). The expression of multiple cytolytic factors in uNK cells not only implies that these cells could be potent cytolytic cells, but also raises the question concerning the physiological targets of uNK cells. Hypothetically, embryo-originated trophoblast cells, which reside in the anatomical frontier facing the maternal environment, could be the targets of any maternal immune attack, including uNK cell-mediated cytolysis. Experimental data pertaining to this issue, however, are sometimes contradictory. In mice, the layer 1 trophoblasts, which are in direct contact with maternal blood, are believed to be poor targets for maternal cytotoxic T lymphocytes since they often do not express classical class I and class II MHC antigens (Redman et al., 1984; Loke, 1989; Redline and Lu, 1989). However, the lack of MHC antigens may theoretically render these cells recognizable by maternal NK cells and thereby susceptible to their attack as “non-self” target cells (Moretta et al., 1992; Karre, 1995; Yokoyama, 1997). Such injury of trophoblasts can potentially jeopardize the survival of the fetus. To ensure the success of pregnancy, therefore, certain mechanism(s) must be operating to protect these trophoblasts from injury. In vitro studies, nevertheless, have shown that uNK cells are capable of lysing freshly isolated placental trophoblast cells (Stewart and Mukhtar, 1988a). This observation is consistent with previous histological studies showing the association of uNK cells with degenerated trophoblasts which lined the maternal blood space in the labyrinthine (layer 1 trophoblasts) (Stewart, 1984). This

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kind of uNK cell-trophoblast interaction was also observed in a cross-species setting in vivo (in the rat-bone-marrow-reconstituted mice) and in vitro, in which rat uNK cells were observed to attach to and kill mouse trophoblasts (Peel and Stewart, 1989). Conversely, rat trophoblasts have been shown to be killed by either rat or mouse uNK cells in vitro (Peel and Adam, 1991). How uNK cells kill trophoblasts in vivo is not well understood. It is of interest to note that the association of uNK cells with degenerating trophoblasts appeared significantly less frequently in perforin knockout mice (Stallmach et al., 1995). This latter finding suggests that perforin released by uNK cells may indeed play a role in the elimination of certain trophoblasts (e.g., trophoblasts that invade the maternal tissues). The possibility of mouse trophoblasts being targets of other cytolytic effector cells has also been examined in several experimental settings. Trophoblasts were shown to be susceptible to LAK cell-mediated cytolysis but resistant to the lysis mediated by either cytotoxic T lymphocytes or conventional NK cells (Zuckerman and Head, 1987, 1988; Drake and Head, 1989; Head, 1989). Based on these results, it is still unclear whether trophoblasts are indeed the main physiological targets for uNK cells in vivo (Head, 1989). Previous studies attempting to characterize the cytolytic capacity of mouse uNK cells in vitro using the NK-sensitive target cells, YAC-1, revealed either no (Croy and Kassouf, 1989) or low (Croy et al., 1991c) degrees of cytolysis when mouse uNK cells were first stimulated with IL-2 to augment their cytolytic activity. These inconsistent results could have been due to the difficulty in obtaining functionally intact uNK cells, as mentioned above. It is, however, possible that uNK cells require appropriate stimulation(s), which might be available only locally in vivo, to express their inherent cytolytic ability. Recent studies investigating the cytolytic capacity of mouse uNK cells have demonstrated that these cells were capable of lysing Wehi 164 fibrosarcoma cells via a TNF-α-mediated mechanism (Stewart and Peel, 1999). In humans, studies investigating the cytolytic activity of uNK cells toward trophoblasts and other target cells have also yielded inconsistent results. Similar to the layer 1 trophoblasts in mice, villous syncytiotrophoblasts bathed in maternal blood do not express classical MHC antigens and thus may evade recognition by maternal T lymphocytes. Whether and how syncytiotrophoblasts are protected from NK cell-mediated attack remains unclear. Extravillous cytotrophoblasts that invade the maternal uterine tissue, on the other hand, are known to express the nonclassical, less polymorphic forms of MHC molecule (e.g., HLA-G) (Kovats et al., 1990; McMaster et al., 1995; Le Bouteiller and Blaschitz, 1999; Le Bouteiller et al., 1999). The HLA-G molecules expressed on these trophoblasts, through engaging specific killer-inhibitory receptors (KIRs) present on the surface of maternal NK cells (Hiby et al., 1997; Verma et al., 1997), can inhibit the cytolytic activity of NK cells and thus protect fetal cells from maternal immune-attack (Munz et al., 1997; Soderstrom et al., 1997; Rouas-Freiss et al.,


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1997; Biassoni et al., 1999; Ponte et al., 1999). Whether a similar fetus-protection mechanism operates in mice is unknown, since mouse MHC molecules analogous to human HLA-G have not been identified. It should be pointed out, however, that although the protection of trophoblasts is critical for the development of placenta and survival of the fetus, it is necessary for the maternal immune system to eliminate trophoblasts that aberrantly intrude beyond the maternal uterine tissue. In this scenario, uNK cells and other maternal NK cells (and probably decidual T lymphocytes) are expected to execute their cytolytic activity promptly on trophoblasts that have wandered too far to prevent their further migration/invasion (King et al., 1997; Zdravkovic et al., 1999). With respect to the cytolytic activity of human uNK cells in general, King et al. (1989a, 1990) reported in several earlier studies that human uNK cells isolated from first-trimester decidua were capable of lysing the human NK cell target, K562 cells, but were unable to kill trophoblasts. The resistance of first-trimester trophoblasts and a choriocarcinoma cell line, JEG-3, to cytolysis, however, did not extend to the killing mediated by IL-2-stimulated uNK cells (King and Loke, 1990b). Ferry et al. (1991) obtained similar results, except that only choriocarcinoma cells (BeWo cell line), but not term trophoblasts, were sensitive to the lysis mediated by IL-2- stimulated decidual CD56+ large granular lymphocytes. Collectively, these results suggest that (1) human uNK cells, under appropriate conditions, could effectively damage certain but not all trophoblasts, and (2) trophoblasts may be protected from maternal immune attack via multiple protective mechanisms. With regard to the former, it should be pointed out that an IL-2-like factor produced by human villous syncytiotrophoblasts has subsequently been identified in human placenta and amnion using anti-IL-2 antibody (Soubiran et al., 1987). This finding, together with another study observing the expression of the IL-2 gene in human syncytiotrophoblasts (Boehm et al., 1989), implies that IL-2-like factor(s) capable of activating human uNK cells is(are) potentially present in the uteroplacental unit. Recent studies by the authors (Ye et al., 1996b) and others (Allen and Nilsen-Hamilton, 1998; Verma et al., 2000) demonstrating the presence of IL-15, a cytokine functionally analogous to IL-2, in the pregnant uterus in mice and humans have lent further support to this postulation. Regarding the immuno-effector role of mouse and human uNK cells, it also remains to be determined whether these cells are involved in preventing vertical transmission of virus from the mother to the fetus. In view of the critical role that NK cells play in halting viral infection, a similar function for uNK cells has been proposed. Investigation in this respect is important since viral infections represent one of the leading causes of birth defects (Hanshaw, 1971; Florman et al., 1973). A recent study using perforin knockout mice, however, showed that lymphocytic choriomeningitis virus infecting the pregnant mouse failed to trespass onto the fetus (Stallmach et al., 1995). Although this latter study does

