Advances in Immunology Volume 58

ADVANCES IN

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

FREDERICK ALT K. FRANK AUSTEN TADAMITSU KISHIMOTO FRITZMELCHERS JONATHAN

w. U H R

VOLUME 5a

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Copyright 0 1995 by ACADEMIC PRESS, INC 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.

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h i r e d Kingdom Edition published by Academic Press Limited 24-28 Oval Road, London NW 1 7DX International Standard Serial Number: 0065-2776 International Standard Book Number: 0- 12-022458-5 PRINTED INTHE UNITED STATES OF AMERICA 95 96 9 7 9 8 99 0 0 B B 9 8 7 6

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CONTRIBUTORS

Numbers in parentheses indicute the puges on which the uuthors’ contributions begin.

Harald von Boehmer (87), Basel Institute for Immunology, CH-4005 Basel, Switzerland Glenn Dranoff (417),Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts 02115 Dominique Dunon (345), CNRS-URA 1135, Universitk Pierre et Marie Curie, F-75006 Paris, France Sankar Ghosh (l),Department of Molecular Biophysics and Biochemistry, Section of Immunobiology, Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06520 Beat A. Imhof (345), Basel Institute for Immunology, CH-4005 Basel, Switzerland Pawel Kisielow (87),Basel lnstitute for Immunology, CH-4005 Basel, Switzerland Elizabeth B. Kopp (l),Department of Cell Biology, Yale University, New Haven, Connecticut 06520 Guido Kroemer (21I), CNRS-UPR 420, F-94801 Villejuif, France Richard C. Mulligan (417), Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142 Gek-Kee Sim (297), Basel Institute for Immunology, CH-4005 Basel, Switzerland David T. Weaver (29), Division of Tumor Immunology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115

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ADVANCES I N IMIIUNOLOCY. VOL. 58

NF-KB and Re1 Proteins in Innate Immunity ELIZABETH B. KOPP* AND SANKAR G H O S H t

t

Department of Cell Biology and Department of Moleculor Biophysics and Biochemistry and Section of Immunobiology, Howard Hughes Medical Institute, role University, New Haven, Connecticut 06520

I. 11. 111. I\’. V. VI. VII.

Introduction Description of NF-KBand IKB The Activation of NF-KB Physiologic Inducers of NF-KB NF-KBand the Inflammatory Responses Inappropriate NF-KBActivation T Cell Activation VIII. Inhibition of NF-KB:Potential for Therapy IX. Conclusion References

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14 18 19

20 21

I. Introduction Vertebrates respond to infection through a combination of adaptive or acquired immunity and innate or natural immunity. The principal feature of acquired immunity is the generation of receptors on B and T cells that can distinguish between selfand nonself, and hence protect the organism from infectious agents such as bacteria or viruses. By contrast, the hallmarks of innate immunity consist of physical barriers and the ability to generate a battery of cytokines upon nonspecific recognition of conserved structures on infectious agents such as bacterial lipopolysaccharides (LPS) (Colten and Ravetch, 1992). The cytokines help to mount an inflammatory response and to recruit specialized cells, such as natural killer cells, to the site of infection (Abbas et al., 1991). A particularly interesting question is whether the rapid induction in the synthesis of these cytokines is coordinated by some common element. Work carried out over the past several years has identified such an element in a transcription factor known commonly as NF-KB.NF-KBis critical for the inducible expression of many genes involved in the immune and inflammatory responses including IL-1, IL-2, IL-2Ra, IL-6, IL-8, TNF-a, TNF-P, P-IFN, GM-CSF, and serum amyloid A protein. In addition, NF-KB has been conserved through 1 Copyright 0 1995 by Academic P r e w lnc All rights of reproduction in any form reserved

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ELIZABETH B. KOPP AND SANKAR GHOSH

evolution. A number of recent reviews have described various aspects of NF-KB function in great detail (Grilli et al., 1993; Baeuerle and Henkel, 1994); therefore, this report will instead focus primarily on the role of NF-KB as a unifying element in the body’s response to infection and injury and thus as an important mediator of natural immunity in vertebrates. II. Description of NF-KB and IKB

NF-KB was first characterized in mature B and plasma cells as a nuclear protein that binds specifically to a 10-bp sequence in the K intronic enhancer (Sen and Baltimore, 1986a,b). The correlation between the activity of this transcription factor and the expression of the K gene suggested that it might be a critical regulator for the tissue and developmental stage-specific expression of this gene (Sen and Baltimore, 1986b; Atchison and Perry, 1987; Lenardo et al., 1987). However, the finding that NF-KBexisted in virtually all cells and could be induced b y treatment with agents, such as LPS or PMA, indicated that it had a significantly broader role. Subsequent studies revealed that a wide variety of inducible genes contained NF-KB-responsive sites in their promoters and enhancers, thus indicating a general role for NF-KB as a rapid response transcription factor in different cells (reviewed in detail recently in Grilli et al., 1993; Baeuerle and Henkel, 1994).In most cells, with the exception of mature B cells, macrophages, and some neurons (Kaltschmidt et al., 1994), NF-KB remains in the cytoplasm by being bound to an inhibitory protein called IKB(Baeuerle and Baltimore, 1988a,b; Baeuerle et al., 1988; Gilmore and Morin, 1993).Treatment of cells with various inducers leads to the dissociation of the cytoplasmic complex and the translocation of free NF-KBto the nucleus (Baeuerle et al., 1988). Therefore, NF-KB serves as a signal transducer by carrying information from external agents directly to the nucleus. NF-KB is classically described as a heterodimer of p50 and p65 subunits; however, the cloning of the genes encoding these subunits revealed that they were members of a much larger family of proteins known as the re1 family of transcription factors (Bours et al., 1990; Ghosh et al., 1990; Kieran et al., 1990; Meyer et al., 1991; Nolan et al., 1991; Ruben et al.,1991; Blank et al., 1992). Now, NF-KBis often more loosely described as a homo- or heterodimer of re1 subunits. In addition to p50 and p65, the re1 family currently includes p52, rel-B, the oncogene v-rel, the corresponding protooncogene c-rel,and the Drosophila morphogen dorsal (Stephens et al., 1983; Wilhelmsen et

NF-KB AND HEL PROTEINS IN INNATE IMMUNITY

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al., 1984; Steward, 1987; Brownell et al., 1989; Neri et ul., 1991; Schinid et al., 1991; Bours et ul., 1992; Mercurio et al., 1992; Ruben et al., 1991; Ryseck et nl., 1992). A conserved, N-terminal, 300-amino-acid segment termed the re1

homology (RH) domain is responsible for the DNA binding, dimerization, activation, and IKBinteractions of the re1 proteins. It is currently believed that the selection of re1 partners in an NF-KBdimer imparts transcriptional activity (or inactivity) on the complex. Thus, the combination of p50 with p65 or c-rel is transcriptionally active as are p65 homodinier and p65/c-rel, whereas p52 and p50 homodimers are transcriptionally inactive and can repress KB-dependent transcription (Bal1992; Lernbecher et al., 1993; Brown et d., 1994; Hansen et lard et d., al., 1994).A PCR-assisted selection of binding sites using recombinant proteins demonstrated that the combination of subunits confers distinct specificities for DNA sequence (Kunsch et al., 1992). The 10bp consensus binding sequence is GGGGYNNCCY, where each re1 protein subunit contacts one-half of the binding site (Urban and Baeuerle, 1990; Urban et al., 1991; see also Baeuerle and Henkel, 1994). The three-dimensional crystal structure of the NF-KBp50 dimer bound to a symmetric binding sequence confirms the basic principles previously established from inutagenesis studies (G. Ghosh et al., unpublished observations). The crystal structure has also revealed a novel DNA binding motif (G. Ghosh et al., unpublished observations). The p50 and p52 proteins are unique in that both of these molecules are derived from larger precursor proteins which are probably cleaved via a novel proteolytic processing mechanism (Fan et al., 1991). The mRNAs for p50 and p52 code for proteins of 105 and 100 kDa, respectively (Bours et al.,1990; Ghosh et nl., 1990; Kieran et al.,1990; Meyer et al., 1991; Neri et nl., 1991; Schniid et ul., 1991; Bours et al., 1992; Mercurio et al., 1992). The N-terminal region of p105 and plOO yields the p50 and p52 molecules. The C-terminal region resembles the NFKB inhibitory molecule, IKB,in that it contains repeats of a sequence motif known as ankyrin repeats. Indeed, intact p105 and plOO do not bind DNA and do not enter the nucleus because the C-terminal region folds back and masks the nuclear localization signal and the DNAbinding domain present in the N-terminus (Beg et al., 1992; Blank et al., 1992; Hatada et al., 1992; Henkel et al., 1992; Liou et al., 1992). As previously mentioned, NF-KB is retained in the cytoplasm of most cells in an inactive form by binding to an inhibitor protein known as IKB(Baeuerle and Baltimore, 1988a,b). Like NF-KB,IKBis a member of a much larger group of proteins. The distinguishing feature of these proteins is the presence of multiple conserved ankyrin repeats

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ELIZABETH B. KOPP A N D SANKAR GHOSH

which are thought to interact with the re1 domain of NF-KB (Davis et al., 1991; Haskill et al., 1991; Inoue et al., 1992b; Franzoso et al., 1992; Gilmore and Morin, 1993; Naumann et al., 1993). The various members of the IKBfamily have preferences for specific combinations of re1 proteins, and the number and spacing of ankyrin repeats appears to determine this specificity (Beg and Baldwin, 1993; Hatada et al., 1993; Naumann et al., 1993). Currently, the IKB family consists of IKB-a,IKB-P, 1 ~ B - 7and , Bcl-3 (Ohno et al., 1990; Davis et al., 1991; Haskill et al., 1991; Tewari et al., 1992; Inoue et al., 1992a; Liou et al., 1992; Bhatia et al., 1991; Hatada et al., 1992; Wulczyn et aE., 1992; Franzoso et al., 1992). With the exception of Bcl-3, the IKBSclearly inhibit NF-KBactivity (Baeuerle and Baltimore, 1988a,b; Inoue et al., 1992b; Zabel and Baeuerle, 1990; Zabel et al., 1993; Beg et al., 1992; Gilmore and Morin, 1993). The putative oncogene bcl-3 (Bhatia et al., 1991) appears to interact specifically with only p50 and p52 homodimers through a rel-ankyrin repeat interaction and can form a nuclear complex. Ironically, bcl-3 has been reported to both inhibit these homodimers and cause transcriptional activation (Franzosoet al., 1992, 1993; Inoue et al., 1993; Fujita et al., 1993; Bours et al., 1993; Kerr et al., 1992; Wulczyn et al., 1992; Nolan et al., 1993; Gilmore and Morin, 1993; Zhang et al., 1994). It may be that bcl-3 prevents the transcriptionally inactive p50 homodimer from binding to DNA thus allowing the binding of a transcriptionally active NF-KB complex to the same site (Franzosoet al., 1993).The Drosophila morphogen dorsal is also inhibited by a protein (cactus) (Geisler et al., 1992; Kidd, 1992) containing multiple ankyrin repeats and therefore reinforces the view that the Re1 homology domain and the ankyrin repeats have remained specific protein-protein interaction motifs throughout evolution. The specific combination of proteins involved determines whether the interaction has a negative or positive regulatory role. 111. The Activation of NF-KB

The mechanism by which NF-KBactivity is induced remains a novel and fascinating process whose molecular details are yet to be fully elucidated. In uitro studies indicated that phosphorylation of NF-KB:IKB complexes causes their dissociation suggesting a direct role for phosphorylation in the signaling pathway (Ghosh and Baltimore, 1990; Shirakawa and Mizel, 1989). It was recently reported that the activation of NF-KB results in the rapid degradation of the IKB protein, thus ensuring a complete release of NF-KBfrom its cytosolic inhibitor (Sun et al., 1993; Scott et al., 1993; Brown et al., 1993; Henkel

NF-KB A N D REL PROTEINS IN INNATE IMMUNITY

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et aE., 1993; Beg and Baldwin, 1993; Beg et aE., 1993). Interestingly, I K B - ~which , is the most thoroughly characterized IKB member, is transcriptionally regulated by nuclear NF-KB (de Martin et al., 1993; Beg et al., 1993; Le Bail et al., 1993; Brown et al., 1993; Sun et al., 1993). The release to the nucleus of NF-KBthus causes the upregulation of IKB-a synthesis which then helps to shut down the NF-KB response. This type of feedback loop is uniquely suited for the role of NF-KB as a transient inducer of responsive genes (Fig 1). A characteristic feature of NF-KB activation is the rapidity with which this protein can be induced. Inducers, such as TNF-a, can cause significant activation NF-KB within minutes (Hohmann et al., 1990b; Henkel et at., 1993; Beg et a!., 1993). This rapid response allows NFKB to function as an effective signal transducer, connecting events occurring in the cytoplasm to responses in the nucleus. This property is therefore similar in principle to some other transcription factors such as the interferon stimulated transcription factor and the steroid receptors. But, as will be discussed, the unique feature of signaling through NF-KBis the diversity of signaling molecules and situations that result in the activation of NF-KBand the types of genes responsive to active NF-KB. Although the nature of NF-KB inducers can be diverse, their common feature is that they all signal situations of stress, infection, or injury to the organism. Therefore, it appears that the Viruses

Reactive Oxygen Intermediates Cytokines

Mitogens

/

FIG.1. NF-KB activation. NF-KB is activated by a variety of agents as shown. The identity and pathway of second messengers is currently unknown. Ultimately, however, 1 ~ B - ais presumed to be phosphorylated and rapidly degraded, leading to the release and translocation of NF-KBto the nucleus. NF-KBthen influences the transcription of selected genes, including that of IKB-a.

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ELIZABETH B. KOPP AND SANKAR GHOSH

primary role of NF-KB is to help coordinate the body’s response to situations of insult by upregulating the synthesis of a wide variety of response genes such as cytokines, adhesion molecules, and acute phase response proteins. IV. Physiologic Inducers of NF-KB

There are many known inducers of NF-KB complexes. NF-KB can be artificially induced in tissue culture by phorbol esters, calcium ionophores, uv light, and various mitogens (Grilli et al., 1993; Baeuerle

and Henkel, 1994).Ofthe physiologic inducing agents, TNFa, sphingomyelin, or other membrane products, LPS, IL-1, viral products including double-stranded RNA, and reactive oxygen intermediates are the most likely to be relevant to NF-KB activation in vivo. These physiologic inducers will be discussed below with regard to their specific roles in innate immunity. OF NF-KB BY BACTERIAL AND VIRAL INFECTION A. INDUCTION The activation of NF-KBupon bacterial or viral infection is a particu-

larly effective way of initiating an immune response to the infection. Since NF-KBpreexists in the cell and can be stimulated without new protein synthesis, its activation can occur quickly. Likewise, the swift activation of this protein promotes the synthesis of many important immune system regulators. It is not surprising then that bacterial and viral products, such as LPS and double-stranded RNA (Visvanathan and Goodbourne, 1989; Lenardo and Baltimore, 1989a; Lenardo et al., 1989), stimulate NF-KB. LPS is known to bind cell surface receptors on important cell types, such as monocytes and macrophages, which can engulf bacteria by phagocytosis. Double-stranded RNA activation of NF-KB has been shown to lead to the production of the important antiviral cytokine, interferon+. The effect of interferon+ is discussed in another section. Other viral products, such as the transactivating proteins of herpes simplex virus, human T cell leukemia virus (HTLVl),(HIV-1), and hepatitis B, can also activate NF-KB (see NF-KB and viral Infection). OF NF-KB BY REACTIVE OXYGEN INTERMEDIATES B. INDUCTION

NF-KB can also be activated through reactive oxygen intermediates (ROIs) (Schreck et al,, 1991). ROIs are produced by macrophages and granulocytes as part of the oxidative burst to destroy bacteria and are also elevated in some pathological situations. The finding that N-acetyl-L-cysteine, an ROI scavenger and glutathione precursor, and

NF-KB A N D REL PROTEINS IN INNATE IMMUNITY

7

dithiocarbamates reduce the activation of NF-KB by many agents (Schreck et al., 1991,1992; Staal et al., 1990; Ivanov et al., 1993; Ziegler-Heitbrock et al., 1993) supports the idea that the redox state of the cell may play a general role in the activity of NF-KB. Although H,O, is reported to activate NF-KB in Jurkat T cells, it is likely that this state (namely, an oxidizing state) merely facilitates the activation of NF-KBb y other physiologic inducers since H,Oz does not activate transcription from &-reporter constructs in all cells without the addition of PMA or TNF (Israel et al., 1992; Ziegler-Heitbrook et al., 1993). OF NF-KB BY CYTOKINES C. INDUCTION

Cytokines are soluble signaling messengers which have a short half-

life and must be induced, which are two properties rendering them

effective for signaling rapid change in homeostasis. NF-KBis critically positioned in a network of cytokines involved in immune system function. Several cytokines induce NF-KBand many cytokines are induced by NF-KBthus establishing an autoregulatory feedback loop. The cytokines known to induce NF-KB are TNFa and IL-I (Grilli et al., 1993; Baeuerle and Henkel, 1994; Beg et al., 1993). IL-1 and T N F a are also transcriptionally regulated by NF-KB (Hiscott et al., 1993).The signalingpathwayof IL-1 has not yet been well characterized. A discussion of the better understood T N F pathway follows (Fig 2). Tumor necrosis factor is a cytokine produced mainly by macrophages which acts on a variety of cell types possessing TNF receptors. T N F induces macrophages in an autoregulatory manner to stimulate the continued production of TNF and of IL-1 and to promote cytotoxic functions of the macrophage. It also stimulates degranulation in neutrophils and the expression of several adhesion molecules in endothelial cells which permits migration and invasion of damaged vessels by immune cells. TNFa activates NF-KB to bind TNF-responsive elements in promoters of a number of genes including its own (Anisowicz et al., 1991; Beget al., 1993; Israel et al., 1989; Hohmann et al., 1990a; Pessara and Koch, 1990; Hohmann et al., 1990b; Yasumoto et al., 1992; also see Section V). The signal transduction pathway leading to the activation of NF-KB by TNFa is currently under investigation. Unlike induction by PMA, the activation of NF-KBby TNFa may function independently of protein kinase C since NF-KB can still be induced by T N F a in cells treated with PKC inhibitors (Meichle et al., 1990). One interesting potential activation pathway of NF-KBinvolves ceramide, the sphingomyelin component. In the T N F a signaling pathway, phospholipase C (PLC) is activated generating diacylglycerol (DAG) and IP3 from PIP2.

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ELIZABETH B. KOPP A N D SANKAR CHOSH

IL-1, TNF-a

FIG.2. NF-KBand the cytokine network. NF-KBparticipates in a cytokine network in which IL-1 and TNFa can activate NF-KBwhich leads to the production of more IL-1 and TNFa as well as other cytokines.

Since DAG is known to activate PKC and PKC can activate NF-KB, it is likely that T N F a stimulates NF-KB through PKC, possibly by phosphorylating IKB. This outcome has been demonstrated in vitro where PKC treatment of NF-KB/IKBcomplexes causes the dissociation of these complexes (Ghosh and Baltimore, 1990). Ceramide, however, is another potential by-product of this cascade since DAG also activates the enzyme sphingomyelinase (Dressler et al., 1992). Indeed, T N F a treatment stimulates sphingomyelinase activity and elevates ceramide levels in Jurkat T cells, HL-60 leukemia cells, and U937 monocytes (Schiitze et al., 1992; Dbaibo et al., 1993; Yang et al., 1993) and the sphingomyelin degradation is effected through the TNF receptor (Weigmann et at., 1992; Yanaga and Watson, 1992). Several groups have now demonstrated that sphingomyelinase and cell-permeable ceramide can independently activate NF-KB in cells suggesting that TNFa could affect NF-KB activation in more than one way (Schutze et al., 1992; Dbaibo et al., 1993; Yang et al., 1993). The mechanism for the ceramide activation of NF-KBmay involve a ceramide-activated protein kinase which, perhaps through Raf (Finco and Baldwin, 1993), may ultimately influence the phosphorylation of I K B(Kolesnick ~ and Golde, 1994).

NF-KB A N D REL PROTEINS I N INNATE IMMUNITY

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It is possible that other membrane products also activate NF-KB through their breakdown. In one recent study, phosphatidylcholine-PLC was shown to activate NF-KB in Jurkat and U937 cells (Arenzana-Seisdedos et al., 1993). This enzyme, added exogenously or overexpressed internally, hydrolyzed phosphatidylcholine specifically without affecting sphingomyelin breakdown or the breakdown of other membrane components. Much more research is still needed to determine the relevance of lipid second messengers to NF-KB signaling and activation. V. NF-KB and the Inflammatory Responses

When faced with insult, inflammatory defense mechanisms quickly respond to repair tissue and destroy invasive organisms in order to reattain homeostasis. The localized response is termed inflammation and is critical for the immediate defense at the site of injury. The systemic reaction to injury or infection is referred to as the acute phase response and is characterized by fever, increased gluconeogenesis, alterations in lipid metabolism, and increased synthesis of several endocrine hormones. Of particular importance for the innate immune response is the production of acute phase proteins, cytokines, and cell adhesion molecules. Interestingly, NF-KB is intricately involved in the promotion of these inflammatory mediators.

A. NF-KB-DEPENDENT INFLAMMATORY MEDIATORS 1 , Acute Phase Response Proteins During the acute phase response, the liver is responsible for the enhanced production of a number of marker plasma proteins referred to as the acute phase proteins (APPs) (Baumann and Gauldie, 1994; Steel and Whitehead, 1994). These proteins provide host protection by scavenging reactive oxygen intermediates, controlling serine proteases, activating complement, and aiding in tissue repair. Several of the acute phase proteins depend on NF-KBfor their efficient transcription including serum amyloid A protein (SAA) (Edbrooke et al., 1989), the C3 component of complement (Darlington et al., 1993), a1 acid glycoprotein (Baumann and Gauldie, 1994), and angiotensinogen (Ron et nl., 1990). The KB binding site renders these genes responsive to mitogen and cytokine stimulation (Edbrooke et al., 1989).The function of many of the acute phase proteins, including SAA and a1 acid glycoprotein, is unknown. The C3 component of complement is important in both the classical and the alternative pathways of complement activa-

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ELIZABETH B. KOPP A N D SANKAR GHOSH

tion (Darlington et al., 1993), and angiotensinogen is the substrate for renin in the renin-angiotensin system for vasoconstriction and sodium retention. Although cell adhesion molecules are not acute phase response proteins, their production during inflammation is critical for the optimum response of migrating immune cells. The promoters of VCAM-1, ELAM-1, and ICAM-1 all contain NF-KB sites (Shu et al., 1993; Degitz et al., 1991; Whelan et at., 1991; Neish et aE., 1992). The activation of NF-KB then can enhance the surface expression of these molecules on a variety of cells thus allowing a specific adherence of immune cells to sites of injury and infection (Table 1).

2. Cytokines Znduced by NF-KB The signals of stress or infection activate NF-KBto allow the upregulation of immediate-response genes. Among the cytokine genes upregulated by NF-KB are IL-1, IL-2, IL-6, IL-8, GM-CSF, G-CSF, TNFa, TNFP, and p-interferon (Grilli et al., 1993; Hiscott et al., 1993). The activation of IL-2 and IL-2 receptor will be discussed with regard to T cell activation. The other cytokines are addressed below with regard to their effect on the inflammatory responses.

TABLE I NF-KB-INDUCED INFLAMMATORY MEDIATORS Cytokines @-Interferon Interleukin-1 Interleukin-2 Interleukin-6 Interleukin-8 CM-CSF (granulocyte/macrophage colony-stimulating factor) C-CSF (granulocyte colony-stimulating factor) T N F a (tumor necrosis factor) LT (lymphotoxin) Acute phase response proteins a1 acid glycoprotein Angiotensinogen Complement factor B (Bf) C 3 component of complement Serum amyloid A protein Cell adhesion molecules ICAM-1 (intercellular cell adhesion molecule 1) ELAM-1 (E selectin) VCAM-1 (vascular cell adhesion molecule 1)

NF-KB A N D REL PROTEINS IN INNATE IMMUNITY

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The hepatic and extrahepatic acute phase response results from exposure of these tissues to cytokines. The so-called inflammatory cytokines consist of IL-1, IL-6, IL-8, and TNFa. The production ofeach of these cytokines results from the activation of NF-KB;as previously mentioned, IL-1 and TNFa also activate NF-KBthemselves (Hohmann et al., 1990a,b; Israel et al., 1989; Collart et al., 1990; Lacoste et al., 1990; Osborn et al., 1989; Nonaka and Huang, 1990; Hiscott et al., 1993) to initiate an autoregulatory pathway. The “early cytokines,” IL-1 and TNFa, are those produced in the first stages of the response. These in turn upregulate the production of the important “late” cytokine, IL-6, and the IL-6 receptor. This upregulation is at least partly mediated by NF-KB proteins binding to regulatory sites in the IL-6 and IL-6 receptor promoter and is discussed further below. NF-KB promotes the production of TNFa by binding to the four KB sites in the TNFa promoter in macrophages and it also activates transcription ofthe TNF-related lymphotoxin gene (Paul et al., 1990).The initiating factors in this inflammatory cytokine cascade are unknown but probable candidates include prostaglandins, free radicals, LPS, and viruses (Koj et al., 1993). As previously mentioned, free radicals, LPS, and viruses have all been shown capable of activating NF-KB. f3-Interferon, GM-CSF, and G-CSF are not strictly defined as inflammatory cytokines. They do, however, participate in the immune and inflammatory responses and thus deserve mention. G-CSF and GM-CSF each are cytokines which stimulate the proliferation and differentiation of macrophages and granulocytes, cells which are critical to the immediate response to infection. Both of these genes contain NF-KB sites which confer inducibility to them. f3-Interferon is a cytokine produced by macrophages and fibroblasts as a result of viral infection. It has an antiviral protective effect on neighboring uninfected cells, stimulates NK activity, and promotes class I MHC expression (Abbas et al., 1991) Double-stranded RNA will induce the production of this cytokine. As previously mentioned, double-stranded RNA will also induce NF-KB activation. This induction may be caused by phosphorylation of I K Bby ~ double-stranded RNA-dependent Ser/Thr protein kinase which has recently been shown capable of phosphorylating and inactivating I K B in ~ vitra (Kumar et al., 1994). These two events are linked in that NF-KB binds a regulatory element known as the PRDII element in the @-interferon promoter. The binding of NF-KB to this site requires the binding of an accessory factor, the high mobility group protein HMG I(Y), which may bend the DNA to allow appropriate binding of NF-KB (Thanos and Maniatis, 1992). The NF-KBbinding site is critical for the inducibility of this gene by

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ELIZABETH B. KOPP A N D SANKAR GHOSH

double-stranded RNA (Visvanathan and Goodbourne, 1989; Hiscott et al., 1989; Lenardo et at., 1989; Fujita et al., 1989).Binding of NFKB to this site then activates production of IFNP RNA. IL-1 is an important inflammatory cytokine involved in the activation and proliferation of lymphocytes and other cell types and in the induction of acute phase response proteins by the liver (see Section 3). IL-1 is also considered to be an endogenous pyrogen, causing fever perhaps by increasing prostaglandins which act directly on the thermoregulatory center of the hypothalamus. IL-1 treatment of cells results in the activation of NF-KBand subsequent enhancement of transcription of a variety of genes including several acute phase proteins such as complement factor B (Bf) (shown for mouse Bf), serum amyloid A2 (SAA2) (Betts et al., 1993), and angiotensinogen (Brasier et al., 1990). The IL-1 induction of these proteins has been shown to depend on an intact KB-like binding site (Nonaka and Huang, 1990; Betts et al., 1993; Brasier et al., 1990) and may require additional factors for maximum expression (Bf, Nonaka and Huang, 1990). The cytokines IL-1 and IL-6 also combine to produce a synergistic activation of SAA2 transcription mediated by NF-KB and NF-IL6 (Betts et al., 1993). IL-8 is another important inflammatory cytokine. It is a chemotactic molecule which activates luekocytes and attracts them to areas of tissue damage and influences the release of histamine by granulocytes. Like T N F a and IL-1, IL-8 is at least partially regulated by NF-KB (Stein and Baldwin, 1993;Yasumoto et al., 1992).Although another important transcription factor, NF-IL6 (see also below) is also necessary for IL8 transcription, it is the NF-KB site that imparts the inducibility to the IL-8 enhancer (Mukaida et al., 1990). NF-IL6 or another C/EBP transcription factor member is able to cooperate with NF-KBto modulate transcription from the IL-8 enhancer (Stein and Baldwin, 1993). Interestingly, in vitro DNA-binding studies and transient transfection assays implicate p65 homodimer as the transcriptional activator binding to the &-like site in this enhancer (Kunsch and Rosen, 1993). p50ip65 did not bind the IL-8 site in vitro and did not transactivate transcription from this enhancer despite its ability to do so in cells transfected with Igrc or HIV KB site reporters (Kunsch and Rosen,

1993).

3. Important Role of ZL-6 IL-6 is an inflammatory cytokine released by a variety of cell types, most notably, activated monocytes, T cells, B cells, endothelial cells, fibroblasts, and glial cells (reviewed in Kishimoto et al., 1994; Wong and Clark, 1988; Van Snick, 1990). The importance of IL-6 in the

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acute phase response cannot be underestimated. IL-6 induces the transcription of virtually all of the APPs either alone or in combination with other cytokines. Indeed, IL-6-deficient mice respond poorly to bacterial and viral infections and tissue injuries, and mRNA levels of haptoglobin, serum amyloid A protein, and a1 acid glycoprotein do not increase in these mice after insult (Kopf et al., 1994). Furthermore, because IL-6 levels are raised in body fluids during many disease states, measurements of IL-6 have prognostic and diagnostic value for various neoplasias, graft versus host disease, trauma, autoimmune diseases, and preterm labor. a. The Production of IL-6 Is Itself Regulated by N F - K B . The IL6 promoter contains single binding sites for the transcription factors NF-IL6 and NF-KB.NF-IL6 is a member ofthe broader class of leucine zipper DNA binding proteins known as the C/EBP family (Akira et al., 1990). In transfection assays, the NF-KBbinding site is necessary for transcription of CAT reporters containing the IL-6 promoter. This transcription is activated when cells are exposed to mitogens or other cytokines including PMA, LPS, IL-1, TNFa, dsRNA, and PHA (Shimizu et al., 1990; Libermann and Baltimore, 1990; Zhang et al., 1990; Dendorfer et al., 1994).Thus, the initial inflammatory event may cause the production of IL-1 and TNFa which in turn can activate NF-KB leading to the production of more IL-1 and of IL-6 (Brouckaert and Libert, 1993).IL-6 induces production of the APPs and may downregulate production of IL-1 and TNFa.

b. Synergy between NF-IL6 and N F - K B . In addition to enhancing the transcription of IL-6, NF-IL6 and NF-KB work cooperatively to increase transcription for a number of other cytokines and acute phase response proteins. There is a synergistic enhancement of transcription from IL-6 pronioter-reporter constructs in the presence of cotransfected NF-KB and NF-IL6 (Betts et al., 1993). This synergism is not limited to pSO/p65 alone as p65 homodimer yields the greatest activation of transcription in the presence of NF-IL6 (Matsusaka et al., 1993).The SAA2 gene was similarly found to respond synergistically to the combination of NF-KB p65 subunit and NF-IL6. The interaction of NF-KB with NF-IL6, although enhancing transcription and DNA-binding from C/EBP promoters and promoters containing both NF-KB and C/EBP sites, can also inhibit transcription from the HIV-1 enhancer containing only KB sites (Stein et al., 1993).Evidence supporting the direct physical contact between NF-KB and NF-IL6 or other C/EBP members demonstrates the ability to crosslink these proteins in vitro in the absence of DNA. Crosslinking using mutant and

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ELIZABETH B. KOPP AND SANKAR GHOSH

wild-type constructs revealed that the putative proteidprotein interaction occurs via the conserved bZIP domain of the C/EBP protein with the re1 homology domain of NF-KB (Matsusaka et al., 1993; Stein et al., 1993; LeClair et ul., 1992). B. EVOLUTIONARY SIGNIFICANCE OF NF-KB AS

AN

INFLAMMATORY MEDIATOR The importance of NF-KB in the innate response to infection and inflammation has been further substantiated by an exciting recent discovery. This discovery involves the characterization of Dif, an insect transcription factor of the re1 family which binds KB-like motifs in the promoters of insect immunity genes ( I p et al., 1993; Sun and Faye, 1992). Dif becomes activated in the fat body of insects as a result of infection. Since the fat body is considered to be the evolutionary ancestor of the liver, it has been suggested that the activation of Dif is analogous to the acute phase response of inflammation in vertebrates ( I p et al., 1993; Hultmark, 1994). This suggests that the NF-KBdependent aspect of innate immunity arose from a primitive form of the same and that this efficient system survived evolution. VI. Inappropriate NF-KB Activation

A. NF-KB AND VIRALINFECTION It is now known that several viruses, including CMV, SV40, and HIV-1, use NF-KB for efficient transcription of their own genes (Grilli et al., 1993). The promoters of these viral genomes contain NF-KB binding sites which influence viral transcription upon activation of NF-KB. Since NF-KB activation is virtually ensured in infected cells, the use of this host transcription factor conveniently enhances viral infectivity. In other situations, the transactivation proteins of other viruses inadvertently activate NF-KBproducing aberrant levels of NFKB-dependent proteins and promoting inappropriate inflammatory responses (Gutsch e t al., 1994; Hammarskjold and Simurda, 1992; Waldmann et ul., 1984; Ballard et al., 1988; Lindholm et al., 1990; Hirai et ul., 1994; Depper et aE., 1984; Ruben et al., 1988). In either case, the innate immune response is exploited to the detriment of the cell. Both types of viral response are discussed below.

1. NF-KB and HIV-1

The HIV-1 LTR contains several cis-acting regulatory sequences (Rosen et al., 1985; Garcia et al., 1987) including two tandem NF-KB sites (Nabel and Baltimore, 1987).Numerous studies have determined

NF-KB AND REL PROTEINS IN INNATE IMMUNITY

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that these NF-KBsites are effective in transcriptional activation of viral genes (Muesing et al., 1987). Indeed, chronically infected cell lines can be induced to produce virus when treated with activators of re1 proteins such as TNFa (Folks et al., 1989; Matsuyama et al., 1989; Duh et al., 1989; LaCoste et al., 1990; Griffin et al., 1989), PMA (LaCoste et al., 1990; Siekevitz et al., 1987), or IL-1. Similarly, transfected cell lines can be stimulated to activate transcription from HIV reporter constructs when treated with the same agents (Kaufman et al., 1987; Osborn et al., 1989; Duh et al., 1989; LaCoste et al., 1990; Nabel and Baltimore, 1987; Siekevitz et al., 1987).It is now becoming clear that effective transcription from the HIV enhancer like many of its cellular counterparts requires several transcription factors of which NF-KB (or the re1 protein family) is one, albeit important, element (Perkins et al., 1993; Muchardt et al., 1992). The relative significance of the multiple transcription factor binding sites depends on the cell type infected with the virus and may rely on the amount of a particular active transcription factor present (Parrott et al., 1991; Ross et al., 1991; Franza et al., 1987; Osborn et al., 1989; Hazan et al., 1990; LaCoste et al., 1990). In addition to the NF-KB sites in the HIV LTR, there are three SP-1 sites and the TAR element, which is responsive to the HIV transactivating product, tat. Recent studies confirm that all of these sites are important (Berkhout and Jeang, 1992) but they may be functionally redundant; that is, the deletion of one or more sites slows transcription but does not halt it (Ross et al., 1991). Cell types that have copious amounts of activated NF-KB (by virtue of infection or stimulation by mitogen or cytokine) are insensitive to deletion of SP1 sites (Parrott et aZ., 1991)or mutation to the TAR element (Harrich et al., 1990). Similarly, deletion of the NF-KB sites but retention of the three SP1 sites and the TAR element will sustain viral production (Leonard et uZ., 1989). Moreover, it appears that NF-KBand SP-1 also physically interact to produce a synergistic response when bound to their respective sites (Perkins et al., 1993).The specific re1 subunits activated during infection may also play a role in the promotion of HIV-1 transcription. For example, the re1 complex, p50B/p65, appears to be particularly effective at stimulating transcription from the HIV1 LTR especially when expressed with the HIV tat protein, whereas c-re1 appears to repress p65-mediated transcription from this promoter (Doerre et al., 1993). This interaction is most likely functional, not physical (Liu et al., 1992; Schmid et al., 1991). Notably, a negative strand RNA transcript has also been detected by R T PCR in acutely and chronically HIV-infected cell lines (Michael et al., 1994), the transcription of which also depends on the NF-KB

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ELIZABETH B. KOPP A N D SANKAR GHOSH

sites. A protein corresponding to this transcript has not yet been described, and the relevance of this RNA to HIV infection remains to be demonstrated.

2 . NF-KB Activation and HZV-1 Infectivity The activation of NF-KBhas been associated with a switch from viral latency to viral productivity (Nabel and Baltimore, 1987; Bachelerie et al., 1991; Griffin et al., 1989).This activation may be caused by cellular signals involved in HIV infection itself, by secondary infection, or by differentiation in HIV-infected cells (Griffin et al., 1989; Bachelerie et al., 1991; Nabel, 1991; Bohnlein et al., 1989; Roulston et al., 1993; Hazan et al., 1990; Tong-Starksen et al., 1987; Paya et al., 1992; Suzan et al., 1991; Hamrnarskjold and Simurda, 1992) positioning NF-KB in a prominent role in maintaining viral production. Human herpes virus8 (HHV6) predominantly infects T cells and other cells involved in HIV-1 infection. Although a common virus for which most humans are seropositive, it exacerbates disease progression for patients suffering from AIDS. Coinfection of T cells with HHVG and HIV-1 accelerates cytopathic degeneration (Lusso et al., 1989).This effect has been investigated transcriptionally in coinfected or cotransfected cells. HHVG isolated from AIDS patients is able to activate transcription from the HIV LTR. This transcription is dependent on NF-KBsites in the HIV enhancer (Ensoli et d.,1989; Gimble et al., 1988; Horvat et al., 1991). Consistent with those observations, HHVG infection has been shown to activate NF-KB. The herpesvirus proteins involved in stimulation of NF-KB have not yet been well defined. An uncharacterized HHVG gene, B701, has been identified which is able to transactivate the HIV-1 promoter contingent on intact NF-KB sites (Geng et al., 1992; Horvat et al., 1991). An HHVG immediate-early protein, ICP4, is also able to stimulate HIV-1 replication although it is unclear whether this stimulation is mediated through the HIV-1 NF-KB sites (Albrecht et at., 1989). Herpes Simplex Virus 1 (HSV-l), HTLV-1, and Epstein-Barr Virus also produce proteins that transactivate NF-KB(Vlach and Pitha, 1992; Albrecht et al., 1992; Hammarskjold and Simurda, 1992). Although the HSV-1 proteins responsible for this transactivation are also not yet adequately characterized, it is known that the HSV-1 immediate-early protein, ICPO, enhances transcription from the HIV-1 LTR in transfected cells. This activity is further increased when NF-KBlevels are high (Vlach and Pitha, 1993). Taken together, all of these results suggest that the HIV-1 LTR is organized for optimal transcription; a complex physical and functional

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interplay between SP1, NF-KB, and tat regulates the effectiveness of this transcription (Jeang e t al., 1988; Ross et al., 1991; Parrott et al., 1991).This transcription can be induced by mitogenic stimulation, by secondary viral infection, or by cellular differentiation. Finally, the tandem NF-KB sites in the HIV-1 LTR confer inducibility of this promoter to cellular signals.

B. INAPPROPRIATE EXPRESSION OF KB-DEPENDENT PROTEINS The HTLV-1 tax protein is a nuclear transcriptional transactivator that induces the production of its own RNAs. Tax does not bind DNA alone; it presumably interacts with other cellular proteins involved in transcription. Tax has been shown to activate NF-KB(re1 proteins) and DNA binding activity in transfected Jurkat T cells and other cells (Li e t al., 1993; Li and Siekevitz, 1993; Bohnlein e t al., 1989; Leung and Nabel, 1988; Ruben et al., 1988; Ballard et al., 1988; Watanabe et al., 1993; Arima e t ul., 1991) although NF-KBdoes not enhance transcription of HTLV-1 genes. In appropriate cells, however, it does inadvertently lead to transcription of some cellular genes responsive to NFKB including the IL-2 receptor gene (Ruben e t al., 1988; Ballard et al., 1988; Leung and Nabel, 1988; Wano e t al., 1988), the IL-2 gene (Wano et ul., 1988), the TNFa gene (Albrecht e t al., 1992),the TNFP gene (Lindholm et ul., 1992), the IgK gene (Lindholm et al., 1992), and the human vimentin gene (Lilienbaum and Paulin, 1993). In fact, T cells infected with HTLV-1 characteristically display unusually high levels of surface IL-2 receptor (Gootenberg et ul., 1981; Waldmann et ul., 1984; Depper et al., 1984). The mechanism of tax activation of NFKB is still unclear. Since tax is predominantly a nuclear protein, it is unlikely that it activates NF-KB by acting directly on the cytosolic NFKB inhibitor, IKB. Notably, soluble tax protein applied extracellularly can also induce NF-KB activity (Lindholm et al., 1990,1992). The role of tax in HIV-1 infection parallels that of the putative HHV-6 transactivator proteins. Since tax is able to activate NF-KB, a secondary infection with HTLV-1 in HIV-infected cells would presumably enhance HIV production. Indeed, the NF-KB sites of the HIV-1 LTR are tax responsive and can convey tax inducibility to a heterologous promoter (Bohnlein et al., 1989). Remarkably, tax appears to interact with the pl00 product of the N F K B gene. ~ This interaction was recently shown to inhibit taxinduced expression from HTLV-1 and HIV-1-CAT reporter constructs cotransfected with plOO and tax in Jurkat T cells. Furthermore, the overexpressed plOO sequestered tax in the cytoplasm where it presum-

18

ELIZABETH B. KOPP A N D SANKAR GHOSH

ably has no activity (Bkraud et al., 1994). This inhibition may be an important factor contributing to the extreme viral latency associated with HTLV-1 infection in uiuo (Bkraud et al., 1994). In another system, overexpressed IKBY,which is an alternatively spliced C-terminal variant of p105, has also been shown to bind and sequester coexpressed tax in the cytoplasm (Hirai et al., 1994). It remains to be seen whether this binding ability is relevant to NF-KBactivation in uivo. VII. T Cell Activation

T cell activation involves engagement of the T cell receptor at the cell surface and transmission of a Ca2+-dependentsignal to the nucleus. This signal activates transcription of a number of genes initiating differentiation, proliferation, and secretion from the cell. Although classical T cell activation requires antigen and thus participates in acquired rather than innate immunity, this process also requires and generates cytokines which depend on NF-KBfor their production. In particular, the production of IL-2 and IL-2 receptor is enormously important in T cell activation and will thus be discussed with regard to NF-KBregulation. A. IL-2 PRODUCTION IL-2 is produced by activated T cells and acts back on these cells to produce more IL-2 and to produce IL-2 receptor. The production of IL-2 is regulated transcriptionally and is associated with differentiation and proliferation of these cells (Brorson et al., 1991). The IL-2 promoter contains binding sites for a number of transcription factors including NF-KB(Lenardo et al., 1988). No one of these transcription factors plays a dominant role; rather, each contributes to the activation of transcription of IL-2 (Serfling et al., 1989; Hoyos et al., 1989). It is now known that activation of IL-2 production by T cells requires calcineurin, a Ca2+-dependent phosphatase. The immunosuppressive activity of the drugs FK506 and cyclosporin A is attributed to these drugs’ ability to bind and inhibit calcineurin. It is notable that in some studies this inhibition does not appear to affect DNA binding and only partially affects transactivation of NF-KB in transformed T cell lines but does affect these properties of other transcription factors suggesting that the induction of NF-KB in T cell activation is independent of calcineurin (Banerji et al., 1991). A more recent study in Jurkat T cells contradicts this conclusion however. Frantz et al. (1994) found that in Jurkat cells transfected with a constitutively active mutant of calcineurin, NF-KB was activated resulting in transcription from the

NF-KB AND REL PROTEINS IN INNATE IMMUNITY

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I L - ~ K promoter. B This affect was enhanced synergistically in the presence of PMA and was sensitive to treatment with FK506. Overexpressed I K B in ~ these cells appears to be phosphorylated, possibly suggesting that calcineurin acts upstream of the release of NF-KBfrom IKB (Frantz et al., 1994). Studies in nontransformed T cells concur indicating that stimulation of transcription from the I L - ~ K B site requires antigen and antigen-presenting cells and is inhibited by cyclosporin A (Kang et al., 1992). Interestingly, this activation appears to be repressed by p50 homodimers and activated by p50/p65 (Kang et al., 1992).