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not necessarily exclude the role of uNK cells in preventing maternal–fetal virus transmission, it appears to argue against a critical role for perforin in this function. One possibility is that uNK cells may prevent virus transmission; if they do so, it is by secreting certain antiviral factor(s) such as IFNγ . Similar to other immune effector cells, mouse and human uNK cells might also be involved in certain pathological conditions. In pathological pregnancy, the survival of the fetus is compromised and abortion takes place. Abortion that occurs during early pregnancy without clear etiology is referred to as spontaneous abortion. In mice, asialo-GM1+ cells (probably uNK cells) have been reported to infiltrate the necrotic fetus (Gendron and Baines, 1988, 1989), and treatment of spontaneous abortion-prone pregnant mice with anti-asialo-GM1 antiserum significantly reduced the frequency of fetus loss (de Fougerolles and Baines, 1987; Gendron and Baines, 1989). In contrast, injection with either polyinosinic cytidilic acid (Poly I:C, a reagent known to induce the production of IFNγ ) or IL-2 and/or IFNγ (TH1 cytokines capable of enhancing NK cell activity) greatly increased the abortion rate (de Fougerolles and Baines, 1987; Chaouat et al., 1990; Lala et al., 1990). These results suggest that uNK cells are probably involved in spontaneous abortion. However, results obtained from our studies using the CBA/J X DBA/2 (spontaneous abortion-prone) model showed that only small numbers of perforin+ cells (probably uNK cells) were detected near the dying fetus and that very few small, perforin+ cells actually infiltrated the dead fetus (Zheng et al., 1993). Although our findings appear to exclude a direct role of uNK cells in spontaneous abortion, they do not formally rule out an indirect involvement of these cells in the demise of the fetus. Similarly, in humans, conclusive evidence for the direct cytotoxic effects of uNK cells against potential target cells within the uteroplacental units in spontaneous abortion remains to be shown (Chao et al., 1995; Vassiliadou and Bulmer, 1998). Based on the brief overview presented above, it is clear that the possible role for uNK cells as effector cytolytic cells is still ambiguous. This ambiguity is likely due to the technical difficulties in obtaining functionally intact uNK cells and the presence of certain physiological factors intrinsic to the decidua. In the latter case, the cytolytic function of uNK cells might be down-regulated by the other decidual cells or their secretory products (Scordas et al., 1990). Other issues related to the cytolytic function of uNK cells, such as the identity of the trophoblast antigen recognized by uNK cells, will also need to be addressed when functional uNK cells become available in large numbers for the implementation of definitive studies. C. NONIMMUNOLOGICAL FUNCTIONS: REGULATION OF THE UTEROPLACENTAL UNIT Several lines of evidence indicate that rodent and human uNK cells may exhibit an endocrinological function. Selye and McKeown (1935) and


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Dallenbach-Hellweg et al. (1965) proposed that rat uNK cells were a source of relaxin. Although the initial study using fluorescent antibodies localized relaxin to uNK cells (Dallenbach-Hellweg et al., 1965), later studies did not support that finding, as the bioactivity of relaxin could not be detected in the uterine tissue extracts prepared from pregnant rats (Larkin, 1974; Anderson and Long, 1978). Subsequently, however, it was reported that relaxin was detected in endometrial granulocytes in hamster uterus (Renegar et al., 1987). In addition to relaxin, two other transcripts: 2ar (osteopontin) and SPARC (osteonectin), whose protein products may act as hormones, have also been identified in mouse uNK cells by in situ hybridization (Nomura et al., 1988). Extracellular matrix in the uterus undergoes dynamic changes during pregnancy and menstrual cycles. Because the development of uNK cells appears to coincide with the late secretory phase of the menstrual cycle (in humans) and implantation of the embryo (in rodents and humans), it is reasonable to postulate that uNK cells may participate in the modulation of extracellular matrix of the uterus. The detection of osteopontin mRNA in mouse uNK cells (Nomura et al., 1988) and the mucinous glycoprotein Muc-1 in the cytoplasmic granules of mouse uNK cells (Braga and Gendler, 1993) has provided evidence supporting this postulated biological function of uNK cells. Moreover, a reduced cellularity has been noticed in the decidua in the pregnant uterus of Muc-1-deficient mice (Croy et al., 1997a). This latter finding implies that Muc-1 secreted by uNK cells may be essential for maintaining the development and viability of decidual stromal cells. Degradation of extracellular matrix and basement membrane by matrix metalloproteinases (MMPs) is an important component of matrix modulation (Matrissian, 1990). In coordination with the production of matrix materials, uNK cells may be involved in the remodeling of uterine tissue by secreting MMPs. Indeed, a recent study has demonstrated that human uNK cells (characterized as CD56+ large granular lymphocytes) secrete significant amounts of MMPs, particularly MMP-9, which is capable of degrading gelatin and collagen (Martelli et al., 1993; Shi et al., 1995). Taken together, these results suggest that one of the biological functions of uNK cells is to regulate the remodeling of the uterine tissue and to maintain a healthy uteroplacental unit which can properly nourish the developing fetus. The importance of uNK cells in the maintenance of healthy decidua and placenta is reinforced by the studies using a panel of transgenic and/or geneknockout mice (Croy et al., 1996, 1997a; Guimond et al., 1997). These studies showed that female Tgǫ26 mice (deficient in T cells and NK cells, including uNK cells) appeared to have normal rates of ovulation and implantation following mating, but they suffered from considerably higher rates of fetus loss during early to mid-gestation than wild-type mice (Guimond et al., 1997). The implantation sites of the remaining viable fetuses were shown to contain small placentae and altered vascular structures at the base of decidua and maternal uterine tissues

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(Guimond et al., 1997). The vascular changes progressed through the mid- to late gestation period and resembled those present in human preeclampsia and hypertension, including thickening of the smooth muscle media, development of foam cells, and deposition of lipid material (Croy et al., 2000). The maternal spiral arteries in the decidua basalis also failed to dilate into the lacunae as occurs in the normal pregnant uterus. As a result of the poorly developed vasculature in the uteroplacental unit, the pups born to the female Tgǫ26 mice were significantly smaller in size compared to the normal litters. Similar histopathological changes have been observed in the pregnant uterus of RAG-2/γ c double knockout mice that are also deficient in uNK cells (Ashkar et al., 2000). The distinctive vasculature changes in the pregnant uterus of uNK-deficient, Tgǫ26 and RAG-2/γ c double knockout mice suggest that uNK cells may be involved in regulating the development of decidual vasculature directly or indirectly via certain yet-unknown mechanism(s). Clues to the possible mechanism(s) have come from recent studies identifying the expression of inducible nitric oxide synthase (iNOS) by uNK cells in mice and rats (Hunt et al., 1997b; Sladek et al., 1998; Burnett and Hunt, 2000). In the pregnant mouse uterus, iNOS was found to be expressed in different types of cells including macrophages, mast cells, and uNK cells. uNK cells appeared to the most prominent of the cell type producing iNOS in the decidua basalis and metrial glands. The expression of iNOS by mouse uNK cells was shown to begin at day 8 and peak at day 10 of gestation, time periods concurring with the development of placental vasculature (lacunae) (Hunt et al., 1997b). iNOS has been shown to be the key enzyme responsible for the synthesis of nitric oxide (NO) in leukocytes (Lowenstein et al., 1994; Sessa, 1994). The production of NO by uNK cells, therefore, can be inferred based upon the expression of iNOS in these cells during the critical period of placental vasculature development. NO generated may induce the relaxation of vascular smooth muscle cells into the formation of blood lacunae, which allow gas and nutrient exchange for the developing fetus. The importance of NO in pregnancy has been demonstrated by studies showing that inhibition of NO synthesis in pregnant rats caused maternal hypertension and intrauterine fetal growth retardation (Molnar et al., 1994; Chwalisz et al., 1999). The temporal production of NO during pregnancy, with the iNOS activity greatly decreased near parturition (Sladek et al., 1993; Hunt et al., 1997b; Sladek et al., 1998), also suggests that the concentration of NO in the pregnant uterus is critical for the maintenance of pregnancy. Therefore, it is conceivable that the absence of uNK cells (and hence NO) in the pregnant uterus of Tgǫ26 mice and RAG-2/γ c double knockout mice may account for the abnormality in the placental vasculature, the impairment in placenta development, and consequently the early demise of the fetus. Interestingly, Hunt and colleagues have shown that uNK cells express a high level of IFNγ , an effective inducer of iNOS, during early pregnancy (Platt and Hunt, 1998). The temporal relationship of the expression of IFNγ (days 6


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to 10 of mouse gestation) and iNOS (days 8 to 10 of mouse gestation) suggests an autocrine regulation of iNOS in uNK cells and perhaps a paracrine regulation of iNOS in decidual macrophages and other decidual cells. These results, collectively, may provide sensible explanations for a recent finding concerning the importance of IFNγ for normal pregnancy in mice (Ashkar et al., 1999). V. Concluding Remarks

The mechanisms underlying the regulation of maternal–fetal immune interactions during pregnancy have undergone extensive scrutiny over the past several decades. This review attempts to cover some of the issues related to uNK cells. Although the origin, morphology, and phenotypes of these cells are relatively well characterized, their physiological functions are, at best, partially understood. It is, however, becoming obvious that the functional capacities of uNK cells are not limited to the immunological ones originally postulated. Recent studies have revealed that uNK cells may be involved in a wide spectrum of biological changes occurring during pregnancy, ranging from immunosurveillance of trophoblasts, to immunomodulation of the local milieu, to promotion of trophoblast growth. Among them, a specific role for uNK cells in maintaining a healthy uteroplacental unit that supports the growth of the fetus is emerging. Studies using genetically modified mice which have been initiated in several laboratories are expected to provide more definitive information in the near future. ACKNOWLEDGMENTS We wish to thank Dr. Li-Mou Zheng and Dr. Paul Lin for performing excellent immunohistochemical and electron microscopic studies for some of the work described here. Part of the work described here was supported by the grants from the National Institutes of Health (CA-47307). C.-C. Liu is supported by an Established Investigator Award from American Heart Association–National Center.

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INDEX

A

neutralization attenuated vaccines, 33 infective vaccines, 32–33 linear epitopes, 33–34 mechanisms, 6–8 structural studies, 8–9 vaccines, 32 viral sensitivity, 23–24 non-Gal determinants, 141–142 response neutralization hepatitis B virus, 26 hepatitis C virus, 26 HIV, 26–27 influenza viruses, 25–26 lymphocytic choriomeningitis virus, 27 virus-neutralizing, 3–4 in xenotransplantation immunosuppression, 162–163 Anticoagulants, in xenotransplantation, 153–154 Antigen receptors, signaling BLNK-associating proteins, 116–117 GEMs role, 110–113 SLP-76 coupling to downstream events, 113–116 Antigens in auto-antibody responses, 20–21 in B cell responses dose, 12–13 organization, 13–15 structure, 15–16 Gal, in xenotransplantation, 137 in interleukin-12 production, 79 VHH binding loop structures, 286–289 immune libraries, 281–284 specific binders from libraries, 280

Acute humoral xenograft rejection antibody role, 136, 141–142 endothelial cell activation, 151–152 macrophages, 146 monocytes, 146 natural killer cells, 143, 145 neutrophils, 148 Adaptor proteins Grb2-related, 98–99, 118–120 in ITAM-coupled receptor signaling key events, 93–95 T cell linker, 95–97 lipopolysaccharide-binding, 231 in PLC-γ 1 activation, 118–120 SLP-76, ITAM-coupled signaling, 98 ADPases, in xenotransplantation, 170–171 AHXR, see Acute humoral xenograft rejection Allogeneic chimerism, in xenotransplantation, 172–173 Animals, in xenotransplantation, 189 Antibodies antidiotypic, in xenotransplantation immunosuppression, 159 anti-Gal, 136–137, 139–141, 157–161 antiviral responses, see Antiviral antibody responses auto-antibody responses, B cell induction, 19–21 in AXHR, 136, 141–142 heavy-chain, see Heavy-chain antibodies infection enhancement, 22 memory maintenance, 29–30 natural in antiviral responses, 9–10 xenotransplantation tolerance, 175–176