B. IL-2 RECEPTORPRODUCTION The expression of the IL-2 receptor is induced during T cell activation and by binding IL-2 contributes to the further production of this important cytokine. IL-2R is made up of two subunits, a! and p, of which the a subunit is inducible concomitant with T cell activation. The IL-2Ra subunit gene contains a KB-like regulatory sequence that is responsive to mitogens, TNFa (Lowenthal et al., 1989), and the HTLV-1 transactivator protein, tax (Pomerantz et al., 1989). This sequence is necessary but not sufficient for efficient transcription of IL2Ra (Pomeratitz et al., 1989; Cross et al., 1989; Freimuth et al., 1989; Hkniar et al,, 1991). Interestingly, it appears that cell-type-specific proteins and/or other sequence elements are also important in the T cell specificity of expression of this gene since the KB site alone does not confer transcriptional activity to heterologous promoters, and nuclear NF-KB which can bind to this site (as in mature B cells) does not necessarily induce transcription (Freimuth et al., 1989; Cross et al., 1989; Pomerantz et al., 1989). VIII. Inhibition of NF-KB: Potential for Therapy

Because the inappropriate activation of NF-KBcan theoretically lead to undesired inflammatory or immune responses, the inhibition of NFKB is now being investigated. As previously mentioned, N-acetylcysteine can block the activation of NF-KBpresumably through interfering with signaling via reactive oxygen intermediates or by altering the redox state of the cell. The protease inhibitor, TPCK, can also inhibit activation of NF-KB (Henkel et al., 1993). Neither of these drugs, however, specifically inhibit NF-KB and therefore would probably not be good candidates for therapy. Interestingly, the common antiinflammatory drugs sodium salicylate and aspirin can inhibit the activation of NF-KBat high doses (Kopp and Ghosh, 1994). Historically, the

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ELIZABETH B. KOPP A N D SANKAR GHOSH

clinical effectivenss of these drugs has been attributed to their ability to inhibit prostaglandin production; however, the prostaglandin hypothesis as the sole explanation for the activity of the salicylates has come into question (Weissmann, 1991). In particular, there appears to be a discrepancy between the high doses necessary to treat chronic inflammatory diseases and the low doses sufficient to inhibit prostaglandin production. Therefore, the inhibition of NF-KBby these drugs may account for their anti-inflammatory activity at high doses. Although the salicylates clearly inhibit other processes beside NF-KB, they are viable drugs as they have been used clinically for hundreds of years (Weissmann, 1991). However, a search for more specific and effective NF-KB inhibitors will surely b e pursued in the future. IX. Conclusion

Although NF-KBwas first discovered as a key regulator for the developmental stage and tissue-specific expression of the immunoglobulin K light-chain gene, subsequent studies as described in this review demonstrate that this inducible transcription factor plays an important role in the expression of many other genes. The remarkable aspects of NF-KB function are its involvement in the expression of so many different target genes and the diversity of signals that activate this transcription factor from its cryptic inactive state. Although such prolific use of one transcription factor for regulating so many genes appears at a first glance bewildering, an examin,ation of these genes suggests that NF-KB is recruited for situations requiring a rapid response to stress, infection, and injury. As many of the gene products regulated by NF-KB(cytokines, acute phase response proteins, and cell adhesion molecules) are integral components of innate immunity, it is not intuitively difficult to regard NF-KBas a crucial element in this protective system. NF-KBplays an evolutionarily conserved role in helping to protect the organism from infection as evidenced by the recent characterization of Dif, an insect transcription factor that regulates innate immunity in Drosophila. The properties of mutant mice that lack the p105 (p50) gene also support the concept of NF-KB as a key player in this system (W. Sha and D. Baltimore, personal communication). These mice, generated by gene targeting, developed normally indicating that NF-KB p50 function is redundant for specifyingoverall development. However, careful examination ofthese mutant mice revealed that their B cells were deficient in their ability to interact with T cells and to respond to LPS. Remarkably, despite containing normal numbers of functional B and T cells, they are far more susceptible to infection than normal littermates.

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ADVANCES IN lMMUNOLOGY, VOL 58

V(D)J Recombination and Double-Strand Break Repair David T. Weaver Division of Tumor Immunology, Dana-Farber Cancer Institute, and Department of Microbiology and Molecular Genetics, Harvord Medical School, Boston, Massachusetts 021 15

I.

Introduction

11. The V(D)J Recombination Mechanism 111. Joining Mechanisms

Genes Involved in DSB Repair and V(D)J Recombination The Ku Autoantigen Human Immunodeficiency and DNA Repair Syndromes ImmunodeficiencylDNA Repair Syndromes Affecting Cell Cycle Checkpoint Mechanisms VIII. Cell Cycle Regulation of V(D)J Recombination and DSB Repair References IV. V. VI. VII.

29 30 39 45

55 62 66 70 74

1. Introduction

The immune system relies on site-specific recombination (V(D)J recombination) for the formation of Ig and TCR genes, the primary antigen-recognition molecules. This complex rearrangement pathway has been investigated on the level of its DNA requirements and the identification of necessary proteins. Mutations in several genes and/ or activation of the mechanism in nonlymphoid cells have identified key players in V(D)J recombination. Some of these factors are lymphoid restricted, but others are ubiquitous and also utilized in DNA repair pathways. Mutations in four complementation groups have the combined defects of V(D)J recombination deficiency, sensitivity to ionizing radiation (IR), and double-strand break repair (DSB repair) deficiency. A subset of human DNA repair syndromes may be relevant to the connection between DNA repair and V(D)J recombination via a DNA damage signal transduction pathway. DNA damage-induced cell cycle arrest is a likely mechanism to monitor V(D)J recombination completion and accuracy. Mutations in cell cycle checkpoints are associated with increased cancer susceptibility, especially in lymphoid tissues . 29 Copyright 0 1995 by Academic Prrs*, Inr. A11 rights 111 reproduction in any form rrsrrvrd.

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DAVID T. WEAVER

II. The V(D)J Recombination Mechanism

The notion that genes rearrange somatically to facilitate immune cell function originated with Tonegawa (Tonegawa, 1983). Since this seminal observation, an extensive investigation of this recombination pathway has ensued. Previous excellent reviews have documented the developmental regulation of V(D)J recombination, the pathway with regard to putative DNA intermediates, and the protein components (Gellert, 1992a,b; Lieber, 1991; Lewis and Gellert, 1989; Schatz et al., 1992; Oettinger, 1992; Alt et al., 1992). Here, I will concentrate on the reaction pathway as it compares with DSB repair. In the ranks of recombination mechanisms V( D)J rearrangement is an illegitimate pathway because the rearranging DNA is not conserved between the substrates and the products of the reaction. This key observation makes the pathway mechanistically intriguing. The principle of junctional variation generated by rearrangement also forms an essential component for antibody and TCR diversity. For simplicity, the discussion of the rearrangement pathway has been divided into two phases: cleavage and joining. Recent advances have offered potential intermediates for the reaction and better described the processes between cleavages and recombination products. Following cleavages, the joining steps bear striking similarities to DNA repair processes. These similarities may include common features of synapsis, processing, and ligation with chromosomal damage or DSB repair.

A. INITIATING V(D)J RECOMBINATION V(D)J recombination is a site-specific process normally restricted to

B and T cell progenitors. Significant progress has occurred toward

proving that the onset of chromosomal gene rearrangements in lymphoid development is regulated by transcription factor assembly and methylation status in cis. The strategy has been to examine several cis-acting sites for enhancer element binding and/or recombinase interaction. Several recent studies with transgenic mice containing knockout mutations of regions of the Ig heavy-chain enhancer, Ig JH, or downstream Ig enhancer elements have illustrated that these cis-acting elements are necessary for gene rearrangement (Kitamura et al., 1991; Chen et al., 1993; Takeda et al., 1993; Oltz et al., 1993).Homozygous mutations of any of these cis-acting elements lead to mice without gene rearrangement and an absence of B cells and Ig. Currently, no human immunodeficiency syndromes correlate with an absence of these elements by mutation. Because a lack of transcription factor binding sites or enhancer assembly elements in the Ig locus can create

V(D)J RECOMBINATION A N D DOUBLE-STRAND BREAK REPAIR

31

immunodeficiency in mouse models, it may also be expected that immunodeficiency diseases may arise where transcription factors are missing. Murine models with transcription factor deficiencies are forthcoming, but a characterization of the syndromes is likely to be complicated by the utilization of these proteins in other tissues and transcription units. The DNA requirements of the reaction have been easiest to examine because of the vast number of independent gene rearrangement events available (see GenBank entries for most complete and up-to-date listing). Also, the advent o f a transient transfection system for monitoring V(D)J rearrangement has made the manipulation of substrate molecules extremely useful. These two experimental strategies provide a means to document the products and substrate parameters for efficient rearrangement. Two cassettes of recombination signal sequences (RSS) are required for V(D)J recombination. Each Ig or TCR gene segment that has been observed to undergo rearrangement i n vivo contains one RSS at its 3' or 5' border. Each RSS is composed of a highly conserved heptamer and a less-conserved nonamer (Fig. 1). The consensus heptamer (CACAGTC) is palindromic and separated from the nonamer by a spacer region in one of two ways. RSS-12 contain a 12-bp spacer and RSS-23 contain a 23-bp spacer. One each of RSS-12 and RSS-23 is needed to signal V(D)J rearrangement, and these are the only essential elements for the reaction (Gellert, 1992a; Akira et al., 1987).Variations in spacer length reduce or eliminate recombination frequency in transient transfection assays in pre-B cell lines (Hesse et al., 1989). Although the spacer sequences are not random, there is also not any strict nucleotide position requirements for this intervening region (Ramsden et al., 1994). In contrast, the heptamer is relatively invariant with close to 100%conservation of the first three residues (CAC----) between all vertebrates examined. The other four residues (---AGTG)

V

5

/

CACAGTG-12-

ACAAAAACC

J

\

GGTTTTTGT-23-CACTGTG 3

FIG.1. Recornhination signal sequence (RSS). Each V, D, and J gene element is flanked by an RSS consisting of a highly conserved palindromic heptamer separated by a spacer from a conserved nonamer. Spacers of either 12 or 23 b p are used together. Triangles of different stippling represent RSS-12 and RSS-23, respectively.

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are conserved at the level of 76437% (see Gellert, 1992a for more details). The molecular features of the recombination products are instructive in delineating mechanism steps. Because the two DNA products are so distinctly different, the joining pathway must diverge at some point following cleavages that initiate rearrangement. Chromosomal signal joints are formed by the precise fusion of the two RSS. Signal joints have been observed from in vivo rearrangements in which the orientation of the gene segments dictates an inversional recombination path rather than deletion. Similarly, recombination templates, such as integrating retroviruses or plasmid transient transfection assays, have also demonstrated that the primary RSS product of gene rearrangements is a fusion without loss or addition of nucleotides. Insertion or deletion of nucleotides is detectable at these junctions, but only considerably less frequently (Lieber et at., 1988b; Boubnov et al., 1994a). The protein requirements for the initiation mechanism are only partly defined. The RAGl and RAG2 genes were cloned based on a functional complementation screen for induction of DNA rearrangement in nonlymphoid fibroblasts (Schatz and Baltimore, 1988; Schatz et al., 1989; Oettinger et al., 1990). Introduction of RAGl and RAG2 into any normal cell type is sufficient to drive V(D)J recombination of cotransfected plasmid substrates (Oettinger et al., 1990). RAG1 and RAG2 are primarily expressed in lymphoid progenitor cells, consistent with the timing of V(D)J recombination (reviewed by Oettinger et al., 1990).Homozygous knockout mutations of either RAGl or RAG2 eliminate all V(D)J recombination, creating immunodeficient mice (Shinkai et al., 1992; Mombaerts et al., 1992). RAG1 and RAG2 may be sufficient to initiate V(D)J recombination, but the biochemistry of how these proteins function is still developing. It has not yet been demonstrated that either RAGl andlor RAG2 are associated with any strand cleavage or strand transfer reactions. Additional proteins may be required to initiate V(D)J recombination to facilitate the action of RAGl and RAG2. RAGl and RAG2 could also potentially be involved in joining steps, but this is becoming increasingly unlikely. Several activities identified by binding to RSS have been discovered and recently reviewed (Gellert, 1992a). Proteins of this type may be used to recognize RSS and assemble recombinase complexes. B. DSB INTERMEDIATES IN V(D)J RECOMBINATION?

A number of observations implicate double-stranded broken ends of DNA as intermediate structures for V(D)J recombination. The cut sites for V(D)J recombination are likely to occur at the borders of RSS

V(D)J RECOMBINATION AND DOUBLE-STRAND BREAK REPAIR

33

heptamers where coding sequence from V, D, or J elements is found. This conclusion has been substantiated by extensive DNA sequencing of gene rearrangements. The RSS junctions themselves are consistent with strand breaks immediately next to the heptamers. Although cleavages at RSS borders may be DSBs, staggered breaks could also be the primary cleavages, where one of the two strand scissions occurs at the heptamer border. Coding sequence junctions, although variable between independent events, are similarly consistent with a sitespecific double-strand break model. New findings suggest that both RSS ends and coding ends pass through double-strand break intermediates in the pathway. RSS ends of TCR 6 rearrangements accumulate in thymocytes sufficiently so that recombination-associated broken DNA of particular configuration can be characterized by Southern blot analysis (Roth et al., 1992a). RSS ends, and not corresponding coding ends, are preferentially seen. More sensitive assays involving the ligation of double-strand broken genomic DNA to oligonucleotide primers for PCR (ligation-mediated PCR, LMPCR) have been recently used (Schlissel et al., 1993; Roth et al., 1993). Free RSS ends can be observed from primary thymocytes, bone marrow, or fetal liver, the most abundant tissue sources of lymphoid gene rearrangement events. RSS ends of J or D elements from the less complex gene rearrangement families were scored. Also, RSS ends from Abelson murine leukemia virus-transformed pre-B cell lines have also been identified for Ig V-JKrearrangements (Schlissel et al., 1993). Thus, RSS ends are a consistent feature of many, and possibly all, of the gene rearrangement families. RSS ends are associated with gene rearrangement because they are not detected in RAG1 -I- knockout mouse lymphoid tissues (Schlissel et al., 1993). It is not yet clear why the RSS ends are preferentially detectable. Perhaps the rate of joining of the “nonessential” RSS junctions discarded from the chromosome is slower than that for coding junctions. Using LMPCR, a more detailed picture of the structure of the RSS ends has also been uncovered (Schlissel et al., 1993; Roth et al., 1993; Fig. 2A). The 5’ RSS strands retain a 5’-phosphate group (5‘-P-CACAGTG-3’) which was demonstrated by blocking of the utilization of oligonucleotides in ligation reactions by pretreatment of the genomic DNA with calf intestine phosphatase. A uniformity of end structures was shown using pairs of oligonucleotides differing by the extent of overlap at the ends. These experiments indicated that RSS ends are blunt ended immediately flanking the RSS heptamers. Considering the consistency with which blunt RSS ends form from primary tissues, it was concluded that RSS ends could well be the immediate products

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DAVID T. WEAVER

A. Cleavage 5’ 3‘

I hairpin

p

e

OH

p

1 hairpin

FIG.2. Pathway of V(D)J recombination. (A) Initiation and cleavage steps of the mechanism. Recombination signal sequences (RSS) are shown as shaded triangles. One RSS containing a 12-bp spacer and one RSS containing a 23-bp spacer is used in the reaction. Protein-DNA intermediates are shown bridged by a phosphate (P) bond. (B) Resolution and joining steps of V(D)J recombination. Resolution of putative coding end hairpins is shown by positions of arrows. Two cases of “resolved’ hairpin ends are shown. Dark lines represent regions of same DNA sequence (DNA homology) between coding ends. Two examples of coding junction formation are illustrated: top, junction occurs in DNA homology; bottom, junction is formed outside of DNA homology. RSS end formation triangles represent RSS.

of cleavage reactions. It stands to reason that RSS ends may be the immediate precursors to RSS joints because they can b e directly ligated. However, it is not yet clear whether RSS ends are normal intermediates in the pathway or are by-products or dead-end products. The latter would not necessarily be informative concerning the mechanism.

V(D)J RECOMBINATION A N D DOUBLE-STRAND BREAK REPAIR

8.

35

Joining RSS ends:

Coding ends:

1 1

1

Deletion

N sddltlon Llgatlon

+

Fill-In

or

FIG.2. Continued

However, because these molecules have some of the anticipated features of V(D)J intermediates, it is tantalizing to speculate that they are involved in the pathway. Broken molecules from coding ends in the reaction have not been detectable by the above methodology (Schlissel et al., 1993).This may be due to a greater degree of heterogeneity amongst the ends, thus reducing the ability to detect any products. Heterogeneity of doublestrand broken coding ends is predicted in models for gene rearrangement. Alternatively, coding ends may be blocked, sequestered, and/or unligatable without processing. To address this issue, the same genomic DNA was pretreated with modifying enzymes prior to LMPCR with coding DNA. The combination of nuclease and DNA polymerase prior to LMPCR did not enhance the recovery and identification of coding ends. An additional explanation would be that coding ends are rapidly repaired in the recombination mechanism such that the short-lived cleavage products from these ends are difficult to detect.

36

DAVID T. WEAVER

In reality, both may be true: coding ends are unligatable without processing and not a stable form compared to RSS ends (see following sections). C. THE HAIRPINRESOLUTION MODELFOR P NUCLEOTIDE FORMATION

V( D)J recombination coding junction products are highly variable in nucleotide content, a consequence of the rearrangement mechanism. Both nucleotide addition and loss were frequently displayed in the products (Tonegawa, 1983). It has been estimated that greater than 95% of all coding junction products contain deletion (Lieber, 1991). Deletion is limited to an average of zero to six residues per coding segment. Therefore, the combined junctional loss of nucleotide is usually 10 b p or less. Coding ends that are not deleted have proved to be informative concerning the reaction. Undeleted coding ends are often associated with the retention of specific nucleotides that appear to be templated by the nucleotide composition of the coding ends. These residues, termed P ( palindrornic) nucleotides, are complementary to the terminal coding segment residues flanking the RSS prior to rearrangement. P nucleotides have been demonstrated from either or both of the RSS in individual reactions (Lafaille et al., 1989; McCormack et al., 1989). P nucleotide formation or retention is more abundant in early stage rearrangements than in developmentally late rearrangements. Tissuespecific and stage-specific variations may be dictated by processing activities rather than by whether or not P nucleotides serve as reaction intermediates. The size of P nucleotides is frequently one or two residues with longer P residues appearing in some TCR rearrangements; exceptionally long P nucleotides have been found for gene rearrangements from scid, V-3, and XR-I mutant cells in culture (see below). Also, sequence analysis of scid TCR rearrangements has revealed extended P nucleotides (Kienker, 1991; Schuler et al., 1991). Nearly all rearranging gene families and plasmid substrates for transient transfection have substantiated the general appearance of P nucleotides as part of the V(D)J mechanism. However, the average occurrence of these residues is on the order of 5% of coding joints (Meier and Lewis, 1993). Thus, the presence or absence of processing, or the level of processing, likely influences the detection of P nucleotides in coding joints. Regarding models for initiation of V(D)J recombination, cleavage events of the reaction may synthesize an intermediate that would be the precursor of P nucleotide formation. As such, hairpin-ended DNA

V(D)J RECOMBINATION AND DOUBLE-STRAND BREAK REPAIR

37

has been proposed as such an intermediate structure (Gellert, 1992b; Lieber, 1991) (Fig. 2B). With a hairpin as a DNA intermediate, coding ends with P nucleotides could be synthesized by the asymmetric cleavage of the hairpin. This process would generate a heterogeneous population of DNA ends at an intermediate stage prior to joining and may thus explain some of the heterogeneity of coding products. Several features of this model are consistent with DNA sequencing data of numerous coding joints. First, most P nucleotide-containing junctions have one or two nucleotides accounted for by a single coding end, indicating that cleavage of putative hairpins would occur near the ends. Thus, overhanging coding ends would usually be only 1 or 2 bp. The terminal residues of hairpins are not base paired due to their thermodynamic instability and may be structurally distinct and recognized by a specific factor(s). Second, measurement of rearrangement of the same sequences numerous independent times shows that the coding joint can include P nucleotides in only a fraction of the junctions. This property is consistent with an unselective resolution of a hairpin. A significant advance was made when hairpins associated with coding DNA sequences were observed in vivo for endogenous gene rearrangements. Roth et al. examined neonatal thymocyte genomic DNA for the appearance of chromosomal breaks associated with TCRG D-J rearrangements (Roth et al., 1992a,b). Scid mouse thymocytes showed an accumulation of broken DNA coding ends that were demonstrated to be hairpin terminated based on two dimensional native-alkaline gel electrophoresis (Roth et nl., 1992b). Hairpin forms do not accumulate in wild-type mouse thymocytes, but linear coding ends from the same genomic region are detectable at low levels. Hairpins associated with Ig, or TCRa or -p rearrangements were not observed. Also, hairpin DNA from rearrangement plasmid substrates following transient transfection into tissue culture cells has not been found (N. Boubnov and D. Weaver, unpublished data). Thus, the generality of hairpins as intermediates in V( D)J recombination is still not proven. Nevertheless, coding end hairpin intermediates of the reaction are thus far consistent with all experimental observations of the recombination reaction and its products. The hairpin model of P nucleotide formation has also gained recent experimental support from plasmid-based recombination assays. The composition of the coding DNA sequences immediately flanking the RSS influences the frequency of P nucleotide formation (Meier and Lewis, 1993; Boubnov et al., 1993). In these studies the composition of the terminal coding DNA sequences (10 bp or less) was the only

38

DAVID T. WEAVER

change in plasmid substrates. An explanation for this effect is that hairpins are processed in alternate ways depending on DNA sequence composition. Exclusively G/C coding ends, or coding ends with a high G/C content, show reduced processing and higher P nucleotide frequencies (Boubnov et al., 1993). Other nonrandom coding ends also show elevated P nucleotide frequencies but the rules for how these frequency levels are determined are unclear (Meier and Lewis, 1993). P nucleotides did not appear in signal junction products, suggesting that hairpin intermediates are not associated with RSS ends in the midst of the reaction. P nucleotides of longer lengths also form in response to the structure of the coding DNA (Meier and Lewis, 1993).

D. A V(D)J RECOMBINATIONCLEAVAGE MODEL V(D)J recombination cleavages are likely to occur in a two-step mechanism that generates an asymmetry between the newly formed coding end and RSS end intermediates. This model has been previously discussed (Lieber, 1991; Gellert, 1992b; Roth et al., 1993). From experimental observations, the RSS ends appear to be blunt ended with 5’ phosphoryl groups. Also, evidence has been generated that coding ends can accumulate as hairpins in scid thymocytes. Furthermore, DNA sequencing of gene rearrangement events shows the presence of P nucleotides in junctions, also consistent with a hairpin mechanism. With the caveat that neither of these structures has been proved as a reaction intermediate for V(D)J recombination biochemically, a compelling model for the cleavage mechanism can be outlined (Fig. 2A). Two cleavage mechanisms are currently consistent with the available data. In one model, DNA-protein intermediates form in a mechanism that is similar to A integration or resolvase site-specific recombination mechanisms [(Stark et al., 1992) and referenced in Roth et al., 1993; Fig. 2AI. This mechanism has been termed “asymmetric cleavage” by Roth et al. (1993). The initial RAGl/RAGe-induced strand breaks are probably single-strand breaks forming a DNA-protein intermediate on the coding end and a 3’-hydroxyl on the RSS end for the two RSS elements in the reaction. The break would occur immediately flanking the RSS on the 3’ strand. Either RAG1 or RAG2 would be the best candidates for the protein covalently bound to DNA at this stage. Hydrolysis of the phosphodiester bond opposite the DNAprotein intermediate would then be promoted, presumably by specific protein contacts in a synaptic complex. Coding end hairpins could then form by an interstrand nucleophylic attack on the DNA-protein phosphodiester bond. Following this cleavage, the two intermediates,

V(D)J RECOMBINATION A N D DOUBLE-STRAND BREAK REPAIR

39

hairpins and 5’-P-RSS ends, would form. An alternate possibility is that protein(s) may also be bound to RSS ends from the initial cleavages, but this was discounted by quantitation of the level of RSS ends (Roth et al., 1993).Thus, binding of protein to only two of the ends as shown may be found. An added benefit of this approach would be that hairpin formation may be favored in the next step, rather than precise sitespecific recombination. Covalently bound protein to all four phosphates would be more consistent with resolvase-type mechanisms, instead of A integrase pathways. If a protein-DNA covalent linkage is used in V(D)J recombination, then it is likely that this bond is via a serine residue in the protein. Tn3 resolvase-type protein-DNA bonds are all through serine residues as 5’ phosphodiester from the DNA; X integrase-type linkages are tyrosine linked to 3’ phosphodiester bonds from the DNA (Stark et al., 1992). Therefore, 5’-P-RSS ends are most consistent with resolvase-type linkages. In sum, potential DNA-protein linkages of V(D)J recombination appear to have some of the features of A integrase and resolvase strategies, possibly indicating that the mechanism wilI be unique. Further tests and refinements of this model will be necessary pending more experiments. In an alternative pathway, no DNA-protein intermediates would be required, and the cleavages would be formed by direct one-step esterification as for Mu transposition (Mizuuchi and Adzuma, 1991; Mizuuchi, 1992). There are no experimental data that are yet to distinguish among these mechanisms. 111. Joining Mechanisms

The mechanism by which joining occurs is dictated by the structures of cleavage products and/or other intermediates. Also, joining steps share extensive similarities to pathways for repairing DSBs in chromosomal DNA either arising spontaneously or induced by DNA damage. A. MICROHETEROGENEITY IN

FORMATION OF CODING JOINTS A striking property of all Ig and TCR gene rearrangements is the imprecision of coding junctions. This heterogeneity has been amply illustrated with the characterization of chromosomal gene rearrangements from lymphoid cells and recombination substrates in lymphoid and nonlymphoid cell culture. For any given V, D, or J element or any RSS, independent joining reactions yield variable deletion and addition of nucleotides in the joint. Thus, the extensive heterogeneity that occurs in the joining mechanism is largely independent of DNA sequence. The processing of these V(D)J coding ends prior THE

40

DAVID T. WEAVER

to joining combines both the addition of nucleotides and the loss of nucleotides. N-type nucleotide addition, attributable to the enzyme TdT, is observed in either the presence or the absence of P nucleotides in the junction. Also, N regions are found regardless of the extent of deletion, including abnormally large deleletions from rearrangements signaled in the presence of the scid mutation (Hendrickson et al., 1988; Malyn et aZ., 1988). Thus, N regions are probably added at a last step prior to ligation in the reaction. Because TdT is largely restricted in expression to specific lymphoid cell stages, it is unlikely to have general significance for the comparable mechanisms of nonhomologous recombination (see below). There are no DNA sequence requirements for codingjunction formation. Unlike strictly site-specific recombination mechanisms, any two DNA sequences can be joined together in V(D)J recombination. These observations would tend to suggest that the enzymology of this process is unique and potentially distinct from the protein requirements for initiation and cleavage. There is no information yet as to preferential use of processed ends in joinings steps in the reaction. Hairpins resolved on either strand by a single-stranded scission near the ends would generate P nucleotides of opposite strand polarity. No experiments have yet addressed whether 5' extended, 3' extended, or blunt ends are more likely to be repaired into coding junctions by the V(D)J recombination machinery. Highly G/C-rich coding ends may be either resilient to processing or have a tendency to cleave at their immediate termini creating blunt coding ends. For example, coding ends ofGloor Clohave a significantly reduced level of deletion (Boubnov et al., 1993). Because deletion is ordinarily observed for each coding end, a tentative conclusion would be that the rate of deletion is faster than the ability of DNA repair polymerases to fill-in gaps. Alternatively, protein contacts holding the recombination synapse together prior to ligation may actually exclude repair enzymes from doing their job. This may mean that repair events continue after the dissolution of the recombination synapse. B. MICROHOMOLOGY IN CODING JOINTS Several laboratories have found a restricted heterogeneity in junctional diversity between certain Ig or TCR V, D, and J gene elements. Characterization of the joints indicated that microhomology between the coding ends may be used in the joining reaction (Gu et al., 1990; Feeney, 1990,1992; Ichara et al., 1989; Feeney, 1991; Asarnow et al., 1989; Aguilar and Belmont, 1991).Small redundant homologies of 15 bp have been found more frequently at the breakpoint in coding

V(D)J RECOMBINATION AND DOUBLE-STRAND BREAK REPAIR

41

joints than would randomly occur. An issue left unresolved by these initial studies was whether immune cell-selection mechanisms may override the distribution or rearrangement products of the basal rearrangement machinery so that recombination products found in mature lymphoid tissues would be skewed by the selection. An indication that selective pressures were not in force was shown with V61-D62 and Vy6-Jy 1 nonproductive rearrangement junctions that showed a preponderance of invariant junctions, consistent with DNA homology sites from the two coding ends (Chien, 1987; Lafaille et al., 1989; Allison and Havran, 1991). Additional evidence for the use of microhomology has also come from recent studies with transgenic mice in which rearrangement of the transgene is scored. In each case the transgene does not produce a protein product so that there is no issue of selection following the rearrangement, but the same rearrangement events can be scored (Asarnow et al., 1993; Itohara et al., 1993). TCR CS-/- mice have a high degree of TCRy and S rearrangements that recruit short homologies in the coding junctions (Itohara et al., 1993). These animals cannot produce TCR y8 on the surface influencing cell-selection mechanisms. Likewise, introduction of frameshift mutations in Vy2,Vy4, and Vy3 obviated the protein production of their rearrangement products which showed an increased utilization of homology stretches (Asarnow et al., 1993). The most compelling examples of microhomology in junctions come from in uiuo gene rearrangements with genetically altered mice. TdT-/- knockout mice use D N A homology once TdT activity is not present. This experiment was done in two ways. TdT-/- somatic chimera mice were generated from RAG2-/- embryos (Komori et al., 1993). In this regimen all of the cells that populate the postrearrangement immune system come from embryonic stem cells with the TdT-/- genetic background (Chen et al., 1993). Alternatively, homozygous mutant TdT- / - mice were generated by breeding (Gilfillan et al., 1993). Gene rearrangements from both studies showed that untemplated N regions are not added in the absence of TdT. Second, the V(D)J coding ends frequently form at regions of microhomology, as illustrated by overlap residues that could have arisen from either coding end partner in the rearrangement (Komori et al., 1993; Gilfillan et al., 1993). These overlaps varied between one and five residues. These experiments are valuable as well because they allow the analysis of V(D)J recombination events in uiuo and at the lymphoid cell stages normally acting for each gene family. Transient transfection assays with recombination substrates have

42

DAVID T. WEAVER

not been so clear cut. In one study, a plasmid substrate with a 4-bp microhomology region located at coding ends gave coding joints in the 4-bp repeat 55% of the time in pre-B cells (Gerstein and Lieber, 1993). However, many of the recombination products observed did not utilize short stretches of D N A homology in the joint. Chinese hamster ovary fibroblasts from this study showed the use of DNA homology at a slightly lower frequency (33%). Alternatively, plasmid substrates with only two nucleotides of overlap between the coding ends are not apparently recruited to any greater extent than random (Boubnov et al., 1993; Gerstein and Lieber, 1993). Interestingly, each of these substrates has the two-nucleotide overlap internal to the coding end. Therefore, it is not clear whether the homology is recruited primarily because it is at the coding termini or because of its size. Because most of the in uivo gene rearrangements that have been implicated to utilize short terminal homology frequently only have 2 bp of overlap, there is a paradoxical difference between these findings. Also, extensive homology of 5-10 b p does not increase the recombination frequency (Boubnov et al., 1993). Perhaps the level of recombination activity is high enough in cell-based assays that there is less dependence on a stage of the reaction at which joining occurs. Because use of DNA homology at coding ends is not universally applied, even in chromosomal rearrangements in viuo, it cannot be considered as an essential part of the mechanism. However, DNA overlaps may be significant for rearranging gene families where a restricted recognition property serves an immunological strategy.

C. A V(D)J RECOMBINATION JOINING MODEL The joining steps of the reaction path allow the formulation of a likely model (Fig. 2B). Coding and RSS junctions differ b y the extent of normal processing. Precise RSS junctions are a logical product of 5’-phosphoryl and blunt RSS ends recently shown (Schlissel et al., 1993; Roth et al., 1993). Therefore, RSS ends may not be processed at all, but are directly ligated. RSS ends could be protected from processing by protein binding. None of the joining steps have any requirement for covalently associated proteins. Cleavage of hairpins by single-strand breaks near the ends would generate fragmented coding ends differing by the position of cleavage. In addition to coding ends of different-overhangs, a varying extent of processing does occur in independent events. DNA homology between the ends may be used to direct joining. The restriction on the extent of deletion is probably limited by the ability to hold the two coding ends in a synaptic complex by protein associations. N region addition is very likely to

V(D)J RECOMBINATION AND DOUBLE-STRAND BREAK REPAIR

43

occur after terminal deletion. The enzymology of coding end processing will require more experiments through the examination of mutants that effect these steps (see below).

D. NONHOMOLOGOUS RECOMBINATION AND ENDJOINING The joining of ends of DNA without regard to DNA sequence is an abundant property of most eukaryotic cells, but the significance of this process for cell metabolism and DNA repair is little understood. Molecular properties of end-joining have been defined for mammalian cells (reviewed in Roth and Wilson, 1988; Roth et al., 1985; Roth and Wilson, 1986; Roth et al., 1989) and Xenopus Zaevis or human cellfree extracts (Pfeiffer and Veilmetter, 1988;Thode et al., 1990; Fairman et al., 1992). Key features of this process bear a striking resemblance to V( D)J recombination coding junction formation, suggesting an overlap in the mechanisms, and a possible recruitment of aspects of nonhomologous end-joining in lymphoid-restricted gene rearrangement. Furthermore, characterization of chromosomal translocation breakpoint junctions associated with a variety of tumor types often has similar molecular features to end-joining. Efficient end-joining can be measured with transfected linearized plasmid DNA in cell culture although similar properties are observed with end-joining from cell-free extracts. Analysis of recovered junctions shows that end deletion/insertion occurs in concert with the joining so that recombinant outcomes of several types may occur. Models that accomodate the formation of all of these observed junctions have been proposed in which there is a combination of nuclease and polymerase functions that leads to the heterogeneity of joints with or without deletion of nucleotides. A sizeable fraction of observed junctions have no loss of DNA, but appear to result from filling-in steps prior to ligation, even if the ends are not cohesive and/or contain 3’ overhanging strands or blunt ends instead of 5’ overhanging strands (Roth et al., 1989; Thode et al., 1990; Pfeiffer et al., 1994b). In addition, “filler” DNA also appears in these junctions (Roth et al., 1989) as is found for V(D)J recombination. To explain the interesting properties of the fill-in reaction, it was proposed that a factor would hold DNA ends together such that repair synthesis could occur from one DNA end to the other. These factor(s), alignment proteins, are proposed to work without ligation between the ends. Evidence for alignment factors in nonhomologous end-joining was reported (Thode et al., 1990).Alignment protein(s) have been suggested to prepare the template so that a DNA polymerase may be

44

DAVID T. WEAVER

primed for repair synthesis across a double-strand gap (Thode et al., 1990). It is interesting that this repair synthesis is itself error prone because mismatched nucleotides between the two ends can be incorporated into the recombinant product rather than excised. Therefore, repair synthesis may occur either before or after ligation so that strands can be ligated in the presence of mismatches or following mismatched nucleotides being repaired (Pfeiffer et al., 1994a). Some rules already apply in this process. Completely nonhomologous ends can be ligated such that the polarity of mismatches relative to flanking matches determines whether or not the mismatches are retained in ligated strands or excised (Pfeiffer et al., 1994a). These parameters offer insight into the processing of DNA ends by matches and mismatches. Similarities between nonhomologous recombination and the sitespecific V(D)J recombination coding joint formation have previously been noted (Roth and Wilson, 1988). One feature of nonhomologous recombination reactions was the finding that short tracks of DNA homology are recruited in the joint a sizeable percentage of the time (approx 50%; Roth and Wilson, 1986; Roth et al., 1989). V(D)J recombination coding joints may share related properties as evidenced by the findings of several laboratories that coding end microhomology frequently dictates the exact nucleotide structure of coding joints. Furthermore, it may be expected that there is a similar use for alignment factors in V(D)J recombination. Because completely nonhomologous ends can be joined in V(D)J recombination (Boubnov et al., 1993), the recruitment of mismatches in the joined ends would be a means to increase junctional heterogeneity. The biochemistry of V(D)J recombination joining has also been explored recently as several aspects of the joining reaction are related to nonhomologous end-joining discussed above. Joining of linear D N A containing RSS elements was observed (Halligan, 1993; Halligan et al., 1994). DNA joining was achieved with a purified protein, VDJP, or crude nuclear extracts from pre-B cells containing VDJP. D N A joining facilitated by VDJP required RSS on both partners of the joining substrates; other DNA homology is nonessential for the reaction. VDJP was cloned from screening a cDNA expression library for binding to the nonamer region of an RSS, and this protein has been demonstrated to bind to the nonamer in uitro. VDJP is identical to a portion of replication factor C (RF-C) but has unique 5’ and 3’ ends. In addition, RNA analysis showed that a 1.7-kb mRNA corresponding to VDJP was expressed in immature lymphoid cells, but not in nonlymphoid cells. In contrast, RF-C has a 4.5-kb mRNA in all cells (Lu et al., 1993; Luckow et al., 1994). Thus, VDJP is possibly a distinct gene from

V(D)J RECOMBINATION AND DOUBLE-STRAND BREAK REPAIR

45

RF-C or a unique gene product that arises by differential splicing or altered RNA processing. The VDJP region of RF-C has homology to bacterial DNA ligases; although the region of homology is not in the vicinity of the ligase active site, it may be a clue to VDJP biochemical activity. VDJP may b e an interesting accessory protein to RAG1 and RAG2 function in V(D)J recombination. This protein may be most involved in RSS junction formation because of the requirement for RSS in VDJP-mediated joining. Another joining activity has been described that could be relevant to V(D)J recombination and DSB repair. The DNA transfer assay (DTA) measures transfer of DNA from a donor to recipient plasmid substrate, dependent on DNA homology (Jessberger and Berg, 1991). Although there is no evidence that DNA homology is required for either V(D)J recombination or DSB repair, DTA may be useful for identifying important biochemical activities for these processes. A high-molecularweight protein complex (RC-1) has been found that permits DTA i n uitro and consists of several DNA ligase and polymerase components (Jessberger et al., 1993).RC-1 activity is highest in CD4-lCD8- immature wild type or RAG2-/-thymocytes and lowest in CD4+lCD8+or single positive thymocytes ( Jessberger et al., 1994). Nuclear extracts from scid lymphocytes have very little RC-1 activity, and deficient scid extracts can be restored by the addition of fractions from wildtype fetal thymocyte extracts ( Jessberger et al., 1994). Using this procedure, a 72-kDa protein has been purified that complements the in uitro deficiency for DTA. It remains to be seen whether or not the 72-kDa protein (Scid recombination stimulatory protein, SRSP) is encoded by the scid gene, but further descriptions of SRSP functions will be keenly interesting. Because V(D)J recombination from start to finish has yet to be accomplished in uitro,the exact correlation with nonhomologous end-joining remains elusive. However, an understanding of mutations that effect both V(D)J recombination and DNA repair may be the most direct route to understanding how these pathways synergize in eukaryotes.

IV. Genes Involved in DSB Repair and V(D)J Recombination

A. THEScid MUTATION The scid mouse was first reported by Bosma et al. (1983).Research on the scid mutation over the past 10 years has defined the nature of the defect in two main arenas: V(D)J recombination and DNA repair. As such, the scid defect serves as a paradigm for the principle that

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there is a recruitment of a number of genes for V(D)Jrecombination and double-strand break repair pathways, and that the two processes overlap mechanistically (Table I). The first noted defect in scid mice was an immunodeficiency that was manifested as a lack of mature lymphoid cells and serum Ig (Bosma et al., 1983).The scid immunodeficiency and the many useful functions of scid mice as cell transfer recipients have been reviewed extensively (Schuler, 1990; Bosma and Carroll, 1991; Hendrickson, 1994). I will review the scid V(D)Jrecombination defect and other molecular properties of the mutation. The V(D)Jrecombination defect of scid mice is responsible for the severe immunodeficiency of the animals (Table I). Whereas normal products of the site-specific recombination pathway give rise to Ig and TCR gene products, these products in w i d cells are aberrant, and usually deleted, such that productive rearrangement only occurs at a small level. This conclusion is supported by molecular evidence from endogenous gene rearrangements in vioo, endogenous rearrangements from derived cell lines, continuing endogenous rearrangements in scid lymphoid cell lines, integrated retroviral rearrangement substrates, and the introduction of plasmid recombination substrates. Schuler et a2. (1986) first showed that scid lymphoid cells are defective by noting the absence of rearrangement alleles of Ig p and TCRD in isolated cell lines. Using scid Abelson murine leukemia virus transformed preB cell lines, several groups demonstrated that ongoing V(D)Jrecombination was defective. The scid pre-B cells did not detectably produce plasmid substrate coding junction products relative to normal controls (Lieber et nl., 1988a). Also, integrated retroviral recombination substrates were aberrantly rearranged in scid pre-B cells. Deletions frequently were extensive enough to include flanking mouse genomic DNA (Hendrickson et al., 1988; Malynn et al., 1988). Whereas coding junction products are absent or defective in scid rearrangements, RSS products are relatively normal. RSS junction plasmid substrates form at a slightly reduced efficiency in scid pre-B cells. Of these products, about 50% have small deletions in the signal junctions and N regions added (Lieber et d . ,1988b). These mutant features are ordinarily characteristic of coding junctions. Integrated retroviral substrates that recombine by an inversional path were informative because the two products of the same rearrangement events could be compared. Although inversions in the absence of extensive deletion are rare in scid pre-B cells, the coding junction products are defective relative to the RSS product (Hendrickson et at., 1990).In these experiments, coding junctions showed aberrant deletions, whereas signal junctions were normal and did not contain deletions. Thus, the scid

TABLE I MUTANTSOF V(D)J R E C O M R t N A T l O N

V(D)J Recombination

IR Mutation Group scid scid

v-3

XR-1

XRCC5 xrs5, xrs6 XR-V79B XR-V15B sxi2, sxi3 sxi 1 rag1 KO rag2 KO tdt KO

Fibro

B and T

DSB Repair

S

S

Deficient Deficient Deficient

S

S S

S

Signal

Coding

Gene Identity

Human Chromosome Location

2

2

Deficient Deficient

Deficient Deficient

Deficient

Deficient Deficient

Deficient

Deficient

p82""

2~134-36

Deficient Deficient wt wt wt

Deficient Deficient 0 0 wt

Deficient Deficient 0 0 No N regions

p70"" RAG 1 RAG2 Tdt

22q3 llp13 llp13

8pll-ql2

5

2

S

S S R R

R

Note. S, sensitive; R, resistant; wt, wild type.

48

DAVID T. WEAVER

defect is selectively aberrant for coding junction formation. A likely interpretation of this data is that the scid mutation does not affect the initiation ofV(D)J recombination, but acts at a later stage in the reaction such as processing or product formation. The signal junction deficiencies observed are possibly secondary consequences of slowing both of the joining reactions down, although only one can be completed. N region addition in the “wrong” junction may be a consequence of the phenomenon of a reduced joining rate. Several experiments indicate that the scid coding junction deficiency may be a processing error. First, yGTCR rearrangements in scid T cells have been shown to have very long P nucleotides (Schuler et al., 1991; Kienker, 1991). Because longer than normal P nucleotides can be generated by changing the DNA sequence composition of coding regions of plasmid substrates for V(D)J joining (Meier and Lewis, 1993), the increased length observed for scid rearrangements may occur by a secondary mechanism. The scid coding junction products can be “rescued” by placement of extended DNA homology (approx 70 bp) flanking the two RSS in inversion plasmid substrates (Lieber et al., 1988a). Formation of normal signal joint products indicates that the reaction being measured is initiated by a RAGURAG2-dependent mechanism; coding joints formed are in the region of DNA homology without any base loss or addition in these sequences. Thus, it appears that rescue by homologous recombination has occurred. With templates containing only 10 bp of DNA homology there is no rescue of the scid coding junction defect (N. Boubnov and D. Weaver, unpublished data), which is consistent with the observations that homologydirected recombination in other systems requires a minimal region of >SO bp. Scid thymocytes accumulate hairpins at TCRG coding ends but not at RSS ends (Roth et al., 1992a). These products are not likely to be aberrant by-products of the reaction because they are detectable in normal mouse thymocytes in parallel and because hairpins are estimated to appear as frequently as signal ends in scid (Roth et al., 1992a). More work will be necessary to confirm that hairpin DNA serves as a V(D)J recombination intermediate. Nevertheless, the findings offer intriguing insight into where scid plays a role in the V(D)J recombination pathway; it is possibly required for resolving hairpins at coding ends. Strand breaks distal to the hairpin in scid may create the extended deletions observed in scid rearrangements. It is possible that thymocytes lack this second function that would allow incomplete scid rearrangements to be rescued by an alternate pathway. On the other hand, cells that break hairpins but do not join the chromosome may

V(D)J RECOMBINATION AND DOUBLE-STRAND BREAK REPAIR

49

be programmed to die by apoptosis. Recent work with bcl2/scid transgenic mice has shown a difference between the T and B cell population (Strasser et al., 1994). At least partial B cell differentiation can be rescued by expression of Bc12, a protein that inhibits cell death (Vaux et al., 1988; Korsmeyer, 1992).Because these B cells presumably can resolve rearrangement-mediated hairpin DNA to continue proliferation and differentiation, there must be a second pathway for hairpin dissolution in B cells. The scidT cells may not have a second resolution pathway so that their proliferation and differentiation is still blocked. These speculations would be consistent with observations that scid thymocytes, but not B cell populations, have been observed to accumulate hairpins. A second primary defect of scid cells is an inability to adequately repair damage from IR (either X rays of y rays; Fulop and Phillips, 1990; Biedermann et al., 1991; Hendrickson et al., 1991; Disney et al., 1992; van Buul et al., 1994). Scid hematopoietic and germ line stem cells, lymphoid cell lines, and fibroblasts all show differential hypersensitivity to IR. IR sensitivity of scid cells is as profound as has been observed with other IR-sensitive mutants. Thus, the scid mutant phenotype extends beyond V( D)J recombination and immune cell function. IR is both mutagenic and lethal in a dose-dependent manner in all cells, and the IR damage that generates chromosomal doublestrand breaks appears to be responsible for the lethality (Radford, 1986).Paradoxically, scid spermatogonial stem cells are IR hypersensitive, but appear to have a significantly reduced level of IR-induced chromosomal translocations (van Buul et al., 1994). One explanation offered for this phenomenon is that translocation-bearing cells are selectively eliminated and thus not scored in the translocation index. Studies with scid fibroblasts illustrate that DSB repair is defective. This conclusion was reached by monitoring DNA fragmentatiodrepair in PFGE or neutral filter elution assays (Biedermann et al., 1991; Hendrickson et al., 1991).These studies are in good agreement, showing that greater than 50% of the broken DNA in irradiated scid cells is not repaired within a time course that fully repairs wild-type cells. Several additional findings have substantiated this correlation. For example, scid fibroblasts are also hypersensitive to IR-mimetic agents such as bleoniycin and another double-strand break inducing agent, neocarzinostatin (Biedermann et al., 1991; Hendrickson et al., 1991; Tanaka et a1,, 1993).Similarly, restriction enzyme introduction, which creates double-strand breaks in cellular chromatin, is selectively toxic to scid cells (Chang et al., 1993). Calcheamycin, an agent that also mimics IR and restriction enzymes by creating staggered double-

50

DAVID T. WEAVER

strand breaks is also a potent toxic agent for scid fibroblasts (Staunton and Weaver, unpublished data). The effects of DNA damage agents that are linked to other types of DNA repair mechanisms generally are repaired normally in scid cells. Scid cells are only slightly sensitive to uv irradiation, and have normal sensitivity to MMS alkylating agents, and mitomycin C crosslinking (Hendrickson et al., 1991; Biedermann et al., 1991; Tanaka et d.,1993). However, there is some sensitivity to the crosslinking by mechlorethamine (Tanaka et aZ., 1993). Because of the involvement of scid in V(D )J recombination and DSB repair, a role for this gene product in other recombination mechanisms has been explored. In fact, these studies may be useful for determining the extent of similarity between the processes. It may be expected that scid cells will have a decreased ability for nonhomologous recombination. Nonhomologous recombination or end-joining is an abundant activity of mammalian cells that can be measured by the joining of noncohesive restriction enzyme sites of a linear plasmid DNA. Furthermore, repair of IR-induced DSBs is thought to occur via nonhomologous end-joining pathways (see above). The scid cells efficiently recircularize transfected linearized plasmid DNAs (Harrington et al., 1992; Lewis, 1994) and efficiently integrate linearized plasmid DNAs into chromosomal sites (Staunton and Weaver, 1994).These aspects of DNA end-joining and integration are unaffected by the scid mutation. Interestingly, hairpin-ended DNA is also efficiently processed and religated in scid transfection experiments. Lewis (1994) showed that recircularized products of linear hairpin-ended plasmid DNA formed efficiently in scid and wild-type cells. The junctions observed contained P nucleotides of one to five bases consistent with similar structures in V(D)J recombination coding joints. Similarly, hairpin-ended plasmid DNAs integrate efficiently into scid and wild-type cells indicating that the processing of these structures is normal (Staunton and Weaver, 1994). Processing and joining of hairpins bears all the features of linear DNA end-joining in in vitro reactions of Xenopus extracts (Beyert et al., 1994)The scid locus is found on mouse chromosome 16 (Bosma, 1989; Miller et al., 1993). Several laboratories have succeeded in identifying a human chromosome that can complement scid defects when introduced into scid fibroblasts. Microcell-mediated chromosome transfer of individual human chromosomes into scid fibroblasts has shown that human chromosome 8 can restore normal resistance to IR damage (Komatsu et al., 1993a; Kurimasa et al., 1993; Kirchgessner et al., 1993). Since an additional hallmark of the scid mutation is aberrant V(D)J

V(D)J RECOMBINATION AND DOUBLE-STRAND BREAK REPAIR

51

recombination, the complementation ofboth of these defects was examined in another study. Transfer of human chromosome 8 complements both scid defects, showing the location of the human SCID gene (Banga et al., 1994).V(D)J recombination coding joints formed in transient transfection assays with RAG1, RAG2, and a plasmid recombination substrate showed a restoration of the wild-type features of coding junctions. Complemented cell lines that have then lost human chromosome 8 by growth in nonselective media were found to be IR sensitive and have the scidV(D)J recombination defective phenotypes. A concordance between IR resistance and retention of human chromosome 8 fragments from radiation hybrid mapping suggests that scid is located in a centromeric region of chromosome 8 ( 8 p l l . l - q l l . 1 ) (Kurimasa et al., 1993). This region is not particularly syntenic with the scid region of mouse chromosome 16; the human Vpre-B and protamine genes that flank scid in the mouse are located on different human chromosomes from scid. Also, this region of human chromosome 8 is not associated with any known immunodeficiency and DNA repair diseases, although Werner’s premature aging syndrome is located on 8p12 (Schellenberg et al., 1992; Goto et al., 1992). Werner’s syndrome fibroblasts are able to execute V(D)J recombination properly in transient transfection assays with RAG1, RAG2, and plasmid rearrangement substrates for coding or signal junction formation (Z. Wills and D. Weaver, unpublished data). The gene for DNA polymerase-@ is located on human chromosome 8pll-12 (Drayna et al., 1993). @-Polymerasehas been implicated in many DNA repair processes and could play a role in V(D)J recombination, as it might be used in joining steps of the reaction. However, P-polymerase maps to another chromosome in the mouse, so @-polymerasecannot be the scid gene. B. THEV-3 MUTATION Chemical mutagenesis of the Chinese hamster ovary cell line AA84 yielded the IR-sensitive mutation, V-3 (Whitmore et al., 1989; Table I). Like scid, this mutant was deficient in repair ofdouble-strand breaks formed by IR. Somatic cell hybrids between V-3 and scid cells remain IR sensitive suggesting that V-3 and scid are in the same complementation group (Taccioli et al., 1994b). Transient transfection of RAG1 and RAG2 into nonlymphoid cells can be used to assess the efficiency of recombination (Oettinger et al., 1990). As exemplified for scid, this strategy has been extremely useful to monitor whether additional cell culture mutants or human DNA repair syndrome disease cell lines may have errors in V(D)J recombination. The V(D)J recombination potential of V-3 cells was fairly similar to scid in that coding joints,

52

DAVID T. WEAVER

but not RSS joints, are most impacted. V-3 coding junctions are generated at very low efficiency and have a preponderance of P nucleotides in the junctions, reminiscent of w i d DJS rearrangements i n vivo (Table I). The V-3 mutation is likely to be different from the scid mutation because V-3 transient transfection assays most frequently give P nucleotides in recovered junctions, whereas scid coding junctions do not. V3 RSS junctions have a similar modest decrease in the frequency of product formation (60%)and a higher incidence of small RSS junctional deletions, like scid pre-B cells and fibroblasts (Taccioli et al., 1994b; Lieber et al., 1988a; Boubnov and Weaver, unpublished data). The combination of each of these V-3 mutant phenotypes is in striking parallel to scid and is strongly supportive of V-3 being another mutant allele of the scid gene.

C . T H E X R - I MUTATION The Chinese hamster ovary cell mutant XR-I was isolated from chemical mutagenesis of CHO-K12 cells in a screen for radiosensitive mutants (Stamato et al., 1983; Table I ) . XR-I cells are also specifically sensitive to double-strand break damage (Giaccia et aZ., 1985),and the effect of IR damage sensitivity can be mimicked by introduction of restriction enzymes as a double-strand break-specific agent (Giaccia et al., 1990).XR-I is a separate complementation group from other IRsensitive mutants (Jones et aZ., 1988). Somatic cell hybrids with scid, V-3, and xrs are all able to complement radiosensitivity. Since the complementing human chromosome for each of these mutants is now known, clearly these mutants are members of unique complementation groups represented by different genes. V(D)J recombination is defective in XR-I cells having reduced RSS and coding junction formation in the transient transfection assay distinguishing this mutant phenotypically from scid in V(D)J recombination (Taccioli et al., 1993). XR-I coding junctions recovered at low efficiency have larger than normal deletions in the products. XR-I RSS junctions also formed at low frequency, and these products show deletion that is not a normal characteristic ofthese junctions. The radiosensitivity of XR-I is complemented by transfer of human chromosome 5 (Giaccia et al., 1990).XR-l:Ch5 cells also revert the V(D)J recombination deficiency for both RSS and coding junction formation (Taccioli et al., 1993).These experiments distinguish XR-I as a separate mutation from scid, but affecting the same pathways (Table I). Recent experiments have elaborated upon the V(D)J joining defect in XR-I cells with an integrated recombination template that undergoes inversional rearrangement (Li and Alt, 1994). The inversional

V(D)J RECOMBINATION AND DOUBLE-STRAND BREAK REPAIR

53

configuration is useful for analysis of both rearrangement products from the same recombination event. Transfection of XR-I G12 cells with RAG1 and RAG2 activates rearrangement that is observed to be largely aberrant by a Southern blotting analysis. Limited deletion occurs some of the time and can be selected in culture. Analysis of the RSS and coding junctions from the same recombination event illustrated that XR-I fails to form precise RSS junctions, whereas the corresponding coding junctions are relatively normal. These coding junctions have a very high retention of P nucleotides of 1-5 bp in size; the highly unusual feature of P nucleotides originating from both strands is also found. Therefore, the junctional properties distinguish XR-I from other DSB repair mutants. However, like each of these mutants, single mutant alleles may not be representative of the phenotypes of a null mutant, and more similarities or differences may arise when it becomes possible to compare more mutants in the same complementation groups. Immune cell dysfunction cannot be analyzed in XR-I because the mutation was generated in a somatic cell line. Once the XR-I gene is cloned, it will be possible to assess the level of immunodeficiency generated by creating a mouse mutant in the gene by knockout. Since XR-I RSS junctions are most defective, perhaps this protein normally functions in RSS junctions. Currently, one candidate for the XR-I gene product is VDJP, the nonamer recognition protein that appears to be able to join DNA ends containing RSS elements (Halligan 1994). D. THEXRCC5 MUTATION Six independent isolates of another complementation group,

XRCC5, were isolated from CHO cells following chemical mutagenesis and a screen for radiosensitivity (Jeggo and Kemp, 1983; Table I). The xrs mutants are severely sensitive to IR and IR mimetic agents, and this radiosensitivity is DSB specific (reviewed by Jeggo, 1990). Other members of this group have been more recently derived by chemical mutagenesis of Chinese hamster lung cells, V79 (Zdzienicka et al., 1988), or by spontaneous mutagenesis of V79 (Lee et al., 1994; Boubnov et al., 1994b; Table I ) . These recently derived mutants (sxi2 and s x i 3 ) were demonstrated to be in the same complementation group as xrs by somatic cell hybrid formation and examination of complementation of radiosensitivity (Boubnov et al., 1994b). V(D)J recombination is also defective for XRCC5 group mutants, as assayed b y transfection above (Taccioli et al., 1993b; Pergola et al., 1993; Boubnov et al., 1994b). Similar to X R - I , xrs cells are deficient for both RSS and coding junction formation measured separately with

54

DAVID T. WEAVER

different plasmids. xrs-6 V(D)J recombination RSS junctions formed inefficiently, and most products that could be recovered had RSS junctional deletion. xrs-6 coding junctions were not detectable b y this assay. The V79 mutants, sxi2 and sxi3, have severely diminished RSS and coding junction product formation (Boubnov et al., 1994b). However, infrequent products that were recovered did show a significant frequency of normal rearrangement junctions. Therefore, sxi2 and sxi3 may be less penetrant mutants of the xrs complementation group. xrs complementation group cells that are reverted to IR resistance also revert the V(D)J recombination phenotypes to normal (Taccioli et al., 1993; Boubnov et al., 1994b). xrs cells containing a transfered human chromosome 2 complement both phenotypes: IR resistance and normal V(D)J recombination (Jeggo et al., 1992; Taccioli et al., 1993). The defect in both signal and coding junction formation for xrs distinguishes this mutant group from the scid V(D)J recombination defect. XRCC5 group mutant cells have additional phenotypes that may reveal the function of this gene product in DNA repair and V(D)J recombination. xrs group mutants have diminished proficiency of integration of plasmid DNA into chromosomes (Moore et al., 1986; Hamilton and Thacker, 1987). Because this process is widely believed to primarily occur in mammalian cells by an end-joining or nonhomologous recombination pathway, the xrs group may well be generally deficient in this process. Similarly, xrs-6 and XR-I cells are hypersensitive to D N A topoisomerase I1 inhibitors, such as etoposide V-16 (Jeggo et al., 1989; Caldecott et al., 1990), whereas scid and V-3 cells are not (Jeggo et al., 1989; Staunton and Weaver, unpublished data). Etoposide-V16 traps DNA topoisomerase I1 bound to DNA (cleavable complex) that is presumed to be processed into some form of a DNA DSB. Thus, xrs may be more generally involved in a variety of endjoininglrepair reactions than scid.

E. THES x i l MUTATION Two other mutants, sxil and sxi4, were isolated by spontaneous mutagenesis of V79-4 Chinese hamster lung cells (Lee et al., 1994). sxil constitutes a new complementation group for IR sensitivity and DSB repair (Boubnov, et al., 1994c; Table 1). sxil is as sensitive to IR damage as any of the other DSB repair mutants discussed. The sxil cells are also hypersensitive to the crosslinking agent mitomycin C, but not hypersensitive to UV irradiation or alkylating agents (Lee et al., 1994). sxil also appeared to form a-new complementation group for DSB repair because it was complemented by V-3, mid, XRCC5 andXR-1 cells in somatic cell fusions tested for IR sensitivity (Boubnov et al., 1 9 9 4 ~ ) .

V(D)J RECOMBINATION AND DOUBLE-STRAND BREAK REPAIR

55

S x i I was also unable to properly conduct V(D)J recombination analyzed by the same methodology as the above mutants (Boubnov et al., 1 9 9 4 ~ )RSS . junction product formation was reduced by approximately 90-fold in s x i l . Of the low frequency of products, a high percentage are normal junctions. Similarly, coding junction formation is reduced significantly, but a high percentage of the joints are in the normal deletional range. These junctions do not retain any elevated frequency or unusual sizes of P nucleotides. Thus, sxil is phenotypically similar to xrs and X R - I with regard to V(D)J recombination defects and distinctly different from scid. V. The Ku Autoantigen

A. Ku An exciting connection has recently emerged for V( D)J recombination and DNA repair from a surprising direction. Autoimmune patients frequently produce antibodies against proteins from their own cells. Many of these intracellular proteins, identified by the antisera that recognizes them, have yielded valuable insights into cellular processes such as transcription, splicing, and chromatin structure. Antisera against Ku antoantigens were originally described from patients with schleroderma-polymyositis overlap syndrome or systemic lupus erytheniatosus (Mimori et al., 1986).Subsequently, it was shown by immunoprecipitation analysis that two proteins, p70 and p82 kDa, were recognized. These two proteins, p70K"and ~ 8 2 ~form " , a stable heterodimeric complex in mammalian cells. The proteins are nuclear and present in moderately high levels. p70 and p82 coprecipitate as a complex since monoclonal antibodies generated against one or the other subunit will stoichiometrically precipitate the other subunit. Ku has also been purified based on its biochemical properties (see below); the purified protein is a 1:l complex of p70 and p82 kDa subunits. Purified Ku heterodimers have the interesting property of binding to DNA ends irrespective of DNA sequence composition. The doublestranded DNA end-binding property is observed with purified Ku protein or in nuclear extracts (Mimori and Hardin, 1986).Ku does not bind efficiently to single-stranded DNA that is unable to self-anneal or to nicks in DNA. With Ku, addition of circular plasmid DNA in excess does not compete with the KU-DNA complex. Addition of DNA with ends binds to KU and competes for the complex. Purified Ku heterodiniers bind DNA ends with a binding constant of 2.4 x loyM-' (Blier et al., 1993).The 70-kDa subunit has the strongest DNA end-binding capacity (Mimori and Hardin, 1986).Ku binding to DNA

56

DAVID T. WEAVER

ends can also be measured in other assays. An easy detection methodology for protein-DNA complexes has been the gel-shift assay, where radiolabeled DNA fragments are altered in electrophoretic mobility by the binding of protein. Ku-DNA complexes are sufficiently stable that characteristic complexes are observed in gel shifts. Also, using DNasel footprinting of Ku-DNA end complexes, both 5' and 3' ends of double-stranded DNA are protected. This end-binding function of Ku prompted early suggestions that Ku heterodimers might have a DNA repair function. The DNA end-binding property of Ku prompted investigators interested in V(D)J recombination to evaluate whether the Ku proteins may be important to this mechanism. Using the DNA end-binding gel-shift assays, extracts from all of the mutant cell lines of V(D)J recombination discussed above were examined. Several of the XRCC5 complementation group cells are deficient in the major DNA endbinding activity attributable to Ku (Rathmell and Chu, 1994; Getts and Stamato, 1994; Taccioli et al., 1994a; Boubnov et al., 1994b).These mutants include xrs-5, xrs-6, XR-V15B, XR-VSB, sxi2, and sxi3 (Table I). The D N A end-binding activity is competed with linear DNA (DNA with ends), but not with circular DNA, as already demonstrated for purified Ku proteins. Azacytidine-induced revertants of xrs-5, human chromosome 2:xrs-6, or sxi2 spontaneous revertants all restore the IRresistant and V(D)J recombination phenotypes and also restored DNA end-binding activity. Interestingly, the sxil and sxi4 mutants also have reduced end binding (Boubnov et al., 1 9 9 4 ~ )In . contrast, scid, V-3, and XR-1 group cells are not defective for the DNA end-binding property. Mutants that are IR sensitive but not DSB specific, such as EM9 and AT5B1, contain normal end-binding capacity (Rathmell and Chu, 1994; Getts and Stamato, 1994; Boubnov et al., 1994b). Bleomycin-sensitive CHO mutants, BL-10 and BL-14, are also normal for this activity. The protein-DNA complexes that are disrupted in the XRCC5 and sxil complementation group mutants were shown to contain Ku normally. The end-binding Ku-DNA complexes are recognized and supershifted by addition of anti-p70K"or a n t i - ~ 8 2antibodies ~~ in gel-shift assays (Boubnov et al., 1994b). Also, immunodepletion of cross-reacting Ku antisera shows that the DNA end-binding complex contains KU (Taccioli et al., 1994a). Anti-p70K"antibody increases the mobility of the major KLI-DNA complex. Higher-order Ku-DNA complexes show extensive changes in mobility. Some a n t i - ~ 8 2 ~ antibodies " supershift the major end binding complex. Other a n t i - ~ 8 2antibodies ~" do not alter the mobility of the major KU-DNA complex, but d o supershift higher-order complexes. An antibody recognizing a shared epitope

V(D)J RECOMBINATION AND DOUBLE-STRAND BREAK REPAIR

57

between p70K" and ~ 8 2 also ~ " shifts the major Ku complex. These criteria show that Ku is responsible for the end-binding activity detected in normal cells. The primary DNA end-binding complex is formed by a Ku heterodimer on DNA. Therefore, an intriguing possibility is that the XRCC5 and sxil complementation groups are mutations in K u subunits. The genes for both subunits of Ku have been cloned (Yaneva et al., 1989; Mimori et uZ., 1990; Reeves and Sthoeger, 1989; Falzon and Kuff, 1992), making it possible to determine whether these DSB repair/ V(D)J recombination-deficient cell lines may be attributable to niutations in Ku. Several experiments argue strongly that the XRCC5 group mutants are defective in ~ 8 2 ~The " . xrs complementation group and p82"" map to the same region of human chromosome 2: 2q33-35 (Ku; Cai et al., 1994) or 2q34-37 (xrs and XR-Vl5B; Jeggo et al., 1993; " expression Hafezparast et al., 1993).Transfection of human ~ 8 2 in~ an plasmid complements the IR sensitivity and V(D)J recombination defects ofsxi2, sxi3, or xrs-6 cells (Taccioli et al., 1994a; Boubnov et al., 19941)). Also, DNA end binding is restored by p82"" in sxi-3 cells. Fui-thermore, radiation hybrids of human chromosome 2 with xrs cells that are IR resistant and V(D)J recombination proficient also have the appropriate region of human chromosome 2 by PCR analysis with primers at particular associated regions. The XRCCS mutations in ~ 8 2 are ~ "not yet fully described, although it is likely that the existing population of mutants contains different types because their repair phenotypes are variable for xrs mutants. Northern blots with a ~ 8 2 ~ " probe indicated that xrs-6 cells produce p82 mRNA indicating that subtle mutations, such as point mutations, are probably the cause (Boubnov and Weaver, unpublished data). Sxi2 and sxi3 d o not have detectable p82"" mRNA (Boubnov et a l . , 199411). p70K"mRNA is also expressed normally in these mutants. There is likely to be some Ku activity in these latter mutants because V( D)J recombination assays on these cells are not identical between xrs-6 and sxi2,sxi3 as discussed above (Boubnov et al., 1994b). N o information is available yet on the human chromosome able to complement the sxil IR sensitivity and V(D)J recombination defects. A likely candidate is human chroinosome 22, where p70"" is located (Cai et al., 1994). sxil may be a mutant in p70"" because it is defective in the DNA end-binding complex associated with KU heterodimers, and because sxil did complement xrs group cells in somatic cell hybrid tests (Boubnov et ul., 1994b,c). Sxil IR', V(D)J recombination, and DNA end-binding is reconstituted by the addition of p70K"(Boubnov et nl., 1 9 9 4 ~ ) .

58

DAVID T. WEAVER

B. How MIGHT Ku STIMULATE V(D)JRECOMBINATION AND DNA REPAIR? The property of DNA end binding of Ku is likely to be highly significant for V(D)Jrecombination. In the joining steps of the reaction, Ku monomers could be associated with each coding and/or RSS ends. This property of Ku may be a means to synapse DNA ends prior to ligation by holding the DNA strands together via protein-protein interactions (Fig. 3). A parallel function may be expected for fragmented or damaged DNA in DSB repair. A.

V(D)J

recornblnatlon

B.

Double strand break repair DNA

J Darnage -~ L--KU

KU

KU KU

+

/ \

end-jolnlng

ceIl cycle arrest

1

end-joining

1

RSS joint

FIG. 3. Similarities between V(D)J recombination product formation and DNA damage-induced double-strand break repair. (A) V(D)J recombination. A putative intermediate structure is shown for V(D)J rearrangement in which a synaptic complex of the four DNA ends in the reaction are sequestered. Coding ends are drawn as hairpins, RSS ends are drawn with terminal RSS (triangles). Protein-protein associations and ~ " ~ 8 2 hetero~ " protein-DNA contacts are shown by shaded boxes. Ku proteins ( ~ 7 2and dimer) are illustrated as joined kiangles associated with double-stranded DNA. Th e property of Ku translocation along DNA and binding to DNA ends and hairpins is discussed in the text. (B) Double-strand break repair. DNA damage, such as IR, creating a DSB in the chromosome is depicted. Ku heterodimers relocated to DNA ends for DNA repair. Two processes are induced: end joining and cell cycle arrest. A DNAdependent protein kinase (DNA-PK) associates with Ku and may b e significant for these processes.

V(D)J RECOMBINATION A N D DOUBLE-STRAND BREAK REPAIR

59

A fascinating biochemical property of Ku is its binding to transitions between double-stranded and single-stranded DNA as effectively as it binds to ends of double-stranded DNA (Falzon et al., 1993). Hairpinended DNA is an efficient substrate and competitor for K u binding (Paillard and Strauss, 1991; Falzon et al., 1993). Hairpins with loop sizes between 4 and 20 bp bound Ku as efficiently as oligonucleotides containing douhle-strand DNA ends (Falzon et al., 1993). Thus, if hairpin coding ends are intermediates for V(D)J recombination, a logical model would be that Ku monomers complex to each hairpin via protein-DNA contacts at the transition between single- and doublestranded DNA at the hairpin base (Fig. 3). Two important functions may be envisioned here. On the one hand, binding at this position may be sufficiently internal to the coding end termini that hairpin resolution and processing by other factors is not interfered with in the presence of KU binding. Second, Ku-Ku associations may b e formed between the two coding ends, allowing continued synapsis and completion of the coding junction rearrangement. Ku may also associate with RSS ends because xrs mutants also affect RSS junction formation. There is an interesting DNA sequence dependence for Ku that has been shown by measurement of DNA end binding in gel-shift assays (Rathmell and Chu, 1994). Similarly, V(D)J recombination is strongly influenced by the DNA sequence composition of coding end DNA (Gerstein and Lieber, 1993; Boubnov et al., 1994a). In particular, stretches of A or T of greater than five nucleotides flanking either one or both RSS inhibitV(D)J recombination by 100-fold or more (Boubnov et al., l994a). A/T-rich DNA is not an efficient competitor for Ku end binding. A/T-rich ends may either be too denatured or contain changes in DNA structure so that KU binding is too far removed from the termini to be effective in facilitating joining. Although these findings can explain the dependence of coding junction formation on DNA sequence composition, they do not explain the reduced efficiency of RSS product formation where there have been no compositional changes. However, if the formation of both products is concerted, then the effects of DNA binding to some of the ends could alter the efficiency of joining of all the products. KU may have a similar ability to recognize and bind to fragmented DNA from IR damage (Fig. 3B). IR-fragmented DNA creates DSBs by two independent ionization events. When these strand breaks occur nearby (<30 bp), broken DNA with overhanging 5' and/or 3' single strands is formed. IR breaks in DNA in oitro frequently contain chemically altered bases, base loss, and other modified DNA structures (Kapp and Smith, 1970; Henner et al., 1983). There is currently no indication that hairpin-ended DNA forms following I R scission of DNA ( J . Staun-

60

DAVID T. WEAVER

ton and D. Weaver, unpublished data). If Ku binds to the doublestranded ends of broken chromosomes then a similar processing region between the site of binding and the single-stranded ends would be expected. Thus, Ku-mediated repair of chromosome damage may yield recombinant joining products by nonhomologous recombination pathways that have small deletions and/or filling-in. More experiments will be required to determine whether the proficiency of Ku binding to hairpin and/or transitions in DNA structure places a limitation on the processing capacity in these V(D)J recombination-directed or damageinduced end-joining steps. Another property of Ku is translocation along DNA. In uitro Ku evidently binds to DNA ends and moves along DS DNA (deVries et aZ., 1989; Paillard and Strauss, 1991; Zhang and Yaneva, 1992). There is also evidence from footprinting analysis that Ku does associate with double-stranded DNA (Gottlieb and Jackson, 1993). An interesting implication of these results would be that Ku can translocate along DNA in uivo to sites of recombination synapsis or DNA damage (Fig. 3). This property could explain how Ku may only be involved in late or joining stages of V(D)J recombination. The rules of Ku involvement for V(D)J recombination product joining may now be better explored with the molecular genetics of the Ku gene products.

C. DNA-DEPENDENT PROTEINKINASE(DNA-PK) Ku also forms a stable complex with a high molecular weight protein kinase [(Gottlieb and Jackson, 1993); reviewed by Anderson, 19931. A novel feature of this serine/threonine protein kinase is that it is activated by binding to DNA, particularly DNA ends (Walker et uZ., 1985). The DNA-PK was purified from HeLa cells and consists of a single polypeptide of approximately 350 kDa (Carter et al., 1990; LeesMiller et al., 1990). The consensus serine/threonine phosphorylation site is simple, SQ or TQ (Less-Miller et al., 1992). DNA-PK is localized to the nucleus, but it is unclear whether its cellular localization is entirely dictated by Ku. The features of DNA-PK thus far observed are consistent with a dual function in cell metabolism: transcription regulation and DNA repair. DNA-PK activates transcription factors by phosphorylation. DNA-PK has been shown to phosphorylate the transcription factors SP1, c-fos, C-Myc,Octl, c-Jun, TFIID, CTF/NFI, and RNA polymerase I1 [see Anderson, 1993 and references within]. Although each of these proteins are phosphorylated in vitro, it is not yet clear what the true substrates of DNA-PK will b e in uiuo. Transitions between DS and

V(D)J RECOMBINATION A N D DOUBLE-STRAND BREAK REPAIR

61

SS DNA binding are presumably found at the region of transcription factor binding in eukaryotic promoters. The model is that DNA conformational changes, possibly caused by transcription factor binding, may be recognized by Ku. The activation of transcription initiation would then be facilitated by localized phosphorylation of transcription factor substrates by DNA-PK. Actually, Ku-DNA-PK has been purified as a stimulatory transcription factor by several groups (Jackson et al., 1990; Falzon et al., 1993; Dvir et al., 1993). Activation of transcription factor function has been clearly documented for S p l (Jackson et al., 1990; Gottlieb and Jackson, 1993)and stimulation of transcription intitiation by RNA polymerase I1 in vitro (Dvir et al., 1993). The tethering of a DNA-dependent protein kinase via Ku heterodimers is likely to have exciting ramifications for V(D)J recombination and DNA repair. Several of the substrates of DNA-PK have been implicated as key players in DNA damage responses and V(D)J recombination. DNA-PK has been shown to phosphorylate SV40 T antigen, p53, Ku subunits, RPA, and topoisoinerases I and I1 (see Anderson, 1993). The tumor suppressor protein, p53, that is part of the G1-S phase DNA damage-induced cell-cycle checkpoint is phosphorylated by DNA-PK at SQ sites (Less-Miller et al., 1990,1992). RPA, which has also been implicated in DNA repair and pathways (Coverley et al., 1991; Liu and Weaver, 1993), is phosphorylated by DNA-PK (Anderson, 1993). Thus, it has been proposed that DNA-PK is an immediate mediator of DNA damage responses by binding to fragmented DNA ends and signaling cell cycle arrest by a p53-dependent pathway (Anderson, 1993). Thus, because of the DNA end-targeting properties of Ku, DNA-PK is likely to be locally available to DNA damage sites. DNA-PK:Ku complexes are anticipated to be immediate responders to damage to DNA because of its availability on the chromosomes. A Ku-associated DNA-PK may be used in V(D)J recombination. A function of Ku-DNA-PK in parallel to DNA damage repair would be in the completion of product formation and end-joining of this pathway. Phosphorylation of Ku may be significant for its V(D)J recombination activity. The DNA-PK is already known to be stoichiometrically associated with a Ku heterodimer (Gottlieb and Jackson, 1993), and p70K" and p82"" are both phosphorylated by DNA-PK in autophosphorylation assays in vitru (Anderson, 1993). DNA-PK is associated with Ku in the DNA end-binding assay because anti-DNA-PK antibodies are able to alter the mobility of some of the Ku:DNA complexes from crude extracts where DNA-PK activity is present (N. Boubnov, V. Liu, and D. Weaver, unpublished data). Investigation is currently under way

62

DAVID T. WEAVER

to evaluate whether phosphorylation of Ku is a requirement for the DNA end-binding activity. An activated DNA-PK may phosphorylate proteins relevant to V(D)J recombination to direct the reaction toward completion of products or to maximize reaction efficiency. Candidate substrates are RAG 1 and RAG2 proteins. RAG2 is phosphorylated at several serine and threonine residues in a cell-cycle-dependent manner (Lin and Desiderio, 1993;Lin and Desiderio, 1994). Ser375 of RAG2 fits a DNA-PK consensus site; however, mutation of this residue has no effect on V(D)J recombination capacity in the transient transfection assay (Lin and Desiderio, 1993). However, phosphorylation of T490 in the COOHterminus of RAG2 regulates its protein stability; a T490A mutation in this phosphorylation site increased RAG2 stability significantly. This phosphorylation site fits the consensus for ~ 3 4 ‘ ~protein ‘~ kinase, as does a related phosphorylation site regulating p53 stabilization (Lin and Desiderio, 1993). Phosphorylation at other residues may also be a means to regulate RAG2 stability, and therefore activity. Likewise, other phosphorylations may activate or inactivate the protein in recombination complexes. If there is a separation between initiation and cleavage steps of the pathway, then Ku binding to hairpin ends may bring DNA-PK into the vicinity of RAG proteins to be inactivated. Other DNA-PK protein substrates could include other proteins in the complex that may regulate the efficiency of recombinational synapses or the completion of products. Finally, it will be interesting to find out whether DNA-PK itself is directly involved in V(D)J recombination. Mutations in DNA-PK may be expected to be defective in gene rearrangement much like Ku mutations if the DNA-PK:Ku complexes are relevant to the pathway. Once the DNA-PK gene is cloned, a reverse genetic analysis will be informative in deciphering the role of this fascinating protein kinase in DSB repair and recombination. VI. Human Immunodeficiency and DNA Repair Syndromes

A. IMMUNODEFICIENCY SYNDROMES WITH V(D)J RECOMBINATIONDEFECTS Severe combined immunodeficiency syndromes in the human population are rare, sometimes exemplified by single patients. Yet, analogous phenotypes with the V(D)J recombination and DNA repair defects found in rodents are anticipated. Severe immunodeficiency diseases in humans are also referred to as “scid” but are not generally synonomous with the murine scid mutant. Approximately one-half of

V(D)J RECOMBINATION A N D DOUBLE-STRAND BREAK REPAIR

63

human scid syndromes are adenosine deaminase deficiencies which are correctable by gene therapy. Only 20% or less of the rare human scid syndromes are classified as T-B- disease (Stephan et nl., 1993). No human scid syndromes have yet been identified that do parallel the scid mouse immunodeficiency phenotypes. T-B- scid could be caused by V(D)J recombination defects because of the common recombinase mechanism between the lineages. Scid diseases that are T’B-, X-linked SCID, or T-Bf are also observed, but most likely are not primarily caused by basal VDJ recombination defects. These diseases more likely reflect differentiation errors that are lineage restricted. Aberrant Ig gene rearrangements have been observed from bone marrow pre-B cells of B- patients, which is consistent with the phenotypic changes for murine scid (Schwartz et al., 1991; Ichikara et al., 1988) (Table 11). Using a PCR assay for Ig DH to JH rearrangement, an assessment of recombination level and product junctions was evaluated in these patients (Schwartz et nl., 1991). A high percentage of the rearrangements observed had aberrantly extended deletions that destroyed coding sequence. Several of these patients were also lacking T cells, in parallel to murine scid, even though TCR rearrangements were not evaluated. Other T-B- patients were examined for IR sensitivity of the granulocyte-macrophage colony-forming unit (GM-CFU)population. In two patients, GM-CFU cells were radiosensitive (Cavazzana-Calvo et al., 1993), another feature of murine scid-type disease. Omenn’s syndrome patients have elevated T cell numbers and hypereosinophilia. Interestingly, T cells from Omenn’s patients are oligoclonal for either cup or y8 TCR rearrangements (Wirt et uZ., 1989; De Saint-Bade et al., 1991). A comparison has been made between this disease and the leaky phenotype of the murine scid mutation (Cavazzana-Calvo et at., 1993).GM-CFU from Omenn’s patients also shows increased radiosensitivity (Cavazzana-Calvo et nZ., 1993). This IR hypersensitivity is comparable to murine scid GM-CFU IR sensitivity (Fulop and Phillips, 1990) and it has been suggested that Omenn’s syndrome behaves as a leaky murine scid-type defect. Because T and B cell rearrangements from Omenn’s can occur as normal, the defect of these cells is not likely to be in the direct mechanism of V(D)J recombination. Cells from Omenn’s syndrome patients have not yet been examined for V(D)J recombination by transient transfection assays as for other mutants. Normal patients show low frequencies ofaberrant Igor TCR recombination detectable by several assays of primary B or T cells (Tycko and

TABLE I1 HUMANDNA REPAIRAND RECOMBINATION SYNDROMES Human Disease Syndrome

Precancer Syndrome

Immunodeficiency

V(D)J Cell Culture

V(D)J Primary BIT wt Ig and TCR transloc. Aberrant Ig D-Jh TCR restricted

Li-Fraumeni Ataxia telangiectasia B-T+

Yes Yes

wt

wt

low Bllow T

wt

?

no B

?

Omenn

?

low Bllow T

Bloom

Yes

low Bllow T

wt

46BR

Yes

low Bllow T

wt

Fanconi's anemia Xeroderma pigmentosa

Yes

wt

wt

wt

Yes

wt

wt

wt

DNA Repair Defect

Mitotic Recombination H yper-rec Hyper-rec

IR' IR'

Gene P53

Gene Location 17~13.1 11~22-23

?

I R' Hyper-rec and elevated SCE H yper-rec (S. cereoisine cdc9)

15

wt

Broad spectrum Broad spectrum MMC'

FACC

uv-induced hyper-rec

uv'

ERCC

DNA Ligase I

19q13.2-3

V(D)J RECOMBINATION AND DOUBLE-STRAND BREAK REPAIR

65

Sklar, 1990). A leading theory for the origins of lymphocytic leukemias is that errors in V(D)J recombination may be aberrantly processed into chromosomal translocations that either predispose or directly facilitate malignancy. Chromosomal translocations with V(D)J recombination site breakpoints are frequently found in lymphoid malignancies, where the translocation alters the expression of genes such as c-myc or bcl2 (reviewed extensively by Korsmeyer, 1992). The same Bcl-2; Ig Jh translocations also are detectable from benign tonsilar tissue of normal individuals (Limpens et al., 1991).Thus, errors in V(D)J recombination that occur spontaneously may facilitate entry into tumor progression pathways. The accurate formation of V( D)J products has been examined in transient transfection assays of EBV-immortalized pre-B cell lymphoma lines and fibroblasts (Gauss and Lieber, 1993; Hsieh et al., 1993; Petrini et al., 1994). In one study, evidence was presented for a bias against coding junction product formation (Gauss and Lieber, 1993), although these data are too preliminary to draw any mechanistic conclusions. Perhaps human progenitor lymphoid cells undergoing V(D)J recombination allow more unrepaired DSBs as by-products of the joining reaction. These ends may serve as the precursors for chromosomal translocations without proper cell signaling of the error.

B. BLOOMSYNDROME AND DNA LICASEDEFICIENCY Candidate proteins for DNA repair and V(D)J recombination deficiency may be found from known proteins with well-characterized properties. Bloom syndrome patients are immunodeficient, have numerous chromosome abnormalities, and have accelerated risk to cancer. Molecular phenotypes of Bloom syndrome (BS) cells have indicated that DNA ligase I activity is reduced and thermolabile (Table 11). Also, BS cells have an inordinately high frequency of sister chromatid exchange and broad sensitivity to a variety of DNA damaging agents. As such, it was anticipated that a DNA ligase activity may also be required for completion of V(D)J recombination and DSB repair, and that defects in these processes niay occur when DNA ligase is mutated. Similarly, a patient with characterized D N A ligase mutations and <5% of the normal DNA ligase I activity (46BR) (Webster et al., 1992) was shown to be immunodeficient, have DNA replication and repair defects, and have a cancer prognosis (Barnes et al., 1992). Both BS and 46BR cells are normal for V(D)J recombination as measured with transient transfections of RAG 1, RAG2, and plasmid rearrangement substrates (Petrini et al., 1994). Therefore, abundant DNA ligase I activity is not required for V(D)J recombination. DNA ligase I is an essential gene for DNA metabolism; gene targeted insertion mutagene-

66

DAVID T. WEAVER

sis of the murine DNA ligase I in mouse ES cells can only be made homozygous for the knockout allele by expression of DNA ligase I ecotypically ( J. Petrini and D. Weaver, unpublished). VII. Immunodeficiency/DNA Repair Syndromes Affecting Cell Cycle Checkpoint Mechanisms

Several human immunodeficiency syndromes have reduced B and T cell populations, symptomatic DNA repair, and recombination alterations, but do not specifically lack accurate V(D)J recombination. These syndromes are variable immunodeficiencies in which the penetrance of the syndromes is not severe. Yet, the molecular basis of these deficiencies is expected to shed new light on control mechanisms of DNA repair and recombination processes with regard to cell-cycle controls. Changes in the regulation of these processes may underly the disregulation in the lymphoid populations leading to increased risk of malignancies.

A.

P53-DEPENDENT

PATHWAYS

The growth and division of eukaryotic cells are highly regulated by diverse growth control mechanisms of positive and negative influence throughout the cell cycle. Cell cycle arrests occur in response to ionizing radiation and other forms of DNA damage (reviewed by Hartwell and Weinert, 1989; Hartwell, 1992). A cardinal feature of these adaptive responses is that they are transitory; cells signaled to arrest by DNA damage reenter the cell cycle following an arrest phase. As such, these checkpoint mechanisms are a response-inducible system in which the genome can be surveyed and repaired for damage that would otherwise be catastrophic prior to important cell cycle events such as DNA synthesis and mitosis. The lack of detection of cell cycle progression signals has gained prominence as a factor in promoting tumorigenesis through new findings with a p53 pathway. The tumor suppressor gene, p53, is the most commonly mutated gene in human neoplasia (Hollstein et al., 1991). Patients that inherit a mutant p53 allele from one parent (Li-Fraumeni syndrome) are susceptible to high rates of tumor formation, particularly lymphoreticular cancers (Malkin et al., 1990; Srivasta et al., 1990; Vogelstein, 1990; Lane and Benchimol, 1990).Although Li-Fraumeni patients are not overtly immunodeficient, the wild-type copy of p53 may be sufficient for checkpoint regulation under most conditions. Immune cell metabolism may be notably vulnerable to the absence of p53 because of the high degree of DNA metabolism in these cells:

V(D)J KECOMBINATION AND DOUBLE-STRAND BREAK REPAIR

67

V(D)J recombination, Ig isotype class switching, Ig VDJ somatic mutation, and D N A replication that must be completed accurately for the expansion, differentiation, and function of lymphocytes. The molecular functions of p53 have been a subject of intense scrutiny. p53 is not required for cell division because knockout mutations of p53 in the germline are not cell lethal and do yield homozygous p53-/- animals as normal. p53-/- mice, however, have a high frequency of spontaneous tumor formation, a property in coninion with Li-Fraumeni patients (Li and Fraumeni, 1969; Li e t ul., 1989; Donehower et al., 1992).p53-l- mice are also susceptible to the onset of lymphoreticular tumors. The presence of wild-type p53 is correlated with the ability of cells to induce a G1-S phase arrest (Mercer et al., 1990; Baker et al., 1990; Diller e t al., 1990; Ginsberg e t al., 1991). Wild-type p53 levels increase in response to DNA damage (for review see Hartwell, 1992; Maltznian and Czyzyk, 1984; Kastan et al., 1991,1992; Kuerbitz et al., 1992). Mutated p53 or an inability to accumulate wild-type p53 both override this arrest mechanism (Kern et al., 1992; Kuerbitz et al., 1992; Dulic et al., 1994). Wild-type p53 is an activator of transcription (Kern e t al., 1991; Funk et d., 1992; Farmer e t d., 1992), and several genes that are negative regulators of growth have been identified that are p53responsive ( p21WAF1.C'P, CADD45, MDMB) (Xiong e t al., 1993; ElDeiry et al., 1993; Harper e t al., 1993; Momand et al., 1992; Kastan et aZ., 1992). Mutated p53 is unable to activate the transcription of downstream effector genes that may either complete the cell cycle arrest and/or allow the DNA repair mechanisms to complete the business of repair prior to reentry into the G l / S transition. Thus, p53 is a key player in the signal transduction mechanism of the G1 DNA damage-induced checkpoint pathway. This signaling pathway must be relevant to regulatory events in the cell cycle that may impact DNA damage repair and V( D)J recombination. B. ATAXIATELANGIECTASIA Ataxia telangiectasia (AT) is an immunodeficiency disease transmitted as an autosomal recessive mutation, where patients are variably immunodeficient, contain increased chromosome instability, and are highly susceptible to cancer (reviewed by Gatti et al., 1991; Gatti, 1993).As many as four separate AT complementation groups are found. The immunodeficiency of these patients affects both T and B cell populations. Ig profiles are most frequently deficient in IgA for AT, although other isotypes are also deficient in other A T patients. A T T cells respond poorly to PHA stimulation in culture. In the spectrum of

68

DAVID T. WEAVER

cancers for which AT patients are at great risk, lymphoid malignancies predominate. Several investigators have examined V(D)J recombination products in A T patient primary lymphocytes or in immortalized AT cell lines in culture. The basal V(D)J recombination mechanism is apparently normal, as assessed by transient transfection of AT cell lines with the V(D)J recombination activating genes RAG1, RAG2, and plasmid substrates (Hsieh et al., 1993; Pergola et al., 1993); N. Boubnov and D. Weaver, unpublished data). AT cells have normal DNA end-binding capacity (Rathmell and Chu, 1994; N. Boubnov and D. Weaver, unpublished data) which is the only other biochemical readout associated with appropriate V(D)J recombination that can currently be monitored. However, AT cells have highly elevated levels of illegitimate interlocus TCR rearrangements between TCRyV regions and TCR JP relative to normal individuals even though other Vy-Jy rearrangements are as normal (Lipkowitz et al., 1990). A clue to the connection between the AT defects and the V(D)J recombination pathway is found with AT lymphocytes where a very high frequency of Ig/TCR chromosomal translocations are observed (Hecht and Hecht, 1987; Rabbitts, 1993). I n particular, translocations and inversions of chromosome 7 and 14 at the sites of Ig and TCR gene rearrangement are found very frequently. A T patients that progress to T cell prolymphocytic leukemia and chronic lymphocytic leukemia often have a characteristic chromosome 14 rearrangement or inversion that first appears as a dominant T cell clone that is premalignant (Hollis et al., 1987; Heppell et al., 1988). These translocations exist prior to the malignant phenotype. Molecular mapping and characterization of these translocation and inversion breakpoints has revealed that they are complex and likely involve sequential rearrangements (Baer et al., 1987; Davey et al., 1988; Russo et al., 1988,1989; Stern et al., 1989). At least some of the translocation breakpoints occur at or near RSS (Russo et al., 1989; Davey et al., 1988; Mengle-Gaw et al., 1988). There is no compelling argument that these chromosomal rearrangements are catalyzed by V(D)J recombination defects per se. Another interesting fact is that these same chromosomal rearrangements are observed in normal individuals at 100-fold or lower frequency. The lymphoproliferative features of AT T cells may artificially augment differences between AT and normal patients. Yet, continual appearance of these aberrant rearrangement-associated translocations in A T patients are suggestive that the AT defects may not sense a chromosomal broken end once it occurs. A T cells are sensitive to ionizing radiation (Taylor et al., 1975). The

V(D)J RECOMBINATION A N D DOUBLE-STRAND BREAK REPAIR

69

IR sensitivity of AT cells is due to the DSBs, as is true for the murine scid mutation and other complementation groups (Table I). A T cells are also sensitive to X-ray mimetic agents, such as bleomycin and streptonigrin, and are sensitive to topoisomerase inhibitors such as etoposide V16. However, unlike these other DSB repair mutant complementation groups, AT cells are capable of repairing DSBs in shortterm assays in which scid, xrs, and XR-I are shown to be DSB repair deficient. These experiments led to the conclusion that AT cells may have the appropriate machinery for DSB repair, but that this mechanism is not properly activated or signaled. AT cells do not appear to recognize cell cycle DNA damage-induced checkpoints at the G1-S transition or in S or G2 phases (Painter and Young, 1980; Kastan et al., 1992). The A T lack of recognition is manifested as entry into the next cell-cycle phase before the DNA damage has been adequately repaired, leading to cell death in subsequent cell cycles. G1 phase AT cells go directly into S phase disregarding damage by IR (Dulic et al., 1994). In S phase AT cells treated with IR, DNA replication continues, termed radioresistant DNA synthesis (RRDS), a process that presumably irreversibly fixes DNA damage and/or chromosome instability (Painter and Young, 1972,1980). Thus, A T cells have several cardinal features indicating that AT complementation groups are checkpoint mutants. Patients have also been identified with some, but not all, of the clinical features of AT. Early studies with Nijmegen Breakage Syndrome (NjBS) patients showed similar immune system impairment as AT and a higher incidence of chromosome 7 and 14 translocations possibly at Ig and TCR rearrangement sites (Jaspers et al., 1987).The patient had no evidence of oculocutaneous telangiectasia or cerebellar ataxia, but did have niicrocephaly and growth delay that is not ordinarily characteristic of AT. NjBS fibroblasts are 1R sensitive and show RRDS, both features of AT. NJBS cells are able to complement A T cells for DNA repair defects which shows that NjBS represents distinct complementation groups. Because of the high phenotypic similarities, perhaps NJBS and AT are both involved in the same signaling pathway for DNA damage recognition. The AT checkpoint defect has significant ramifications for the immune system composed of rapidly proliferating cells undergoing DNA rearrangements. This property of several immune deficiency syndromes may also explain the variable nature of the immunodeficiencies in patients, the predelection of these cell populations to malignancy, and an inability to respond as part of the immune defense mechanism.