331

332

INDEX

Antigens (continued) xenotransplantation genetic engineering, 167–168 pharmacotherapy, 181–182 Anti-inflammatory hormones, 80–81 Antimicrobial genes, in immune response differential expression, 248–249 GATA factors, 247–248 immune deficiency pathway, 245–247 induction and regulation, 238–240 Relish pathway, 244–247 Toll pathway, 240–244 Antiviral antibodies, V(D)J rearrangements, 3 Antiviral antibody responses complement role, 10–12 Fc-receptors role, 12 mechanisms, 5 natural antibody role, 9–10 overview, 2–3 protection, 5–6 ATPases, in xenotransplantation, 170–171 Auto-antibody responses, B cell induction, 19–21 Autoimmunity, B cell induction, 19–21

B Bacteria, interleukin-12 induction, 62–63 B cell linker protein associating proteins, 116–117 deficient B cells, 108–110 ITAM-coupled signaling, 99–100 B cells autoimmune induction, 19–21 BLNK-deficient, 108–110 interleukin-12 production EBV-transformed cells, 59–60 normal cells, 60 responses, antigen role dose, 12–13 organization, 13–15 structure, 15–16 unresponsiveness, viral studies, 18–19 in viral infections, 16–17 in xenotransplantation pharmacotherapy, 181–182 Biotechnology, VHH role, 289–290 BLNK, see B cell linker protein Bombyx mori, immune response, 229

C Cactus, in Drosophila immune response, 243–244 Camelidae, heavy-chain antibodies antigen-specific, isolation dromedary immunization, 275–276 monoclonal VHH, 277–281 proteolyzed HCAbs, 276–277 H chain CH1 domain removal, 265–266 variable domain, 266–269 L chain, 269–270 natural occurrence, 261–264 ontogeny, 272–275 VHH antigen-binding loop structures, 286–289 antigen-specific, immune libraries, 281–284 biotechnological role, 289–290 gene phylogeny, 270–272 structure, 285 VL-side conformation, 285–286 Cardiovascular system, xenotransplantation barrier, 185 CCR5, see Chemokine receptor 5 CD47, in interleukin-12 production, 79 Cell differentiation granulated metrial gland cells, 306–307 human uNK cells, 310 Cell proliferation, human uNK cells, 310 Cellular responses, in xenotransplantation acquired response, 148–150 immunosuppression, 163–164 innate, see Innate cellular responses CH1 domain, in heavy-chain antibody H chain, 265–266 Chemoattractants, in xenotransplantation, 147 Chemokine receptor 5, in interleukin-12 production, 77 Chimerism, mixed, xenotransplantation hematopoietic cell, 171–177 pig-to-primate model, 171–177 Clinical trials, in xenotransplantation, 190 CLNK, see Cytokine-dependent hematopoietic cell linker Cloning, VHH immunized Camelidae, 277–279 naive and synthetic banks, 279 Clotting, in xenotransplantation, 155

333

INDEX

Coagulation systems, xenotransplantation clotting, 155 disordered thromboregulation, 152–153 disseminated intravascular coagulation, 153 fibrinolytic abnormalities, 154–155 natural anticoagulants, 153–154 platelet agonists, 152 platelets, 155 prevention, 169–171 regulators, 153–154 thrombotic diathesis, 152 xenogeneric molecular incompatibilites, 153 Collectins, in insect immune response, 232 Colony stimulating factor-1, 311–312 Complement in antiviral responses, 10–12 in interleukin-12 production, 78 xenotransplantation humoral responses, 135 xenotransplantation immunosuppression, 161–162 Complement regulatory proteins, 166–167 Crystal cells, phenoloxidase, 236 CSF-1, see Colony stimulating factor-1 Cytokine-dependent hematopoietic cell linker, 100 Cytokines interleukin-12 production, 73–77 uNK cell production, 311–314 Cytolytic mediators, human uNK cells, 309–310 Cytolytic T lymphocytes, 303, 305 Cytoplasmic adaptor protein, SLP-76, 98

D DCs, see Dendritic cells Decidua, uterine natural killer cell role, 319–320 Dendritic cells, interleukin-12 production, 61–62, 77 DIC, see Disseminated intravascular coagulation Digestive system, xenotransplantation barrier, 184 Diptericin A, in immune response, 248–249 Disseminated intravascular coagulation, 153

DNA, bacterial, interleukin-12 induction, 63–64 Dose, antigens in B cell responses, 12–13 Dromedaries epitope recognition, 284 immunization, 275–276 Drosomycin, in immune response, 248–249 Drosophila melanogaster immune response antimicrobial genes, 238, 248–249 C-type lectins, 233 GATA factors, 247–248 gram-negative binding protein, 231–232 humoral response, 226–227 immune deficiency pathway, 245–247 model, 227–228 overview, 226 peptidoglycan recognition proteins, 229–230 phenoloxidase, 236 Relish pathway, 244–247 scavenger receptors, 233–234 thioester proteins, 237 Toll pathway, 240–244 pathogen exposure, 227

E ELISPOT assays, xenotransplantation tolerance, 175 Endocrine system, xenotransplantation barrier, 185–186 Endothelial cells, in xenotransplantation AHXR, 151–152 HAR, 150 Endotoxin tolerance, 80 Epidermal growth factor, 312 Epitopes antibody neutralization, 33–34 dromedaries, 284 Gal, 136–139 Ethics, in xenotransplantation, 188–190 Extracellular matrix, uterus, 319

F Fc-receptors in antiviral antibody responses, 12 Fcγ , in interleukin-12 production, 78–79

334

INDEX

α-Fetoprotein, in pregnancy, 298 Fibrinogen, in xenotransplantation, 170 Fibrinolytic pathways, 154–155 Functional domains, SLP-76 coupling, 113–116

G Gads, see Grb2-related adaptor protein Gal anti-Gal antibodies, 136–137, 139–141, 157–161 antigens in xenotransplantation, 137 epitopes, 136–139 gene transfer, 165 knockout technology, 168 non-Gal determinants, 141–142 GATA factors, in immune response, 247–248 GEMs, signaling protein recruitment, 110–113 Genes antimicrobial, see Antimicrobial genes Diptericin A, 248–249 Drosomycin, 248–249 Gal, 165 Metchnikowin, 249 p35, 56–58, 71–72 p40, 56–58, 67–71 VHH, see VHH genes VHH-D-J, 272–274 Genetic engineering, xenotransplantation donor modification, 166–169 pig, 133 recipient modification, 164–165 β-1,3-Glucan-binding protein, 230–232 Glycosaminoglycans, 64–65 Glycosylation, xenotransplantation genetic engineering, 167–168 GMG cells, see Granulated metrial gland cells GNBP, see Gram-negative binding protein Gram-negative binding protein, 230–232 Granulated metrial gland cells, rodents cell differentiation, 306–307 morphology and phenotypes, 302–305 origin, 300–302 overview, 299–300 Grb2-related adaptor protein ITAM-coupled signaling, 98–99 PLC-γ 1 activation, 118–120 Guinea pig, xenotransplantation, 156

H Hamster, xenotransplantation, 156 HAR, see Humorally mediated rejection HBV, see Hepatitis B virus HCAb, see Heavy-chain antibodies H chain, see Heavy chain HCV, see Hepatitis C virus Heart, xenotransplantation, 130–131 Heavy chain, in H chain antibodies CH1 domain removal, 265–266 variable domain, 266–269 Heavy-chain antibodies, Camelidae antigen-specific dromedary immunization, 275–276 monoclonal VHH, 277–281 polyclonal VHH, 276–277 VHH immune libraries, 281–284 H chain CH1 domain removal, 265–266 variable domain, 266–269 L chain, 269–270 natural occurrence, 261–264 ontogeny, 272–275 VHH antigen-binding loop structures, 286–289 biotechnological role, 289–290 gene phylogeny, 270–272 structure, 285 VL-side conformation, 285–286 Hematopoietic cells, xenotransplantation allogeneic engraftment, 177–180 mixed chimerism, 171–177 xenogeneic engraftment, 177–180 Hemocytes, insects, 227 Hepatitis B virus, 26 Hepatitis C virus, 26 Histocompatibility antigens, pig, 133 HIV, see Human immunodeficiency virus Hormones, in interleukin-12 production, 80–81 Human BLNK-deficient, 108–110 uterine natural killer cells, 307–310, 316–318 Human immunodeficiency virus, 26–27 Humorally mediated rejection endothelial cell activation, 150 innate responses, 134–135 Humoral mediators, in xenotransplantation, 152

INDEX

Humoral response acquired xenotransplantation anti-Gal antibodies, 136–137, 139–141 Gal antigen, 137 Gal epitopes, 136–139 non-Gal determinants, 141–142 overview, 135–136 Drosophila, 226–227 innate xenotransplantation, 133–135 Hyaluronan, interleukin-12 induction, 64–65 Hypermutation, in virus-neutralizing antibodies, 3–4

I IFN-γ , see Interferon-γ IL-1, see Interleukin-1 IL-12, see Interleukin-12 Immune deficiency pathway, in immune response, 245–247 Immune effector cells, 315–318 Immune responses antimicrobial genes differential expression, 248–249 GATA factors, 247–248 immune deficiency pathway, 245–247 induction and regulation, 238–240 Relish pathway, 244–247 Toll pathway, 240–244 Drosophila model, 227–228 insects C-type lectins, 232–233 function, 225–226 β-1,3-glucan recognition, 230–232 gram-negative binding proteins, 230–232 hemocytes, 227 peptidoglycan recognition proteins, 229–230 scavenger receptors, 233–234 toll-like receptors, 234–235 mammals scavenger receptors, 233 toll-like receptors, 234–235 phenoloxidase role, 236 thioester protein role, 236–237 vertebrates, 226 Immune system, virus outside, 21–22 Immunization, llamas and dromedaries, 275–276

335

Immunobiology, pregnancy, 297–299 Immunoglobulin, in xenotransplantation immunosuppression, 159 Immunological memory antibody memory maintenance, 29–30 biological relevance, 29 overview, 28 T cell memory, 30–31 Immunoreceptor tyrosine-based activation motif-coupled receptor signaling BLNK-deficient B cells, 108–110 BLNK protein, 99–100 CLNK protein, 100 Gads, 98–99 key events, 93–95 LAT-deficient T cells, 101–103 LAT- and SLP-76-deficient mice, 105–108 linker for activation of T cells, 95–97 SH2-domain-containing leukocyte protein, 98 SLP-76-deficient T cells, 103–104 Immunosuppression virus neutralization, 22–23 xenotransplantation accomodation, 160 antidiotypic antibodies, 159 anti-Gal antibodies, 157–161 cellular response, 163–164 complement, 161–162 human immunoglobulin, 159 induced antibody response, 162–163 Immunosuppressive factors, 298 Inducible nitric oxide synthase, 320–321 Infection, antibody-dependent enhancement, 22 Infectious agents, in xenotransplantation, 187–188 Influenza virus, neutralizing antibody response, 25–26 Informed consent, in xenotransplantation, 189–190 Innate cellular responses, xenotransplantation gamma–delta T cells, 142–143 macrophages, 146–148 monocytes, 146–148 natural killer cells, 143–146 neutrophils, 148 NK/T cells, 142 iNOS, see Inducible nitric oxide synthase

336

INDEX

Insects, immune response antimicrobial genes differential expression, 248–249 GATA factors, 247–248 immune deficiency pathway, 245–247 induction and regulation, 238–240 Relish pathway, 244–247 Toll pathway, 240–244 C-type lectins, 232–233 function, 225–226 β-1,3-glucan recognition, 230–232 gram-negative binding proteins, 230–232 hemocytes, 227 peptidoglycan recognition proteins, 229–230 phenoloxidase role, 236 scavenger receptors, 233–234 thioester protein role, 236–237 toll-like receptors, 234–235 Integrins in interleukin-12 production, 77–78 in xenotransplantation, 170 Interferons IF-γ , 68–69, 313–314 type I, 75 Interleukin-1, 312–313 Interleukin-1 receptor-associated kinase, 241–242 Interleukin-12 biological functions, 58 discovery, 55–56 endotoxin tolerance inhibition, 80 induction activated T cells, 65–66 hyaluronan, 64–65 pathogens, 62–64 mitogen-activated protein kinase role, 70–71 molecular structure, 56–58 p35 gene, 71–72 p40 gene interferon-γ priming, 68–69 promoter elements, 67–68 promoter polymorphisms, 71 transcriptional factor PU.1, 69–70 production anti-inflammatory hormones, 80–81 CD47 role, 79 chemokine receptor 5 role, 77 complement role, 78 cytokine role, 73–77 dendritic cells, 61–62