70

D A V I D T. WEAVER

VIII. Cell Cycle Regulation of V(D)J Recombination and DSB Repair

A. V(D)J RECOMBINATION AND THE CELLCYCLE V(D)J recombination may preferentially occur in G1 phase of the cell cycle. Recent experiments with antibodies against murine RAG2 indicate that RAG2 protein is 20-fold more abundant in G 1 cell cycle phase than in S, G2, or M for either pre-B cells or thymocytes (Lin and Desiderio, 1994). Both cell sources are active for V(D)J recombination. RAG2 protein is actively degraded by a mechanism similar to p53 inactivation, and p53 and RAG2 may be influenced by the same degradation pathway. The implications of these experiments are that V(D)J recombination probably occurs in G1 phase of the cell cycle. Future experiments will have to sort out whether the increased RAG2 protein levels in G1 are consistent with elevated recombinase activity in the cell. Studies with the transient transfection assay with plasmid gene rearrangement substrates in cell culture have illustrated that V(D)J recombination is not dependent on DNA replication. Nonreplicating plasmids, when transfected into RAGl/RAG2+ cells, are as efficient at V(D)J recombination as replicating plasmids (Hsieh, 1991; Petrini et al., 1994). Activation of recombination by heat-shock induction of RAGl and RAG2 expression vectors in murine B cells (Oltz et al., 1993) has also been used to show that plasmid V(D)J recombination occurs prior to DNA replication (N. Boubnov and D. Weaver, unpublished data). These experiments are consistent with a G 1 cell cycle window for the initiation and completion of V(D)J recombination. It seems logical that V(D)J recombination events would be completed in the same cell cycle stage as they were initiated because DNA replication through broken DNA at V(D)J recombination sites may well be mutagenic or lethal (Fig. 4). Broken DNA ends have been implicated as an intermediate in V(D)J recombination based on the correlation oftheir appearance with RAGl and RAG2 activation (Schlissel et al., 1993; Roth et al., 1993). Interestingly, Ig Jh RSS ends are observed in cells that are in GO/Gl of the cell cycle, and not S or G2 (Schlissel et al., 1993), which is consistent with RAG2 protein abundance. Also, Jh RSS ends are observed in cells from scid bone marrow at wild-type levels, which is consistent with the notion that scid V(D)J recombination events initiate properly but cannot form coding junction products. Coding ends are not detectable by this assay from wild-type cells (Schlissel et al., 1993).

V(D)J RECOMBINATION AND DOUBLE-STRAND BREAK REPAIR

S

G1

w t

--

71

J

V

arrest

--x scid adaplatlon

deletlons and translocations

FIG.4. Cell-cyle checkpoint mechanism and cell mutant errors in V(D)J recomhination. Th e appearance of V(D)J recombination intermediates in G1 phase of the cell cycle is diagrammed. Wt, wild-type cells that produce broken DNA at low levels in the V(D)J rearrangement pathway. Errors of this type presumably cause a cell cycle arrest (X) in parallel to IR chromosome damage. Scid,the murine scid mutation may accumulate hairpin intermediates at V(D)J recombination coding ends. Hairpins either generate a cell cycle arrest or by adaptation, progress to the next cell cycle phase, S ( D N A synthesis). DNA replication forks in the vicinity of hairpin-ended DNA are shown. xrs, AT, mutations in the xrs complementation group or in ataxia telangiectasia (AT) may be unable to recognize broken DNA resulting from V(D)Jrecombination. Either mutant allows passage to S phase.

B. V(D)J RECOMBINATION/DSB REPAIRMUTANTSAND CELLCYCLEARREST Scid cells are highly sensitive to IR damage, but these cells appear to recognize cell cycle IR-induced checkpoints as normal. In S phase scid fibroblasts arrest DNA synthesis as do wild-type cells (Komatsu et al., 1993). V-3 cells, in the same complementation group as scid, have been examined for the cell cycle position of their radiosensitivity (Whitmore et al., 1989). V-3 cells are sensitive to IR in GI but not in S/G2. In contrast, rrs cells appear to vary in the level of sensitivity to IR damage with regard to checkpoint control in S phase. rrs-1,2,4,6, and -7 mutants have a prolonged inhibition of DNA synthesis following IR damage in S phase (Jeggo, 1985). The rrs-5 mutant is indistinguishable from the parental CHOKl cells with regard to RRDS. xrs-5 and other xrs mutants are similar in other DNA repair and recombination

72

DAVID T. WEAVER

phenotypes. A prolonged inhibition of DNA synthesis is the opposite phenotype to that which is observed in cells from AT and NjBS patients. Athough cell cycle arrest points have not specifically been examined for the XR-I mutation, XR-I cells are extremely sensitive to IR damage in the G1 cell cycle phase (Giaccia et d., 1990).G1 phase of the cell cycle has greater than a 10-fold hypersensitivity to IR as opposed to late S phase. It is not yet clear whether or not XR-I cells will bypass a DNA damage checkpoint. Synchronized cells at these two stages also differ significantly in ability to repair y-ray-induced DSBs as measured by a short-term neutral filter elution assay (Giaccia et d.,1990). Late S phase cells, that may be indistinguishable from G2 by these assays, have the same kinetics of DSB repair as the parental cell controls. xrs mutants also examined for DSB repair in short-term assays are indistinguishable from the controls (Kemp et al., 1984).Thus, there may be a distinction between the machinery for DSB repair at different cell cycle times. In S and G2, a likely mechanism would be recombinational repair (from sister chromatids). In G1, a sister chromatid is not present, and the efficiency of utilization of the chromosome homolog may not be high enough to allow efficient recombinational repair. Under G1 “conditions” DSB repair by a nonhomologous recombination or end-joining process may be prominent. The G1-radiosensitive phenotype of XR-I and scid may be indicative of the enzymology required for this process. Two of the xrs mutants that show prolonged S phase arrest also have been examined for other checkpoints by FACS analysis of asynchronous populations. xrs-2 and xrs-4 show a pronounced G2 arrest that may be irreversible under the conditions of the assay (Weibezahn et al., 1985). The G2 arrest of xrs mutants may point to defects in the recognition properties of the DNA damage checkpoint. The absence of ~ 8 2 would ~ ” not be expected to allow DNA-PK to associate with DNA ends. Barring tethering of DNA-PK association, phosphorylation of yet unidentified DNA-PK substrates in G2 may prevent processing of the DNA damage signaI to arrest cell-cycle progression. The G2 checkpoint may not be significant for V(D)J recombination. C. How MIGHTV(D)J RECOMBINATION/DSBR MUTANTSLEADTO CHROMOSOME ERRORS? Cell cycle checkpoint errors could broadly explain the origin of defects resulting from V(D)J recombination for several of the V(D)J recombination/DSB repair mutants identified in mice, cell culture, or human syndromes (Fig. 4). Recognition of broken DNA as a stage in

V(D)J RECOMBINATION AND DOUBLE-STRAND BREAK REPAIR

73

the V(D)J recombination mechanism may be a means to signal the cell to arrest the cell cycle for a sufficient time to allow DSBs to be repaired. If this process was interrupted b y mutation, then chromosomal breakage initiated by the correct initiation of V(D)J recombination may lead to errors eventually resulting in translocations. In this model, DNA breaks associated with V(D)J recombination intermediates and/or errors would occur in G1 phase of the cell cycle (Fig. 4, wt). The presence of broken molecules would lead to cell cycIe arrest until repair can be completed. This process would be expected to be mediated by Ku complexes. Three mutant outcomes can be considered. In the scid case, an enzymatic step of V( D)J recombination is modified or lost, whereas the signaling mechanism is normal (Fig. 4). Scid rearrangement events may generate, but not resolve, hairpins as discussed above. Consequently, hairpin ends may or may not be recognized by a DNA damage checkpoint mechanism. This issue is yet to be answered by experimentation. If hairpins do arrest the cell cycle, then the scid cells will be irreversibly blocked unless hairpin DNA is resolved by another means. Extensive deletion may result by default utilization of a secondary and less-favored pathway that leads to chromosome deletion. Usage of a secondary pathway to overcome a cellcycle checkpoint (adaptation) is a standard feature of checkpoint pathways. In the specialized case of precursor lymphocytes undergoing gene rearrangement, an excessive loss of DNA in Ig or TCR loci is not deleterious since no essential genes are localized in these regions, and the deletion can be tolerated. For repair of IR damage, extensive deletion of chromosomal material may not be tolerated. Alternatively, chromosomal hairpin ends may be unrecognizable as a DNA damage arrest signal. Thus, unresolved hairpins will pass into S phase; DNA replication through these structures would presumably be accompanied by excessive deletion. In the A T case, the enzymology of V(D)J recombination is intact, but the means of signaling mistakes might be mutated (Fig. 4). In this scenario, no unusual DNA products are involved. Instead, broken DNA ends that ordinarily arise in a low level in the normal pathway become recombinogenic for translocations by being unable to signal cell-cycle arrest in a p53-dependent manner. In the xrs case, DNA ends may not be properly held together (Fig. 4). The xrs defect would potentially be compounded by also losing the signaling pathway of a cell cycle arrest checkpoint from G1-S. Checkpoint mutants, such as xrs, do lead to faulty processing of double-strand breaks of coding and signal ends. Because the same protein is involved in both the regular

74

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joining of these ends and the cell cycle arrest at DNA damage checkpoints, a dual error would be invoked. Ku binds to hairpins in uitro as efficiently as to DNA ends (Paillard and Straws, 1991; Falzon et ul., 1993), but no experiments have yet examined whether there is a DNA-PK activation that may actually serve as the signal transducer for DNA chromosomal breaks.

ACKNOWLEDGMENTS I thank menihers of the laboratory for many fruitful discussions and ideas. Many thanks to colleagues in the field who have shared results and manuscripts prior to publication and a special thanks to John Petrini for his critical reading and helpful comments.

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ADVANCES I N IMMUNOLOGY. VOL 58

Development and Selection of T Cells: Facts and Puzzles PA WE^ KISIELOW~AND HARALDVON BOEHMERZ Basel Institute for Immunology, CH-4005 Basel, Switzerland

I. Introduction 11. Thymus-Derived Lineages of‘T Cells 111. Extrathymically Derived Lineages of T Cells

87 102 110

and Communication with the Antigen-Presenting Cells V. Intrathyinic Development and Selection of T Cells VI. Coiicluding Remarks References

112 139 174 175

IV. T Cell Surface Molecules Involved in an Antigen Recognition

1. Introduction

Mature T cells are essential components of the immune system. They represent phenotypically and functionally heterogenous populations of lymphocytes that are equipped with unique, clonally distributed, heterodimeric (a@ or yS) T cell receptors (TCR), which unlike antigen-specific immunoglobulin (Ig) receptors on B cells, are designed to recognize exclusively cell-bound antigens and cannot be blocked by free antigen. To cope with various kinds of subversive agents exhibiting different biological properties, several lineages of T cells evolved which are programmed to respond differently to different types of antigens. For example, virus-specific antigens on the surface of infected cells recognized by “killer” T cells will stimulate them to release molecules lysing the infected target cells, whereas viral or bacterial antigens recognized by “helper” T cells will stimulate them to release growth factors that regulate the activity of other cells of the immune system, including antibody production by B cells. Recent years have witnessed an enormous progress i n elucidating mechanisms b y which self and foreign proteins (antigens) are pro-

’ ’

On leave of absence from the Institute of Immunology and Experimental Therapy, Polish Academy of Science, 53-114Wroclaw, Poland. Affiliated with the Faculte d e Medicine Necker Enfants Malades, Paris, France. 87 Cupyrigllt 0 1995 bv Academic Press, Inr. ( 1 1 rr~prrrductioni n d n ) form rewrred.

hll riglit,

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PAWEL KISIELOW A N D HARALD VON BOEHMER

cessed and made “visible” to T cells, especially to those that express ap TCRs and “see” antigen in the context of the major histocompatibility complex (MHC) encoded molecules. The main discovery was that during their intracellular assembly, MHC molecules bind peptides and transport them to the cell surface for T cell recognition (reviewed by Yewdell and Bennink, 1992; Germain, 1994). As a rule, class I MHC molecules bind peptides derived from intracellular proteins and class I1 MHC molecules bind peptides derived from extracellular, ingested proteins. The development and programming of the majority of T cells to recognize foreign antigens, and to perform their different functions, occurs predominantly in the thymus. T lymphocytes learn during development to disregard peptides derived from the body’s own or selfproteins at their physiological concentrations with the exception of those that are not present in the thymus. In that sense the T cell immune system can distinguish between self and nonself. Understanding how functionally different lineages of T cells develop from multipotential hemopoietic stem cells has been a great challenge. The goal is very ambitious and to reach it, it will be necessary to identify all different T cell lineages, to trace back their origin to hemopoietic stem cells, and to elucidate molecular mechanisms of cellular communication and signaling which are responsible for divergence, selection, differentiation, and migration of various cellular stages with progressively restricted developmental potential. For many years the paucity ofavailable markers distinguishing different T cell subsets and/or their developmental stages, combined with the unknown molecular nature of the antigen-specific TCR molecules and their ligands, was preventing any significant progress in this field. The stage for the present era in the study of T cell development, which began about 10 years ago with the discovery of TCR molecules and genes encoding them (reviewed by Allison and Lanier, 1987), was set b y a number of methodological breakthroughs. First, monoclonal antibodies helped to discover TCR molecules and equipped investigators with reagents of great specificity and potential to identify and purify protein molecules. Second, the advances in solubilizing and crystallizing cell membrane proteins coupled with the progress in peptide chemistry permitted the description of the structure of TCR ligands. Third, the polymerase chain reaction technique, which allows the amplification of a single copy of any nucleotide sequence, enriched the armamentarium of gene cloners by a powerful tool. Finally, the

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89

generation of transgenic and gene deficient (“knockout”) mice created new possibilities to unravel the physiological role of individual genes and their products during development of a whole animal and/or of a particular cell lineage. The widespread application of these new technologies generated data that have been advancing our knowledge and their impact on understanding the development of T cells was periodically reviewed (Adkins et al., 1987; von Boehmer, 1988; Raulet, 1989; Fowlkes and Pardoll, 1989; von Boehmer, 1990; Allison and Havran, 1991; Rothenberg, 1992; Haas et al., 1993). Despite this recent progress, however, the extent of phenotypic and functional heterogeneity of T cells is not yet fully appreciated. Along with the identification of new surface markers and the methodological refinement of multiparameter analysis of cell populations in normal and mutant mice, minor subsets of T cells with unexpected phenotypes and unknown ftinctions continue to be discovered. In the present review, we will focus mainly on the important results of recent years of research that was dominated by the analysis of T cell development in transgenic and gene-deficient mice. Genes and proteins, whose role in T cell development has already been analyzed in knockout mice, are listed in Table I, which also summarizes the obtained results. In our review we will discuss the mechanisms of positive and negative selection of T cells within the framework of the concept of programmed cell death (Glucksmann, 1951; Lockhin and Williams, 1965), which strongly influenced the thinking about T cell development in recent years. Programmed cell death could be viewed as a ticking time bomb, which kills developing cells at a predetermined hour unless its clock is set back by signals that positively select “wanted” cells or, if needed, is set forward by apoptosis-inducing signals that precipitate negative selection of “unwanted” cells. It is now well established that during the intrathyinic development TCRs play a central role in transmitting signals that induce proliferation, maturation, or cell death. Understanding the cellular and molecular mechanisms of these events has been a major goal of the ongoing research. We will begin our review by a survey of the heterogeneity of mature T cells, which will be followed by the description of our still sketchy knowledge concerning the mechanisms of T cell development from hemopoietic stem cells.

TABLE I EFFECTSOF NATURALAND ARTIFICIALMUTATIONSOF SOMESELECTED GENESON T CELLDEVELOPMENT Gene

scid

RAG-1 or RAG-2

TdT ip

c

TCRp

TCRa

Phenotype and Conclusions Complete block of development at the CD4-8-44-25' stage. N o mature T and B cells. TCR gene rearrangement is observed but signal sequences are not required. Conclusion: Appropriate rearrangement of TCR and Ig genes is essential for lymphocyte development. Complete block of development at CD4-8-44-25' stage. N o mature T and B cells. No V(D)J recombination. Conclusion: Both RAG genes and their products are indispensable for V(D)J recombination and development of lymphocytes. Cellularity and T cell subset composition in the thymus and peripheral lymphoid organs is not affected. T and B cells are capable of mounting normal responses to complex antigens. Lymphocytes have no o r few N nucleotides in their variable region genes which show extensive evidence of homology-directed recombination (a characteristic of Ig and TCR genes of newborn mice, limiting junctional diversity of rearranged genes). Conclusion: TdT is responsible for N region additions to rearranging antigenreceptor genes. Reduced numbers of CD4'8' cells. N o mature CD4+8- and CD4-8+ cells. Some TCRa rearrangement. Apparently normal development of y6 T cells. The origin and nature of a few CD4'8- and CD4-8' cells present i n these mice is unknown. Conclusions: (1) TCRp gene rearrangement and expression is not essential for CD4/8 expression but important for the expansion of CD4'8' cells. (2) TCRp rearrangement is not needed for generation of y6 T cells. Normal development up to the CD4'8' stage. The numbers and phenotypes of CD4-8- thymocytes appear normal. No effect on rearrangements of TCRP, -6, and -y loci. Apparently normal development of y6 T cells. Small numbers of CD4'8-TCRa-p' cells whose nature and function is unknown are present in the periphery of older mice

Reference Bosina and Carroll, 1991; Rothenberg et ul., 1993

Mombaerts et ul., 1992; Shinkai et al., 1992 Komori et ul., 1993; Gilfillan et ul.. 1993

Mombaerts et ul., 1992

Mombaerts et ul., 1992; Phillpot et ul., 1992

TCRa and TCRp TCRG

5

TCRp and TCHG

CD3c

Conclusions: (1) TCRa chain is not required for the progression of thyniocytes from the CD4-8- to CD4'8' stage and the expansion of CD4'8' cells but is required for further differentiation to CD4'8- and CD4-8' cells. ( 2 ) TCRa expression is not needed for the generation of y6 cells. The phenotype of these mice is identical as that of TCRP mutant mice. Conclusion: Development of yti cells is independent of rearrangement and expression of the TCR genes encoding a p TCR. Complete lack of T cells bearing TCR yG chains while development of ~$3 T cells is unaffected. N o difference in the pattern of y and 6 gene rearrangements and i n generation of restricted junctional sequences between TCRG mutant mice ( i n which only the CG gene segment was disrupted) and wild-type animals. Mice are unable to resist liver infection with Plosmodium yoelli. Conclusions: (1) Development of ap T cells is independent of yG T cells. (2) Rearrangement of TCRy and TCRG genes is developmentally programmed. ( 3 )y6 cells can provide immunity to infectious disease. The phenotype of these inice represents a combination of phenotypes of TCRp and TCHG mutants. Development of a@ T cells is arrested at (7134-8-44-25' stage and i n contrast to TCRp mutants, virtually no CD4'8' cells are generated. yG cells are also absent i n these mice. I n contrast to single mutants (TCRaP- or TCRyG-) the TCRp x TCRG double mutants are sensitive to disease caused b y Listet-iu nronocytogenes. Conclusions: ( 1 ) The yG cells can provide immunity to infectious disease. (2) In TCRp single mutant mice the small numbers of cells expressing CD4 and/or CD8 molecules represent cells of y6 lineage and/or yG cells can induce CD4/ CD8 expression i n cells of other lineages. Complete block of development at CD4-8-44-25' stage. N o mature T cells. Normal rearrangement of TCRp locus. N o TCRa rearrangement. Conclusion: CD3.s is not required either for TCRp rearrangement or for generation of CD44-25' thyniocytes but is required for further development of these cells, resulting i n immature and mature ap T cells.

hloinbaerts et a / . , 1992 Itohara et u l . , 1993; Tsuji d., 1994

Pt

Mombaerts et al., 1992,1993

B. hlalissen, personal communication

(continued )

TABLE I-Continued Gene CD36

5

Phenotype and Conclusions Normal development up to the CD4-8' stage. CD4+8- and CD4-8' cells are virtually absent in the thymus and considerably reduced in the spleen and lymph nodes. Apparently normal development of y8 cells. Conclusions: (1) CD36 is not required for the progression of thyniocytes from the CD4-8- to CD.1-8' stage and the expansion of CD4'8' cells but is required for further differentiation to CD4'8- and CD4-8' cells. (2) CD36 is not required for the generation of y6 cells. The HSA'CD44-25- subset of immature CD4-8- thymocytes is missing in these mice. The numbers of CD4+8+(which differentiate directlv from CD44-25' cells), CD4+8-, and CD4-8ap' thymocytes and peripheral ap T cells are greatly reduced as well as the expression levels nf u1p TCR. y6 [ELs are not affected. The crPTCR complexes arc associated prudomiiiaiit,ly with r ) homodimers. Only vory small amounts of FcyR transcripts are detected. Proliferative responses to TCR stimulation are impaired. Conclusions: (1)<-chain is required for the normal level of assembly and surface expression of the TCR complex in immature thymocytes and this function cannot be fully compensated by r ) or FcyR. (2) 6 chain has a critical role in thymocyte development and signal transduction of T celIs in ciao: the absence of {-chain does not abrogate the differentiation of CD4-8- to CD4+8+cells but inhibits their proliferation and downmodulation of CD25. The generation of CD4'8- and CD4-8ap' cells is also not completely abrogated. Cellularity and T cell suhset composition in the thymus and peripheral lymphoid organs is not affected. hlloprolifcrative and CTL responses are rlonriaj. Positive arid negative selection of transgenic ;inti-HI' TCli is not chaiiged. Conclusion: The product of q gene is not necessary for T cell development and selrctiurl.

Reference D. Kappes, personal communication

Ohno et al., 1993; Love et al., 1993; Crompton et ul., 1994

Koyasu et ul., 1994; Ohno et ol., 1994

FcRy chain

CD4

CD8a

CD8B

The phenotype is very similar to (-only mutants. Rearrangement of TCRa and TCRp genes is unaffected. Numbers of IELs are normal but surface levels of TCRs on both ap and y6 cells are reduced. TCRs on y6 and ap IELs that express CD8a homodinier are associated with homodimers of the FceRIy chain. CD4+8- and CD4-8ap' IELs are CD3-. Conclusions: (1) 5 gene product is essential for efficient generation and/or survival of CD4'8+ thymocytes. (2) TCRs associated with FceRIy chain appear able to sustain the maturation of gut IELs. Thymic and peripheral populations of ap T cells appear normal. up and y6 IELs were not investigated in these mice. Conclusion: FcRy chain is not essential for the development of thymus-derived ap T cells. No atrophy of lymphoid organs. T/B cell ratio is normal. The production of IL-2 in response to class I1 MHC differences and T cell-dependent antibody responses are greatly reduced but not all class I1 MHC restricted responses are affected. Antiviral class I MHC restricted cytotoxic responses are not significantly affected in these mice. The defect in helper T cell responses can be corrected by expression of CD4 transgene. Conclusions: (1)CD4 is needed for efficient development of some but not all T helper cell functions. (2) Development of cytotoxic CD4-8' T cells is independent of CD4 expression. No atrophy of lymphoid organs. T/B cell ratio is normal. Not only CD8a' but also CD8p' cells are missing in these mice. Cytotoxic T cell responses are dramatically decreased but helper functions appear unaffected. Conclusions; (1) CD8 (either CD8a or CD8p or both) is needed for development of cytotoxic T cells but not for helper T cells. (2) CD8a is limiting the surface expression of CD8ap heterodimers. Development of CD4+8+and CD4'8- thymocytes as well as CD4'8- T cells is nomial. The numbers of CD4-8' thymocytes and T cells are strongly reduced. The number of CD4-8+ T cells was restored by transfer of an exogenous CD8p gene into CD8p-deficient T cells. No functional studies reported.

Malissen et al., 1993

Takai et ul., 1994

Rahemtulla et al., 1991; Locksley et al., 1994

Fung-Leung et al., 1991

Nakayama et al., 1994

(continued )

TABLE I-Continued ~

Gene

CD4 and CD8

CD45-exon6

CD28

CD2

Phenotype and Conclusions Conclusion: CD8p is required for efficient positive selection of some CD4-8' T cells. Cellularity of the thymus is normal but the numbers of mature apTCRh1gh, HSA- cells (also in the periphery) are greatly reduced. Alloreactive but not antiviral cytotoxic responses can be generated. Conclusion: CD4 and CD8 are essential for maturation of immature (aPTCR'"" HSAhlgh)thymocytes. More than 90% of T cells lack expression of CD45 but some (about 9%) remain CD45 positive. The development of T cells is impaired as manifested by ) reduced numbers of increased numbers of CD4-8- thymocytes ( 2 ~and CD4'8' (1/3), CD4+8-, and CD4-8+ (1110) thymocytes as well as peripheral T cells: CD4'8- (1/3)and CD4-8' (1/5). Proliferative responses to TCR stimulation of both CD45- and CD45+ cells are strongly reduced and cytotoxic T cell responses to LCM virus are absent. CD4'8' but not CD4'8and CD4-8' thymocytes express elevated levels of CD4 and CD8 molecules. Clonal deletion of superantigen-reactive T cell is not impaired. Conclusions: (1) CD45 is required for efficient transition of CD4+8+to mature CD4'8- and CD4-8' T cells and possibly (to some extent) for transition of CD4-8- cells to CD4+8+thymocytes. (2) Negative selection of superantigen reactive T cells does not require CD45 expression. Development of T and B cells appeared normal. Impaired responses to lectins: decreased IL-2R (CD25) expression and IL-2 production. Reduced helper activity. Apparently normal CTL and DTH responses. No defect in the clonal deletion of self-reactive T cells specific for Mls-1 and I-E. Conclusion: CD28 is not essential for T cell development and repertoire selection and it is dispensable for the development of some effector cells. Development of crp and yti T cells in CD2 mutant mice and their investigated functions are indistinguishable from those of wild-type mice.

Reference

Schilham et al., 1993

Kishihara et ol., 1993

Shahinian et al., 1993

Killeen et al., 1992b

~

IL-2Ry chain

IFNyR

TNFRp55

W

cn

p56Ick

p59fun

Conclusion: CD2 is dispensable for the development and function of T cells I n humans mutations of the IL-2R-y gene result in X-linked severe combined immunodeficiency (XSCID) that is characterized by absence or markedly reduced numbers of thymocytes and T cells. Conclusion: IL-2Ry chain plays a vital role in intrathymic development of T cells in humans. All major subsets of thymocytes and peripheral T cells are normal as revealed by cell count and staining with CD3, CD4 and CD8 antibodies. CTL and T helper cell dependent antibody responses against vaccinia, LCMV and VSV viruses were normal. Conclusion: Lack of IFNyR seems to interfere neither with normal development of T cells nor with their function. Thymic and peripheral populations of T lymphocytes appear normal and clonal deletion of T cells reactive to endogenous viral superantigens is not impaired. Also, the CTL responses against vaccinia and LCMV viruses are normal. Conclusions: 55-kDa receptor for tumor necrosis factor does not play a role in the development of thymus-derived (YP T cells and is not essential for clonal deletion of potentially self-reactive T cells or for CTL responses. Drastic reduction of CD4'8' as well as CD4'8- and CD4-8' cells but not CD4-8-44-25+ cells in the thymus. Few peripheral CD4'8- and CD4-8' cells express strongly reduced levels of CD4 and CD8, respectively. T h e expression of (YP TCR on thymocytes is elevated. O n e percent of the lymph node population is y6 T cells. Proliferative responses of peripheral T cells to TCR crosslinking and to IL-2 alone are almost normal. Conclusions: (1) p56Ickplays a critical role in early stages of intrathyniic T cell development, which cannot b e compensated for by other tyrosine kinases. (2) p56ICkis dispensable for some of the TCR-CD3 and for the IL-2 signaling pathways. The development of all T cell subsets is normal both in terms of the numbers and cell surface phenotypes. Phenotypically mature thymocytes but not

Noguchi et ol., 1993

Huang et al., 1993

Pfeffer et ol., 1993; Rothe et al., 1993

Molina et al., 1992

Appleby et al., 1992 (continued)

TABLE I-Continued Gene

ZAP-70

(D 0)

IRF-1 (Interferon regulatory factor-1)

IRF-2 B2m

Phenotype and Conclusions

Reference

peripheral T cells are compromised in their ability to mobilize intracellular calcium and to proliferate in response to TCR-mediated stimulation. The ability of CD4+8+thymocytes from p59fY" deficient mice to mobilize intracellular calcium in response to TCR stimulation is unaffected. Conclusion; p59fYn plays a pivotal role in TCR signal transduction but loss of p5@' signaling leaves T cell development intact. Arpaia et al., 1994 Thymi of human patients carrying mutation of zap-70 resulting in loss of activity of ZAP-70 kinase have CD4+8+and CD4'8- thymocytes but no or few CD4-8+ thymocytes or T cells. CD4'8- T cells do not respond to TCR stimulation by mitogens or anti-TCR antibody. Conclusion: ZAP-70 kinase appears to be indispensable for the development of CD4-8' T cells as well as for signal transduction in CD4'8- T cells. The phenotype is similar to that of p2m, TAP-1, and ZAP-70 mutants. Thymi of Matsuyama et al., 1993 IFR-1 mutant mice contain normal total numbers of thymocytes but are specifically (10-fold) deficient in CD4-8' thymocytes and peripheral T cells. The proportion of CD4'8- T cells was elevated but yti T cells were not affected. MHC class I expression was not changed. Antiviral cytotoxic responses of residual CD4-8' T cells are normal. CD4'8- helper activities are not affected. Conclusions: (1)IRF-I is involved in the development of CD4-8' but not CD4+8- T cells. (2) Reduced expression of MHC class I molecules does not appear to be responsible for the effect of IFR-1 mutation. Matsuyama et al., 1993 T cell development and function is not affected. Zijstra et al., 1990; Koller et Cells in these mice are severely deficient in the surface expression of most if ol., 1990 not all class I MHC molecules but a low level of class I molecules on the cell surface can be detected with serological and/or functional tests (reviewed by Raulet, 1994b). Thymi of mutant mice contain normal total numbers of thymocytes but are specifically deficient in CD4-8' cells. The residual

I-AP (MHC class 11)

I-i (MHC-I1 associated invariant chain)

p2m and I-A/3

CD4-8' cells in the thymus and periphery constitute only 1-5% of the normal pool of TCRaP'CD4-8' T cells. Cytotoxic responses are drastically reduced but not totally absent. The development and function of CD4'8cells appears not affected. y6 T cells are normal. Conclusions: (1) MHC class I molecules are crucial for positive selection of most TCRaP+CD4-8+ T cells. (2) Development of most y8 T cells does not require class I MHC molecules, at least at their normal expression levels. Complete absence of class I1 MHC molecules on the cell surface. Thymi of mutant mice contain normal total numbers of thymocytes but are specifically deficient in CD4'8- cells. A sizeable proportion of immature cortical thymocytes express CD4'8''" phenotype. CD4'8' thymocytes display elevated levels of CD4 and af3 TCR. The residual CD4'8- cells in the thymus and periphery constitute about 5% of the normal pool of TCRaP'CD4'8- T cells and many of them are larger, express CD4 and ab TCR at reduced levels, and are found at distinct anatomical sites. The mice are unable to mount T cell-dependent antibody responses. Conclusion: Class I1 MHC molecules are required for positive selection and functional maturation of most CD4'8- T cells. The absence of the I-i chain results in a reduction of cytoplasmic and cell surface expression of I-Ab and in surface reduction of I-A' and I-Ek molecules which have an aberrant conformation. T cell development in these mice is affected similarly as in I-AP mutants but reduction of CD4+8- cells is less pronounced. In contrast to the wild-type animals the CD4'8- cells contain significantly higher proportion of I-E reactive VPll' cells. Primary but not secondary antibody responses to protein antigens are impaired. Conclusion: Lack of I-i chain results in deficient expression of class I1 and deficient positive and negative selection of CD4% cells. The phenotype of these double mutants is a combination of the phenotypes of single mutants. Thymi contain normal total numbers of thymocytes but are strongly deficient in CD4+8- and CD4-8' cells both in the thymus and in the periphery. The CD4'8' thymocytes express equivalent levels of CD4 and

Cosgrove et ul., 1991; Grusby et ul., 1991

Viville et d., 1993; Bikoff et ul., 1993

Grusby et al., 1993

(continued )

TABLE I-Continued Gene

Phenotype and Conclusions CD8 molecules: cells with CD4'""8' and CD4'8'"" phenotypes are practically absent. y8 cells appear to develop normally. Cells from these mice exhibit very poor cellular immune responses in uitro but can reject imcompatible skin grafts efficiently. Whether in uiuo responses are due to the activity of residual ap T cells or to non-ap T cell-mediated mechanisms is unknown. Conclusions: (1) Development of most up T cells is dependent on the presence of MHC molecules. (2) Development of most y8 T cells does not require MHC molecules, at least at their normal expression levels. The effect TAP-1 mutation on T cell development and function is similar to that of the mutated p2m gene.

TAP-1 (peptide transporter gene 1) LMP-7 (proteasome Despite clear reduction (10-40%) in cell surface expression of the MHC class I subunit) molecules no obvious effect on T cell development and function was noticed. Antigen presentation is less efficient. ICAM-1 All major subsets of thymocytes and peripheral T cells are normal. Infammatory responses are impaired. Mutant cells provide negligible stimulation in mixed lymphocyte reaction but proliferate normally as responders. Conclusion: Lack of ICAM-1 does not seem to impair T cell development. The development of major subsets of thymocytes and peripheral T cells is IL-2 unperturbed in these mice but they have altered serum levels of immunoglobulin isotypes and in uitro T cell responses are dysregulated. In oiuo, however, the ability to mount antiviral cytotoxic responses is preserved. Conclusion: IL-2 is dispensable for T cell development and (at least for some) functions. IL-4 There is no deficiency in absolute cell numbers or in the proportion of major T cell subsets in the thymus and in the periphery. The serum levels of IgGl and IgE are strongly reduced and Th2 cytokine responses (IL-5 and IL-10) are significantly impaired.

Reference

van Kaer et al., 1992 Fehling et al., 1994 Sligh et al., 1993

Schorle et al., 1991; Kundig et al., 1993

Kuhn et al., 1991; Kopf et al., 1993

IL-4 and 1L-2. IFNy

IL-6

IL-10

Perforin

Conclusion: IL-4 is not essential for development of major T cell subsets but is required for the generation of the The-derived cytokines. All major T cell subsets are normal. Conclusion: IL-2 and IL-4 are not essential for development of T cells. All major subsets of thymocytes and peripheral T cells are normal as revealed by cell count and staining with CD3, CD4, and CD8 antibodies. Splenic T cells respond by uncontrolled proliferation to mitogens and allaontigens. In oitro-generated CTL responses against alloantigens are enhanced. Conclusion: IFNy is not essential for the development of T cells but is essential as a regulator of T cell function. Numbers of thymocytes and peripheral T cells are reduced by 20-40% compared to controls but no difference in the representation and distribution of major subsets defined by TCRa, -p, -y, and -6 chains, CD4, CD8, CD44, and HSA is observed. CTL function against some (vaccinia) but not other (LCMV) viruses is strongly reduced and T cell-dependent antibody response against VSV virus is impaired. Conclusion: T cell development is not significantly affected by the absence of IL-6 but IL-6 is important as regulator of T cell functions. T lymphocytes appear to develop normally. T cell-dependent antibody responses are normal. Following infection with nematode N . brasiliensis develomient of a The remonse is not affected but in contrast to normal mice development of T h l response is not supressed. The mice develop chronic enterocolitis. Conclusion: IL-10 is not required for development of T cells but is needed to inhibit T h l cell responses. T lymphocytes appear to develop normally. No significant differences in the number and distribution of thymocyte and T cell subsets were observed. CD4-8+ T cells do not lyse virus-infected or allogeneic fibroblasts and the mice fail to clear LCMV virus. Conclusion: Perforin is a key molecule for T cell-mediated cytolysis.

Sadlack et ul,, 1994 Dalton e t al., 1993

Kopf et al., 1994

1993 Kuhn et d.,

Kagi et ul., 1994

(continued )

TABLE I-Continued

O

Gene

Phenotype and Conclusions

L I F (Leukemia inhibitory factor)

Thymocyte numbers are slightly reduced compared to control animals but no differences in major thymocyte and T cell subsets are observed. The Con Ainduced proliferative response of thymocytes but not peripheral T cells is reduced both in homo (LIF-’-)- and heterozygous (LIF+’-)mice. Conclusion; LIF plays no major role in T cell development but has some poorly defined effect on thymocyte proliferation. T cell development is unaffected. Like in normal mice, CD4+8+thymocytes are susceptible to apoptosis induced by TCR engagement or by glucocorticoids but unlike in normal mice are resistant to apoptosis induced by ionizing radiation and etoposide. Conclusion: p53 is dispensable for T cell development but is required for apoptosis induced by agents that cause DNA strand breakage. The development of T cells is initially normal. Until 3 weeks of age thymus and peripheral T cells show normal subset composition but later undergo massive apoptotic involution. Peripheral T cells have short life span and increased sensitivity to glucocorticoids and ionizing radiation. Stimulation with anti-CD3 antibody inhibits death of these cells.

P53

bcl-2

Reference Escary et al., 1993

Donehower et al., 1992; Lowe et al., 1993; Clarke et al., 1993

Veis et al., 1993b; Nakayama et al., 1993

c-jun

c-fos

Conclusions: (1)Bcl-2 is dispensable for T cell development but is required for the resistance of mature T cells to glucocorticoid and y-irradiation-induced apoptosis. ( 2 ) Rescue from programmed cell death of developing thymocytes may involve Bcl-2-independent signaling mechanism. Thymus and peripheral T cells show normal subset composition as assessed by staining with C D ~ ECD4, , CD8, aP TCR, y6 TCR, Thy-1, IL-2Ra, CD5, and CD45R but thymocyte numbers are small (10% of normal), mostly due to depletion of CD4'8' as well as CD4'8- andCD4-8' cells. In contrast, the number of peripheral T cells which show normal activation responses (expression of IL-2R, IL-2 secretion, and proliferation) is significantly increased in some mice. Conclusion: c-jun expression is dispensable for T cell development in oioo but its absence causes some unexplained quantitative perturbations of the process. T cell development and function is unaffected.

Chen et al., 1994

Jain et al., 1994

102

PAWEX: KISIELOW A N D HARALD VON BOEHMER

II. Thymus-Derived lineages of T Cells

The thymus is a major site of T lymphocyte production that starts during the late phase of embryonic life and continues until puberty. Thereafter, the role of the thymus as a source of newly generated T cells subsides and it can be removed without major consequences for the functioning of the immune system. Genes encoding TCR chains are formed in immature thymocytes by rearrangement of V (variable), D (diversity), J boining), and C (constant) gene segments, similarly to immunoglobulin genes (Tonegawa, 1983; see Section 1V.A.la). The majority of T cells produced in the mature thymus expresses ap TCRs, while a minority bears y6 TCRs. During ontogeny cells with y6 TCRs appear earlier than ap T cells (Bluestone et al., 1987; Havran and Allison, 1988; Pardoll et al., 1987a) and until Day 17 of gestation y6 cells comprise the major population of TCR positive cells in the murine thymus (reviewed by Fowlkes and Pardoll, 1989). A. THYMUS-DERIVED y6 T CELLS Detailed discussions of the heterogeneity, specificity, and function of thymus-derived as well as nonthymus-derived y6 T cells (Section II1,A) have been recently published (Allison and Havran, 1991; Haas et al., 1993) and we will only briefly summarize the most important points.

1 . Heterogeneity Subsets of the thymus-derived y6 T cells can be distinguished according to the TCR-V gene segment usage, anatomical location, and time of appearance in ontogeny. Although thymus-derived yi3 T cells were reported to express CD4 (Itohara et al., 1989; Morita et al., 1991; Spits et al., 1991) or CD8 molecules (Bucy et al., 1989; Groh et al., 1989; Itohara et al., 1989), which on two major subsets of thymusderived ap T cells function as coreceptors during antigen recognition (see Section IV.B.l), the overwhelming majority of y6 cells are CD4-CD8-. The function of CD4 and CD8 molecules on y6 cells is unknown. The yi3 cells which appear in the first two waves that colonize the fetal thymus have monomorphic TCRs. They express TCRy chains encoded by rearranged V5JlC1 or V6JlC1 gene segments, respectively, paired with TCRG chains encoded b y rearranged VlD2J 1C-8 gene segments (for explanation of nomenclature and genomic organization of TCR loci, see Fig. 1). These receptors exhibit essentially no junctional diversity (Section IV,A,lb) (Elliott et al., 1988; Laffaille et

DEVELOPMENT AND SELECTION OF T CELLS: FACTS AND PUZZLES

t

___L

Wl

V B k m m x 25)

TCRp

103

itlittk

JBll-17

W2

CB2

CB2

JP21-27

*

VP14

+

c a pom 7Ovb+lOV6

&DSlM2&1

J62

M

v65 qJa

J o (approx 801

Ca

TCRo-6

t-

C v 4 Jy4

TCRv

Vvl Vy2

-e

Jv2 Cv2

CV3

Jv3

Vv3

+fB&k4

Cvl

Jvl

et Vy5 Vv6

tt

Vv4 Vy7

+Et+-tt---

FIG.1 . Genornic organization ofthe murine TCRP, TCRdG, and TCRy loci. Arrows indicate transcriptional orientation; * indicate pseudogenes. VG segments are distinguished by open squares. 8 rec and $ Jo are elements mediating deletion of the TCRG locus. A11 of the C genes are composed of multiple exons (not shown). Drawings are not to scale. Nomenclature of TCR-Vy gene segments is according to Heilig and Tonegawa (1986).

ul., 1990).Vy5-expressing cells migrate to the skin and Vy6-expressing cells to the epithelia of the tongue and the female reproductive tract (uterus, vagina). The next waves of y6 cells consist of cells expressing TCRs with extensive junctional diversity. Cells with TCRs encoded by V1J4C4y and V6fV4fVS or V7-6 are abundant in newborn thymus and spleen and can be found in the skin and in the gut. y6 cells expressing Vy2 are infrequent and can be found in different locations. The predominant population of y6 cells in the thymus, spleen, and lymphnodes of adult mouse expresses a highly diverse repertoire of TCRs encoded by V4JlC1-y and V5-6fV4-6fV6-6 or V7-6. The origin (thymic versus extrathymic) of y6 cells residing in the liver, lung, and mammary gland is unclear. The predilection of y6 cells with different TCRs for particular anatomical sites suggested the possibility that the specificity of y8 TCRs determines the homing pattern. This, however, is not the case because in y6 TCR transgenic mice the correlation between anatomical site and y6 TCR broke down (Bonneville et al., 1990).

2 . Speci5city Murine and human y6 T cells (clones, lines, and hybridomas) recognizing classical class I and class I1 MHC proteins, nonclassical MHC class I-like (TL region encoded) proteins, mycobacteria, and heat shock proteins have been described (reviewed by Haas et al., 1993) but the

104

PAWEE KISIELOW AND HARALD VON BOEHMER

nature of the natural target antigens (ligands) of y6 T cells remains elusive and the general rules of antigen recognition by these cells and antigen presentation have yet to be established. Two lines of evidence suggest that the molecular nature of y6 T cell recognition may be fundamentally different from that of a/3 T cells (reviewed by Raulet, 1994a). First, it was found that most y6 T cells develop normally in mice lacking MHC class I and class I1 molecules (Bigby et al., 1993; Correa et al., 1992; Grusby et al., 1993) which questions the involvement of these MHC molecules in the normal function of y6 T cells. Second, analyses of y6 T cell hybridomas specific for classical MHC class I1 molecule (IE) and for TL class I molecules were interpreted to indicate that the topology of the y6 T cell receptor’s interaction with the MHC is distinct from that of a/3 T cells and that MHC bound peptides do not influence TCR binding (Schild et al., 1994). These results could suggest that some yS TCRs bind to MHC molecules not from the top but from the side, away from the peptide-binding groove. Comparative measurements of the length of the third complementarity determining regions (CDR3) of 6, y, a,and /3 TCR chains and immunoglobulin heavy and light chains suggested the possibility that y6 TCRs may be more immunoglobulin like in their antigen-recognition properties and therefore recognize native antigens without a requirement for degradation and presentation by professional antigen-presenting cells (Rock et al., 1994; Schild et al., 1994). The monospecific Vy5 expressing cells residing in skin epithelium were shown to react specifically to cultured keratinocytes and/or to living or fixed fibroblasts treated with tryptic digests of cultured keratinocytes (Havran et al., 1991)suggesting that the Vy5 subset is specific for a stress-induced, keratinocyte-specific self-peptide (Allison and Havran, 1991). Since the third complementarity determining regions of the canonical TCR of the Vy6 and Vy5 subset are identical it was proposed that the two TCRs recognize the same endogenous peptide in the context of different tissue-specific peptide-presenting molecules (Haas et al., 1993).