EBV-transformed B cells, 59–60 Fcγ receptor role, 78–79 integrins role, 77–78 mast cells, 61 normal B cells, 60 phagocytic cells, 60 small molecules, 80–81 Interleukin-12 receptor signaling, 58–59 IRAK, see Interleukin-1 receptor-associated kinase ITAM-coupled receptors, see Immunoreceptor tyrosine-based activation motif-coupled receptor signaling

K Kidney, xenotransplantation, 130, 185

L LAT, see Linker for activation of T cells LBP, see Lipopolysaccharide-binding protein L chain, see Light chain Lectins, C-type, in insect immune response, 232–233 Leukemia inhibitory factor, 313 LIF, see Leukemia inhibitory factor Light chain, heavy-chain antibodies, 269–270 Linker for activation of T cells deficient mouse, 105–108 deficient T cells, 101–103 Gads association, 98–99 ITAM-coupled signaling, 95–97 in PLC-γ 1 activation, 118–120 signaling protein recruitment, 110–113 Lipopolysaccharide-binding protein, 231 Lipopolysaccharides, 230–232 Liver, xenotransplantation, 131 Llamas, immunization, 275–276 LPS, see Lipopolysaccharides Lungs, xenotransplantation barrier, 185 Lymphocytic choriomeningitis virus, 27 Lymphokine-activated killer cells, 303, 305

M Macrophages interleukin-12 production, 60 in xenotransplantation, 146–148

337

INDEX

Mammals, immune response antimicrobial genes differential expression, 248–249 GATA factors, 247–248 immune deficiency pathway, 245–247 induction and regulation, 238–240 Relish pathway, 244–247 Toll pathway, 240–244 scavenger receptors, 233 thioester protein role, 236–237 toll-like receptors, 234–235 uterine natural killer cells, 310 Mast cells, interleukin-12 production, 61 Membrane-anchored adaptor protein, 95–97 Mesometrial triangle, granulated metrial gland cells, 299 Metchnikowin, in immune response, 249 Mitogen-actived protein kinase, 70–71 Models Drosophila immune response, 227–228 mixed chimerism, 171–177 xenotransplantation, 155–157, 180–181 Monocytes interleukin-12 production, 60 in xenotransplantation, 146–148 Morphology, rodent granulated metrial gland cells, 302–305 Mouse BLNK-deficient, 108–110 granulated metrial gland cells morphology and phenotypes, 302–305 origin, 300–302 LAT- and SLP-76-deficient, 105–108 uterine natural killer cells, 316–318 xenotransplantation, 156–157 μ–γ switch, Camelidae heavy-chain antibodies, 275

N Natural antibodies, in antiviral responses, 9–10 Natural killer cells, xenotransplantation NK cells, 143–146 NK/T cells, 142 pharmacotherapy, 181–182 tolerance, 176–177 Neutralization attenuated vaccines, 33 hepatitis B virus, 26

hepatitis C virus, 26 HIV, 26–27 immunosuppression, 22–23 infection enhancement, 22 infective vaccines, 32–33 influenza viruses, 25–26 linear epitopes, 33–34 lymphocytic choriomeningitis virus, 27 mechanisms, 6–8 outside immune system, 21–22 structural studies, 8–9 vaccines, 32 viral sensitivity, 23–24 Neutrophils, in xenotransplantation, 148 NFκKB, interleukin-12 production, 80–81 Nitric oxide synthase, 320–321 Nuclear transfer, xenotransplantation genetic engineering, 169

O Ontogeny, Camelidae heavy-chain antibodies, 272–275

P p35 gene encoding gene, 56–58 transcriptional control, 71–72 p40 gene encoding gene, 56–58 interferon-γ priming, 68–69 promoter elements, 67–68 promoter polymorphisms, 71 transcriptional factor PU.1, 69–70 PACAP, see Pituitary adenylate cyclase-activating polypeptide Papillomaviruses, outside immune system, 21 Pathogens Drosophila exposure, 227 interleukin-12 induction, 62–64 in xenotransplantation, 187 Pathology, uterine natural killer cells, 318 Patient confidentiality, in xenotransplantation, 190 Peptidoglycan recognition proteins, 229–230 PERVs, see Porcine endogenous retroviruses PGRP, see Peptidoglycan recognition proteins Phagocytic cells, interleukin-12 production, 60

338

INDEX

Pharmacotherapy, in xenotransplantation, 181–182 Phenoloxidase, in immune responses, 236 Phenotypes, rodent granulated metrial gland cells, 302–305 Phylogeny, Camelidae VHH genes, 270–272 Pigs Gal-knockout technology, 168 non-Gal determinants, 141–142 uterine natural killer cells, 310 as xenograft donor, 131–133, 180–181 Pituitary adenylate cyclase-activating polypeptide, 81 Placenta, uterine natural killer cell role, 319–320 Platelets, in xenotransplantation, 152, 155, 169–171 PLC-γ 1 adaptor protein role, 118–120 LAT coupling, 101–103 tyrosine kinase interaction, 103–104 Polymorphisms, in interleukin-12 gene promoter, 71 Porcine endogenous retroviruses, in xenotransplantation, 187–188 Pregnancy α-fetoprotein, 298 immunobiology, 297–298 immunosuppressive factors, 298 uterine changes, 298–299 uterine natural killer cells colony stimulating factor-1 production, 311–312 cytolytic activity, 315–318 epidermal growth factor production, 312 interferon-γ production, 313–314 interleukin-1 production, 312–313 leukemia inhibitory factor production, 313 uteroplacental unit, 318–321 Primates, as xenograft recipients, 180–181 Promoters, interleukin-12 p40 gene, 67–68, 71 Protection, antiviral antibody responses, 5–6 Proteins adaptor, see Adaptor proteins B cell linker, see B cell linker protein BLNK associating protein, 116–117 complement regulatory protein, 166–167 α-fetoprotein, 298 β-1,3-glucan-binding protein, 230–232 gram-negative binding protein, 230–232

mitogen-actived protein kinase, 70–71 peptidoglycan recognition proteins, 229–230 Ras, 101–103 SH2-domain-containing leukocyte protein, see SH2-domain-containing leukocyte protein signaling proteins, 110–113 SLP-76 coupling to downstream signaling, 113–116 thioester proteins, 236–237