3. Function The great diversity ofy6 TCRs, achieved mainly by junctional diversification, strongly suggests that the majority of y6 T cells is concerned with protection against microbial infections. Recent experiments in mice deficient for a/3 T cells provide direct evidence that y6 T cells can provide immunity to infectious diseases (Mombaerts et al., 1993; Tsuji et al., 1994): mice deficient for both a/3 and y6 T cells were found to be extremely sensitive to disease caused by the intracellular

DEVELOPMENT AND SELECTION OF T CELLS: FACTS AND PUZZLES

105

bacterium Listeria monocytogenes, whereas mice deficient for either a@ or y6 T cells were found to be similarly resistant to primary infections (Mombaerts et al., 1993). In another study (Tsuji et al., 1994) yS T cell-deficient mice, unlike a@ T cell-deficient mice, were shown to be unable to resist liver infection with Plasmodium yoelli. The role of the monospecific subsets of y6 cells is more puzzling. It is suspected that some of them may recognize self-antigens such as stress-induced proteins (Allison and Havran, 1991; Asarnow et al., 1988). The ability of stimulated yS cells to secrete various lymphokines and to lyse target cells has been amply documented (reviewed by Haas et al., 1993). Interestingly, analysis of rare CD4' yS T cell clones indicated that they produce lymphokines at high levels but have little or no cytolytic activity (Spits et al., 1991), suggesting the existence of similar correlation between function and expression of CD4 and CD8 molecules among y8 T cells as observed among a@ T cells (see Section II.B.l and 2). B. THYMUS-DERIVED a@ T CELLS On the basis of cell surface expression of CD4 and CD8 molecules (see Section IV, B, 1) one can distinguish at least three different lineages of T cells with a@TCRs, namely the CD4+8-, the CD4-8', and the CD4-8- cells that are exported from the thymus with these phenotypes (Kelly and Scollay, 1990). Recently, the export of CD4'8cells that do not bear TCR (neither a@ nory6) was described in neonatal mice (Kelly and Scollay, 1992). The fate of these cells is, however, not known and therefore they will not be discussed further here. Small numbers of CD4+8+cells, apparently representing recent thymic emigrants, can also be found in peripheral organs (Bonomo et al., 1994; Hosseinzadeh and Goldschneider, 1993) where they may undergo post-thymic maturation. This population is especially prominent in lymph nodes of 3 day old mice (Bonomo et al., 1994). Some experiments suggest that in the absence ofa thymus these cells can differentiate into autoimmune effector cells that may be responsible for organspecific autoimmune disease observed in neonatally thymectomized mice (Nishizuka and Sakakura, 1969). 1 . CD4'8- a@ T Cells a . Heterogeneity and Function. The CD4+8- T cells, originally characterized as possessing helper activity in humoral and cellmediated immune responses (Cantor and Boyse, 1975; Kisielow et al., 1975; Dialynas, et al., 1984; Rheinherz and Schlossman, 1980), do

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not represent a homogenous population of cells. The analysis of the spectrum of lymphokines produced by long-term clones of CD4+8- T cells led to the distinction of functionally different subsets of T helper (Th) cells (Mosmann et al., 1986; Street et al., 1990; for reviews, see Mosmann and Coffman, 1989; Swain et aZ., 1991; Paul and Seder, 1994). Cells producing IL-2, IFNy, and tumor necrosis factor beta (TNFp) have been designated as T h l ; cells producing IL-4, IL-5, IL6, IL-10, and IL-13 as Th2; and cells producing IL-2, IFNy, and IL4 as Tho. It is believed that T h l and Th2 cells arise via an intermediate Tho stage (Kamogawa et al., 1993) as a result of antigenic stimulation of naive CD4+8- precursors (pTh),which can produce IL-2 but neither IL-4 nor IFNy (reviewed by Paul and Seder, 1994). T h l and Th2 cells are involved in regulation of cellular immunity and antibody production, respectively, as well as in the mutual regulation of their activities, which appears important not only for mounting the appropriate response to different pathogens but possibly also for regulating autoimmune responses. With regard to the latter possibility it was found that lymphokines produced by Th2 cells can regulate the activity of T h l cells (Liew et al., 1989), which in some cases of prolonged activation may b e responsible for local inflammation and tissue damage. Consistent with the observations of inhibitory influences of Th2 cells on T h l cells was the finding that IL-10-deficient mice develop chronic enterocolitis (Kuhn et al., 1993) which could result from uncontrolled response of Thl cells to normal antigenic stimulation. It was also found that IL-12 is able to promote the differentiation of T h l cells while inhibiting Th2 formation (Hsieh et al., 1993). Hayakawa and Hardy defined four distinct subsets of CD4'8- T cells on the basis of expression of cell surface markers called 6C10 and 3 G l l (Hayakawa and Hardy, 1988), which can be further subdivided by different levels of CD45, L-selectin (Mel-14), and CD44 molecules on the cell surface (Kariv et al., 1994). Analysis ofanti-TCRinduced production of lymphokines by individual cells in each subset from normal, healthy, unmanipulated mice revealed that the surface phenotype defined by the above markers largely overlaps with clusters ofcells showing uniform and distinct cytokine profiles. In these studies only cells corresponding to pTh and Tho subsets were detected. As discussed by the authors the absence of typical T h l , i.e., IL2+IIFNy+/ IL4-, cells and typical Th2, i.e., IL-2-/IFNy-/IL4+, cells in healthy nonimmunized animals may indicate that potent antigenic stimulation as it occurs in infected or intentionally immunized animals may be required to generate cells exhibiting a more extreme bias in cytokine production. In fact, the avidity ofTCR for their ligands may also contribute to development into T h l or Th2 subsets.

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Recently, two additional distinct minor subpopulations of CD4'8cells exhibiting restricted TCR V gene utilization were described. One was designated Thy0 (Hayakawa et al., 1992) and was of the CD4'8-HSA (heat-stable antigen)'""'- 6C10-3G11- phenotype. This cell type is found predominantly in the thymus and consists of autoreactive class I1 MHC-specific or restricted cells (Kariv et al., 1992). Another one, identified by expression of NK1.l marker, has the HSA~OW/-Ly6Ct3G11'"" phenotype (Arase et al., 1992,1993; Bendelac et al., 1994). The CD4'8-NKl.1' cells are found in the thymus as well as in the periphery and are particularly potent producers of IL4 (Bendelac et at., 1992; Yoshimoto and Paul, 1994). Their function is unclear but it was speculated that they may enhance differentiation of CD4+8- cells into Th2 cells (Paul and Seder, 1994). As a rule CD4'8- ap T cells are not directly cytotoxic but in some instances direct cytotoxicity of these cells can be demonstrated (Barnaba et al., 1990; Macphail and Stutnian, 1987; Muller et al., 1992).The mechanism of direct killing by CD4+8- cells is, however, unknown.

b. S p e c i j k i t y . Most CD4'8- T cells (pTh, Tho, T h l , Th2, and cytolytic CD4 cells) respond to peptides presented by self or foreign class I1 MHC molecules that also ligate CD4 molecules (Doyle and Stroniinger, 1987; Konig et al., 1992; see Section IV,B,l). Coengagenient of the ap TCR and the CD4 coreceptor by the same ligand is required for most efficient activation of CD4+8- cells (reviewed by Miceli and Parnes, 1993),which may give the illusion that the ap TCR of these cells can only bind to class I1 MHC molecules. Since binding assays of soluble TCRs are not readily available the possibility remains that ap TCRs of CD4'8- T cells can in fact bind to a much larger variety of antigens but that this is not revealed in T cell activation because ligands other than class I1 MHC molecules cannot coligate the CD4 coreceptor. Experiments in TCR transgenic mice expressing receptors specific for peptides presented by class I (Pircher et al., 1989; Sha et al., 1988; Teh et al., 1988)or class I1 (Berg et al., 1989; Kaye et al., 1989) MHC molecules as well as in mice deficient in class I (Koller et al., 1990; Zijlstra et al., 1990) or class I1 (Cosgrove et al., 1991; Grusby et al., 1991)MHC molecules demonstrated that the development ofthe majority of CD4'8- ap T cells required interaction with class I1 MHC molecules, whereas the development of the majority of CD4-8' ap T cells required interaction with class I MHC molecules (see Section V,C,2). However, CD4+8- ap T cells with specificity for class I MHC antigens have been described (De Bueger et al., 1992; Macphail and Stutman, 1987; McKisic et al., 1991). They could represent cells se-

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lected by class I1 MHC molecules that cross-react with class I MHC molecules or alternatively could be selected by class I MHC molecules in the thymus. Recently, it was found that some CD4'8- ap T cells can develop in class II-deficient mice (Bendelac et al., 1994; Cosgrove et al., 1991) but not in class I1 as well as class I-deficient mice (Chan e t al., 1993a). It was also proposed that CD4+8- cells expressing the NK1.l marker are selected by interaction with class I MHC molecules on hematopoietic cells (Bendelac et al., 1994).

2 . CD4-8' ap T Cells a. Heterogeneity and Fzirtction. Thymus-derived CD4-8+ a@T cells express CD8 molecules as heterodimers consisting of a and p chains (Parnes, 1989). Like CD4+8- T cells, CD4-8' T cells consist of heterogenous subsets. The predominant function of the majority of CD4-8' ap T cells is commonly believed to be the generation of cytolytic effector cells after antigenic stimulation but CD4-8+ cells with helper activity were also described (Horvat et al., 1991; Swain, 1981)(see review by Kemeny e t al., 1994). Cytolytic activity may result either from the release of perforin molecules together with granzyme molecules (Griffith and Muller, 1991) or from the recently discovered interaction of Fas protein with the Fas-ligand (Rouvier et al., 1993; Vignaux and Golstein, 1994; see Section IV.B.6). The importance of perforin-mediated cytotoxicity was documented in perforin-deficient mice that were severely compromised in their ability to cope with viral infections (Kagi et al., 1994). However, as mentioned above, cytotoxicity is not the only effector function of CD4-8+ T cells. These cells can, at least for some time, grow in an autocrine fashion resulting from the production of small amounts of IL-2 (von Boehmer et al., 1984)and when antigenically stimulated can also secrete IFNy, TNFa, and TNFp (reviewed by Rothenberg, 1992). In one particular line of TCR transgenic mice, CD4-8+ cells with a receptor for an HY peptide presented by class I MHC molecules could be induced to proliferation and lymphokine secretion but only to poor cytolytic activity (von Boehmer et al., 1991). These cells failed to reject skin grafts but could reject hemopoietic grafts from male mice (unpublished results). It is possible that the type of effector function of CD4-8' cells is, at least in part, determined by the avidity of the TCRs for their ligand. Recently, it was reported that CDw6O antigen on human CD4-8+ T cells allows discrimination between helper T cells and T cells with cytotoxic and suppressor activity (Rieber and Rank, 1994). The alloantigen-specific cytotoxic activity and B cell differentiation sup-

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pressing activity resided entirely in the CDw60-population, whereas the CDw60' population provided substantial help to B lymphocytes.

b. Specijicicity. As mentioned above, CD4-8+ T cells are, as a rule, selected in the thymus by interaction with class I MHC molecules (Teh et al., 1988; Kisielow et al., 1988a; Zijlstra et al., 1990) (see Section V.C.2) and exhibit specificity for peptides presented by class I MHC molecules, which represent specific ligands for CD8 molecules (Rosenstein et al., 1989; Salter et al., 1990). Again, it is not clear whether the observed specificity of CD4-8+ T cells for class I MHCassociated peptides is due to the fact that their ap TCRs do not bind to peptides presented by class I1 MHC molecules or to the fact that in order to induce CD4-8+ cells efficiently, the TCR must be coligated with CD8 molecules (Emmerich et al., 1986). Some CD4-8+ T cells or T cell clones can be activated either by allogeneic class I1 MHC molecules or by peptides presented by self-class I1 MHC molecules (Schilham et al., 1986). In most cases it is not known whether this simply reflects cross-reactivity of class I MHC-restricted T cells selected by thymic class I MHC molecules or whether some of these cells were in fact selected by class I1 MHC molecules. Recently, CD4-8+ T cells that expressed exclusively a transgenic, class I1 MHCrestricted TCR, have been shown to be selected by thymic class I1 MHC molecules (Kirberg et al., 1994; see Section V.C.2). This may occur regularly with cells that express TCRs of relatively high affinity for self-MHC ligands and that can be selected without the coengagement of TCRs and CD4/CD8 coreceptors by the same MHC ligand (see Section V.C.2). 3 . CD4-8- ap T Cells CD4-8- a@ T cells are found in the thymus and in peripheral lymphoid organs where they account for less than 1% of all a@ T cells. The characteristic feature of this population is the predominant expression of Vp8 TCR chains (Fowlkes et al., 1987) which is present on approximately 60% of CD4-8- ap T cells. The identity of progenitor cells of ap CD4-8- cells is mysterious as well as is their selection, function, and specificity. Studies of the methylation patterns of the CD8 gene in these cells may suggest that at least some of them passed through CD8+ stage (Takahama et al., 1991; Wu et al., 1990). CD4-8aP T cells can be stimulated by engagement oftheir TCRs to proliferate and secrete various lymphokines (Fowlkes et al., 1987; Zlotnik et al., 1992). A fraction of these cells expresses the NK1.l marker (Levitsky et al., 1991),and recent studies were interpreted to indicate that some

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Vp8+CD4-8- thymocytes are positively selected by class I MHC molecules expressed b y hematopoietic cells (Bix et al., 1993). 111. Extrathymically Derived lineages of T Cells

The existence of extrathymic lineages of T cells has been suspected ever since it was found that cells with the characteristics similar to thymus-derived lymphocytes were not completely absent in athymic mutant (nulnu)mice and rats. More recent studies excluded the possibility that all these cells are generated in abnormal thymic rudiments of nulnu animals (Rocha, 1990; Speiser et al., 1992a) and provided strong evidence that some yS and some ap TCR-expressing cells may be generated extrathymically, particularly in the gut and probably in the liver and in the lungs (reviewed by Haas et al., 1993; Hayday, 1993; Lefrancois, 1991a; and Rocha et al., 1992).While considering the issue of extrathymic pathways of T cell differentiation it is important to keep in mind that the results of experiments addressing this question do not exclude the possibility that at least some extrathymically derived T cells are actually thymus dependent, i.e., that their development is influenced indirectly by thymus-derived cells or factors.

A. EXTRATHYMICALLY DERIVED yS T CELLS The main population of extrathymically derived yS T cells is believed to reside in gut epithelia (Guy-Grand and Vassalli, 1993; Lefrancois, 1991a; Rocha et al., 1992). Most of these cells are characterized by the predominant usage of TCR-Vy7 and TCR-Vy1 chains, multiple TCR-VS chains, and high junctional diversity of their TCR repertoire (Bonneville et al., 1988; Kyes et al., 1989; Takagaki et al., 1989) as well as by expression of CD8a homodimers rather than CD8aP heterodimers (see Section IV.B.l)(Guy-Grand et al., 1991). They are first detected during the first weeks of life and represent the dominant population among the gut intraepithelial lymphocytes (IELs) of nude mice (Bandeira e t al., 1991; Guy-Grand et ul., 1991).They also appear in the gut epithelia after T cell-depleted bone marrow or fetal liver reconstitution of irradiated, thymectomized mice (Bandeira et al., 1991; Guy-Grand et al., 1991; Lefrancois et al., 1990).Consistent with the possibility that yS IELs are ofthe extrathymic origin are the observations that Vy7 rearrangements can be detected in Day 11 fetal gut and liver (i.e., prior to colonization of the thymus by T cell precursors) (Carding et al., 1990) and that transcripts of recombination activating gene-1 (RAG-1) have been detected in TCR-IELs (Guy-Grand et al., 1991,1992). That the gut itself has the ability to induce extrathymic T

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cell development is suggested by recent experiments in which grafts of fetal intestine, implanted under the kidney capsule of athymic radiation chimeras, were shown to have the capacity to repopulate peripheral lymphoid organs with T cells (Mosley and Klein, 1992). Other y6 T cell populations of possible extrathymic origin are found in the lungs (Augustin et al., 1989; Sim and Augustin, 1991) and in the liver (Otheki et al., 1991). The y6 IELs were reported to produce IFNy and IL-5 (Taguchi et al., 1991) but otherwise very little is known about the function and specificity of extrathymic y6 cells.

B. EXTRATHYMICALLY DERIVED ap T CELLS The gut epithelium is believed by some (Hayday, 1993; Mosley and Klein, 1992; Poussier et al., 1992) but not others (Guy-Grand and Vassalli, 1993; Rocha et ul., 1992) to be the major extrathymic site for the generation of not only yi3 but also various types o f a P TCR-expressing T cells, which in adult animals constitute the major population of IELs (Lefrancois, 1991b). On the basis of differential expression of CD4, CD8a, and CD8p molecules, the ap TCR-expressing IELs can be divided into five subsets: CD4+CD8ap-, CD4-CD8ap+, C D 4 + C D 8 a a + , CD4-CD8aat, and CD4-CD8aP-, of which CD4-CD8aa+ is the most prominent one (Guy-Grand et al., 1991; Poussier et al., 1992,1993; Rocha et al., 1992). Three of the above subsets, namely CD4+CD8ap-, CD4-CD8ap+, and CD4-CD8&, are phenotypically indistinguishable from conventional thymus-derived ap T cells, whereas the remaining two (CD4+CD8aat and CD4-CD8aa+) appear as unique to intestinal epithelium. The CD4+CD8+IELsdiffer from the majority of intrathymic CD4+CD8+ cells not only b y expression of CD8a homodimers instead of CD8ap heterodimers but also by high expression levels of ap TCR, characteristic for mature T cells. The developmental relationship between those various subsets of ap IELs and whether they are ofthymic or extrathymic origin is unclear and controversial. While several lines of evidence (reviewed by GuyGrand and Vassalli, 1993; Rocha et al., 1992) suggested that CD4'CD8- and CD4-CD8ap+ a@TCR' IELs may be thymus derived, it was claimed in recent reports that the intestinal epithelium represents the site of extrathymic development of all above-mentioned subsets (Mosley and Klein, 1992; Poussier et d., 1992). It is also not entirely clear whether the majority of C D 8 a a + IELs in normal mice is produced in situ or whether some thymus-derived ap T cells can assume this phenotype. Most recent results indicate that in lethally

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irradiated thymectomized (or RAG-2 deficient) mice reconstituted with

T cell-depleted hemopoietic stem cells one finds very much reduced numbers of CD4-CD8aaf cells in the gut epithelium suggesting either that these cells require help from thymus-derived cells for expansion or alternatively that most of the CD4-CD8aat cells in normal mice originate in the thymus (B. Rocha, personal communication). The liver is considered by some authors to represent another autonomous extrathymic site of a p T cell development, especially in older mice (Otheki et al., 1992; Okuyama et al., 1992) and others believe that it is also a site of T cell destruction (Crispe and Huang, 1994). The predominant intrahepatic a/3 T cells are CD4-CD8-TCR”lt and express the B220 epitope of the CD45 molecule (Huang et al., 1994) and are thus similar to the abnormal CD4-8- cells that accumulate in Zprllpr mutant mice (Davidson et al., 1987) suffering from lymphoproliferative disorder caused by defects in Apo-1lFas antigen that mediates apoptosis (see Section IV,B,6) ( Watanabe-Fukunaga et al., 1992; Steinberg, 1994). Because large proportions of intrahepatic B220+CD4-CD8-a/3 T cells undergo apoptosis it was proposed that the Zpr defect results in the accumulation of these cells in lprllpr mice (Huang et aZ., 1994). Strong support for the existence of extrathymic development of some ap T cells was obtained by a recent study (Makino et al., 1993) providing molecular evidence indicating that contrary to rearrangements of V a l . 1 TCR gene, which were detected in the thymus, Peyer’s patches, and spleen but not in other tissues, Val4 TCR gene rearrangements could be detected in bone marrow, liver, and intestine but not in spleen of normal and athymic mice. Little is known about the specificity and physiological function of the (rp TCR+ cells in extrathymic sites such as the gut and the liver. The strong reduction of ap TCR+ IELs in class I MHC-deficient mice (Correa et al., 1992) suggests that their numbers are regulated by binding of their TCRs to class I MHC molecules. Many a/3TCR+ IELs appear to be activated, contain granules rich in perforin and granzymes, and cytotoxicity can be revealed in certain assays (Guy-Grand et al., 1991). By these criteria they resemble terminally differentiated cells. IV. T Cell Surface Molecules Involved in an Antigen Recognition and Communication with the Antigen-Presenting Cells

Clonally distributed a@and y8 TCR heterodimers responsible for the antigen binding are integral parts of larger multisubunit protein complexes, which in addition to variable, disulfide-linked a and p or

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y and 6 TCR chains, consist of five invariant, noncovalently associated proteins ( C D ~ ECD3y, , CD36, 6, and q), believed to be involved in signal transduction (see Section IV,A,3c). The antigen-induced TCR complex-mediated signaling itself as well as the cellular response to it are positively and negatively regulated by a number of additional invariant T cell surface molecules which interact with their respective ligands on antigen-presenting cells (see Section 1V.B).The responses of antigen-inexperienced, naive T cells are generally thought to be more dependent on the regulatory influences of such ancillary molecules than the responses of antigen-experienced effector or “memory” T cells (Luqman and Bottomly, 1992; Sagerstrom et al., 1993; Sanders et al., 1988). The list of the molecules that have been shown to influence, in one way or another, the response of T cells to antigenic stimulation is long and growing (Janeway and Golstein, 1993) and includes molecules such as CD2, CD4, CD8, CD28, CD44, CD45, LFA-1, HSA, and the receptor for IL-2, just to mention a few (see Section IV,B). Antigen recognition is accompanied by changes in expression and function of several ancillary molecules on both T cells and APCs. For example, as far as T cells are concerned, it has been shown that TCR engagement increases the avidity of LFA-1 for its ICAM ligand (Dustin and Springer, 1989), induces expression of CD40L (Armitage et d., 1992; Hollenbaugh et al., 1992), CD44, and IL-2R molecules, results in downmodulation of MEL14 (L-selectin),and changes the expression of CD45 molecule from the high to the low molecular form (reviewed by Swain and Bradley, 1992). Induction of expression of B7 molecules (the ligand for CD28) on antigen-presenting B cells following antigen recognition by T cells (Nabavi et a l . , 1992; Ranheim and Kipps, 1993) may involve crosslinking of peptide-presenting MHC molecules by the T cells (Koulova et al., 1991). Thus, antigen recognition by T cells may have consequences for both the T cells as well as the antigenpresenting cells. In recent years a better understanding of the mechanisms of T cell development was obtained by studying the expression and function of various surface molecules on developing thymocytes. Bezore discussing the results of these studies it is convenient to describe some of these molecules as well as their function in mature T cells.

A. THE a/3 AND y6 TCR COMPLEXES Following the discovery of TCR molecules the progress in understanding genomic organization, rearrangement, and expression of TCRa, -/3, -6, and -y genes, as well as structure, biochemistry, a: sembly,

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function, and specificity of TCR complexes, was extensively discussed in a number of reviews (Allison and Lanier, 1987; Alt et al., 1992; Ashwell and Klausner, 1990; Benoist and Mathis, 1992; Clevers et al., 1988; Davis, 1990; Davis and Bjorkman, 1988; Exley et al., 1991; Frank et at?., 1990; Jorgensen et ul., 1992; Kronenberg et al., 1986; Leiden, 1993; Malissen et al., 1992; Malissen and Schmitt-Verhulst, 1993; Raulet, 1989; Rudd et al., 1994; Schatz et al., 1992; Toyonaga and Mak, 1987; Weiss and Littman, 1994). Therefore, in this section we will only briefly summarize the main findings, which are relevant for the following discussion of the involvement of molecules forming TCR complexes in the control of T cell development (see Section V). 1 . Organization, Rearrangement, and Expression of TCR-a, $3, -y, arLd -6 Genes a. Genomic Organization of TCR Genes. The germline organization of the murine T cell receptor loci is shown in Fig. 1. The TCRp locus is on chromosome 6 (in humans on chromosome 7 (Isobe et al., 1985; Barker et al., 1984)).The TCRa locus, which contains the TCRS locus (Chien et al., 1987), is on chromosome 14 (both in mice and in humans)(Dembicet at., 1985; Collins et al., 1985), and the TCRy locus is on chromosome 13(in humans on chromosome 7)(Kranz et al., 1985). The overall organization of TCRP, -a,and -6 loci shows remarkable evolutionary conservation, whereas the organization of TCRy locus displays greater heterogeneity in different species. Each TCR locus contains V, J, and C gene segments. TCRP and TCRG loci also contain D segments. Rearranged V(D)J elements encode the variable domains, whereas C elements encode the constant domains of TCR chains. In contrast to the TCRa and -6 loci, which contain only one C segment (Ca and CS), the TCRP locus contains two and the TCRy locus contains four C segments, one of which (Cy3) is a pseudogene. The two Cp segments, in contrast to Cy segments, are nearly identical and both are used to encode functional TCRP proteins that are expressed in all subsets of aP T cells. Upstream of each C p segment there is a cluster of seven J segments (six functional and one pseudogene) and one D segment. A pool of approximately 25 Vp genes is located upstream from the duplicated DJC clusters. T h e Vp14 element is found 3‘ of the Cp2 gene. T h e TCRa locus comprises a pool of approximately 70 V a genes, which are separated from C a by a large cluster of at least 50 J a segments and by the TCR S locus located upstream of J a cluster. The TCRS locus contains about 10 VS genes (distinct from Va), which are partially interspersed with V a genes and two JS and two

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D6 segments located upstream of C6. Although theoretically the organization of TCRa/G locus should permit the usage of the same V genes to encode TCRa and TCRG chains, in practice this does not seem to occur, at least not frequently. The molecular mechanism responsible for the usage of V6 genes i n TCRG chains and V a genes in TCRa chains is not understood. Each of the four Cy segments is associated with a single Jy and Vy segment with the exception of Cyl, which is associated with four Vy segments. The rearrangements occur almost invariably within particular VJC clusters which therefore couId be viewed as separate TCRy miniloci. Each germ line V, D, and J segment is flanked by conserved “heptamer-spacer (12 or 23 bp)-nonamer” recognition sequences recognized by the “recombinase” (see Section 1V.A.lc) during initiation of gene rearrangement, which occur via deletion of intervening DNA sequences or via inversion depending on the location and orientation of the rearranging V segment in relation to other rearranging gene segments . The studies on the transcriptional regulation of TCR genes indicated that the expression of each of then1 is controled by T cell-specific enhancers that bind partially overlapping sets of ubiquitous and lymphoid-specific transcription factors (see review by Leiden, 1993). Some ofthese factors, like TCF-1 (Oosterwegel etul., 1991) and GATA3 (Ho et ul., 1991; Joulin et al., 1991), appear to be T cell specific, are expressed very early in ontogeny (Oosterwegel et al., 1992), and are considered to be good candidates for T cell lineage determining factors. In many cases, the location of the known regulatory elements, which include transcriptional promoters, enhancers, and silencers, has been identified (reviewed by Leiden, 1993). 13. Gmerution of Diuersity. From the above description it is clear that there are significant differences in the organization and number of different gene segments from which genes encoding different TCR chains are assembled. The reasons for this are not clear. However, the different numbers of V, D, and J segments, which are available for different TCR chains, do not represent the only factor affecting the degree of diversity of the antigen-combining sites created by variable domains of ap and yG heterodiniers. The mechanisms generating diversity are also diverse and, in addition to combinatorial associations of different V, D, and J elements and conibinatorial pairing of TCR proteins forming ap and y6 heterodimers, they involve “junctional diversity” created by removing or adding nucleotides at V(D)J junctions.

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Template-dependent additions are called P nucleotides and random additions are called N nucleotides or N regions. N nucleotides are rare in V(D)J junctions from newborn mice, but contribute substantially to the diversity of TCRs from adult mice (Bogue et al., 1991; Elliot et al., 1988; Laffaille et al., 1989). In contrast to the immunoglobulin genes, there is no evidence for the contribution of somatic mutations to the generation of diversity among the TCR genes, but T cells also have a mechanism contributing to diversity of TCRs that is not available for B cells: D segments can be joined together and can be translated in different reading frames.

c. Rearrangement and Expression of TCR Genes. The early studies on the ontogeny of rearrangement and expression of murine TCR genes (reviewed by Fowlkes and Pardoll, 1989) established that the TCR loci rearrange in a distinct order such that full-length y and 8 transcripts are detected on Day 14 of gestation, i.e. 1 day before fulllength @ transcripts and 3 days before full-length a transcripts (Raulet et al., 1985; Snodgrass et al., 1985). Germ-line transcripts of the unrearranged Cy and C p segments can be detected between Days 12 and 13 (Pardoll et al., 198717) and p transcripts representing incomplete D-J rearrangements can be found between Days 14 and 15 of gestation. TCRa transcripts are detectable by Day 16. Understanding the molecular mechanisms of the initiation and completion of rearrangement at different TCR loci and of the regulation of transcription will be required to explain (1) how the ordered sequence of expression of TCR genes is established during ontogeny, (2) how T cell progenitors become commited to y8 and aj3 Iineages, and ( 3 ) how only one of the two alleles of the y , 6, and p, but not the a,TCR loci (see below) with the potential to encode TCR chain is functionally rearranged and expressed. In addition, detailed knowledge of the molecular constraints of the rearrangement and expression of TCR genes as well as of the assembly of heterodimeric TCR molecules (see Section IV,A,2) will be required to determine the relative contribution of molecular versus cellular selection of TCR molecules to the repertoire of specificities expressed in mature T cells. The exons encoding variable domains of TCR chains are assembled during lymphocyte development from V, (D),and J gene segments by a site-specific recombination reaction known as V(D)J recombination. The V(D)J recombination is believed to be mediated by a so far unidentified enzymatic machinery called recombinase, whose activity has been detected only in immature lymphocytes (Desiderio and Wolff, 1988; Lieber et al., 1987).The recombinase is thought to be endowed

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with multiple functions involving such diverse activities as recognition of RS sequences, cutting DNA strands, removing and adding nucleotides at coding junctions, and ligating rearranged gene segments (reviewed by Blackwell and Alt, 1989).In recent years important progress has been made in identifying genes encoding putative components of this complex enzymatic machinery. A few years ago two closely linked and evolutionary conserved genes (RAG-1 and RAG-2) were discovered on the basis of their ability to activate V(D)J recombination of artificial substrates in transfected fibroblasts (Oettinger et al., 1990; Schatz et al., 1989; Schatz and Baltimore, 1988).Although the precise function of the as yet unidentified products of these recombination activating genes is not known, indirect evidence strongly favors the view that they encode lymphoid-specific components of the V(D)J recombinase (Alt et al., 1992; Schatz et al., 1992). The essential role of both RAG genes in the rearrangement process has been proven in both RAG-l-deficient and RAG-2-deficient mice that are unable to initiate V(D)J rearrangement (Moinbaerts et al., 1992b; Shinkai et al., 1992). Another important component of V(D)J recombinase involved in DNA repair is believed to be encoded by the gene affected by the scid (severe combined immunodeficiency) mutation (Biedermann et al., 1991; Fulop and Phillips, 1990; Hendrickson et al., 1991).In scid mice, unlike in RAG-deficient mice, V(D)J rearrangement is initiated but due to a defect in forming coding joins (Malynn et al., 1988), rearrangements cannot be properly completed. The enzyme terminal deoxynucleotidyl transferase (TdT), for a long time suspected (Alt and Baltimore, 1982) and recently proven (Gillfilan et al. 1993; Komori et al., 1993) to be responsible for the addition of N nucleotides at VDJ junctions, can be considered as yet another component of the recombinase system. Some time ago it was postulated that susceptibility of TCR gene segments to rearrangement is controlled by transcriptional regulatory elements that determine which gene becomes accessible to the recombinase (Yancopoulos et al., 1986). It was also proposed that transcription and/or translation of productively rearranged genes prevents further rearrangement by making that locus on both homologous chromosomes inaccessible to the recombinase. Experiments in transgenic mice provided evidence supporting both suggestions: the contribution of transcription regulating elements to the control of recombinase activity was shown in mice, in which rearrangement of a transgenic TCRP minilocus was observed only when an additional enhancer element was present (Ferrier et al., 1990).

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Experimental support for the hypothesis that the product of a functional TCR gene may feed back and further inhibit rearrangement of the locus was obtained for the TCRP locus: it was shown that thymocytes that express a functional TCRP transgene fail to rearrange endogenous VD elements (Uematsu et al., 1988). It appears that this mechanism is, however, not operating at all or operating far less effectively for the TCR a locus. Rearrangement and expression of functional TCRa genes does not prevent further rearrangement, and the expression of different functional TCRa chains in and on the surface of a single cell has been observed (Malissen et al., 1992; von Boehmer, 1990). The observations that in TCR-VylJy4Cy4 transgenic mice rearrangement of endogenous TCRy but not TCRS, -P or -a loci was partially inhibited (Ferrick et al., 1989) and in yS TCR transgenic mice rearrangements of endogenous TCRy as well as TCRS loci were inhibited (Ishida et al., 1990) suggest that the mechanism of allelic exclusion of TCRy and -6 loci involves feedback inhibition by the products of functional TCR genes. Whether rearrangement of either the y or the S locus is terminated when the yS TCR binds to a selfligand, as in the case of the TCRa locus, is unclear at present. Recent evidence indicates that allelic exclusion of the TCRP locus and regulation of rearrangement and expression of the TCRa locus involves the p56lCktyrosine kinase (see Section V). Anderson and colleagues (1992) have shown that overexpression of ~ 5 6 ' in " ~developing thymocytes results in strong reduction of VP-DP rearrangement but does not interfere with and may in fact initiate TCRa rearrangement, suggesting that feedback inhibition of TCRP rearrangement by the TCRP protein involves ~ 5 6 ' " In ~ . support of this notion, a catalytically inactive dominant negative ~ 5 6 ' transgene "~ (Levin et al., 1993b) was shown to prevent allelic exclusion by a TCRP transgene (Anderson et al., 1993a)as observed in TCRP transgenic mice (Uematsu et al., 1988). Also, thymocytes expressing this dominant negative ~ 5 6 ' "mutant ~ failed to rearrange the TCRa locus (Levin et al., 1993b). In view of the above clear-cut ef'fects of transgenes encoding catalytically active and inactive forms of p56lCk,it may be surprising that rearrangements of TCRa locus were observed in thymocytes of gene targeted, p56lCk-'- (Molina et al., 1992), as well as in TCRP-'- (Mombaerts et al., 1992a) mutant mice, suggesting that a-chain gene rearrangements may be independent of TCRP proteins and of ~ 5 6 ' " ~ kinase. However, as discussed by Levin et al. (199313) the possibilities that in former mice a small amount of functional kinase was produced from the mutant transcript or that in the absence of p561ckkinase other protein kinases assumed the normal function of this molecule cannot

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be excluded. On the other hand, detection of a-chain gene rearrangements in TCRP-'- mice suggest that while a TCRp protein may be sufficient it may not be absolutely essential for a-chain gene rearrangement (see Section V). Interestingly, $-bearing cells were not absent from transgenic mice expressing a dominant-negative mutant of ~ 5 6 "indicating '~ that in contrast to TCRa genes, rearrangement and expression of TCRG and TCRy genes could occur in these mice, suggesting that different mechanisms are involved in controlling TCRG and -a-chain gene rearrangement. This conclusion is consistent with the previously mentioned observation that a TCRG transgene inhibited rearrangement of endogenous 6 gene segments (Ishida et al., 1990), whereas a TCRa transgene did not inhibit rearrangement of endogenous a gene segments (von Boehmer, 1990).The rearrangement status of TCRy and TCRG loci in transgenic mice overexpressing the catalytically active form of ~ 5 6was ~ 'not ~ reported; thus, a comparison between the influence of an active ~ 5 6 ' on " ~ap- and on $-bearing cells in these mice cannot be made.

d. y6 versus ap Lineage-Specific Expression of Functional T C R Genes. At present, the contribution of rearrangement controlling and transcription controlling mechanisms to the lineage-specific expression of TCR proteins and to allelic exclusion at different loci is not clear. With regard to commitment to the y6 or ap lineage the identification of lineage-specific transcriptional silencers in the TCRa (Winoto and Baltimore, 1989a) and TCRy genes (Ishida et al., 1990) may help to elucidate molecular mechanisms involved in this process. Early results concerning the ontogeny of TCH gene expression as well as TCR gene rearrangement in mature ap and y6 T cells (reviewed by Fowlkes and Pardoll, 1989) could not distinguish between different models accounting for their developmental relationship. One of the initial possibilities considered (Pardoll et al., 1987a), namely that precursors of cup T cells represent "dropouts" from the ys lineage that have failed to rearrange their y and 6 loci productively, has been seriously undermined (although not rigorously excluded, at least for some a p T cells) by experiments using y6 TCR transgenic mice (Bonneville et ul., 1989; Ishida et al., 1990). It was shown by these authors that in transgenic mice expressing functionally rearranged y and 6 genes, development of ap T cells (in which at least one o f t h e transgenes was transcriptionally silenced) was not impaired, as long as the regions flanking the y transgene were of appropriate length (Ishida et al., 1990; Bonneville et ul., 1989). In contrast, removal of the flanking sequences from the transgene resulted in mice in which

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development of ap T cells was strongly inhibited (Bonneville et al., 1989). On the basis of these results a model was proposed according to which the lineage-specific expression of functional TCR genes is regulated by the TCRy gene silencer (Ishida et al., 1990). The factors activating or suppressing the y silencer element would determine whether the cell will express y and 6 genes or go on to rearrange TCRP and finally TCRa genes. Thus, the presence or absence of factors activating y silencer would be a distinguishing marker for separate y6 and aP T cell precursors. The involvement of silencer in supressing rearrangement of TCRa genes in yG but not in a/3 T cell precursors was also postulated (Winoto and Baltimore, 1989a). D e Villartay and colleagues (1991) obtained results in humans suggesting that deletion of the 6 locus is a prerequisite of a/3 T cell development. The normal development of a/3 T cells in yG TCR transgenic mice, however, argues against a model in which commitment to the a/3 T cell lineage requires the absence of TCRG proteins. In another approach to the question of the progressive versus independent rearrangements of TCR genes during ontogeny of yG and C Y lineages, two groups have analyzed the rearrangement status of TCRG locus in the excision products of rearranged TCRa genes in aP T cells (Winoto and Baltimore, 1989b; Takeshita et al., 1989).The results of Winoto and Baltimore indicated that the G-chain gene in the TCRa circles had a germline configuration consistent with the model of independent regulation of rearrangement in distinct lineages. The opposite results were obtained by Takehita et al. (1989) which supports the progressive rearrangement model of the a/G locus. The issue thus remains unsettled.

e . Evidence for Intracellular (Molecular)Selection of TCR Proteins lndependent of Cellular Selection. The question to what extent the repertoire of TCR specificities expressed on T cells is the result of molecular selection or cellular selection has been clarified to some extent in recent studies. In the past, many studies were concerned with cellular selection mechanisms. The reason was not only the lack of appropriate technology that would permit analysis of molecular mechanisms in the absence of cellular selection but also the generally held assumption that the molecular processes generating diversity are essentially random. Along with the better understanding of the molecular constraints imposed on the rearrangement and expression of TCR genes there is an increasing appreciation of the importance of molecular selection mechanisms independent of cellular selection in shaping the repertoire ofTCR specificities. A good example is provided

~

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b y some papers concerned with the lack of diversity of the early expressed TCRy chain. While earlier studies were interpreted to indicate that this resulted from cellular selection (Lafaille et al., 1990), a more recent study analyzing TCRy and -6 genes in mutant mice with a disrupted C6 gene segment, preventing expression of any complete TCR 6 chain but not V6D6J6 recombination (Itohara et al., 1993), provided clear support of programmed rearrangement occurring in the absence of cellular selection. In these mutants which cannot express the y6 receptor on the cell surface, the developmental pattern of rearrangement and the junctional sequences of y and 6 genes were virtually indistinguishable from those in wild-type mice, which strongly supports the view that rearrangement of the various Vy and V6 gene segments as well as homogeneity of Vy5Jy1, Vy6Jy1, and V61DJ62 junctional sequences in fetal cells are controlled by an intracellular mechanism largely independent of TCR-mediated cellular selection. These mechanism may consist of programmed rearrangements, and “short sequence homology”-mediated joining of variable gene segments, which in the absence of TdT-mediated N region additions, occur with high frequency (Gillifan et ul., 1993; Komori et al., 1993). Other examples of nonrandom rearrangement of TCR gene segments (reviewed by Benoist and Mathis, 1992) possibly occuring in the absence of cellular selection were provided by experiments analyzing TCRa and TCRP loci (Candeias et ul., 1991; Roth et al., 1991; Thompson et al., 1990).

2. Structure und Assembly of TCR Complexes As far as we know, the ap and y6 TCR complexes differ only with regard to the antigen-recognizing heterodimers which are encoded by different genes but the rest of both complexes can be composed of the same invariant components encoded by CD3.9, CD3y, CD36, and [/v genes (van Neerven et al., 1990). 5 and 7 proteins are alternatively spliced products from a common gene (Jin et al., 1990; Clayton et al., 1991). Recently, another member of 5/77 family, termed 8, which is generated by alternative splicing event, has been described (Clayton et al., 1992).The CD3e, CD3y, and CD36 polypeptides are evolutionarily related and belong to the Ig superfamily of molecules, whereas 5 and 7,together with the y-chain of the high-affinity IgE receptor (FceRI), form a distinct family of molecules (reviewed by Samelson and Klausner, 1992). The disulfide-linked ap and y6 TCR chains are anchored in the cell membrane by transmembrane spanning portions ending by short (3-5 amino acids) intracytoplasmic C terminal tails. The N terminal extracel-

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lular part of each TCR chain consists of two (membrane proximal C and distal V) Ig-like domains, the distal ones together forming the antigen-binding site. Transmembrane CD3e, -y, and -6 chains have single extracellular Ig-like domains but their cytoplasmic regions are considerably longer (44-81aa) than those of the TCRa, -p, -y, and -6 chains. The 5 and 1) polypeptide chains have short (9aa) extracellular regions and long (113and 155aa)cytoplasmic tails. The physical association of TCR heterodimers with CD3 components in the cell membrane was demonstrated by coprecipitation, comodulation, and other methods (reviewed by Clevers et al., 1988). The stoichiometry of the TCR subunits has not yet been precisely elucidated but results of several studies suggested that two copies of C D ~ and E one copy of CD3y and CD36 are present per (YPTCRcomplex (Blumberg et al., 1990; de la Hera et al., 1991; Jin et al., 1990; Manolis et al., 1991). The results of experiments of Alarcon and colleagues (1991) and Kappes and Tonegawa (1991) suggest the possibility, however, that the stoichiometry of the TCR complex is not ySe252, but that CD36 and CD3y subdivide TCR complexes into two separate types: 6 4 2 and ye52. The 5 molecule exists primarily as a disulfide-linked homodimer (in about 90% of TCR complexes) or {/r, heterodimer (in about 10% TCR complexes). Recent evidence suggests that F c ~ R 1 ymay also be a part of TCR complex and form homodimers and heterodimers with 5 or 1) (Malissen et al., 1993; Mizoguchi et aZ., 1992; Orloff et ul., 1990; Rodewald et al., 1991). Thus, several isoforms of TCR complexes may coexist simultaneously on a T cell. In ontogeny, CD3.5, -y, and -6 proteins are synthesized before the ap and y6 TCR proteins. Initially it was believed that they remain inside the cell until TCR chains are produced and form the TCR complex, which is then transported to the cell surface. However, some murine (Ley et nl., 1989) and human (Carrel et al., 1987) T cell lines were found to express low levels of CD3 proteins in the absence ofthe TCR chains and TCR-independent expression of CD3.c was recently detected on early immature thymocytes in normal and TCRp chaindeficient, including RAG-l-deficient, mice ( Jacobs et al., 1994; Levelt et al., 1993a,b).The significance of these observations for understanding the mechanisms controlling differentiation of early, immature thymocytes will be discussed under Section V. In contrast to CD3 molecules, TCR chains in mature T cells appear unable to reach the cell membrane alone and their surface expression (with the possible exception of glycosyl-phosphatidylinositol (GP1)linked forms (Groettrup and von Boehmer, 1993a) requires intracellular association with CD3 components. The mechanism and kinetics

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of this association are not precisely established but it was suggested to occur in the following sequence: CD3sIy or CD3s/6 dimers interact with TCRa and TCRP to form ScaP and ycaP tetramers which can then interact with dimers of 6 or homologues (q and FcsRy chain) to obtain a complete TCR complex (Mallabiabarrena et al., 1992). Unassenibled or partially assembled subunits are unable to exit from the endoplasmic reticulum and are rapidly degraded (reviewed by Exley et al., 1991; Klausner et al., 1990). The mechanisms regulating the kinetics of assembly and those responsible for quantitative changes in surface expression level of ap TCR complexes, which occur during intrathymic development (see Section V), are not yet well understood but the existing evidence indicates that one regulatory factor may be the availability of (-chains because, with the exception of 6 family proteins, all other components of TCR complex (i.e., TCRa, TCRP, CD3s, CD36, and CD3y proteins) are synthesized in great excess over the quantity that is expressed on the cell surface (Minami et al., 1987). Recent evidence, however, suggests that the level of aP TCR may be regulated by posttranslational modification of TCRa (Kearse et al., 1994). The early studies on the minimal requirements for surface expression of TCR complex in mature T cells suggested that both TCRa and TCRP chains are indispensable (reviewed by Klausner et ul., 1990). Therefore, it came as a surprise when 3 years ago it was found that in immature thymocytes the TCRP chain can be expressed on the cell surface in the absence of TCRa chains (Kishi et al., 1991; Groettrup et al., 1992; Groettrup and von Boehmer, 1993a). Recent results indicate that in early immature thymocytes the TCRP chain forms a heterodimer with the newly discovered glycoprotein (gp33) (Groettrup et ul., 1993), forming a pre-T cell receptor that associates with CD3 proteins and is involved in regulation of the early intrathymic stages in T cell development (Groettrup and von Boehmer, 199317; von Boehmer, 1994) (see Section V,B). It has been hypothesized (Malissen and Schmitt-Verhulst, 1993)that different isofornis of TCR complexes may be responsible for transmitting different signals during both development and activation of mature T cells. Some experimental support for this possibility has been emerging (Bauer et ul., 1991; Mercep et al., 1989) and it will be an important task to firmly establish whether and what distinct roles in T cell development and/or function different isoforms might play. Such studies have recently been initiated by analyzing the effects of targeted inactivation of genes encoding different components of CD3 complex (Koyasu et al., 1994; Love et al., 1993; Malissen et al., 1993;

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Ohno et al., 1993,1994). The results obtained so far (see Table I ) indicate that inactivation of genes encoding different components of CD3 complex has very different effects on T cell development. The results obtained by analysis of [-deficient mice confirmed a role of [-chain in the assembly and surface expression of a/3 TCRs on thymocytes and the majority of peripheral T cells whose numbers were drastically reduced in [-deficient mice (Love et al., 1993; Malissen et al., 1993; Ohno et al., 1993). Interestingly, the populations of CD4-8a(rfTCRa/3’ and CD4-8aa+TCRyti+ IELs were only minimally affected (if at all) and expressed somewhat reduced levels of TCRs, which were found to be associated with homodimers of FccRIy chain. These observations indicate that in the absence of [-chain, the FccRIy chain appears able to sustain the development of gut IELs but not other T cell populations (Malissen et al., 1993). In contrast to [- or [/q-deficient mice, mice deficient only in 7-chain appeared completely normal and did not display any noticeable defect in T cell development, phenotype, or function (Koyasu et al., 1994; Ohno et al., 1994). In CD3c-deficient mice that also lack other CD3 components, T cell development is blocked at the early CD44-25’ intrathymic stage (see Section V ) and no mature T cells can develop (Malissen, personal communication), indicating that CD3c is essential for the development of all T cell lineages. In contrast, in CD38-deficient mice (Kappes, personal communication) development of y8 T cells seems unaffected but development of a/3 T cells is blocked at the stage of immature CD4+8+thymocytes. These results indicate that the CD38-containing isoforms of TCR complexes are dispensable for development of y6 T cell lineage as well as for the transition from CD4-8- to CD4’8’ stage during development of a/3 T cell lineage, but are essential for further differentiation of CD4’8’ thymocytes to CD4’8- and CD4-8+ T cells (see Section V).