R Rabies virus, outside immune system, 21–22 Ras protein, LAT coupling, 101–103 Rat, xenotransplantation from guinea pig, 156 from hamster, 156 wild-type rat, 157 Relish pathway, 244–247 Risk–benefit ratio, in xenotransplantation, 189 Rodents, granulated metrial gland cells cell differentiation, 306–307 morphology and phenotypes, 302–305 origin, 300–302 overview, 299–300

S Scavenger receptors, in insect immune response, 233–234 SH2-domain-containing leukocyte protein, SLP-76 analog BLNK, 99–100 analog CLNK, 100 coupling to downstream signaling, 113–116 deficient mouse, 105–108 deficient T cells, 103–104 Gads association, 98–99 ITAM-coupled signaling, 98 in PLC-γ 1 activation, 118–120 Signaling pathways interleukin-12 receptors, 58–59 ITAM receptor, see Immunoreceptor tyrosine-based activation motif-coupled receptor signaling PLC-γ 1 activation, 118–120 proteins, LAT recruitment, 110–113

339

INDEX

SLP-76 antigen receptor coupling, 113–116 TEC tyrosine kinases, 120–121 Silkworm, see Bombyx mori SLP-76, see SH2-domain-containing leukocyte protein Small molecules, in interleukin-12 production, 80–81 Surface molecules, human uNK cell studies, 308–309

T T cell antigen receptor, LAT interaction, 101–103 T cells B cell activation, in viral infections, 16–17 interleukin-12 induction, 65–66 LAT-deficient lines, 101–103 memory, 30–31 SLP-76-deficient lines, 103–104 in xenotransplantation gamma–delta T cells, 142–143 NK/T cells, 142–143 tolerance, 174 TCR, see T cell antigen receptor TEC tyrosine kinases, in SLP-76 signaling, 120–121 TEPs, see Thioester proteins Thioester proteins, in immune responses, 236–237 Thromboregulation, in xenotransplantation, 152–153 Thromboregulatory factors, in xenotransplantation, 170 Thrombotic diathesis, in xenotransplantation, 152 Thymus, xenotransplantation, 182–184 T lymphocytes, in GMG cell–NK cell relationship, 303, 305 Toll-like receptors, immune response, 234–235 TRAF, see Tumor necrosis factor-receptor-associated factors Transcription, interleukin-12 p35 gene control, 71–72 Transcriptional factor, PU.1, 69–70 Trophoblast cells, 315–317 Tropism, viral sensitivity, 23–24

Tumor necrosis factor-α, 75–77 Tumor necrosis factor-receptor-associated factors, 241–242 Tyrosine kinases, PLC-γ 1 interaction, 103–104

U uNK cells, see Uterine natural killer cells Uterine natural killer cells human, 307–310 mammalian species, 310 in pregnancy colony stimulating factor-1 production, 311–312 cytolytic activity, 315–318 epidermal growth factor production, 312 interferon-γ production, 313–314 interleukin-1 production, 312–313 leukemia inhibitory factor production, 313 uteroplacental unit, 318–321 rodent GMG cell relationship cell differentiation, 306–307 morphology and phenotypes, 302–305 origin, 300–302 overview, 299–300 Uteroplacental unit, 318–321 Uterus, changes during pregnancy, 298–299

V Vaccines, antibody neutralization, 32–33 Variable domain, in heavy-chain antibody H chain, 266–269 Vasoactive intestinal peptide, 81 V(D)J rearrangements, 3 Vertebrates, immune response, 226 VHH-D-J genes, 272–274 VHH genes antigen-binding loop structures, 286–289 antigen-specific, isolation immune libraries, 281–284 monoclonal VHH, 277–281 polyclonal VHH, 276–277 biotechnological role, 289–290 overview, 270–272 structure, 285 VL-side conformation, 285–286 VIP, see Vasoactive intestinal peptide

340

INDEX

Viruses B cell unresponsiveness, 18–19 infections, T-cell-dependent B cell activation, 16–17 neutralization immunosuppression, 22–23 infection enhancement, 22 outside immune system, 21–22 neutralizing antibody response, 3–4 hepatitis B virus, 26 hepatitis C virus, 26 HIV, 26–27 influenza virus, 25–26 lymphocytic choriomeningitis virus, 27 sensitivities, antibody neutralization vs. tropism, 23–24 von Willebrand factor, 170

X Xenograft rejection acute humoral, see Acute humoral xenograft rejection humorally mediated, 134–135, 150 Xenotransplantation acquired cellular responses, 148–150 allogeneic hematopoietic cells, 177–180 coagulation disturbances clotting, 155 disordered thromboregulation, 152–153 disseminated intravascular coagulation, 153 fibrinolytic abnormalities, 154–155 natural anticoagulants, 153–154 platelet agonists, 152 platelets, 155 prevention, 169–171 regulators, 153–154 thrombotic diathesis, 152 xenogenic molecular incompatibilities, 153 concordant vs. discordant, 133

early attempts, 130 endothelial cell activation AXHR, 151–152 HAR, 150 ethical issues, 188–190 future research, 190–192 genetic engineering donor modification, 166–169 recipient modification, 164–165 heart, 130–131 humoral responses acquired, 135–142 innate, 134–135 overview, 133–134 immunosuppression accomodation, 160 antidiotypic antibodies, 159 anti-Gal antibodies, 157–161 cellular response, 163–164 complement, 161–162 human immunoglobulin, 159 induced antibody response, 162–163 infectious agents, 187–188 innate cellular responses gamma–delta T cells, 142–143 macrophages, 146–148 monocytes, 146–148 natural killer cells, 143–146 neutrophils, 148 NK/T cells, 142 kidney, 130 liver, 131 mixed hematopoietic cell chimerism, 171–177 models, 155–157 necessity, 129–130 pharmacotherapy, 181–182 physiological barriers, 184–187 pig donor, 131–133 pig-to-primate model, 180–181 thymus, 182–184 xenogeneic hematopoietic cells, 177–180

CONTENTS OF RECENT VOLUMES

Volume 73

Murine Models of Thymic Lymphomas: Premalignant Scenarios Amenable to Prophylactic Therapy EITAN YEFENOF