3. Antigen (Ligand) Recognition and Signal Transduction by the a@ TCR Complex a. Specijcity of the TCR a/3 Heterodimer. Direct insight into the molecular details of the specific interaction between the a/3 TCR and the peptide/MHC complex, which can by now both b e obtained in soluble form, requires crystalization of the TCR-MHC-peptide complex. In the absence of such crystals several indirect approaches were devised to analyze the question, what does the a/3 TCR “see” and how? The results obtained have recently been extensively discussed by Davis and colleagues (Chien and Davis, 1993; Jorgensen et al., 1992).

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The emerging picture can be summarized as follows. The threedimensional structure of TCR heterodimers, deduced from the amino acid sequence comparisons, could resemble the structure of a Fab fragment of an antibody molecule (Chothia et al., 1988; Claverie et al., 1989). By analogy to the antigen binding site ofantibody molecules, at least three hypervariable regions, called CDR1, CDR2, and CDR3, can b e distinguished in TCRa and -0 chains. The most variable CDRS region is encoded by the V(D)J junctions, whereas C D R l and CDR2 are encoded by germline sequences of the V gene segments. The results of many studies, which analyzed the effects of single amino acid substitutions of peptide or MHC residues (predicted to interact with TCR) on recognition of the complex by various T cell clones indicate that TCR heterodimers contact both peptide as well as MHC residues (Chien and Davis, 1993).Other observations seem to be consistent with the proposition (Chothia et al., 1988; Claverie et al., 1989; Davis and Bjorkman, 1988)that the CDRS region is principally responsible for peptide contact, whereas C D R l and CDR2 regions contact the MHC molecules. The analysis of the crystalized peptide/MHCclass I (Fremont et al., 1992; Young et al., 1994) and peptide/MHCclass I1 complexes (Stern et al., 1994) revealed that only about 30% of the peptide surface is potentially available for interaction with the TCR. Substitution for peptide or MHC residues buried in MHC grooves, which do not affect peptide binding, can also alter T cell recognition (Boehncke et al., 1993; Busch et al., 1991; Ong et al., 1991) probably by altering the peptide surface that directly contacts the TCR. Conformational changes in class I MHC molecules induced by different peptides and affecting allorecognition have been reported (Bluestone et al., 1992; Catipovic et al., 1992; Fremont et al., 1992; Sherman et al., 1993), but how general and significant these changes are for selfMHC restricted peptide recognition by TCR is not clear. Altogether, the results suggest that the TCR-peptide contacts and TCR-MHC contacts are dependent on each other but that the former are more “rigid” and appear to have stronger influence on the ultimate configuration of the trimolecular complex than the latter, which are more “flexible” (Chien and Davis, 1993).

b. The Role of the TCR a0 Heterodimer in Signal Transduction. The aim of studies concerned with the signal transduction in T cells is to understand how TCR occupancy is sensed and translated into biochemical reactions that stimulate T cells to divide, to differentiate, or to die. For convenience, the whole process of signal transduction can be divided into three compartments: the cell surface proximal

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events, the cell surface distal cytoplasmic events, and the nuclear events. In recent years there has been significant progress in identifying and connecting important components in each of these compartments but we are still a long way from understanding the entire cascade and even further from understanding how these events are related to T cell development or to the immune response. In the following discussion, we will briefly outline our knowledge of the major events that occur in various compartments following TCR triggering. We will begin by considering the role ofthe TCR ap heterodimer. Because the cytoplasmic tails of antigen-binding heterodimers are short it is unlikely that they have any enzymatic activity or serve as a substrate. The signal transducing function is thought to rely on other components of the TCR complex, namely the CD3 and 5/77 proteins, whose cytoplasmic tails appear suited to transduce signals to intracellular signaling pathways (see below). Until recently, the role of (rp (or yS) heterodimers in initiating T cell responses was generally believed to be passive and limited to the binding of the ligand, whose only function would b e to crosslink TCR complexes, an event considered sufficient to activate signal transduction machinery. This view is now being revised and the possibility that the ap TCRs have an active role in signal transduction received a lot of attention. The present interest in the possible active role of the ap heterodimer stems from observations which indicate that various modes of T cell responses to TCR occupsincy can b e achieved by subtle modifications of immunogenic peptides (DeMagistris et al., 1992; Evavold and Allen, 1991; Evavold et al., 1993a; Jameson et al., 1993; Sloan-Lancaster et al., 1993; Racioppi et al., 1993; Ruppert et al., 1993; reviewed by Evavold et al., 1993b). The observations indicate that the occupancy of the TCR does not result in the “all or none” type of T cell response but that it can induce several qualitatively different responses. For example, single amino acid substitution in the antigenic peptide can result in the inability to stimulate proliferative response of the responding T cell clone without affecting the ability to induce IL-4 production (Evavold and Allen, 1991). The modified peptides, which selectively affect different types of responses, have been called partial agonists (Evavold et al., 199313). The modified peptides may also behave as antagonists, in which case they inhibit the response to the wild-type peptide without delivering any identifiable positive signal to the cell (DeMagistris et al., 1992; Jameson et al., 1993). The mechanisms by which partial agonists and antagonists mediate their effects are unknown but one possibility is that they induce different conforma-

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tional changes in the a@ heterodimer which are then differentially "sensed" by components of the CD3 complex. This possibility has some experimental support in the earlier studies suggesting that the TCR may undergo conformational changes upon reaction with antiTCR antibodies (Rojo and Janeway, 1988; reviewed by Janeway and Bottomly, 1994).

c. The Role of CD3-.5, -6, -y, and 517) lnvariant Chains. Recent evidence suggests that the CD3.5, -y, -6, and -5 subunits may form at least two autonomous signal transduction modules (Wegener et al., 1992; Letourneur and Klausner, 1992). The signal transducing functions of the invariant chains of the TCR complex are thought to result from the presence of a single (CD3.5, -y, and -6 chains) or triple (5chain) tyrosine-containing sequence motifs (Reth, 1989) (called TAM by Samelson and Klausner, 1992; ARAM by Weiss and Littman, 1994; and ARHl by Cambier and Jensen, 1994) that couple these proteins to tyrosine kinases (PTKs). One of the first signaling events involves TAM phosphorylation by Src family PTKs: p59fy", which is physically associated with the TCR complex (Samelson et al., 1990), and ~ 5 6 ' " ~ at least some of which is associated with the cytoplasmic domains of CD4 or CD8 molecules (Rudd et al., 1988; Veillette et al., 1988; and see below). The activity of both p59fY" and ~ 5 6 ' was ' ~ shown to increase following TCR stimulation (Danielian et al., 1992; Tsygankov et al., 1992).The events initiated by the phosphorylation of TAMS have been recently discussed in detail by several authors (Cambier and Jensen, 1994; Rudd et al., 1994; Weiss and Littman, 1994) and although the sequence of interactions between PTKs and their substrates is not yet precisely defined, the proposed scenario can be briefly summarized as follows. The phosphorylation of TAMS by Src family PTKs results in recruitment and binding of ZAP-70, a member of another family of PTKs, whose structural features, unlike those of Src family PTKs, does not allow for constitutive association with the plasma membrane. The biochemical reactions that follow after association of ZAP-70 with the TCR complex are not clear but it is believed that the amplified PTKs activity results in association and phosphorylation of downstream effector molecules such as y l isoenzyme of phospholipase C (PLC) and SH2-domain-containing adaptor proteins like Vav (Gulbins et al., 1993), Grb-2, mSOS, or Shc (Ravichandran et al., 1993) implicated in linking receptors (via PKC independent pathway, see below) to the activation of Ras, a GTP-binding protein with GTPase activity. It may be interesting to make a digression here and note that quite recently

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an isoform of Grb-2 with apoptotic properties (see Section V,D,2) has been identified and cloned (Fath et al., 1994). Activated PLC catalyzes the hydrolysis of phosphoinositide 4,sdiphosphate to generate diacylglycerol and inositol triphosphate, thereby stimulating PKC and mobilizing intracellular Ca2+,respectively. Sustained increase in Ca2+concentration results in the activation of calcineurin, a calcium/calmodulin-dependentserine phosphatase. One of the targets of calcineurin activity is a cyclosporin A-sensitive component of the NF-AT transcription factor, which regulates interleukin-2 (IL-2) gene expression. PKC activation leads to serine phosphorylation of multiple cellular substrates and induces activation of Ras and c-raf kinase, which in turn activate Mek and MAP kinases implicated in regulating the activity of transcription factors, including the nuclear component of NF-AT composed of the c-fos and c-jun related proteins. Other transcription factors affected by the TCR signal transduction pathways that are currently intensely investigated because oftheir possible role in determining the fate (death or survival) of activated T cells include c-myc and Nur-77 (see Section IV,C). When trying to understand the role of TCR-mediated signaling in the development and function of T cells it is worth keeping in mind that altogether well over 100 genes are activated in T cells following antigenic stimulation (Ullman et al., 1990). B. T-CELLSURFACE MOLECULESTHAT REGULATE THE FUNCTION OF THE TCR COMPLEX AND/OR THE CELLULAR RESPONSE TO ANTIGENICSTIMULATION As already mentioned, the TCR complex does not act alone in antigen recognition. It is assisted directly and indirectly by a number of cell surface molecules, some of which regulate its signal transducing function, and others that are believed to play an important role in determining whether the induced cell will perform its function (i.e., divide, differentiate, kill, or secrete lymphokines), will become anergized (i.e., will persist in a quiescent state refractory to further stimulation), or will die by apoptosis. By functional criteria such ancillary molecules are usually classified as (1)coreceptors (e.g., CD4 and CD8), (2)costimulators (e.g., CD2, CD28, HSA), (3)adhesion molecules (e.g., LFA-1, CD44, VLA-4, VLA-5, VLA-6), and (4) receptors for growth factors (e.g., IL-2R). Coreceptors interact with the same peptidepresenting MHC molecules that are specifically recognized by the a@ TCR and amplify the TCR-mediated signals. Costimulators interact with their own specific ligands and behave as independent signal

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transducing molecules influencing the cellular response by a TCRindependent signaling pathway. Adhesion molecules facilitate and stabilize the contact between T cells and APCs. Growth factor receptors react with soluble ligands andare responsible for control ofcell proliferation and differentiation via TCR-independent signals. The above classification is quite arbitrary and emphasizes the main functions of these molecules as we understand them but it is clear that coreceptors and costimulators also act as adhesion molecules, whereas adhesion molecules are also signal transducers (Hynes, 1992; O'Rourke and Mescher, 1993; Seventer et al., 1991). In addition, T cells express other surface molecules with specific functions, like CD45 or Apo-1/Fas (see below), which cannot be easily assigned to any ofthe above categories. We will now briefly summarize the current view on the role of some of these molecules in modulating TCR-mediated signal transduction and in influencing its outcome in mature as well as developing T cells.

1 . CD4 and CD8 Coreceptors (Reviewed by Micelli and Parnes, 1993) CD4 and CD8 molecules are transmembrane proteins whose extracellular domains can specifically interact with conserved, nonpolymorphic regions ofclass I1 and class I MHC molecules, respectively (Konig et al., 1992; Salter et al., 1990). CD4 is a single polypeptide with an extracellular part consisting of four Ig-like domains. CD8 is a dimer of polypeptides-either a-chain homodimers or ap heterodimers-each having an amino-terminal Ig-like domain. The great majority of ap T cells expresses the CD8ap heterodimer. The crystal structure of fragments of CD4 molecules (Ryu et al., 1990; Wang et al., 1990) and CD8-a homodimers (Leahy et al., 1992) that are implicated in the interactions with MHC molecules was determined and their binding sites on class I1 (Cammarota et al., 1992; Konig et al., 1992) and class I (Connolly et ul., 1990; Salter e t a l . , 1990) MHC proteins, respectively, have been mapped. As already mentioned, the cytoplasmic domains of CD4 and CD8a chains interact with p56lck, which may account for their ability to influence the TCR complex-mediated signaling. Several years ago it was noticed that coaggregation of either CD4 or CD8 with the TCR complex results in greatly enhanced T cell growth (Emmerich et al., 1986; Ledbetter et al., 1988)accompanied by increased tyrosine phosphorylation of cellular proteins (Ledbetter et al., 1990)when compared to that induced by aggregating the TCR complex only. The mechanism responsible for this effect is not clear (see below) but it may result, at least under conditions when ~ 5 6 ' is " ~limiting, from the juxtaposition

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of p56lCkto signal-transducing invariant chains of the TCR complex, leading to tyrosine phosphorylation of TAMs. On the other hand, ligation of CD4 alone (Newel1 et al., 1990; Tite et al., 1986),but according to Julius and colleagues (1993)not CD8 alone, inhibits T cell activation triggered via the TCR complex. Since one possible mechanism of the inhibitory effect of the aggregation of CD4 may be the sequestration of ~ 5 6 ’from ” ~ TCR complex (Julius et al., 1993), the difference between the effect of CD4 and CD8 aggregation on subsequent stimulation through the TCR complex may be due to the different stoichiometry of their association with p56’“. The proportion of cellular ~ 5 6 ’ ” ~ associated with CD4 was shown to be 10- to 20-fold greater than that associated with CD8 (Veillette et ul., 1989). However, the ability of CD8 to negatively regulate TCR-mediated signaling, possibly through sequestration of p561Ck, was demonstrated in a system in which CD4-8thymocytes from TCR transgenic mice were reconstituted with a transgenic CD8a molecule (van Oers et al., 1993). The above interpretations of positive and negative influences of CD4 and CD8 molecules on TCR-mediated signaling are consistent with a model of antigen-induced activation, in which crosslinking of the TCR with coreceptors by the same MHC molecules provides the primary stimulus responsible for the amplification of an otherwise weak signal generated by the TCR alone, possibly by recruiting ~ 5 6 ’ “ ~ into close proximity of TAMs. Recent observations, however, indicate that this mechanism represents only one side of the coin and suggest that as much as the crosslinking of coreceptor with TCR may regulate the TCR-mediated signaling, the TCR-mediated signal itself may regulate the ability of coreceptors to “concentrate” on the same MHC which is being “seen” by the TCR, thus increasing the avidity of the interaction and facilitating signaling. Xu and Littman (1993) have found that under certain conditions, deletion of the catalytic domain from CD4-associated p56lck did not impair but rather improved the coreceptor function of the CD4 molecule, provided that the SH2 domain mediating the interaction of lck with other proteins was preserved. The authors offer one likely interpretation of this observation-that the SH2 domain of CD4-associated ~ 5 6 ‘ engages “~ with tyrosine-phosphorylated residues of the TCR complex, the phosphorylation being the result of the encounter with antigen that may or may not involve crosslinking with coreceptors. Such a mechanism would result in anchoring coreceptors to the TCR complex and ensure that they will be brought into the proximity of MHC/peptide complex recognized by the TCR via an intracellularly regulated mechanism. O’Rourke et al. (1990) and O’Rourke and Mescher (1992)have shown

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that the increase of the avidity of CD8 molecules for class I MHC molecules following activation of the TCR with TCR antibodies could be blocked by the tyrosine kinase inhibitors herbiniycin A and genistein, providing another example of the regulatory influence of an PTK-dependent pathway of TCR signaling on the function of coreceptors. The model emphasizing the importance ofTCR/coreceptor crosslinking for T cell activation does not necessarily imply that in the resting state CD4 and CD8 molecules are dissociated from the components of TCR complex. In fact, recent observations suggest that the majority of CD36 chains may be associated with CD4 or CD8 molecules on resting T cells (Suzuki et al., 1992), which is compatible with a model in which recognition of antigen alters the configuration of the existing association of coreceptors with TCR such as to optimize the phosphorylation of the components of the TCR complex rather than causing de novo association of previously dissociated components. CD4 and CD8 proteins may also function as tailless adhesion molecules completely independent of ~ 5 6 ' (Killeen "~ and Littman, 1993; Chan et al., 1993a) (see Section V,C,2a). Although CD4 and CD8 coreceptors perform many analogous functions on CD4+8- and CD4-8+ T cells, respectively, there are several differences between them in addition to their distinct molecular structure and differential abilities to bind to and activate ~ 5 6 ' "The ~ . significance of these differences (Micelli and Parnes, 1993) for the physiological role of CD4 and CD8 molecules is not clear. The importance of CD4 and CD8 coreceptor molecules for T cell development and function has been confirmed by analysis of mice generated by targeted disruption of CD4 (Rahemtulla et al., 1991) or CD8a (Fung-Leung et al., 1991) genes. These mice showed selective and strong impairment of helper or cytotoxic T cell responses, respectively. In mice deficient for both CD4 and CD8 (Schilham et al., 1993), both functions were strongly affected. Recently, it has been reported that in CD8p knockout mice, deficient in CD8ap heterodimers, but not CD8aa homodimers, development of CD4-8' thymocytes and T cells is inhibited, indicating that CD8p may be important for the maturation of CD4-8+ T cells (Nakayama et al., 1994).

2 . The CD45 Glycoprotein (for Review See Thomas 1989) CD45 is a transmembrane glycoprotein with intrinsic tyrosine phosphatase activity (Tonks et aZ., 1989; Charbonneau and Tonks, 1992) and is found on all nucleated cells of hemopoietic origin. It exists in many isoforms, which are the result of alternative splicing of the exons

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4,5, and 6. The other CD45 exons are expressed in all isoforms, which have the same cytoplasmic domain consisting of two tyrosine-specific phosphatase (PTPase) domains in tandem. On T cells, different CD45 isoforms display a complex pattern of expression, which appears to be regulated by poorly understood developmental and antigen-driven mechanisms. The majority of developing thymocytes and activated T cells express low-molecular-weight CD45 isoforms, while mature thymocytes and naive T cells express highmolecular-weight isoforms. Expression of CD45 is essential for TCR-mediated signaling in T cells. This was shown by studies of T cell lines deficient in CD45 expression which display a specific defect in early PTK-dependent events of TCR-mediated signaling (Koretzky et al., 1990; Pingel and Thomas, 1989). While the identity of the physiological ligand for extracellular domains of CD45 is uncertain, the target of its cytoplasmic domain is believed to be the negative regulatory site of tyrosine phosphorylation in the PTKs ~ 5 6 ' and " ~ p59fYn. By dephosphorylating this site, CD45 is thought to activate the catalytic function of PTKs thus allowing their participation in TCR-mediated signaling. The negative regulation of the catalytic function of ~ 5 6 ' and " ~ p59fgn is believed to depend on another cytoplasmic tyrosine kinase, called ~ 5 0 "(Chow '~ et al., 1993), which phosphorylates their negative regulatory sites. The function ofthe extracellular domains of CD45 is not clear (Alexander et al., 1992; Julius et al., 1993); nevertheless, it was suggested that different isoforms may be responsible for different arrangements of the TCR and its coreceptors thus controlling the quality of the generated signal at different stages o f T cell development and in different T cell subsets ( Janeway, 1992).Recent studies with chimeric receptor PTPase suggest that the dimerization of the CD45 molecule by its ligand may negatively regulate TCR-mediated signaling (Desai et al., 1993). The severely compromised immune system ofmice deficient in exon 6 of the CD45 gene (Kishihara et al., 1993) provided evidence for the importance of CD45 molecules in both T cell development and T cell function.

3. Molecules Znvolved in T Cell Costimulation Some time ago it was noticed that TCR-mediated signaling is not sufficient to induce naive T cells to respond optimally to antigen stimulation and that an obligatory second signal delivered by antigenpresenting cell is required (reviewed by Schwartz, 1990). In the

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absence of costimulation, TCR signaling may result in long-lasting unresponsiveness to antigen stimulation (anergy) (Jenkins et al., 1987) or may lead to cell death (Liu and Janeway, 1990). In recent years the role of CD28 molecule in transducing such costimulatory signals in T cells, following binding of its ligand (for review see Linsley and Ledbetter, 1993) thereby preventing the induction of anergy in T cell clones (Harding et al., 1992), has become apparent. CD28 is a homodimeric, disulfide-linked transmembrane glycoprotein with a single Ig-like domain in the extracellular region and a short cytoplasmic domain. It is expressed on all murine CD4'8- and CD4-8+ T cells as well as on CD4+8+thymocytes (Gross et al., 1992). y6 T cells also express CD28. The natural ligands for CD28 are the B7-1 (Linsley et al., 1990,1991)molecules expressed on activated B cells, dendritic cells, and monocytes, and B7-2 (Freeman et al., 1993; Hathcock et nl., 1993) molecules that are also expressed on resting monocytes. Interaction of CD28 with B7 molecules generates two kinds of signals: calcium-independent/cyclosporinA-resistant and calcium-dependent/cyclosporin A-sensitive signals. Thus, at least one arm of the CD28-mediated signaling pathway appears to be distinct from that used by the TCR. Both CD28-induced pathways, i.e., calcium dependent and independent, seem to be initiated by PTK activity and can be ablated by coclustering with CD45 (Lu et d., 1992; Vandenberghe et nl., 1992) suggesting that dephosphorylation of a CD28associated substrate is responsible for this effect. CD28 signals are not effective when delivered before TCR stimulation (Linsley and Ledbetter, 1993). The main result of CD28-mediated signaling is the stabilization of lymphokine mRNAs and an increased rate of lymphokine gene transcription (Lindsten et al., 1989). Recently, new insights into the mechanism of signaling via CD28 and its integration with TCR-mediated signaling were obtained by the observation that the lipid kinase PI-3 kinase, implicated in a putative phosphorylation-independent signaling pathway (see Rudd e t a1., 1994), binds to a specific phosphorylated motif within the cytoplasmic tail of CD28 (Prasad et al., 1994) following CD28 ligation. On the basis of these and other results, the following two-step scenario integrating the initial events in TCR-mediated signaling with CD28mediated signaling was suggested (Rudd et ul., 1994): step 1 intiated by the activated TCR complex and mediated by ~ 5 6 "and ~ p5gYn kinases involves binding of ZAP-70 as well as other TCR(/CD3 binding proteins and leads to the activation of PTKs that phosphorylate the CD28 pYMXM motif, thus providing minimal conditions for the binding of the PI 3-kinase to CD28. In step 2, ligation of CD28 by B7

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would induce further recruitment of the PI-3 kinase, thereby providing the second signal needed for IL-2 production and onset of DNA synthesis. CTLA-4 is a second T cell receptor for B 7 that is closely related to CD28 (reviewed by Linsley and Ledbetter, 1993), but unlike CD28 it is expressed only on antigen-experienced T cells and not on naive T cells. On activated T cells, CD28 and CTLA-4 are coexpressed and synergize in delivering costimulatory signals (Linsley et al., 1992). Considering the above-mentioned evidence for the important role of CD28 in regulating T cell responses to TCR stimulation in vitro, it was somewhat surprising to find that in CD28 knockout mice development of T cells appeared normal and only some helper functions of CD4'8- T cells were impaired (Shahinian et al., 1993). The in vitro responses to B 7 stimulation are influenced b y the interactions of LFA-1 with ICAM-1 (Damle et al., 1992; van Seventer et al., 1990) which, unlike CD28, signal the T cell via the same biochemical second messengers as those generated through TCR engagement (van Seventer et al., 1992), while signaling via CD28 may modulate T cell adhesion (Shimizu et al., 1992). Other receptor-ligand pairs enhancing T cell activation include CD2-LFA-3 (Bierer et al., 1989), VLA-4(5)-fibronectin, and VLA-6/ laminin (Burkly et al., 1991; Davis et al., 1990; Shimizu et al., 1990a). Heat stable antigen (HSA) expressed on dendritic cells and B cells has also been reported to provide costimulatory signal for CD4'8- T cells (Liu et al., 1992). The nature of the T cell receptor that interacts with the costimulatory HSA molecule is not known.

4 . The CD44 Glycoprotein (for Review See Lesley e t al., 1993) CD44, also known as Pgp-1, is composed of a family of alternatively spliced transmembrane glycoproteins that bind hyaluronate. It is expressed on lymphoid progenitors. It is primarily a cell adhesion molecule with poorly understood function during the earliest stages of intrathymic T cell development. The level of CD44 decreases as T cell precursors mature and then increases again when mature T cells are activated. 5. The IL-2R Complex (for Review See Minami et al., 1993) The IL-2R is a growth-factor receptor made up of at least three transmembrane chains: IL-2Ra (CD25), IL-BRP, and IL-2Ry. Genes encoding IL-2Ra and the IL-2 are silent in resting T cells but are efficiently induced upon T cell activation. The IL-2RP gene is constitutively expressed in some but not all T cells, whereas IL-2Ry is constitu-

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tively expressed in all T cells. The IL-2Ra chain is dispensable for signal transduction but is involved in the formation of the high-affinity IL-2R complex. The other two chains are required for formation of the high-affinity complex as well as for signal transduction, although the role of IL-2Ry in IL-2 signaling remains unclear. Expression of the high-affinity IL-2R complex is induced by TCR stimulation. The IL-2R components lack intrinsic protein tyrosine kinase activity and their signal-transducing function, like that of the TCR complex, is dependent on association of IL-2RP with p56lCk,but the signaling mechanisms associated with the IL-2R are distinct from those activated by the TCR-recognizing antigen (Cantrell et al., 1993). In the end both induce activation of Ras and PI-3 kinase but they do this by different mechanisms. IL-2R-mediated signals do not induce phosphatidylinosito1 hydrolysis, increases in intracellular calcium, or activation of PKC. Ligation of the IL-2R induces rapid expression of immediate-early genes, including protooncogenes bcl-2, c-fos, c-jun, c-myc, and others. As several of these genes encode DNA-binding proteins, it is believed that these gene products are involved in regulating expression of the later genes responsible for cellular differentiation and specific function. An important insight into the possible role of IL-2Ry has been obtained by the observation that a nonsense mutation of the IL-2Ry gene in humans results in X-linked severe combined immunodeficiency that is characterized by the absence of thymocytes and mature T cells (Noguchi et al., 1993). This observation, and the fact that in IL%deficient mice the development of T cells is unperturbed (Schorle et al., 1991; Kundig et al., 1993; Sadlack et al., 1994), have led to the speculation that the IL-2R complex, in particular IL-2Ry, may have an additional function to those recognized today (Mills, 1993; Taniguchi and Minami, 1993).

6. Apo-1 lFas Apo-1 (Trauth et al., 1989) and Fas (Yonehara et al., 1989) are the names of identical cell surface molecules identified on human and mouse cells, respectively. Apo-11Fas is a membrane-spanning protein homologous to tumor necrosis factor and nerve growth factor receptors (Itoh et al., 1991; Oehm et al., 1992). It is the only known cell surface molecule whose function appears to be specifically linked to the induction of programmed cell death, but recently it has been reported that it may also play a role in stimulating cell proliferation (Alderson et al., 1993).A region ofthe intracellular part ofApo/Fas shows homology with the “death domain” of the p55 T N F receptor and may be important for transmission of the apoptotic signal (Tartaglia et al., 1993).

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The intrathymic developmental pattern of expression of Apo-1IFas appears to be inversely correlated with expression of Bcl-2 protein, an intracellular inhibitor of apoptosis (see below). It is expressed on a minority of immature CD4-8- thymocytes (Andjelic et al., 1994),then at high levels on CD4+8+thymocytes (Andjelic et al., 1994; Debatin et al., 1994; Drappa et al., 1993), and downregulated in mature thymocytes (Debatin et al., 1994). The signaling pathway activated through Apo-l/Fas and its relationship to TCR-mediated signal transduction pathways are not known. The natural ligand of Apo-1/Fas molecule, which also belongs to the tumor necrosis factor family, has recently been described (Suda et al., 1993). C. INTRACELLULAR MOLECULES IMPLICATED IN T CELL FATEDETERMINATION In addition to cell surface molecules which are important for effective cell-cell interactions and regulation of TCR-mediated signaling, a growing number of genes encoding intracellular proteins attract attention as possible regulators of the cellular response to TCR triggering. Below we will briefly describe some of those that have been implicated to play a role in determining the fate (death or survival) of activated T cells.

1 . Bcl-2 The bcl-2 gene, first described as an oncogene in a human B cell lymphoma (Tsujimoto et al., 1985), encodes an intracellular, membrane-associated protein that was shown to be involved in protecting various cell types, both in uitro and in uioo, from many hut not all forms of programmed cell death (apoptosis) (Allsop et al., 1993; Hockenberry et al., 1990; Nunez et al., 1990; Vaux et al., 1988). The bcl-2 gene shares sequence similarity with the nematode Caenorhabditis elegans gene ced-9 (Hengartner and Horvitz, 1994) which suppresses ced-3 and ced-4 gene-dependent programmed cell death in that worm. It was found that the human bcl-2 gene can suppress programmed cell death in C. elegans (Vaux et al., 1992) indicating that the regulation of programmed cell death has been highly conserved during evolution. Recently, the protease interleukin-lp-converting enzyme was found to be a mammalian homologue of the ced-3 gene product, which was shown to be required for programmed cell death in C. elegans (Miura et al., 1993; Yuan et al., 1993). Recently, a number of bcl-%related genes have been identified in mammalian cells, including bax (Oltvai et al., 1993) and bcl-x (Boise et al., 1993), whose products may regulate bcl-2-dependent or bcl-2-

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independent pathways of apoptosis (see Section V.D). In the thymus, the expression level of bcl-2 mRNA and protein is developmentally regulated, suggesting that it is involved in thymocyte selection (Andjelic et ul., 1993b; Veis et al., 1993a) (see Section V,C). It has been reported that the Bcl-2 protein acts as an antioxidant (Hockenberry et ul., 1993) but the molecular mechanism by which bcl-2 exerts its apoptosis-preventing effect is not known. Considering the generally accepted view that crucial events responsible for induction and regulation of apoptosis occur in the cell nucleus (King and Ashwell, 1993), a recently reported finding that bcl-2 protection from apoptosis can be demonstrated in anucleate cytoplasts ( Jacobson et ul., 1994) is surprising and shows how little is understood about the mechanisms involved. The general physiological importance of Bcl-2 protein has been documented in bcl-2-deficient mice generated by gene targeting (Veis et al., 1993b). Mice lacking Bcl-2 appeared normal at birth but displayed growth retardation and early postnatal mortality accompanied b y widespread apoptotic death in lymphoid organs. The latter developed until 3 weeks of age without any distortion of the proportions of thymocyte subsets or T cells. Similar observations were made in mice, in which bcl-2 was selectively inactivated in lymphoid cell lineage only (Nakayama et al., 1993): intrathymic development of T cells also appeared normal but mature T cells displayed increased sensitivity to glucocorticoid and y-irradiation and disappeared by 4 weeks of age. Interestingly, in uitro treatment with CD3 antibody inhibited death of these cells. 2. Nur-77 The Nur-77 protein (Hazel et al., 1988)is a nuclear hormone receptor whose function was unknown until recently. This year it was reported (Liu et al., 1994; Woronicz et ul., 1994) that Nur-77 is present at high levels in an apoptotic T cell hybridoma and in apoptotic thymocytes but not in growing T cells, suggesting that Nur-77 may be critically involved in determining the fate of a T cell responding to TCR engagement.

3. Glucocorticoid Receptor Immature and mature T cells have about the same number of cytoplasmic glucocorticoid receptor (GR) molecules but their sensitivity to glucocorticoids is very different (Homo et ul., 1980). Whereas cortical thymocytes are killed at glucocorticoid concentrations within the physiological range, most T cells are resistant. Glucocorticoid-induced death

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of cortical thymocytes is the classical model of apoptosis (Wyllie, 1980; reviewed by Cohen, 1992). Recently, there has been a renewed interest in the role of glucocorticoids as regulators of T cell development (see Section V,D,2).

4 . p53

The p53 tumor suppressor gene encodes a DNA-binding protein (Kern et al., 1991) implicated in regulating normal cell proliferation by controlling a checkpoint during the G1 phase of the cell cycle that may monitor the status of DNA before entry into S phase (Lane, 1992). The association of p53 with apoptosis in cultured cell lines has been known for some time (Yonish-Rouch et al., 1991)but its role in controlling some forms of apoptosis of T cells has only recently been demonstrated. By analyzing thymocytes from mutant mice lacking p53, it was found that p53 is required for radiation and etoposide-induced death of CD4'8' thymocytes (Clarke et al., 1993; Lowe et al., 1993).

5. C-M~JC The c-Myc protein is a transcription factor implicated in the control of normal cell proliferation. Recently, c-Myc was shown to induce apoptosis in cultured fibroblasts (Evan et al., 1992) stimulating interest in its possible involvement in regulating death of developing thymocytes (see Section V,D,2). 6. c-Jun and c-Fos The c-Jun and c-Fos proteins are members of the ubiquitous AP-1 family of a rapidly inducible transcription factors involved in regulation of transcription of cytokines in activated T cells, including IL-2. The c-Jun and related proteins form homo- or heterodimers with one another or with c-Fos and related proteins. Such dimeric AP-1 factors bind to consensus DNA sequences, the AP-1 sites within the IL-2 regulatory sequences (Angel et al., 1991).c-Jun can also form a complex with Oct-1, which can transactivate the ARRE motif in the IL-2 enhancer (Ullman e t al., 1993), and plays a role (together with c-Fos) in the formation ofthe lymphoid-specific transcription factor NF-AT ( Jain et al., 1993). Recent results indicate that inducibility of NF-AT and AP-1 complexes by calcium ionophore and phorbol esters correlates with the developmental stages of thymocytes (Chen and Rothenberg, 1993); they can be induced in mature CD4+8-, CD4-8+, as well as in immature CD4-8- thymocytes, but not in immature CD4+8+thymocytes. The involvement of c-fos and c-jun in regulating cell death is sug-

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gested by the observation that apoptosis induced in lymphoid cell lines by growth factor deprivation is preceeded by the upregulation ofc-fos and c-jun expression and can be blocked by antisense oligonucleotides which decrease their levels (Collotta et al., 1992). In c-jun-deficient mice (Chen et al., 1994) generated by RAG-2deficient blastocyst complementation method (Chen et al., 1993), which allows investigation of the lyniphocyte-specific roles of genes whose inactivation causes embryonic lethality, thymuses are small but have normal subset composition, and peripheral T cells showed normal activation responses. In c-fos-deficient mice some authors observed a deficit of immature CD4+8+thymocytes (Wang et al., 1992), while others did not (Jain et al., 1994). However, the development and function of mature T cells was not affected. These results suggest that other members of Fos and Jun families are able to functionally substitute for c-Fos and c-Jun during T cell development. V. lntrathymic Development and Selection of T Cells

Functionally distinct subsets of thymus-derived T cells are the end products of several thymocyte lineages branching out from common precursors with progressively restricted developmental potential. During T cell ontogeny, the expression ofvarious genes and their products, some of which were described in previous sections, is turned on and off in a defined order allowing to distinguish several stages in intrathymic development of blood-borne precursors. On the basis of the developmental order of the cell surface expression of TCR molecules it is convenient to divide the whole process of the intrathymic development into three phases: the early phase, controlled by mechanisms independent of surface expression of the variable chains of TCR complex; the intermediate phase, controlled by mechanisms dependent on the expression of the TCRP chain; and the late phase, controlled by mechanisms dependent on the surface expression of the LYPTCR complex. Since we know little about the existence of different stages in the development of various lineages of y8 T cells, in the following we will focus almost exclusively on the development of the major (CD4+8and CD4-8') thymus-derived (YP T cell lineages.

A. FROM THYMUS COLONIZING CELLSTO TCRP EXPRESSING CELLS

The earliest thymocytes are CD2-CD8-CD25- and express c-kit receptor tyrosine kinase (Matsuzaki et al., 1993), CD16 (low-affinity FcyR) (Rodewald et al., 1993), CD44, and low levels of CD4 (Wu

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et al., l99la). They have TCR genes in the germ line configuration (Rodewald et al., 1993; Wu et al., 1991b) and following adoptive transfer can give rise not only to y6 and ap T cells but also to B cells (Matsuzaki et al., 1993;Wu et al., 1991b)natural killer cells (Matsuzaki et al., 1993; Rodewald et al., 1992) and dendritic cells (Ardavin et ul., 1993). Virtually no myeloid cells could be derived from these precursors. Whether or not these cells are pluripotent i s not certain because reconstitution assays with single cells could not be performed and it is thus possible that different precursors were residing in this population. This becomes an important question in the light of recent findings that fetal blood contains precursors of T cells that can give rise to T cells only (Rodewald et al., 1994). These precursors were of the Thy-l'c-kit'""CD3- phenotype. When injected intravenously or intrathymically these cells gave rise to different developmental stages of ap T cells but failed to reconstitute B lymphocytes, myeloid, and erythroid lineages. At present, it is not clear whether these Thyl+ckit'""CD3- cells can give rise to cells of the y6 lineage. Thus, it is still an open question whether the thymus is colonized by pluripotent or restricted T cell precursors and whether the divergence of precursors restricted to a@ and y6 lineages can occur prethymically. It is also not excluded that the thymus in addition to being colonized by restricted T cell precursors is also seeded by multipotential cells that can give rise to lineages other than T cells. The initial steps in the intrathymic differentiation of c-kitfCD16+ CD44+CD41""CD8- progenitors (CD44'CD25-CD4'""CD8-, in short) are associated with downregulation of CD4 and expression of CD2 and CD25 (IL-2Ra) molecules (Rodewald et al., 1993), which may be preceeded by transient expression of the IL-SRP chain (Falk et al., 1993) that is never coexpressed with CD25 in fetal thymic ontogeny (Takeuchi et al., 1992). A fraction of the IL-2Rp+CD25- cells express y6 TCRs on their surface (Falk et al., 1993). Upon reconstitution of fetal thymic lobes in uitro, IL-2Rp'TCR- cells develop into both ap TCR' and y6 TCR+ cells. The data also show that about 50% of the IL-2Rp+ cells express intracellular CD3.9, and about 25% express the TCRG chain, whereas TCRP chains are not detected (Falk et al., 1993). (From here on we will no longer be concerned with the CD2 molecule since its expression does not change in further development). We know very little about the cellular interactions and signals controlling these initial steps in intrathymic development. Recent results suggest that SCF, the ligand for c-kit, may play a role. It was shown that SCF enhances IL-7-dependent proliferation of early thymocytes and that SCF antibodies inhibit development of T cells in fetal thymic organ

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culture (Godfrey e t al., 1992). CD44'CD25'CD8-CD4cells (CD44'CD25', in short) still have TCRP and TCRa loci in germ line configuration (Godfrey et ul., 1993;Rodewald et al., 1993)but, similar to CD44'IL-2RPtCD25- cells, a fraction of fetal CD44+CD25+cells expresses y6 TCRs on the surface (Rodewald e t al., 1993). Eventually, CD44+CD25' cells become c-kit-CD16-CD44-CD25' (CD44-CD25+,in short). The majority of CD44-25' cells show TCRP but no TCRa rearrangement and produce full-length TCRP transcripts (Pearse et al., 1989; Rodewald et uZ., 1993). These cells also transcribe the RAG-1 and RAG-2 genes (Wilson e t al., 1994) and express CD3.5 proteins on the cell surface (Levelt et ul., 1993a; Jacobs e t al., 1994). In addition, they express high levels of bcl-2 mRNA and protein (Andjelic e t al., 1993b). The level of HSA molecules, which also shows a characteristic developmental pattern, is low on CD44'CD25- cells but high on all subsequent stages until the CD4'8' stage after which it declines again (Howe and MacDonald, 1988; Wilson e t al., 1988). Thus, the probable sequence of a phenotypic maturation can be described as: HSA-CD44' IL-2R@-CD25-TCRaoTCRpCD3E-RAG-+ HSA-CD44 'IL-2RptCD25-TCRa"TC~CD3~ 'RAG- + HSA'CD44' I L - ~ R ~ - C D ~ ~ + T C R ~ " T C R ~ C D-+ ~ C HSAtCD44-IL-2RP'RAG-

CD25+TCRa"TCRPRCD3&'RAGt.

In an abbreviated form the above developmental sequence among early CD4-CD8- thymocytes of the a@lineage can be delineated as: CD44+CD25- -+ CD44'CD25' + CD44-CD25+ + CD44-CD25-. CD44-CD25-CD4-CD8- cells apparently represent the latest developmental stage which can give rise to y6 T cells (Petrie et al., 1992) suggesting that the final point of divergence of y6 and ap lineages may lie just before the onset of CD4 and CD8 expression (see later). This of course does not exclude the possibility that separate, precommited pathways for a@and y6 cells exist that cannot be distinguished until the late CD44-CD25- stage. At present, it is not clear whether C D ~ E is associated with any receptor proteins on the surface of CD44-CD25' thymocytes: immunoprecipitation studies employing digitonin lysates failed to reveal any association of CD3 components with proteins other than CD3, but CD3.5 antibodies did coprecipitate CD3y and 5, but little CD36 proteins (Jacobs et d., 1994). Thus, at the moment it is not clear in what way these CD3 components reach the cell surface and whether they are associated with a putative pro-T cell receptor. It is clear, however, that these CD3 components require neither of the TCR proteins that are encoded by rearranging gene loci since they are present on CD44-CD25+ thymocytes in scid mice as well as in RAG-l-deficient

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mice (Levelt et ul., 1993b). Expression of CD3c or CD36 protein is not essential for the generation of CD44-CD25+CD4-8- thymocytes because these cells are present in normal numbers in CD3edeficient (-’-) (Malissen, personal communication) and CD36-’- mice (Kappes, personal communication). Thus, a putative pro-T cell receptor associated with CD3 is not required to generate CD44-25’ cells nor is it required for TCRP rearrangement since this occurs normally in CD3c-I- mice (Malissen, personal communication). Recent in uitro experiments analyzing the effects of several lymphokines suggested that interleukin-7 may be involved in the regulation of TCRP (Muegge et al., 1993) and TCRy (Appasamy et ul., 1993) gene rearrangement. It is, however, not clear whether the role of IL-7 is, as proposed, to induce rearrangement or to allow the recombination machinery to continue in vitro. Other lymphokines, i.e., GM-CSF, IL-1, IL-2, IL-3, IL-4, IL-6, IL-10, and IL-12, which have been shown to act directly on lymphoid cells, have been implicated in one way or another in the regulation of early intrathymic development (for review see Carding et al., 1991) but at present their physiological relevance remains unclear. B. CONTROL OF INTRATHYMIC DEVELOPMENT BY THE PRE-T CELLRECEPTOR 1 . Development Znduced by the TCRP Chain We owe our knowledge concerning control of T cell development by proteins encoded by rearranging TCR genes to the analysis of rearrangement defective mice that were selectively reconstituted with TCR transgenes. The first experiment of this type was conducted with a natural mutant, the scid mutant, that is defective in DNA repair in general, and TCR and Ig rearrangement in particular. Development of thymocytes in these mice is arrested at the CD44-25+ stage of development (Rothenberg et al., 1993). Genetic reconstitution experiments showed that in scid mice T cell development failed because of a rearrangement defect since introduction of a productive TCRP gene bypassed and partially relieved the developmental arrest (von Boehmer, 1990; Kishi et al., 1991). As a consequence, the number of thymocytes increased significantly and up to 70% of the cells expressed CD4 and CD8 coreceptors (von Boehmer, 1990; Kishi et at., 1991; Groettrup et al., 1992). These experiments indicated that the TCRP protein was sufficient to propagate T cell development since full size TCRa message could not be found in the CD4+8+ cells (Kishi et al., 1991). These experiments were repeated in artificially produced,

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143

rearrangement-deficient mutants, namely RAG-l-’- and RAG-2-lmice, that were reconstituted with productive TCRP transgenes. Basically the same effect as in the natural scid mutant was observed except that the increase in cell numbers was more pronounced (about 10-fold higher than that in TCRP transgenic scid mice), which most likely has to do with the nature of the mutation in the different mice (Mombaetrs et nl., 1992a; Shinkai et al., 1993). In this regard, it is interesting to note that the introduction of both TCRP and TCRa transgenes into scid mice considerably increased the number of CD4+8+thymocytes compared to TCRP transgenic scid mice. It was discussed that this may represent a scid-specific effect in that the a@ TCR could in some way interfere with the defective DNA repair andlor rearrangement machinery (von Boehmer 1990).This hypothesis appeared to be correct as TCRp transgenic RAG-2-’- mice possess very similar numbers of CD4+8+thymocytes as TCR aP transgenic RAG-2-/- mice (Shinkai et al., 1993). While the TCRP transgene reconstitution experiments of rearrangement-deficient mice indicated that the TCRp protein was szfficient to propagate T cell development as well as CD4 and CD8 expression, analysis of TCRp-’--deficient mice indicated that TCRP expression was riot necessary for CD4 and CD8 expression, as such mice contained a relatively small thymus with variable proportions of CD4+8+thymocytes (Mombaerts et ul., 1992a). Because in TCRG-’-, TCRP-’- mice almost no CD4+8+ thymocytes could be detected it was argued by the authors that CD4+8+cells in TCRP-’- mice could represent cells of the y6 lineage. From this study it was concluded that TCRP rearrangement or expression is required for the transition from CD4-8- to CD4+8’ stage in the (YP T cell lineage. There are, however, no available data that support this view. On the contrary, other data would indicate that developing T cells do not need to express y,6 or a,P TCR proteins in order to become CD4+8+cells: experiments in scid mice reconstituted with bone marrow cells (Shores et al., 1990) or y6 T cells (Lynch and Shevach, 1993) indicate that cells with TCRs of some sort can induce CD4 and CD8 expression in other cells that are TCR protein negative. Similar experiments in RAG-’mice have supported this view (H. R. Rodewald, personal communication). It appears, however, that this intercellular communication does not lead to the accumulation of large numbers of CD4+8+thymocytes. This notion is supported by various experiments which showed that there is a strong bias for productive TCRP genes in thymocytes of TCRa-’- mice (Mallick et al., 1993)and for TCRP proteins in CD4+8+ cells from fetal thymuses (Levelt et ul., 1993a): much more than ex-

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PAWEK KISIELOW A N D HARALD VON BOEHMER

pected if factors that work by intercellular mechanisms would lead to massive expansion of CD4'8' thymocytes irrespective of productive TCRP genes. Expansion of CD4'8' cells was not the only consequence observed after introduction of TCRP transgenes: TCRP transgenes could also increase the transcription of the TCRa locus (Kishi et al., 1991) which probably correlates with efficient rearrangement of this locus. In addition, as already mentioned under Section IV,A,l, introduction of a TCRP transgene into rearrangement-competent mice interfered with TCRP rearrangement such that most T cells expressed the TCRP transgene and contained either DJ rearrangements or no rearrangement of the endogenous TCRP locus at all (Uematsu et al., 1988; Fenton et al., 1988). In various transgenic mice that were produced with different TCRP genes it was shown that the phenotypic changes required a TCRP protein since nonproductive TCRP genes were ineffective ( Jacobs et al., 1994; Krimpenfort et al., 1989). A complete TCRP protein was, however, also not required as a TCRP protein, from which most of the V region was deleted, could still produce both the developmental progression in TCR rearrangement-deficient mice ( Jacobs et al., 1994) as well as the allelic exclusion of the TCRP locus in rearrangementcompetent mice (Krimpenfort et al., 1989). 2 . Structure and Function of the Pre-T Cell Receptor For a long time it was not clear in which way a TCRP protein could mediate the described effects in immature T cells since it was known that in mature T cells the TCRP protein is relatively quickly degraded in the endoplasmic reticulum in the absence of other TCR proteins encoded by rearranging TCR genes (Klausner et al., 1990). This was apparently not true for immature T cells as in TCRP transgenic scid as well as TCRP transgenic RAG-'- mice TCRp protein could be easily detected on the cell surface of CD4-8- as well as CD4'8' thymocytes (Groettrup et al., 1992; Kishi et al., 1991; Mombaerts et al., 1992a; Shinkai et al., 1993; von Boehmer et al., 1988). The earliest experiments in TCRP transgenic mice already indicated that there were at least two forms of the TCRP protein on the surface of thymocytes, namely a monomer of 40 kDa as well as a disulfide-linked form of 80 kDa (Groettrup et at., 1992; Kishi et al., 1991; von Boehmer et aZ., 1988).It was likely that some TCRP expression representedan unphysiological event because in TCRa-'- mice very little TCRP protein could be detected on the surface of thymocytes (Groettrup et al., 1993; Mombaerts et al., 1992a; Phillpot et al., 1992). This difference in the data from TCRP transgenic mice, on the one hand, and nontransgenic

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145

mice, on the other hand, was explained by Gropettrup and von Boehmer (1993a) who showed that much of the protein on the surface of TCRP transgenic scid thymocytes occurred in the form of a GPIlinked monomer that was not associated with CD3 signal-transducing molecules. This most likely represented a transgenic artifact as this form was not detected in any significant amount on the surface of fetal thymocytes, immature T cell lines, and thymocytes from normal and TCRa-’- mice (Groettrup et at., 1993; Groettrup and von Boehmer, 1993a; Jacobs et al., 1994). In fact, it was shown that in the various cell types only the disulfide-linked 80-kDa form could be detected which was associated with CD3 components: C D ~ E-y, , -6, and low amounts of 5 in the case of pre-T cell lines (Groettrup et al., 1992), and C D 3 5 --y, and probably others in TCRP transgenic RAG-’- mice (Shinkai et al., 1993) and TCRa-’- mice (Jacobs et al., 1994). The partner protein(s) of the TCRP complex in the 80-kDa form escaped detection when precipitations were performed with surface iodinated material (Groettrup et al., 1992) but a partner chain was eventually detected in lysates from surface-biotinylated cell lines and was found to represent a heavily glycosylated protein with an average molecular mass of 33 kDa, hence named gp33 (Groettrup et al., 1993; Groettrup and von Boehmer, 1993b). In the meantime, the gene encoding gp33 has been cloned and shown to encode a transmembrane Iglike protein that is expressed in immature thymocytes but not in mature T cells and has a relatively long cytoplasmic tail with two potential phosphorylation sites. Even though this TCR chain has little homology to the TCRa it is named pre-TCRa chain (pTa). Formation of the preTCR-a chain gene does not require rearrangement since it is expressed in CD4-8-, CD44-CD25+ thymocytes from RAGB-’- mice (St. Ruf et al., 1994) (Fig. 2). Currently, we have some clues by which mechanism this pre-T cell receptor complex may signal the developing T cell that a TCRP protein has been translated from a productive gene. Experiments by Levelt et al. (1993a,b) and Jacobs et al. (1994) have shown that the effect of a TCRP transgene on thymocytes from scid or RAG-’- mice can be mimicked in vitro and in uico by CD3 antibodies that under the appropriate circumstances generate as many CD4+8+cells as does a TCRP transgene. This could suggest that under physiological conditions the TCRPIgp33 complex that is associated with CD3 proteins signals through the CD3 complex but because of the cytoplasmic tail of the pre-TCRa chain the signal transduction mode of the pre-TCR may be different from that of the ap TCR. At present, it is not clear whether the pre-TCR has to bind to an intrathymic ligand or whether the insertion or the assembly of the TCR complex in the cell membrane is

146

PAWEG KISIELOW A N D HARALD VON BOEHMER

CD4-8-

I

bc12tBret

1 differentiation expansion

CD44;CD25CD4+8+

bc12t are4

FIG.2. Developmental control by the pre-T cell receptor. CD4-CD8-CD44-CD25+ thymocytes rearrange the TCRp locus while expressing the gp33 gene encoding the invariant chain of the pre-T cell receptor. T h e pre-TCR complex associated with C D 3 signal transducing molecules is inserted in the membrane. SignaIing by the pre-TCR, that may or may not involve binding to a intrathymic ligand, results in massive cellular expansion as well as differentiation. Differentiation events may include CD4/8 expression, suppression of TCRp and enhancement of TCRa rearrangement, downregulation of the Bcl-2 protein, and upregulation of the Fas protein.

sufficient to generate the signal(s). That the pre-TCR-CD3 complex is competent to generate a signal was shown in the pre-T cell line described by Groettrup et al. (1992): crosslinking of the pre-TCR induced strong Ca2+ mobilization. It is possible that the { protein is also involved in the signal transduction by the TCR even though this protein was found to be only weakly associated with the pre-TCR complex (Groettrup et al., 1992; Shinkai et d.,1992). In {-deficient mice (Love et al., 1993; Malissen et al., 1993; Ohno et al., 1993) the number of CD4+8+thymocytes was severely reduced (see Table 11) and the CD44-CD25- subset of CD4-8- cells was almost entirely missing (Crompton et ul., 1994). On the other hand, CD36 appears to be dispensable for signaling through the pre-TCR because in CD36-'- mice development of immature thymocytes, including CD4+8+cells, is unaffected (D. Kappes, personal communication) (see

TABLE I1 MUTATIONSAND TRANSGENES AFFECTINGEARLY INTR4THYMIC

STAGES IN

T-cell Receptor

Thymocytes (% of Normal Numbers) Gene K.O. scid scid

Immature

Endo. Gene Rearr.

Mature

p

Cell Surface

TG

All

-

2

100

0

0

20

100

20

0

2

100

0

0

100 2 10

100 100 100

100 0 5

0 0 0

0 0 0

( + ) - + +

-

2

loo

0

0

0

( + ) - + +

-

2

100

0

0

0

TCRp

RAG-1 or -2 RAG-2 RAG-2 TCRp (DP-CP2) TCRp x TCRG CD~E

T CELL DEVELOPMENT

TCRp TCRa

CD44-25'

CD4'8'

CD4'8-

CD4-8'

a

-

-

-

-

-

-

-

+

6

y

?

-

-

?

ap

y6

p only -

?

?

References Rothenberg et al., 1993 von Boehmer, 1990 Kishi et al., 1991 Shinkai et al., 1992 hlonibaerts et al., 1992 Shinkai et al., 1992 Shinkai et a/., 1992 Mombaerts et a / . , 1992 Mombaerts et ul., 1992 Malissen, personal communication (continued )

++

U

+ +

h

+

v

+

+ I

g

h

Q

I

a.

+

+

a.

+

m

o

2

a

m

g

+

+

+

( 1 .

+ + +

+

+a. +

I

+". +

I

o

+

+

+

o

I

+ + + +

w

m

+

+ m

m

w

m

O O O O g g

g

LD

2

0

2 2

I

F

8

kJ

LJ-

148

TABLE 111 MUTATIONSAFFECTINGPOSITIVE SELECTION AND/VH FUNCTION OF MATURET CELLS Mature Cells (% of Normal Nos.)

Gene K.O.

Transgene

CD4+8-

CD4-8+

ap TCR anti-HY/Db

0 0 100

TAP-1 MHC class I1

100

TCRa CD4 CD8a CD8p P2m

Scid (H-zb)

Scid (H-zd)

a0 TCR anti-HY/Dh

P2m

I

$

MHC class I1 C D36

ZAP-70 IRF-1 CD28 Perforin

CD8

Function DTH

Prolif.

200 0 3

? ? ?

+

3

3 200

? ?

? ?

4 0 150 100 100

5 200 0 10 25

? ? ? ? ?

?

10

100

?

?

0

0

?

?

5

20

?

2

100

0 10 100 100

? ?

-

100 100 100

Help

CTL

+ -

?

? ? ?

?

+ +

''

References Scott et al., 1989 Scott e t al., 1989 Zijlstra et al., 1990 Koller e t al., 1990 van Kaer et al., 1992 Cosgrove et al., 1991, Grusby et al., 1991 Mombaerts et al., 1992 Rahemtulla et al., 1991 1991 Fung-Leung et d., Nakayama et al., 1994 Davis et al., 1993; Baron et al., 1994 Chan et al., submitted for publication Kappes, personal communication Ohno et al., 1993; Love et al., 1993 Arpaia et al., 1994 Matsuyama et a!., 1993 Shahinian et al., 1993 Kagi et al., 1994

150

PAWEL KISIELOW A N D HARALD VON BOEHMER

Table 111). It is very likely that further in the signaling cascade the ~ 5 6 ' "tyrosine ~ kinase plays an important role: thymocytes in ~ 5 6 ' " ~ - ' - mice have a developmental arrest that really does not look all that different from that seen in TCRP-'- mice (Molina et al., 1992). Also, mice carrying a catalytically inactive form of ~ 5 6 ' "as~ a dominant negative mutation can have even fewer double-positive cells depending on the amount expressed (Levin e t al., 1993b). In these mice the TCRP but not the TCRa loci were extensively rearranged, indicating that TCRa rearrangement is regulated by the pre-TCR. When a TCRP transgene was introduced in mice carrying a dominant negative p56lCkmutation, the usual effect of TCRP protein, i. e., the induction of developmental progression, as well as exclusion of endogenous TCRP loci from rearrangement (Uematsu et al., 1988) were not observed (Anderson et al., 1993a). Finally, mice overexpressing active ~ 5 6 ' generated "~ CD4+8+thymocytes that lack VP rearrangements but express V a J atranscripts (Anderson et aZ., 1992). It appears that these early effects of p56Ickhave very little to do with the ability to bind to CD4 and CD8 coreceptor molecules: a transgene that encodes ~ 5 6 ' " ~ protein unable to bind to CD4 or CD8 acts just the same as a transgene encoding the wild-type form (Levin et al., 1993a). All of this strongly suggests that a TCRP protein associates covalently with pTa in immature thymocytes and forms a complex with CD3 signaling proteins that in turn interact with the ~ 5 6 ' tyrosine "~ kinase independently of CD4 and CD8 coreceptors. Signals thus generated lead to progression in development including CD4 and CD8 coreceptor expression, cellular expansion, accelerated TCRa rearrangement, and possibly other developmental changes. While it is clear from experiments in TCRp-'- mice that the pre-TCR complex is not absolutely essential for the induction of CD4 and CD8 expression. There appears to b e some requirement for a signal-transducing receptor because in CD3&-'mice no CD4 and CD8 expression is observed and TCRP but no TCRa rearrangement can be detected (B. Malissen, personal communication). Perhaps the TCRa rearrangement observed in TCRP-I- mice is brought about by a similar mechanism that induces CD4/8 coreceptor expression in these mice, i. e., possibly by intercellular factors generated by yS cells. The latter may be sufficient to induce expression of CD4 and CD8 on some cells but not to generate the large number of CD4+CD8+cells that are present in a normal thymus. It is actually possible that any mechanism that helps surviving CD4-/8- cells (rescue from programmed cell death) will result in CD4 and CD8 expression and some TCRa rearrangement. It is clear, however, from the experiments in TCR transgenic, rearrangement-deficient mice that ap

DEVELOPMENT A N D SELECTION OF T CELLS: FACTS A N D PUZZLES

151

or y8 TCRs are not required for CD4/8 coreceptor expression and TCRa rearrangement and that expression of the pre-TCR is sufficient to initiate these events (Fig. 2 ) .

C . CONTROL OF INTRATHYMIC DEVELOPMENT BY THE (rp TCR 1 . The Generation and Turnover of CD4+8+ Thymocytes Once the pre-T cell receptor has delivered its signal large numbers of CD4'8' cells are generated that effectively rearrange the TCRa locus. Experiments analyzing the level of RAG-1 and RAG-2 genes in thymocyte subsets have indicated that there are relatively high levels of these enzymes in CD44-CD25+ cells correlating with TCRP rearrangement, intermediate levels in CD44-CD25- cells, and again a higher levels in CD4+8+blasts, correlating with TCRa rearrangement (Wilson et al., 1994). When TCRa rearrangement is most efficient the expression of the pre-T cell receptor will be downregulated through diminished expression of the TCRP partner chain gp33 such that the TCRP protein can now pair effectively with TCRa proteins. Another event of potential physiological importance for the development and selection of thymocytes, which occurs during transition of CD4-8- cells into CD4+8+thymocytes, is the downregulation of Bcl2 protein levels (Andjelic et al., 1993b; Veis et al., 1993b)and increased expression of APO-l/Fas (Andjelic et al., 1994). This may have to do with the limited life span of CD4+8+cells and their increased susceptibility to the induction of apoptosis (see below) (Fig. 2). The transition of CD4-8- cells into CD4+8+thymocytes occurs in such a way that either CD8 is expressed before CD4 (more common) (Kisielow et al., 1984) or in some mice CD4 is expressed before CD8 (Hugo and Petrie, 1992). This is of practical importance because the early CD4 or CD8 single-positive cells need to be distinguished from mature single positive T cells in the medulla of the thymus. An easily utilized criterion is the large size of these immature single positive cells that upon culture in vitro (von Boehmer et al., 1986) or upon intrathymic injection (Nikolic-Zugic and Bevan, 1988) become CD4+8+cells within hours. CD4+8+thymocytes go through a limited number of divisions and begin to express ap TCRs on the cell surface, some cells at higher levels and others at relatively low levels, when compared with mature T cells. The fate of CD4+8+blast cells with high TCR levels is not clear. It could be that TCR levels will be downregulated in this population, i.e., that some cells that have relatively quickly made a productive TCRa rearrangement express the receptor at high levels and then TCR expression is downregulated by

152

P A W E t KISIELOW AND HARALD VON BOEHMER

an unknown mechanism probably related to the stability of TCRa protein (Kearse et al., 1994). Alternatively, it could be that these CD4+8+blasts are undergoing positive selection and upregulate the TCR as a consequence of binding to an intrathymic ligand. While the latter possibility cannot be excluded, data in TCR transgenic mice are better compatible with the first notion: early CD4-8' and CD4+8+ blasts with relatively high receptor levels were found in mice that cannot positively select the transgenic TCR (Swat et al., 1992). Most CD4+8+double positive blasts and small cells in normal mice express the TCR at low levels and some do not express any detectable TCR on the cell surface. It was noted that TCR-negative cells were as frequent among CD4+8+blasts as they were on small CD4+8+cells (see review by Rothenberg, 1992), while in TCR transgenic mice all CD4+8+cells were TCR positive (Berg et al., 1989; Kaye et al., 1989; Kisielow et al., 1988b; Sha et al., 1988). The latter result indicated that TCR-negative CD4+8+cells in normal mice were cells that lacked productive TCRa rearrangements and that they did not represent a distinct, TCR-negative subset of thymocytes. It was initially noted in TCR transgenic mice (Teh et al., 1988) that TCRa rearrangement did not cease in cells that expressed a transgenic aP on the cell surface, much in contrast to TCRP rearrangement that was almost completely shut off by a TCRP transgene (Uematsu et al., 1988). In the TCR transgenic mice the ongoing TCRa rearrangement resulted in mature T cells that expressed at the level of RNA and protein two different TCRa chains that could both be surface expressed in disulfide linkage to the same transgenic TCRP chain (Borgulya et al., 1992, von Boehmer, 1990).Other studies actually showed that this represented not a peculiarity of transgenic animals but that in normal individuals up to 30%of T cells can express two productive TCR rearrangements at the level of RNA (Casanova et al., 1991) and protein (Padovan et al., 1993). The fact that this was due to continuing rearrangement in CD4+8+ thymocytes that already expressed an a@TCR was well supported b y the notion that both TCR cup-positive and -negative CD4+8+ thymocytes expressed relatively high levels of RAG-1 and RAG-2 (Turka et al., 1991a).Thus not only the outer cortex that contains dividing blasts, but the entire thymic cortex containing small and large cells is occupied with generation of T cell receptor diversity. In nontransgenic mice the ongoing rearrangement can of course delete productive rearrangement and thus it is possible that TCRa+ cells again become TCRa-. The small CD4+8+TCR+cells from the thymic cortex are peculiar in that unlike mature aP T cells, they could not be activated to produce

DEVELOPMENT AND SELECTION O F T CELLS: FACTS A N D PUZZLES

153

IL-2. In this context it is interesting to note that PMA plus ionomycin could induce IL-2 secretion in the precursor cells of CD4+8+thymocytes, i. e., in the CD4-8- cells, but that the same agents that bypass the TCR signal-transducing cascade and activate PTKs do not induce IL-2 production in cortical CD4+8+thymocytes. Likewise, CD4+8+ thymocytes cannot be induced by TCR ligands to develop effector filnction-like cytolytic activity (reviewed by Rothenberg, 1992). Thus, it appears that in CD4+8+thymocytes TCR signaling results in very different cellular changes when compared to mature T cells. The CD4+8+thymocytes represent an interesting population of cells because they have a relatively short, programmed lifespan, i.e., these cells will die in a cell autononious fashion after about 3.5 days of their life. This was analyzed in continuous labeling studies with DNA precursors that labeled more than 95% of these cells in a linear fashion in 3.5 days (Egerton et ul., 1990; Huessnian et ul., 1991). No other cell type in the thymus was labeled with similar kinetics suggesting that CD4+8+thymocytes died rather than changed their phenotype intrathymically. Thus, CD4+8+thyniocytes, much like cells in the developing worm C . elegans (Ellis et ul., 1991), are programmed to die unless they interact with their environment in a specific way that can result in either prolongation or shortening of their life span.

2. The Generution of CD4+8-und C D 4 - 8 + Thymocytes from C 0 4 8 Preczi rsors +

+

Initially, a relationship between C D 4 + 8 +thymocytes and CD4+8and CD4-8+ cells could not be established: while most C D 4 + 8 +thymocytes were labeled in a few days with continuously supplied DNA precursors, single-positive cells took up the label much more slowly (Egerton et al.,1990; Shortnian and Jackson, 1974). The first labeling studies on single positive cells indicated a linear increase of labeled cells from the start of the experiment (Egerton et ul., 1990) indicating that they behaved as an independent population of cells that expanded slowly rather than being derived from another nondividing precursor population. This was so because these initial labeling experiments did not distinguish between the early immature single positive cells that were precursors ofthe C D 4 + 8 +thymocytes from the mature single positive cells. Because the former were dividing they took up the label immediately. When care was taken to exclude the immature single positive population from the analysis it was shown that mature single positive cells labeled with a lag indicating that they were mostly derived from a population of nondividing precursors (Egerton et al., 1990; Huesmann et d.,1991; Shortman et ul., 1991).

154

PAWEY. KISIELOW AND HARALD VON BOEHMER

Before these labeling experiments were conducted it had already become evident that CD4+8' thymocytes were precursors of single positive cells: some experiments in which the generation of CD4+8cells could be inhibited with CD8 antibodies (Smith, 1987) were at least suggestive that some precursors of CD4+8- cells expressed CD8. Additional evidence was obtained in TCR transgenic mice with a transgenic TCR expressed on CD4-8+ but not on CD4+8- cells (Teh et al., 1988). The latter cells were generated from C D 4 + 8 +precursors that initially expressed the transgenic TCR only. By continuing TCRa rearrangement some of CD4+8 cells eventually expressed endogenous TCRa chains that could displace the transgenic TCRa from the transgenic TCRp protein and some of these cells could be selected by class I1 MHC molecules to become CD4+8- cells (reviewed by Kisielow and von Boehmer, 1990). Such CD4+8-/3TaE cells were at least 10-fold more frequent in mice that positively selected cells with the transgenic TCR than in mice that negatively selected C D 4 + 8 + cells with the transgenic TCR. Since negative selection required the CD8 coreceptor (Killeen et al., 1992a) it was clear that in these mice CD4+8-/3TaE cells were missing because their CD4+8+/3TaTprecursors had been eliminated at an early stage of development, thus linking the CD4+8+ and the C D 4 t 8 - compartment. Finally, another, still indirect, indication was obtained in mice in which antibodies against CD4 interfered with deletion ofMls-reactive CD4'8+ cells. This interference permitted the generation of mature CD4-8+ cells with TCRs specific for Mls determinants that could no longer be deleted (Fowlkes et al., 1988). More direct evidence was obtained in experiments that involved purification of CD4+8+ cells and intrathymic injection: initially such experiments yielded results suggesting that large CD4+8+ blasts, but not small CD4+8+ thymocytes, could develop into single positive thymocytes after intrathymic injection (Guidos et al., 1989). Thus, these experiments came up with the surprising notion that most of the cells in the thymic cortex were beyond repair, i.e., destined to die only and not rescuable by TCR-ligand interactions. This seemed rather wasteful since the small CD4+8+thymocytes continued to rearrange TCRa genes (Borgulya et al., 1993; Brandle et al., 1992; Petrie et al., 1993), i.e., continued to generate TCR diversity even though they were apparently destined to die. Since this made little sense, the original experiments were repeated in TCR transgenic as well as in normal mice (Lundberg and Shortman, 1994; Swat et al., 1994b). Both approaches came to the conclusion that small CD4+8+thymocytes are not dead end cells but can be rescued to become single positive cells after intrathymic injection. In the transgenic system CD4+8' cells +

DEVELOPMENT A N D SELECTION O F T CELLS: FACTS A N D PUZZLES

155

with the transgenic TCR were prepared from mice that cannot positively select cells with the transgenic TCR and the purified cells were injected into a thymus containing positively selecting ligands. It was found that the generation of single positive cells from these precursors was about as effective as that from large C D 4 + 8 + precursors. With cells from normal mice C D 4 + 8 +blasts were more efficient than small cells (Lundbertg and Shortman, 1994). Whether this difference is due to different receptor expression by the various subsets is not known. The fact that the generation of single positive cells requires rescue from cell death of C D + 8 +precursors was postulated by von Boehmer (1986) and subsequently documented in TCR transgenic mice. The hypothesis was proposed in order to explain earlier experiments in hemopoietic chimeras that suggested that MHC molecules had some impact on the T cell repertoire (Bevan, 1977), that MHC molecules in the thymus were important in shaping the repertoire (Zinkernagel et ul., 1978), and that so-called immune-response genes, which determine whether a mouse will make a high or low response to a given antigen, encoded the MHC molecules expressed in the environment rather than in the T cells (Kappler and Marrack, 1978; von Boehrner et id.,1978). Because these early experiments did not permit analysis other than activation of mature T cells it was impossible at that time to determine whether and in which way positive selection could occur and whether the observed phenomena reflected some bending of the T cell repertoire rather than an essential selection step in the maturation o f T cells. The advent ofclonotypic antibodies and the generat'ion of TCR transgenic mice as well as the availability of natural as well as artificial mutants has very much facilitated the attempts to support the hypothesis that positive selection represents rescue from programmed cell death and that positive selection is in fact essential for the generation of mature aP T cells. The data showed that generation of single positive cells with a transgenic aP TCR specific for a certain peptide presented by a certain MHC molecule required the presence of the MHC molecule but not the specific peptide in the thymus (reviewed by von Boehmer and Kisielow, 1990). In the absence of the appropriate MHC molecule development of cells expressing the transgenic TCR only did not go beyond the C D 4 + 8 +stage (Kisielow et ul., 1988b; Teh et nl., 1988). This was most clearly shown in TCR a@transgenic, rearrangement-deficient scid mice that contained T cells exclusively with the transgenic TCR (Scott et ul., 1989). The notion that positive selection represents rescue from programmed cell death was confirmed and extended in a variety of different TCR transgenic mice (Berg et ul., 1989; Kaye et ul., 1989; Pircher et al., 1989; Sha et

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al., 1988). The data in TCR ap transgenic mice also showed that class

I or class I1 MHC-restricted TCRs ended up, as a rule, on positively selected CD4-8+ and CD4+8- thymocytes or T cells, respectively. These data obtained in TCR transgenic mice were well in accord with studies that showed inhibition of the generation of CD4 ‘8- cells by class I1 MHC antibodies (Kruisbeek et al., 1985), diminished numbers of CD4+8- cells in class I1 MHC gene-deficient mice (class I1 MHC-/-) (Cosgrove et al., 1991; Grusby et ul., 1991), as well as lack of significant numbers of CD4-8+ T cells in class I MHC-deficient mice, which lacked class I MHC at the surface because of a deficient p2-microglobulin gene (P2m-l- mice) (Koller et al., 1990; Zijlstra et al., 1990). The studies in the TCR ap transgenic mice, however, were required to show that the specificity of the ap TCR determined the CD4/CD8 phenotype of mature T cells (Berg et al., 1989; Kaye et al., 1989; Sha et al., 1988; Teh et al., 1988). The analysis of TCRa-’- mice corroborated the finding that an ap TCR was essential for positive selection (Mombaerts et al., 1992; Phillpot et al., 1992) even though some of these mice contained CD4+8- T cells with a TCRP protein on their surface in peripheral lymph nodes. The exact nature of this TCRa-less surface complex needs to be determined. The discovery of the principles of positive selection in TCR transgenic mice raised a number of interesting questions with regard to the mechanisms of this selection as well as the thymic MHC ligands that induce positive selection. Below, we will address two questions: one concerned with the lineage commitment of CD4+8+T cells that undergo positive selection and the other concerned with the ligands as well as the cells that induce positive selection. u. Commitment of CD4+8+ Thymocytes To Either CD4 or CDB Lineages ( F i g . 3). When comparing a thymus of a TCR transgenic mouse that can positively select the transgenic TCR with that of another mouse that cannot, a clear-cut difference was found with regard to a subset of small C D 4 + 8 + thymocytes which express high TCR levels in the former type of mouse and that were absent in the latter (Borgulya et al., 1991; Ohashi et al., 1990). Thus, the TCR-MHC ligand interaction clearly affected thymocytes before they had downregulated either CD4 or CD8. Some of these cells expressed the activation marker CD69 indicative that a signal had been transmitted, even though this signal does not result in proliferation or induction of effector function (Bendelac et al., 1992; Swat et al., 1993). It could further be shown that C D 4 + 8 +cells undergoing positive selection, i.e., cells

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no binding

t

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12 hrs

MHC

+

specific peptide

t

FIG.3. Developmental control by the ap T cell receptor. CD 4 + 8 +cells with ap TCRs have a life span of 3 or 4 days during which TCRa rearrangement continues and cells produce new receptors. Ifduring this time the ap TCRs d o not bind to intrathymic ligands the cell will die a programmed death (left). If the receptor binds to the specific .peptide cell death will b e precipitated and the cell will be eliminated by apoptosis within hours (right). I f the receptor binds to a positively selecting ligand different from the MtlC’-specific peptide, the cell will be rescued from programmed cell death and begin to downregulate either CD4 or C D 8 coreceptors (center).This may b e an entirely or partly stochastic event. As a rule, only cells with “fitting” ap TCR-CD4/CD8 coreceptor conibinations, able to bind to the same class I or class I1 MHC molecule, will fully mature. Cells with a mismatched combination will die. Positive selection will terminate ongoing TCRa rearrangement and induce higher expression of Bcl-2 protein while downregulating Fas protein levels.

with higher TCR levels, contained less RAG-1 and RAG-2 RNA than CD4’8’ cells with low TCR levels indicative that TCRa rearrangement was terminated at that time (Borgulya et al., 1992; Brandle et al., 1992). At this stage the CD4+8+thymocytes begin to downregulate either the CD4 or the CD8 coreceptor and upregulate the level

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of Bcl-2 protein (Gratiot-Deans eta,!.,1993; Veis et al., 1993).It appears that as a rule these events are initiated by binding of both the TCR as well as one of the CD4/8 coreceptors to thymic MHC ligands. This was analyzed in class I1 MHC-deficient mice (II-'-) that normally produce some CD4+8'"" cells that express levels of C D 3 higher than most CD4+8+cells. Some of these cells also express CD69 molecules (Chan et al., 1993a). Since such cells are not seen in MHC-negative, i.e., class I and class I1 (I-'-, II-'-) mice it was argued that generation of these cells in II-'- mice required binding of the a/3 TCR to class I MHC molecules. In double-mutant mice (II-'-, CD8-'-) the CD4+CD3highCD69+ cells were in fact very much reduced, much like in (I-'-, II-'-) double-mutant mice, and hence it was infered that as a rule positive selection begins by binding both TCR as well as CD4/ 8 coreceptor to thymic MHC molecules (Chan et al., 1993a,1994). On the basis of these and other data it was argued that CD4 and CD8 downregulation occurred stochastically, i.e., independent of the MHC specificity of the TCR and coreceptor, because CD4+8'"" and CD4'""8+ cells were detected in the thymus of II-'- and I-'- mice respectively. For technical reasons it was difficult to show that in fact CD4+8'"" and CD4'""8+ cells that had higher TCR level than CD4+8+ thymocytes and expressed intracellularly the bcl-2 gene at higher levels than CD4+8+thymocytes could produce only mature CD4+8- and CD4-8+ cells, respectively (Chan et al., 1993a,1994; van Meervijk and Germain, 1993). If CD4 and CD8 downregulation occured stochastically in CD4+8+ cells undergoing positive selection, and the final selection required a "fitting" expression of TCR and coreceptor, one should be able to rescue CD4+8- cells with a class I MHC-restricted TCR and CD4-8+ cells with a class I1 MHC-restricted TCR by CD4 and CD8 transgenes that are expressed in all T cells, respectively. Such an experiment initially failed (Borgulya et al., 1991; Robey et al., 1991). However, in a series of later experiments, rescue of CD4-8+ and CD4+8- subsets with class I and class I1 MHC-restricted receptors, respectively, by CD4 and CD8 transgenes could in fact be observed (Baron et al., 1994; Chan et al., 1994; Davis et al., 1993; Itano et al., 1994). The mechanism by which CD4 and CD8 coreceptors function during positive selection is not clear. Recent experiments indicate that association with ~ 5 6 ' may " ~ not be essential since overexpression of mutant CD4 and CD8 molecules, which are unable to bind p56lCk,could rescue development of CD4+8- and CD4-8+ lineages in CD4-deficient and CD8-deficient mice, respectively (Chan et al., 1993b; Killeen and Littman, 1993). Independent experimental evidence appears to support the no-

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tion that positive selection, at least of a subset of CD4+8+ cells, can involve consecutive receptor-ligand interactions: in class I1 MHCrestricted-TCR transgenic RAG-2-’- mice, i.e., mice that can express only a single TCR, positive selection generated in addtion to CD4+8cells a significant number of CD4-8+ cells with the class I1 MHCrestricted TCR that could be induced by class I1 MHC presented peptides to become potent killer cells. Both subsets required the expression of the relevant class I1 MHC molecules in the thyinus for their positive selection. While this was sufficient for the full maturation of CD4+8- cells with the transgenic TCR, it was not for full maturation ofthe CD4-8+ cells because in TCR transgenic, class I MHC-’- mice, CD4-8+ cells in the thymus expressed high levels of HSA (characteristic of immature cells) and were not found in the periphery, i.e., full maturation of the CD4-8+ cells required an additional interaction with class I MHC molecules (Kirberg et nl., 1994). The transgenic TCR of these mice is available in soluble form (Weber et al., 1992) and could be shown to bind to class I1 but not class I MHC molecules in the absence of the specific peptide (Karjalainen, personal communication). This may suggest that full maturation of the CD4-8+ cells with the class I1 MHC-restricted TCR required only the binding of the CD8 coreceptor to class I MHC molecules and binding of the a@ TCR to class I1 MHC molecules, i.e., a@TCR and coreceptor were not ligated by the same MHC molecule. Whatever the mechanism of this type of selection, these experiments indicate that positive selection of ap T cells can require two consecutive or even continuous interactions of MHC ligands with receptors, thus again supporting a model in which CD4/8 downregulation occurs stochastically. In the reported (probably exceptional) case the selection did not require a combination of TCR and coreceptor with “matched” specificity that can engage the same MHC molecule (Kirberg et nl., 1994) perhaps because this particular TCR has relatively high affinity for MHC molecules (Weber et al., 1992). At present, it is not clear how frequent CD4-8+ cells with class I1 MHC-restricted receptors, inducible by class I1 MHC-presented peptides, occur. These cells possibly represent important regulator cells because they can effectively destroy cells presenting peptides from endogenous proteins like macrophages and B cells. Perhaps it is not a coincidence that this type of selection was found with an IE-restricted clone because IE has been implicated in the suppression of various immune responses (Wassom et al., 1987). Perhaps such suppression is mediated by class I1 MHC-restricted, cytolytic T cells. While the experiments discussed so far could suggest that lineage

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commitment occurs at a time when the (YPTCR on CD4+8 thymocytes binds to MHC molecules in the thymus and induces apparently random downregulation of CD4 and CD8 coreceptors, there are other experiments that have been interpreted to suggest that lineage commitment may occur even earlier: it was found that a particular transgenic TCRP protein could not be positively selected on CD4+8- cells by a particular class IIb molecule but apparently could be selected on CD4+8- cells by class IId molecules. The authors found that in H-2b mice most cells already expressed at the C D 4 + 8 +stage endogenously encoded TCRP proteins, while in H-2d or H-2bxdmice, they did not. Furthermore, H-2d antibodies increased the expression of endogenous TCRP proteins when injected into H-2d mice. It was concluded from these experiments that lineage commitment could already have occured, at a very early stage and if the TCR could not engage with MHC molecules this resulted in the generation of different receptors (Crompton et al., 1993).It remains to be seen how generalizable these conclusions are. In summary, much of the recent experimental evidence appears to favor a stochastic/selective model of lineage commitment with less evidence in favor of an instructive model as originally proposed by von Boehmer (1986). The only note of caution with regard to entirely dismissing an instructive model at this stage is that the supposedly intermediary subsets of cells with “mismatched” TCRs and coreceptors, as well as those rescued by CD4/8 transgenes, are relatively small when compared to those with matched TCR and CD4/8 coreceptor combinations. One could come up with the extreme view that these small subsets are representative of errors that are made when developing CD4+8+T cells are instructed to develop into mature CD4-8+ and CD4+8- cells by a specific signal generated by the binding of TCR and coreceptor to either class I or class I1 MHC molecules, respectively. Two recent observations are indicative that the generation of CD4-8+ and CD4’8-cells in either the instructive or the selective/ stochastic way does involve different signals. First, it was found that in ZAP-70-deficient humans the generation of CD4’8- cells was much less impaired than that of CD4-8+ cells (Arpaia et al., 1994). Second, mice deficient in interferon regulatory factor-1 have drastically reduced numbers of CD4-8+, but not CD4+8-, thymocytes and T cells (Matsuyama et al., 1993). +

b. Positively Selecting Ligands. An issue that is similarly intensely debated as the question of CD4/CD8 lineage commitment is the ques-

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tion concerning the exact nature of the MHC ligand which is inducing positive selection. While it is clear that the polymorphic parts of MHC molecules determine positive selection, the role of peptides in this event has only recently begun to be analyzed. A series of experiments studying the selection of single, most often transgenic, TCRs by mutant MHC molecules has concluded that mutations in the peptide binding grooves of MHC molecules strongly affect positive selection (Berg et al., 1990; Jacobs et al., 1990; Nicolic-Zugic and Bevan, 1990; Sha et al., 1990). Since it was considered unlikely that the mutations affected the overall structure of MHC molecules (but see Section IV,A,3a), it was concluded that peptides were involved in positive selection, an almost unavoidable conclusion because MHC molecules without peptides are highly unstable at the cell surface (Townsend et al., 1989; van Kaer et al., 1992) and peptides contribute to the shape of the surface of MHC molecules (Fremont et al., 1992; Stern et al., 1994; Young et al., 1994). If one ignores the possibility that mutations in the groove could conceivably alter the shape of the MHC molecules themselves, two mutually not exclusive possibilities could be considered to account for the contribution of peptides to positive selection: either the peptide could interfere with the binding of the TCR to MHC molecules, thereby preventing positive selection, or the peptide could bind directly to the TCR and thereby contributing to positive selection (Jacobs et al., 1990). The most extreme version of the latter possibility was that the TCR would bind only to peptides and not to the MHC surface and that positive selection basically required the same peptide as T cell activation (Kourilsky and Claverie, 1986). This proposal raised the dilemma of how the thymus could generate almost all possible peptides and if so, how some of the peptides could positively but not negatively select. Possible solutions were that a large variety of peptides was generated by mistakes in translation and that most of these peptides occurred in too low of a concentration to induce negative, but in a sufficient concentration to induce positive, selection. At the other extreme was the proposition that the thymus epithelium produced “special” peptides sitting deep in the grove of MHC such that the TCR could easily bind to the surface of MHC without influence by the peptide (Marrack and Kappler, 1987). The comparison of the peptide content of class I1 MHC molecules from thymic stroma and other tissues, however, failed to reveal major differences (Marrack et al., 1993) and thus there was no evidence supporting the latter possibility. The role of peptides in positive selection was again addressed in more recent experiments in which positive selection was studied in

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embryonic thymic organ culture employing P2m-I- (Hogquist et al., 1993,1994; Sebzda et al., 1994) or peptide transporter gene-1 (TAP1) (Ashton-Rickard et al., 1993,1994) mice that were defective in class I MHC expression. When organ cultures from both types of mice were incubated with peptide(s) as well as P2 microglobulin some increased expression of MHC (in the case of P2rn-l- mice not detectable by antibody staining, i.e., at least 100 fold less than in normal mice) was observed. At the same time the proportion of single positive CD4-8’ (and to some extent CD4+8-) increased during organ culture. Different peptides varied in their ability to increase the number of single positive cells and in their ability to restore class I MHC expression: one particular single peptide was relatively efficient in restoring class I MHC expression to some degree and in increasing the number of single positive cells with diverse receptors in organ culture (AshtonRickardt et al., 1993). As a rule, however, it appeared that while single peptides were as efficient in restoring class I MHC expression as mixtures of diverse peptides, they were less efficient in increasing the number of single positive cells. From this it was concluded that the function of peptides was more than to stabilize MHC expression and that they contributed to positive selection (Ashton-Rickardt et al., 1993).A similar but somewhat less extensive study was reported by Hogquist and colleagues (1993). These data thus seemed compatible with the data in MHC-mutant mice that suggested that peptides could play a role in positive selection but they left open the question whether they would contribute to or interfere with positive selection. In further experiments TCR transgenic P2m-l- or TCR transgenic TAP-1 - mice were studied by the same approach. It was shown that with one particular TCR the number of CD4-8+ cells could be increased by antagonist peptides (see Section IV,A,3b) when given at relatively low concentrations. Some of these peptides induced deletion of CD4+8+ cells when given at relatively high concentrations. Again, these experiments were carried out under conditions in which peptides did not produce detectable changes in class I MHC expression because all of these effects were observed at MHC surface densities below the levels of detection. Nevertheless, these studies were interpreted to indicate that positive selection of immature thymocytes with a transgenic TCR can be produced by peptides that fail to induce cytolytic activity in mature T cells bearing the same transgenic TCR, indicating that positive selection does not require the same peptide as T cell activation (Hogquist et al., 1994). These experiments were also consistent with the notion that deletion of C D 4 + 8 +cells requires a higher avidity of a TCR-ligand interaction than positive selection even though, strictly

-’-

’

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speaking, this is an extrapolation which can only be made if it can be shown that there are indeed more ligands on the cell that induces negative selection that on the cell that induces positive selection. The studies in the TCR transgenic TAP-l-’- mice came up with the notion that peptides that can induce proliferation and deletion at high concentrations could seemingly produce positive selection at low concentrations (Ashton-Kickardt et al., 1994). The same notion was made in another report (Sebzda et al., 1994) even though in these experiments rnuch lower concentrations of the specific peptide were required. While Bevan and colleagues (Hogquist et al., 1994) did not observe positive selection b y the specific peptide at any concentration in initial experiments they noted in later experiments that low concentrations of the specific peptide yielded CD4-8+ T cells with lower levels of CD8 but these cells failed to respond properly to the specific peptide (Jameson et nl., 1994). The authors drew attention to the fact that cells selected by the specific peptide in the other reports had likewise low CD8 levels and that their responsiveness was not tested. This report makes the type of “positive selection” obtained with specific peptides questionable. In fact it was reported in earlier experiments that CD4-8+ T cells with a class I MHC-restricted TCR and low levels of CD8 were present in mice that contained the specific peptide (Kisielow et d., 198813; Teh et al., 1989). Selection of these cells, however, did not require the expression of selecting ligands on thymic epithelium and these cells were likewise not responsive to the specific peptide (von Boehmer et al., 1991). These cells could actually represent a transgenic artifact like cells of the y8 lineage that express an aP transgenic TCR. Thus at present the various interpretations of these recent experiments that aim to identify positively selecting ligands are highly questionable. The only safe conclusion that can be made at the moment is that ligands inducing positive selection differ from ligands that induce T cell activation. Whether this is so because they bind with different avidity and/or induce conformational changes rather than crosslinking of the TCR is not clear. The suggestion that different TCR-ligands are responsible for positive and negative selection (Fig. 3) is perhaps not surprising in the light of earlier findings that the same subset of CD4’8’ cells can be affected by positive and negative selection (Kisielow et al., 1991,1992; Lundberg and Shortman, 1994; Spain and Berg, 1992; Swat et al., 199lb,1994b; Vasquez et al., 1992). The experiments addressing the role of peptides in positive selection do not provide a satisfactory answer to the question of the purpose of positive selection: while they indicate that peptides can influence

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positive selection they do not indicate whether it is the peptide rather than the MHC molecule itself that determines positive selection at physiological densities of MHC molecules. It seems obvious that positive selection should select T cells that can be efficiently induced by foreign peptides presented by self-MHC molecules. Whether this “MHC-ness” of T cell recognition is dependent on the upward pointing residues of the MHC molecules itself or is imprinted in the shape of the peptide is, at the moment, also not clear. In other words, despite the experiments showing that peptides can influence positive selection under certain experimental conditions it is still possible that under physiological conditions and with the majority of TCRs it is the MHC molecule itself rather than the peptide that determines positive selection. The fact that peptides that cannot induce T cell immune responses and are in effect antagonistic, can nevertheless be sufficient to mediate positive selection may in fact be the most significant result of these recent experiments. Despite these experiments it is quite possible that with many TCRs and under more physiological conditions MHC molecules may b e able to positively select irrespective of their peptide content. In other words, we are getting better ideas of what can positively select under nonphysiological conditions but we would like to know what does positively select under physiological conditions.

c . Cells Inducing Positive Selection. The issue concerned with the question of which cells can induce positive selection and whether they generally differ from those inducing negative selection has been addressed in many different ways. From experiments in various hemopoietic chimeras it is clear that hemopoietic cells are far less efficient, if at all, than radioresistant epithelial cells (Kisielow et al., 1988a; Lo and Sprent, 1986; Sprent et al., 1988). The ability of thymic epithelial cells to induce positive selection was also demonstrated by the intrathymic injection of cell lines derived from thymic epithelium (Hugo et al., 1992; Vukmanovic et al., 1992). Nevertheless, some reports indicated that either hemopoietic cells (Bix and Raulet, 1992)or even fibroblasts (Hugo et al., 1993; Pawlowski et al., 1993) could induce positive selection (of CD4-8+ T cells) in uivo, while all attempts to achieve positive selection in uitro in the absence of thymic epithelial cells have failed (Anderson et al., 1993b).Also, in contrast to CD4-8+ cells, positive selection ofCD4 8- cells on class II-positive hematopoietic cells was not observed (Markowitz et al., 1993).One way to reconcile these apparent discrepancies is to assume that thymic epithelial cells and their product(s) are indeed essential for positive selection but that the ligands do not necessarily need to be presented by epithe+

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lial cells, i.e., one could imagine a C D 4 + 8 +cell undergoing positive selection by its TCR binding to a ligand on some fibroblast while other accessory molecules of that CD4+8 thymocyte would interact with epithelial cells or their products. If this were really true one still needs to explain why, in many experiments (Kisielow et al., 1988a; Markowitz et al., 1993),potential selection by hemopoietic cells in the thymus was so inefficient. One could argue that in this case appropriate ligands, i.e., MHC molecules and peptides, were not expressed by the hemopoietic but only by epithelial cells. Alternatively, one could argue that the fibroblast injection experiments (Hugo et al., 1993; Pawlowski et al., 1993) were grossly unphysiological and that it was something in the transformed cell lines and/or the presentation of large amounts of ligands that permitted positive selection to proceed. Unfortunately the nature of molecules, with the exception of some TCR ligands which are required for positive selection, is unknown at present; thus, it is not very useful to further discuss in which way thymic epithelial cells may be special. +

D. NEGATIVE SELECTION OF THYMOCYTES The purpose of selective events in the thymus is the separation of T cells that are potentially useful for the organism from those that are useless or could be potentially harmful (von Boehmer et al., 1989). This is achieved by rescuing from programmed cell death (positive selection) cells by one type of MHC ligand and by inducing death of cells (negative selection) by another type of MHC ligand (von Boehmer and Kisielow, 1990). The concept of programmed cell death, recently reviewed by Raff (1992), assumes that survival of cells depends on the suppression of genetically programmed, suicidal machinery that leads to cell disintegration by apoptosis, i.e., by a poorly understood process of a natural cell death that is dependent in most cases on the synthesis of new macromolecules and is accompanied by characteristic morphological features that include cell shrinkage, segmentation of the nucleus, membrane blebbing, and endonuclease-mediated DNA fragmentation (reviewed by Cohen, 1992). It is now generally believed that apoptosis represents the main mechanism of cell death for thymocytes whose receptors do not engage in interaction with intrathymic ligands as well as for thymocytes whose receptors bind with high avidity to intrathymic ligands. Below we will review the evidence supporting this belief and discuss the possible mechanisms controlling the induction of apoptosis at different stages of intrathymic development.