Mechanisms of Exogenons Antigen Presentation by MHC Class I Molecules in Vitro and in Vivo: Implications for Generating CD8+ T Cell Responses to Infectious Agents, Tumors, Transplants, and Vaccines JONATHAN W. YEWDELL, CHRISTOPHER C. NORBURY, AND JACK R. BENNINK

INDEX

Signal Transduction Pathways That Regulate the Fate of B Lymphocytes ANDREW CHAXTON, KEVIN OTIPODY, ALMIN JIANC, AND EDWARD A. CLARK

Biochemical Basis of Antigen-Specific Suppressor T Cell Factors: Controversies and Possible Answers KIMISHICE ISIHZAKA, YASUYUKI ISHII, TATSUMI NAKANO, AND KATSUJI SUGIK

Oral Tolerance: Mechanisms and Therapeutic Applications ANA FARIA AND HOWARD L. WEINER Caspases and Cytokines: Roles in Inflammation and Autoimmunity JOHN C. REED T Cell Dynamics in HIV-1 Infection DAWN R. CLARK, BOB J. DE BOER, KATJA C. WOLTHERS, AND FRANK MIEDEMA Bacterial CpG DNA Activates Immune Cells to Signal Infectious Danger HERMANN WAGNER Neutrophil-Derived Proteins: Selling Cytokines by the Pound MARCO ANTONIO CASSATELLA

Volume 74

The Role of Complement in B Cell Activation and Tolerance MICHAEL C. CARROLL Receptor Editing in B Cells DAVID NEMAZEE Chemokines and Their Receptors in Lymphocyte Traffic and HIV Infection PIUS LOETSCHER, BERNHARD MOSER , AND MARCO BACCIOLINI Escape of Human Solid Tumors from T-Cell Recognition:Molecular Mechanisms and Functional Significance FRANCESCO M. MARINCOLA, ELIZABETH M. JAFFEE, DANIEL J. HICKLIN, AND SOLDANO FERRONE

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342


The Host Response to Leishmania Infection WERNER SOLBACII AND TAMAS LASKAY INDEX

Volume 75 Exploiting the Immune System: Toward New Vaccines against Intracellular Bacteria ¨ JURGEN HESS, ULRICH SCHAIBLE, ¨ BARBEL RAUPACH, AND STEFAN H. E. KAUFMANN The Cytoskeleton in Lymphocyte Signaling A. BAUCH, F. W. ALT, G. R. CRABTREE, AND S. B. SNAPPER TGF-␤ Signaling by Smad Proteins KOHEI MIYAZONO, PETER TEN DIJKE, AND CARL-HENRIK HELDIN MHC Class II-Restricted Antigen Processing and Presentation JEAN PIETERS T-Cell Receptor Crossreactivity and Autoimmune Disease HARVEY CANTOR Strategies for Immunotherapy of Cancer CORNELIS J. M. MELIEY, RENE E. M. TOES, JAN PAUL MEDEMA, SJOERD H. VAN DER BURG, FERRY OSSENDORP, AND RIENK OFFRINGA Tyrosine Kinase Activation in the Decision between Growth, Differentiation, and Death Responses Initiated from the B Cell Antigen Receptor ROBERT C. HSUEH AND RICHARD H. SCHEUERMANN The 3′ IgH Regulatory Region: A Complex Structure in a Search for a Function AHMED AMINE KHAMLICHI, ERIC PINAUD, CATHERINE DECOURT, CHRISTINE CHAUVEAU, AND MICHEL COGNE´ INDEX

Volume 76 MIC Genes: From Genetics tok Biology SEIAMAK BAHRAM CD40-Mediated Regulation of Immune Responses by TRAF-Dependent and TRAF-Independent Signaling Mechanisms AMRIF C. GRAMMER AND PETER E. LIPSKY Cell Death Control in Lymphocytes KIM NEWTON AND ANDREAS STRASSEN Systemic Lupus Erythematosus, Complement Deficiency, and Apoptosis M. C. PICKERING, M. BOTTO, P. R. TAYLOR , P. J. LACHMANN, AND M. J. WALPORT Signal Transduction by the High-Affinity Immunoglobulin E Receptor FceRI: Coupling Form to Function MONICA J. S. NADLER , SHARON A. MATTHEWS, HELEN TUHNER , AND JEAN-PIERRE KINET INDEX

Volume 77 The Actin Cytoskeleton, Membrane Lipid Microdomains, and T Cell Signal Transduction S. CELESTE POSEY MORLEY AND BARBARA E. BIERER Raft Membrane Domains and Immunoreceptor Functions THOMAS HARDER Human Basophils: Mediator Release and Cytokine Production JOHN T. SCHROEDER , DONALD W. MACGLASHAN, JR., AND LAWRENCE M. LICHTENSTEIN Btk and BLNK in B Cell Development SATOSHI TSUKADA, YOSHIHIRO BABA, AND DAI WATANABE


Diversity and Regulatory Functions of Mammalian Secretory Phospholipase A2 s MAKOTO MURAKAMI AND ICHIRO KUDO The Antiviral Activity of Antibodies in Vitro and in Vivo PAUL W. H. I. PARREN AND DENNIS R. BURTON Mouse Models of Allergic Airway Disease CLARE M. LLOYD, JOSE-ANGEL GONZALO, ANTHONY J. COYLE, AND JOSE-CARLOS GUTIERREZ-RAMOS Selected Comparison of Immune and Nervous System Development JEROLD CHUN INDEX

343

Attractions and Migrations of Lymphoid Cells in the Organization of Humoral Immune Responses CHRISTOPH SCHANIEL, ANTONIUS G. ROLINK, AND FRITZ MELCHERS Factors and Forces Controlling V(D)J Recombination DAVID G. T. HESSLEIN AND DAVID G. SCHATZ T Cell Effector Subsets: Extending the Th1/Th2 Paradigm TATYANA CHTANOVA AND CHARLES R. MACKAY MHC-Restricted T Cell Responses against Posttranslationally Modified Peptide Antigens INGELISE BJERRING KASTRUP, MADS HALD ANDERSEN, TIM ELLIOT, AND JOHN S. HAURUM

Volume 78 Toll-like Receptors and Innate Immunity SHIZUO AKIRA Chemokines in Immunity OSAMU YOSHIE, TOSHIO IMAI, AND HISAYUKI NOMIYAMA

Gastrointestinal Eosinophils in Health and Disease MARC E. ROTHENBERG, ANIL MISHRA, ERIC B. BRANDT, AND SIMON P. HOGAN INDEX


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