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1 , Apoptosis as a Mechanism of Negative Selection of T Cells From early and later studies on the kinetics of generation of thymocytes and their export to peripheral organs it was suspected that the great majority of newly formed cortical thymocytes die in situ (Matsuyama et al., 1966; McPhee et al., 1979; Scollay et al., 1980). It was, however, difficult to support this notion by direct evidence. Although a series of earlier studies, comparing the relative retention in the thymus of iododeoxiuridine and thymidine after their incorporation into thymocyte DNA, was interpreted as providing direct evidence for the intrathymic death of thymocytes (Joel et al., 1977; McPhee et ul., 1979), it turned out that deiodination, rather than cell death and nucleoside reutilization, was the real explanation of these results (Quackenbush and Shields, 1988; reviewed by Rothenberg, 1990). Thus, the evidence for the intrathymic death of cortical thymocytes was based on studies of the kinetics of their turnover and was therefore indirect. Another indirect evidence of intrathymic cell death was the extreme sensitivity of cortical thymocytes to changes in the level of glucocorticoids (Blomberg and Andesson, 1971; Shortman and Jackson, 1974) (reviewed by Cohen, 1992).Elevated levels of glucocorticoids resulted in rapid elimination of CD4+8+thymocytes, whereas decreased levels led to an increase of thymus size. It was shown by Wyllie (1980) that glucocorticoid-treated cortial thymocytes died by apoptosis. The interest in apoptosis as the mechanism of thymocyte death was revived following the observation that immunological self-tolerance involved deletion of immature cortical C D 4 + 8 + thymocytes that expressed a self-antigen-specific a/3 TCR (Kisielow et al., 1988b; Sha et al., 1988). It was shown that apoptosis of immature C D 4 + 8 +thymocytes could be induced not only by glucocorticoids but also by CD3 antibodies (Shi et al., 1991; Smith et al., 1989a) or antigen (Murphy et al., 1990; Swat et al., 1991b).These results identified another physiological trigger of programmed cell death of thymocytes and indicated that apoptosis of C D 4 + 8 + cells represents not only a mechanism for disposal of useless cells but also serves the purpose of eliminating cells whose receptors bind self-antigen with high avidity. In vitro experiments, analyzing the responses of purified populations of thymocytes bearing a transgenic TCR of known specificity (Kisielow et al., 1991; Pircher et al., 1992; Spain and Berg, 1992; Swat et al., l99lb; Vasquez et al., 1992), convincingly demonstrated that apoptosis of C D 4 + 8 +thymocytes resulted from specific, TCR-mediated interaction of these cells with antigen-presenting cells. These experiments also showed that identical conditions of antigenic stimulation led to dele-

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tion of CD4 '8 thyniocytes and to proliferation of mature T cells (Kisielow et d., 1991). Thus, these results directly showed that CD4+8' thymocytes express functional aP TCRs that can transduce antigen-specific signals, which instead of inducing proliferation and differentiation induce death by apoptosis. The idea that the response of immature and mature lymphocytes to antigen is intrinsically different and that self-antigen-induced deletion of immature lymphocytes is one mechanism of immunological selftolerance was proposed many years ago by Burnet (1959) and Lederberg (1959).Evidence compatible with the concept of clonal deletion but suggesting that it occurs at later developmental stages of thymocytes was also obtained in normal mice by showing that certain TCRP chains conferring specificity to determinants encoded by mouse tumor viruses (Acha-Orbea et ul., 1991; Choi et d., 1991; Woodland et al., 1990,1991) (reviewed by Acha-Orbea and Palmer, 1991) were present in immature C D 4 + 8 +and mature single positive cells of mice lacking these determinants but absent from single positive, but not C D 4 + 8 + , immature cells in mice expressing MTV-encoded proteins (Kappler et al., 1987,1988; MacDonald et al., 1988a). In subsequent analyses it was also found that C D 4 + 8 +thymocytes could serve as targets of socalled "superantigen"-induced deletion (White et al., 1989). In order to more directly investigate the question of the susceptibility of different intrathymic stages to TCR-mediated induction of apoptosis, and to determine the stage at which the cells become refractory to deletion and start to respond by proliferation to TCR triggering, the in uitro assay of clonal deletion (Swat et al., 1991a,b) was modified such as to allow for estimation of the responsiveness of different subsets in a semiquantitative way (Swat et al., 1993).The results indicated that in the absence of exogenous growth factors all intrathymic stages expressing low or high levels of c.P TCR (i.e., CD4+8+ thymocytes and their CD4-8' precursors as well as CD4-8+ progeny) were succeptible to TCR-mediated deletion induced by anti-TCR antibody or antigen (Kisielow et al., 1991; Swat et al., 1993,1994a; W. Swat and P. Kisielow, unpublished results). In contrast to C D 4 + 8 +thymocytes, TCR-mediated deletion of later stages in thymocyte development could be largely but not completely prevented by addition of exogenous IL-2 (Lavetzky et al., 1991; Nieto et al., 1990; Swat et al., 1993). From these results it appears that positively selected thymocytes remain susceptible to TCR-mediated deletion and that the switch from deletion to proliferation in response to TCR triggering occurs at the late C D 4 + 8 - and CD4-8+ stage of intrathymic development. Of course this does not mean that response of mature T cells to antigen never results in apoptosis. It was shown that under certain conditions, +

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antigen recognition by mature T cells may lead to deletion after a brief phase of cell proliferation (Lenardo, 1991; Rocha and von Boehmer, 1991; Russell et al., 1991). The above examples of suicidal death of a6 TCR-expressing thymocytes in response to antigen recognition provided good evidence for the role of so-called “activation-induced apoptosis” in negative selection. The by then generally accepted view that unselected C D 4 + 8 + thymocytes, whose TCR failed to interact with intrathymic ligands, die b y apoptosis was supported by observations that their spontaneous as well as glucocorticoid-induced death in vitro and in vivo is inhibited by a bcl-2 transgene (Sentman et al., 1991; Strasser et al., 1991). The role of apoptosis during the early developmental stages that preceed the expression of the a/3 TCR is not obvious. Strasser et al. ( 1994) have reported that in bcl-2 transgenic scid mice, thymocyte numbers are not increased and immature thymocytes undergo rapid apoptotic death in vitro despite expressing high levels of bcl-2 protein, which contrasts with the above-mentioned effect of bcl-2 overexpression in CD4+8+thymocytes. On the other hand, Andjelic et al. (1993b) reported that CD4-8-CD25+ thymocytes from normal mice survive well in uitro, express high levels of bcl-2 mRNA, and display a selective resistance to ionomycin (Ca2+)-mediatedapoptosis (but not to steroid-induced apoptosis) which is lost as soon as the cells begin to express CD4 and CD8 molecules and downmodulate bcl-2 expression. These observations are consistent with the possibility that cells unable to rearrange or to express TCR genes die by apoptosis and that bcl-2 is not sufficient to ensure survival in the absence of TCRP chain. However, more experiments would be required to clarify this issue. Whether the pre-T cell receptor, like the aP TCR, has the dual potential of transmitting death-inducing and differentiation-inducing signals is not known. So far the only documented effects of the expression of TCRP in the absence of TCRa chain are related to survival, differentiation, and expansion. A recent report (Dent et al., 1993) suggests that self-reactive yi3 thymocytes may also die b y apoptosis but its role in development of y8 T cells compared to that of a@T cells is much less clear. In fact, the notion of apoptotic cell death of self-reactive yi3 cells was contested by other authors (Haas et al., 1993).

2 . Control of Apoptosis during Zntrathymic Development From the discussion throughout this review it should be clear that the possible reasons for natural intrathymic death by apoptosis are manyfold and may be different for different developmental stages of thymocytes. Some of the causes have been identified and include

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TCR-dependent (antigen) as well as TCR-independent (glucocorticoid, irradiation) factors; others remain to be elucidated. There exists some evidence that different intracellular molecular mechanisms control the susceptibility of thymocytes to different apoptotic stimuli. This evidence stems from the studies on the role of bcl2, Nur-77, p53, and c-myc gene products in controlling programmed cell death. For example, several studies have shown that overexpression of bcl-2 in CD4+8' thymocytes of normal and TCR transgenic mice protected these cells against glucocorticoids, radiation, and CD3 antibody-induced apoptosis but not, or only marginally, against antigen- or superantigen-induced deletion (Sentman et al., 1991; Siege1 et al., 1992; Strasser et al., 1991; Tao et al., 1994). On the other hand, inhibition of Nur-77 expression was shown to prevent the induction o f apoptosis by CD3 antibodies but not by glucocorticoids (Liu et al., 1993; Woronicz et al., 1993). Siniilar effects were observed by analyzing the influence of retinoic acid (Yang et al., 1993), which inhibited C D 3 antibody-induced as well as antigen-induced apoptosis of CD4+8' thymocytes but was ineffective in protecting against glucocorticoid-induced death. Experiments with p53-deficient mice have shown that the induction of apoptosis in cortical thymocytes b y y-irradiation and etoposide required the presence of the p53 tumor suppressor gene but apoptosis induced by glucocorticoid or phorbol ester and calcium ionophore is independent of p53 (Clarke et al., 1993; Lowe et al., 1993).It seems possible that the physiological function of p53 during T cell development could have to do with the elimination of cells which damaged DNA, which in the thymus could occur with higher frequency due to the ongoing process of TCR gene rearrangement. The evidence for the involvement of c-myc in regulation of thymocyte apoptosis is conjectural. It is based on the observation that activated CD4+8+thymocytes show a substantial increase in c-myc mRNA levels (Riegel et al., 1990) and that c-myc antisense oligonucleotide inhibits apoptosis in T cell hybridomas treated with TCR antibodies but not with steroids (Shi et al., 1992).Interestingly, apoptosis induced by c-myc in cultured nonlymphoid cells was shown to be sensitive to inhibition by bcl-2 (Bissonnette et al., 1992; Fanidi et al., 1992). However, the involvement of such mechanisms in regulating apoptosis in thymocytes has yet to be established. Finally, as already mentioned, bcl-2 overexpression in CD4-8- thymocytes from scid mice does not inhibit apoptosis in these cells (Strasser et al., 1994). Thus, it appears that multiple death pathways, which are regulated by distinct molecular mechanisms, may control intrathymic development of thymocytes. Because cortical thymocytes are sensitive to glucocorticoid-induced

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apoptosis within the physiological range of the hormone concentration, it was suggested that this could be the mechanism of negative selection of thymocytes whose antigen receptors failed to bind to MHC molecules (Cohen, 1991)and that positive selection would depend on inhibition of glucocorticoid-induced apoptosis by TCR-mediated signal of appropriate “strength” (King and Ashwell, 1993).Results showing that thymocytes could be rescued from glucocorticoid-induced death by stimulation via the TCR/CD3 complex (Iwata et al., 1991; Zacharchuk et al., 1990) seem to support the model of selection based on the mutual antagonism between TCR- and GR-mediated signaling pathways. However, GR-mediated potentiation of TCR-mediated apoptosis of thymocytes has also been reported (Jondal et al., 1993), indicating the need for further studies on the relationship between TCR-mediated signals and GR. a . Cells Znducing Deletion of Thymocytes. Early in vivo studies concerned with the question of the identity of thymic stromal elements responsible for induction of immunological tolerance, using various kinds of chimeric animals, indicated that bone marrow-derived cells were very efficient, whereas the role of epithelial cells was controversial (reviewed by Sprent and Webb, 1987; Sprent et al., 1988). Since that time, the use of direct in vitro assay for clonal deletion of CD4+8+ thymocytes (Swat et al., 1991a,b) and better-defined in uivo systems employing transgenic mice and cloned cell lines provided convincing evidence that tolerance induction does not require “dedicated” antigen-presenting cells. It was shown both in vitro (Iwabuchi et al., 1992; Pircher et al., 1992; Tanaka et al., 1993) and in oivo (Bonomo and Matzinger, 1993; Gao et al., 1990; Speiser et al., 1992) that not only bone marrow-derived cells, including thymocytes themselves, but also thymic epithelial cells are able to delete immature, self-antigen reactive thymocytes, albeit with different efficiency. It was also shown that clonal deletion of C D 4 + 8 +thymocytes can be induced by the same epithelial cell lines that mediate positive selection (Hugo et al., 1994; Vukamnovic et al., 1994), which definitively ruled out the possibility that negative and positive selection of thymocytes requires different antigen-presenting cells. Of course, this does not mean that under physiological conditions positive and negative selection is not mediated b y different thymic stromal elements.

b. TCR Ligands Znducing Deletion of Thymocytes. In contrast to the controversy and uncertainty surrounding the nature of the ligand for positive selection and its relation to the antigenic MHC/peptide

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complex activating mature T cells, the question concerning the nature of the ligand inducing deletion and its relation to antigen has never incited much controversy. It was clear from a series of experiments in normal and TCR transgenic mice that ligands able to induce activation of mature T cells can also induce apoptosis in immature thymocytes. Subsequent studies using cells from transgenic mice expressing TCR specific for antigenic complex of MHC with defined peptides (Ashton-Rickardt et al., 1994; Hogquist et al., 1994; Iwabuchi et al., 1992; Mamalaki et al., 1992; Murphy et al., 1990; Page et al., 1994; Pircher et al., 1992; Sebzda et al., 1994; Spain and Berg, 1992, Spain et ul., 1994; Vasquez et al., 1992) provided direct evidence for the ability of antigenic and antagonistic peptides to induce clonal deletion of C D 4 + 8 +thymocytes. The obtained results suggest that sensitivity of immature thymocytes to peptide-induced deletion is greater than the sensitivity of mature T cells to peptide-induced activation but lower than sensitivity of immature thymocytes to ligands inducing positive selection (recently reviewed by Allen, 1994; Janeway, 1994).

c . Role of Coreceptors arid Other Cell Surface Molecules in Negative Selection of Thymocytes. To learn about the involvement of various accessory T cell surface molecules and their ligands on thymic stromal cells during the process of negative selection several experimental approaches were used which included: blocking with antibodies, transgenic and gene knockout technologies, and a combination of them. Below we will briefly summarize the results of the studies concerned with the role of the following molecules: CD4, CD8, CD45, CD28, CD2, LFA-1, and Apo-l/Fas. cl . CD4 and CD8. The involvement ofCD4 coreceptor in negative selection was initially suggested by the fact that in viuo treatment with anti-CD4 antibodies inhibited the deletion of precursors of CD4-8 T cells, expressing receptors specific for superantigen (Fowlkes et al., 1988; MacDonald et al., 1988b). Subsequent experiments in transgenic mice expressing MHC class I molecules mutated in the CD8 binding domain (Aldrich et al., 1991; Ingold et al., 1991; Killeen et al., 1992a) confirmed that coreceptor participate in clonal deletion of most selfantigen reactive thymocytes. However, the requirement for CD4/CD8 coreceptor in negative selection is not absolute. It was shown that deletion of some class I MHC-restricted self-antigen-reactive and class I1 MHC-associated superantigen-reactive thymocytes can occur in the absence of CD8 (Fung-Leung et al., 1994) and CD4 (Wallace et al., 1992), respectively. It was also shown that overexpression of CD8 on the T cell surface may result in deletion of immature thymocytes that +

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are normally positively selected (Lee et al., 1992; Robey et al., 1992a). On the other hand, decreasing the affinity of the interaction with CD8 on cells expressing TCRs of high affinity for self-antigen may lead to the escape from deletion (Sherman et al., 1992). The differential requirement for CD4 and CD8 in negative selection of thymocytes could explain why in some cases it was claimed that clonal deletion " ~ Oers et al., of thymocytes is dependent on activation of ~ 5 6 (van 1992), whereas in other cases it is not (Nakayama and Loh, 1992). However, despite association of coreceptor with p56lCk,the mechanism underlying the requirement for CD8 and CD4 molecules in the deletion process is not clear because, as already discussed, CD4 and CD8 molecules can also function in the p56lCk-independentfashion (Chan et al., 1993a; Killeen and Littman, 1993). c2. CD45. The role of CD45 in negative selection is not clear. It was reported that in CD45 exon6-deficient mice clonal deletion of superantigen reactive T cells was not affected suggesting that negative selection may be independent of CD45 (Kishihara et aZ., 1993). On the other hand, in a study using TCR transgenic mice, it was shown that thymocytes with increased levels of CD45 molecules were hyperresponsive to TCR stimulation resulting in enhanced TCR-mediated apoptosis, suggesting some kind of regulatory involvement of CD45 during negative selection (Ong et al., 1994). c3. CD28. At least two lines of evidence may suggest that CD28 does not participate in intrathymic selection processes: first, the development and selection of thymocytes in CD28-deficient mice appears to be unperturbed (Shahinian et al., 1993);second, neither high-affinity soluble CTLA4-Ig ligand nor anti-CD28 Fab fragments are able to block thymocyte deletion (Jones et al., 1993; Page et al., 1993). c4. CD2. In CD2-deficient mice, development of thymocytes appears normal (Killeen et al., 1992b), indicating that interaction of CD2 with LFA-3 is not essential for selection processes occuring in the thymus. c5. LFA-I. Using anti-HY TCR transgenic mice it was shown that antibodies against LFA-1 or its ligand ICAM-1 could specifically block the antigen-dependent deletion of CD4+8+ thymocytes by dendritic cells, suggesting an important role for LFA-1/ICAM interaction in negative selection (Carlow et al., 1992; Fine and Kruisbeek, 1991). c6. Apo-1 /Fas. Although the developmental pattern of Apo-l/Fas expression of thymocytes suggests its role during selection there is so far no functional data demonstrating that it is involved in regulating apoptosis in the thymus. On the contrary, the results indicating that lack of functional Fas in lpr mutant mice does not preclude induction

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of thymocyte apoptosis by corticosteroids or by TCR-mediated mechanism (Cohen and Eisenberg, 1992; Sidman et al., 1992) indicate that Apo-1/Fas is dispensable for these processes.

d . TCR Signaling in CD4+8+Thymocytes. Superficially, the conspicuous lack of evidence for the involvement of known costimulatory interactions in negative selection of thymocytes, except LFA-l/ICAM, which may simply reflect the need for stabilizing physical contact between interacting cells, appears to be consistent with a “two-signal” concept, according to which engagement of TCR in the absence of costimulation leads to cell death. There is, however, little direct biochemical evidence to support this possibility. On the contrary, a recent report suggested that two signals, one delivered by antigen and another by unidentified costimulator on antigen-presenting cells, may be required for in uitro deletion of C D 4 + 8 +thymocytes (Page et al., 1993). The signaling pathways induced by the crp TCR in C D 4 + 8 +thymocytes are very poorly understood and the results concerning the role of the calcium and PKC-dependent events in activating cell death are controversial (reviewed by King and Aswell, 1993).Early observations indicated that triggering of TCR in C D 4 + 8 + thymocytes results in phosphorylation of <-chain (Nakayama et al., 1989) and elevation of intracellular calcium (Finkel et al., 1987; Havran et al., 1987; Weiss et al., 1987),which due to impaired activation of PLC (Sancho et al., 1992), is weaker than that in mature T cells. Several in uitro studies have shown that induction of apoptosis in immature thymocytes by anti-TCR antibodies is dependent on an early calcium flux (McConkey et al., 1992) and elevated levels of intracellular calcium in thymocytes of TCR transgenic mice undergoing deletion in uiuo (Nakaymam et al., 1992) indicate that it is also important for negative selection occuring under physiological conditions. In contrast to the general agreement concerning the role of early calcium fluxes in activating cell death, the role of calcineurin, a downstream component of the Ca2’ signaling pathway, is not clear because according to some authors cyclosporin inhibits TCR-induced death of thymocytes (Shi et al., 1989), but according to others it does not (Vasquez et al., 1992). The main reason for the uncertainty in this area has been the lack of physiological models for studying the responses to CD4+8’ thymocytes to intrathymic TCR-ligands. Thus far the most suitable system for such studies is based on suspension culture of thymocytes expressing transgenic TCRs in the presence of antigen-presenting cells (Swat et al., l991a,b). Recently, Vasquez et al. (1994) have used such a culture system to systematically analyze intracellular signals that medi-

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ate thymic negative selection. The results of this study suggest that antigen-induced apoptosis of CD4+8+thymocytes is dependent on phosphatidylinositol hydrolysis, early intracellular calcium flux, and activation of PKC, but is independent of calmodulin/calcineurinmediated effects (Vasquez et al., 1994). The results obtained in these systems are handicapped, however, by the fact that no positive selection is observed under those conditions and therefore one cannot distinguish differentiation-inducing signals from death-inducing signals as both could lead to cell death under those conditions. On the other hand, the fetal thymus organ cultures used to investigate the positive selection are not suitable for analyzing intracellular signals responsible for negative or positive selection of CD4 + 8 +thymocytes. VI. Concluding Remarks

Looking back on experiments and results concerned with T cell development over the past decade it is quite apparent that natural and artificially generated mutant mice as well as trangenic mice have contributed substantially to our understanding of cellular and molecular processes that govern the generation ofan effective immune system. We have learned that the pre-TCR, in the form of the TCRp chain, which is paired with a developmentally regulated, disulfide-linked protein drives expansion and differentiation of small numbers of early immature thymocytes and we seem to understand at least part of the signaling cascade of this receptor. We have also realized that the specificity of the Cup TCR, i.e., its ability to bind differently or not at all to thymic ligands, decides whether a developing cell will die rapidly by apoptosis, will be rescued from programmed cell death, or will die by programmed cell death after several days. We are only beginning to understand the molecular mechanisms that are behind these selective events. Without the help of mutant mice our thinking about T cell development would still be in its infancy and we would have no clues as to which genes and developmental steps are essential for T cell maturation and which ones are not. It is probably fair to conclude that the advantages of the studies in genetically altered and therefore somewhat artificial experimental animals are outweighting the disadvantages as long as we make sure that the newly discovered principles and mechanisms are really operative under physiological conditions. It seems only obvious that a better understanding of the role of various gene products as well as the molecular and cellular mechanisms governing T cell development will significantly contribute to our under-

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standing of disease that are associated with malfunctioning immune systems. Already, studies in mutant mice have made possible the identification of defective genes causing immunodeficiency or autoimmunity in humans. Mutant mice will not only serve as tools to study development and function but they will also be useful in the future as disease models to explore therapeutic strategies.

ACKNOWLEDGMENTS We thank Drs. Richard Hardy, Dietinar Kappes, Klaus Kajalainen, Bernard Malissen, Diane Mathis, Benedita Rocha, Hans Raimer Rodewald and Ken Shortman for sharing unpublished data with us and for permission to cite them. We also thank Hanspeter Stahlberger for artwork and Malgorzata Szyniocha and Jan Kisielow for help in preparation of the manuscript. T h e Basel Institute for Immunology is founded and supported by Hoffniann-L;i Roche Ltd.

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pathways are mutually antagonistic. J. Irnmuno[. 145, 4037-4045. Zijlstra, M., Bix, M., Simister, N. E., Loring, J . M., Raulet, D. H., and Jaenisch, R. (1990). /32-microglobulin deficient mice lack CD4-8’ cytotoxic T cells. Nnture (Landon)344, 742-746. Zinkernagel, R. M., Callahan, C. N., Althage, A., Cooper, S., Klein, P. A., and Klein, J. (1978). On the thymus in the differentiation of “H-2 self-recognition” by T cells: Evidence for dual recognition? j . E x p . Mad. 147, 882-896. Zlotnik, A . , Godfrey, D., Fischer, M., and Sutla, T. (1992). Cytokine production by mature and immature CD4-CD8- T cells: alpha/beta T cell receptor+ CD4-CDK T cell produce IL-4. J. Zrnrnunol. 149, 1211-1215.

ADVANCES IN IMbIUNOL0C;Y. VOL .58

The Pharmacology of T Cell Apoptosis GUIDO KROEMER CNRS-UPR420, F-94801 Villeiuif, France

I. Introduction 11. Induction of T Cell Death 111. Inhibition of T Cell Death IV. Theoretical Insights Gained by Apoptosis Modulation V. Functional Consequences of Apoptosis Modulation VI. Conclusions References

21 1 215 240 265 272

280 283

L’inferno dei viventi non e qualcosa che sari; se ce n’e uno, 6 quello che 6 gia c4ui, I’inferno che abitianio tutti i giorni, che formiamo stando insieme. D u e niodi ci sono per non soffrirne. I1 primo riesce facile a molti: accettare l’inferno e diventarne parte fino a1 punto di non verderlo piB. I1 secondo t rischioso ed esige attenzione e apprendimento continui: cercare e saper riconoscere chi e cosa, in mezzo all’inferno, n o n e inferno, e farlo durare, e dargli spazio. ltalo Calvino. Le citti invisibili T h e hell o f t h e living is not something that will arrive in the future; if hell exists, then it is that we are already living in day by day, that w e form being together. There are two ways for not suffering from hell. The first results easy for many people: accept hell and become part of it to the point that it you will not see it any more. The second way is risky and requires continuous attention and learning: find out and recognize what, in the middle of hell, does not belong to hell, and help it to last and give room to it. Italo Calvino, The invisible cities

1. Introduction

The irruption of programmed cell death (PCD, apoptosis) into immunology illustrates how concepts acquired in fundamental, nonapplied 211 Copyright 0 199.5 l ~ yAcademic h e \ , Inc. All ilrhtr of rrpniduction in .iny form rr\erved.

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science may fecundate a medically oriented discipline. Apoptosis as a mechanism of cell death was discovered in the early seventies (Kerr et al., 1972) but was neglected for a certain period. Nonetheless, today it is clear that PCD is a cardinal element of homeostatic regulation of the normal immune system, and attempts are being undertaken to modulate it by external agents, thus correcting disease states coupled to deficient cell death regulation. This survey will focus on the pharmacology of apoptosis of T lymphocytes. PCD is a widespread phenomenon that probably concerns any nucleated cell type and not only lymphocytes (Cohen 1992b;1993; Golstein et al., 1991; Martin, 1993a; Schwartz and Osborne, 1993; Williams and Smith, 1993). It may be defined either morphologically or biochemically. In most cases, PCD is accompanied by characteristic ultrastructural alterations (cell shrinkage, cytoplasmic compaction, membrane blebbing, nuclear chromatin condensation) (Kerr et al., 1972) and/or the internucleosomal “ladder-type” fragmentation of nuclear DNA by endogenous endonucleases (Peitsch et al., 1994; Willie, 1980).Classical PCD requires de nouo gene expression and protein synthesis, i.e., active collaboration of cells in the realization of a “death program.” However, at least in some cases, PCD only requires activation of a preexisting suicidary enzymatic machinery. Many immunologists employ the terms “apoptosis” (that refers to the morphology of death) and PCD (that refers to the biochemistry of death) as quasisynonyms because lymphoid PCD in uitro is usually accompanied by the morphological picture of apoptosis. Both terms, apoptosis and PCD, may be defined by opposing them to another fundamental type of cell death, necrosis. Whereas necrosis exemplifies passive cell death caused by exogenous stimuli (e.g., mechanical trauma, heat, toxins, hypotonic osmotic pressure, detergents) causing disrupture of internal and external membranes, PCD involves the active participation of endogenous cellular enzymes in the mediation of death before membranes lose their integrity. Lytic necrosis entails the liberation of denatured proteins and DNA fragments into the intercellular space, the action of oxygen radicals, as well as local inflammatory responses with production of cytokines, thus causing potentially dangerous secondary responses within the organism. In contrast, cells undergoing PCD exhibit near-to-intact external membranes until late phases of the metabolic suicide, allowing adjacent cells to engulf the dying cell and to eliminate it before it releases its content (Savill et al., 1993). Fullblown apoptosis probably constitutes a default pathway of PCD, and in uiuo most cells undergoing PCD are removed b y phagocytosis before the death process culminates in low-molecular-weight DNA frag-

PHARMACOLOGY OF T CELL APOPTOSIS

213

mentation and apoptotic morphology of the nucleus. Consequently, apoptotic cell death avoids disrupture of the tissue architecture, damage of cells and matrix proteins by lysosomal enzymes, and subsequent inflammatory responses caused by invading specialized phagocytes. From a teleological point of view, PCD appears more advantageous for removal of cells than necrosis (Bursch et al., 1992). Throughout their life span T lymphocytes and their precursors are notoriously prone to undergo apoptotic cell death. At any moment, a T cell is confronted with the choice of continuing its existence or committing suicide. According to current understanding, programmed death and/or phagocytic elimination constitutes the natural fate of T cells. In a steady-state system in which the total number of lymphocytes remains stable, each mitosis occuring during the generation and expansion of lymphocyte precursors and mature T or B cells should be compensated for by programmed death of two cells. As will be discussed in detail in this paper, a plethora of different stimuli cause T cell apoptosis: antigen receptor-mediated stimulation, cell contactdependent signals, triggering of cytokine or steroid receptors, as well as mild physical and chemical damage. It is important to state that there are no physiological stimuli that always provoke the death of T cells. On the contrary, a putative death-inducing stimulus will only trigger PCD in dependence of a determined context: the differentiation and activation states ofthe T cell, the presence ofother (co)stimulatory signals, the metabolic environment, the presence of antigen, etc. Thus, T cells fulfill a semiotic function in the sense that they continuously integrate stimuli arriving via multiple receptors that receive information from antigen-presenting cells, bystander cells, cell matrix proteins, cytokines, hormones, and the metabolic microenvironment (Kroenier et al., 1993a).Only a determined combination of signals will cause PCD. In general terms, it appears that any kind of aberrant stimulation of T cells causes PCD. Thus, T cells obey the maxim “better dead than wrong” in order to minimize the risk of expanding self-reactive clones and of developing leukemias or lymphomas. The cardinal importance of PCD regulation for the normal function of the immune system is unraveled by pathological states in which the control of PCD is compromised (Table I). Two different possibilities may be taken into account. Either T cells are fragile, thus leading to an enhanced apoptotic turnover and a state of functional and/or numeric immunodeficiency, or T cells are abnormally resistant to PCD. Consequently, the decision whether a lymphocyte will mount an unwarranted (autoaggressive) or desired (antitumor, antiparasite) response or undergo malignant transformation depends on the functional

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TABLE I PATHOLOGICAL CONSEQUENCES OF DISTURBANCES IN Apomosis REGULATION IN T CELLCOMPARTMENT Abnormal Resistance to Undergo Apoptosis

THE

Elevated Apoptotic Decay of T Cells

Lymphocytosis Lymp hadenopathy Splenomegaly Accumulation of self-reactive T cells Autoimmune disease Lyniphoma/leukemia development

Lymphopenia Systemic immunodeficiency Specific immunodeficiency Septic shock?

Note. For details, examples. and references see text.

state of its (anti)apoptotic machinery. Accordingly, abnormalities in the regulation of lymphocyte PCD have been implicated in different diseases. An increase in the susceptibility to PCD induction causes immunodeficiency. Mice carrying a genetic defect of the PCDinhibitory protooncogene bcl-2 spontaneously develop a severe numeric immunodeficiency (Nakayama et al., 1993; Veis et al., 1993). An excessive apoptotic decay of lymphoid cells is involved in the pathogenesis of acute transient virus-induced immunodepression, e.g., infectious mononucleosis (Uehara et al., 1992), and infection with cytomegalo virus (Meyaard and Miedema, 1994). More importantly, an enhanced tendency to undergo PCD is observed in lymphocytes from human immunodeficiency virus (H1V)-infected donors and is thought to contribute to the development of chronic AIDS-associated lymphopenia (Gougeon et aZ., 1991; Groux et nl., 1992; Meyaard et nl., 1992). Enhanced PCD may also participate in tumor-mediated immunosuppression. An esophageal squamous carcinoma cell line has been shown to release a suppressive factor that arrests T ceIls at the G l / S interphase of the cell cycle and induces apoptosis (O’Mahony et nl., 1993). If PCD concerns all T cells irrespective oftheir specificity, it causes a systemic immunodepression. In contrast, the elimination of a part of the T cell repertoire will only cause a more specific immunodeficiency. Deficient PCD may also participate in the disease process. Abnormal resistance to the induction of PCD is coupled to the development of leukemias and lymphomas, lymphoid hyperplasia, and autoimmune diseases (Table I). Thus, defective expression ofthe gene encoding the PCD-regulatory CD95 (Fas/Apo-1) protein (the so-called Zpr mutation) (Watanabe-Fukunaga et al., 1992), a point mutation in the ligand of

PHARMACOLOGY OF T CELL APOPTOSIS

215

Fas (the so-called gld mutation) (Takahashi et al., 1994), a defect in the apoptosis-regulatory bcl-2 protooncogene ( b ~ 1 - 2(Garchon ~ ~ ~ ) et al., 1994), as well as transgene-enforced hyperexpression of the bcl-2 gene (McDonnell et al., 1989; Strasser et al., 1991a) cause an abnormal resistance ofperipheral T cells to PCD induction linked to the development of generalized lymphoproliferative disease, an overexpansion of T cells in the spleen (in the case of the NOD strain), and spontaneous autoimmune diseases. In addition, mice homozygous for deletion mutations of the apoptosis-inducing antioncogene p53 develop lymphomas (Donehower et al., 1992), and transgene-enforced overexpression of the antiapoptotic protooncogene bcl-2 gene may cause tumors (Strasser et al., 1990). In view ofthe pathological role ofabnormal PCD regulation, pharmacological interventions on T lymphocyte apoptosis will constitute a welcome addition to the clinician’s armamentorium. On one hand, it can be attempted to induce PCD in cells that failed to do so. On the other hand, pharmacological treatments are being designed with the purpose of suppressing excessive apoptosis. In this article different strategies for the induction or inhibition of peripheral T cell apoptosis will be described with special emphasis on the mechanism of action of such interventions, as well as on their functional impact on the immune system.

II. Induction of T Cell Death

T cells succumb to PCD in a variety of different circumstances. (i) Positive transmembrane signaling can trigger death in cells that

would continue their existence in the absence of such stimuli.

(ii) Some T lymphocytes undergo apoptosis when they lack a deter-

mined survival signal. (iii) Apoptosis can occur as a consequence of cell damage. In this section, the multiple stimuli causing cell death in T cells will be discussed briefly. On one hand, apoptotic cell death is induced by stimuli targeted to the T cell receptor (TCR)-CD3 complex, thus providing a model for antigen-driven negative selection processes. On the other hand, a plethora of different non-TCRmediated, i.e., nonspecific” (in the immunological sense of nonantigen-specificity), stimuli provoke PCD of T cells. “

A. TCR-MEDIATED INDUCTION OF T CELLDEATH 1 . In Vitro Models of Peripheral Deletion Stimuli delivered via the TCR that under most conditions would activate T cells for proliferation induce PCD in determined circum-

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stances (Table 11). For example, thymocytes exposed to immobilized anti-CD3c antibodies in vitro tend to undergo apoptosis. This applies in particular to the CD4+CD8+fraction of mouse thymocytes (Smith et al., 1989a). Similarly, purified CD4+CD8+human thymocytes that are exposed to sequential stimulation with plastic-bound anti-CD3c antibodies and soluble anti-alp TCR antibody undergo apoptosis in uitro (Ramirez et al., 1994). In organ cultures, the bacterial superantigen Staphylococcus aureus enterotoxin B (SEB) causes apoptosis of SEB-reactive thymocytes (Jenkinson et al., 1989). A human CD4+ T cell clone also reacts to bacterial superantigens by undergoing apoptosis (Kabelitz and Wesselborg, 1992). Moreover, T cell hybridomas obtained by fusing peripheral T cells with immortal leukemia cell lines are highly prone to undergo PCD upon activation via the TCR/CD3 complex (Odaka et al., 1990; Shi et al., 1990). Mature peripheral CD4+ or CD8+ T lymphocytes undergo PCD upon stimulation with immobilized (not soluble)anti-CD3 or anti-TCR antibodies, provided they have been preexposed to specific antigen (Russell et al., 1992) or phytohemagglutinin (PHA) (Wesselborg et al., 1993a). Freshly isolated peripheral blood lymphocytes from humans are resistant to the induction of apoptosis by PHA and anti-CDSE. However, cells stimulated with PHA and PMA and further expanded in interleukin-2 (IL-2) become progressively susceptible to the induction of PCD with these agents. Hence, stimuli that activate resting T cells initiate death by apoptosis in activated T cells (Wesselborg et ul., 1992a). Religation of the TCR after primary activation also induces apoptosis of mature mouse T cells (Radvanyi et aZ., 1993). Priming to apoptosis induction could be associated with repeated passage through the cell cycle (Radvanyi et al., 1993; Russel et al., 1991; Wesselborg et al., 1993a). It appears that human peripheral T cells expressing a memory phenotype (CD45RBd"") are more prone to undergo PCD after stimulation with phytohemagglutinin in the absence of IL-2 than naive T cells (Salmon et al., 1994). This correlates with reduced expression of the antiapoptotic protooncogene bcl-2 and an elevated expression of the apoptosis-inducing surface molecule Fas/Apo-1 (Salmon et al., 1994). Whether these changes are responsible for an enhanced susceptibility to undergo apoptosis remains elusive. The susceptibility of T cells to undergo apoptosis upon TCR/CD3 ligation can be considerably augmented by providing additional stimuli. In a CD4' T cell line, ligation of the a/@TCR and CDllaICD18 (LFA-1/@2integrin) by insoluble ICAM-1 or CD29C/CD49d (VLA-5/ 01 integrin) by VCAM-1 causes a higher incidence of apoptotic cell

INDUCTION OF

Substance

TABLE I1 APOPTOslS VIA THE TCRiCD3 COMPLEX

Probable Active Principle

Cell Type

Reference

Ant i-CD3

Ligation of TCRiCD3 complex

Anti-CD3

Religation of the TCR after primary activation

CD4'CDB' thyniocytes T cell hybridomas Peripheral mouse T cells Peripheral human cells

Exposure to nominal class Irestricted peptide in citro Exposure to nominal class Irestricted peptide i n uitro Injection of class 11- or class Irestricted peptides Injection of antigen-expressing s t i ni u I ator ce 11s Superantigen

Cell contact-dependent activation Stimulation of transgeneencoded TCR on most cells Stimulation of specific TCRtransgene-expressing cells Stimulation of TCR- transgeneexpressing T cells Activation-induced cell death

Smith et ol., 1989a; Odaka et al., 1990 Russell et al., 1992; Radvanyi et ol., 1993; Wesselborg e t al ., 1993a Walden and Eisen, 1990; Moss et ol., 1991 Swat et al., 1991

F23.1 anti-VB8

Activation i n the ab\ence of APC Macrophage-dependent deletion of VP8+ T cells Superantigen-like effect

Bivalent anti-TCR VP8-anticlass I1 antibody

Specific CTL lines CD4+CD8+thyniocytes Thymocytes in uiuo Peripheral T cells

Murphy et ul., 1990; Marnalaki et ol., 1993 Carlow et a / . , 1992

CD4+ and CD8' T cells i n rjico Thyinocytes in cirjo CD4' human T cell lines

Kawabe and Ochi, 1991

Peripheral T cells and th ymocytes Peripheral T cells

Gonzalo et al., 1994a

D'Adamio et aZ., 1993 Kabelitz and Wesselborg, 1992

Yagi et al., 1994

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death than crosslinking ofthe TCR alone (Damle et al.,1993a).Pretreatment of freshly isolated splenic CD4+ T cells with anti-CD4 inhibits proliferation in response to anti-TCRalP antibodies, but not to antiC D ~ ESimultaneous . independent crosslinking of CD4 and TCRaIP results in programmed cell death of the T cell (Newel1 et al., 1990). Similar to mouse T cells, human T lymphocytes exposed to anti-CD4 antibody are primed to undergo PCD in response to stimulation of the TCR. A similar effect is obtained by ligation of CD4 and gp120 from HIV, followed by crosslinking with anti-gpl2O antibodies (Banda et ul., 1992). Another molecule that is able to program T cells for apoptosis is CD2. Both CD4+ and CD8+ human T cells that are preactivated with mitogenic pairs of CD2 antibodies in uitro and kept in culture for several days become susceptible to anti-CD3-induced cell death (Rouleau e t al., 1994). Similarly, simultaneous crosslinking of the TCR/CD3 complex and the a3 domain of class I molecules causes PCD (Sambhara and Miller, 1991). These data can be interpreted to mean that abnormal combinations of stimuli, e.g., separate triggering of the TCR/CD3 complex and the CD2 or CD4 molecules, cause apoptotic T cell death. Although antigen-driven activation can cause T cell apoptosis, antigen may also cause apoptotic deletion of T cells, which would imply T cell activation. In at least two cases, T cells are deleted passively, i.e., without an activation signal delivered via the TCR/CD3 complex. First, antigen can be presented by MHC molecules on the lymphocyte surface and provoke the recognition and lysis by other T cells. Thus, cytotoxic T lymphocytes (CTL) proceed to their fratricidal deletion via apoptosis when they are specific for a self-peptide presented by MHC class I molecules. Accordingly, CTL specific for a peptide from influenza virus (Pemberton e t al., 1990), ovalbumine (Walden and Eisen, 1990), or Epstein-Barr virus (Moss et al., 1991) will undergo PCD in uitro upon exposure to high concentrations of the antigenic peptide. Induction of PCD requires cell contact (Moss et al., 1991; Su et nl., 1993b) and is inhibited by anti-CD8 and anti-LFA-2 antibodies (Dutz et al.,1992).Second, it is theoretically conceivable that superantigens form a molecular bridge between T cells and class II-expressing macrophages, thereby triggering superantigen-dependent lysis of the T cell (Dohlsten et al.,1991). Although this mechanism appears to be irrelevant to the superantigen-mediated deletion of T cells in uiuo (uide infra), it has been shown that TCR-specific antibodies can give rise to macrophage-mediated deletion of T cells without prior activation (Gonzalo et al., 1994a). In synthesis, thymocytes, T cell hybridomas, and T cell lines, as well as ex uiuo-activated primary T cell cultures, are susceptible to

PHARMACOLOGY OF T CELL APOPTOSIS

219

the TCR/CD3-mediated induction of PCD. The differentiation stage (thymocytes are more susceptible than peripheral T cells), the activation stage (memory cells are more susceptible than naive and nonactivated cells), the presence of cytokines, or additional costimuli determine whether TCRICD3 ligation will cause productive T cell activation or apoptosis.

2. 1n Vivo Models ($CD3iTCR-Mediated Peripheral T Cell Deletion A number of different stimuli targeted to the CD3iTCR complex are known to delete thymic T cells in oivo (Table 11): anti-CD3 antibodies (Smith et al., 19891; Tadakunia et a / . , 1990);exogenous superantigens of bacterial origin (White et al., 1989; D’Adamio et al., 1992; Lin et al., 1992); infection with retroviruses of the mammalian mammary tumor virus (Mtv) type that encode superantigens (Marrack et al., 1991); transfer of Mtv-infected cells ( W a n d e r s et al., 1993); and vertical transmission of Mtv superantigens incorporated into the germline (Kappler et al., 1990; MacDonald and Lees, 1990). Similarly, injection of relevant peptides causes the apoptotic deletion of thymocytes expressing specific transgene-encoded a l p TCR. This has been shown for both class I- and class II-restricted peptide antigens. Transgenic mice injected with large doses of a peptide recognized by a class IIrestricted TCR (specific for a chicken ovalbumine peptide presented by I-Ad) exhibit signs of apoptosis in their thymus (Murphy et al., 1990). Similarly, in mice transgenic for a TCR specific for a class I-restricted influenza virus nucleoprotein-derived peptide, a single injection of nominal antigen causes a rapid (3 hr) internucleosomal DNA fragmentation among thymocytes (Mamalaki et al., 1993). Confrontation of specific thymocytes expressing a male antigen-specific a1 p TCK with the class I-restricted endogenous self-peptide also induces apoptosis (Swat et al., 1991), and similar results have been reported for thymocytes expressing a self-specific transgenic y / S TCR (Dent et al., 1993). These data are believed to exemplify physiological intrathymic clonal deletion. Although deletion of self-reactive cells has been first observed in the thymus at a relatively immature stage of thymocyte differentiation (mostly CD4+CD8+cells), it appears that post-thymic mature T cells are also constantly subjected to clonal deletion processes. One of the best studied models is the superantigen-induced deletion ofperipheral T cells in the mouse. Parenteral administration of bacterial superantigens causes the deletion of reactive cells in peripheral lymphoid compartments (Kawabe and Ochi, 1991; MacDonald et d.,1991,1993; McCormack et a l . , 1993).This has first been shown for SEB, a superanti-

TABLE 111 is BY A SUPERANTICENAND DIFFERENTIAL MODEOF ACTION OF A ~ o p ~ o sINDUCTION

AN

ANTIBODYTO

THE

SAMETCR Vp DOMAIN

Consequences of Intravenous Injection of 50 p g Parameter ~

Staphylococcus Aureus Enterotoxin B (SEB)

F23.1 (Anti-Vp8.1, 2, and 3; IgC2a Mouse Antibody)

Biphasic depletion interrupted by a phase of proliferation (24-72 hr after injection); only partial deletion of Vp8' T cells 8-12 and 72-96 hr after injection Yes Stimulation of T h l , Th2, and monocytespecific cytokine production that is detectable in serum 60 to 240 min after injection Yes (maximum 90-180 min after injection) Induction of a lethal septic shock-like syndrome Yes

Monophasic depletion leading to the nearto-complete deletion of VP8' T cells 72 hr after injection

At least partial inhibition of T cell depletion Enhanced depletion of VP8' T cells No effect

No effect

~~~~~

Kinetics of deletion of peripheral VP8' T cells Maximum of apoptotic DNA fragmentation Mitogenic effect in uiuo Induction of cytokine release syndrome (IL-la, IL-2, IL-4, IL-6, IFNy, TNF) Induction of glucocorticoids after injection Acute toxicity upon injection together with D-gahCtOSamine or RU-38486 Induction of anergy in surviving VP8' T cells Effect of apoptosis inhibition by retinol, RU-38486, or linomide Effect of exogenous glucocorticoids Effect of i n viuo macrophage depletion Prophylactic effect on the development of lupus in MRLlMp-lprllpr mice

Yes, one single injection at 2 months of age greatly reduces later disease manifestation

12 to 24 hr after injection No No detectable cytokine induction in uiuo

No No acute toxicity

No

No effect Reduced depletion of antibody-binding cells Yes, continuous application every week is necessary to obtain prophylactic effect

Note. Data from Kawabe and Ochi (19913, de Alboran et a / . (1992). Tarazona e t ol. (1994). Gonzalo et ol. (1994a,d), Yagi et ol. (1994). and unpublished observations.

22 1

PHARMACOLOGY OF T CELL APOPTOSIS

genic exotoxin that specifically stimulates VPS' T cells. Intravenous injection of SEB exerts pleiotropic effects in v i m (Marrack and Kappler, 1990; Kotzin et al., 1993; MacDonald et al., 1993) causing a biphasic change in the frequency of specific peripheral T cells. A first wave of deletion (minimum of VPS' cells at 12-24 hr) is followed by a phase of expansion (36-92 hr) and a second phase of deletion (>4 days) (MacDonald et al., 1991,1993; D'Adamio et al., 19Y2,1993a,b; Kawabe and Ochi, 1991; Wahl et al., 1993) (Table 111, Fig. 1). Since the expansion and deletion of VPS' cells also occur in thymectomized animals, they are truly peripheral in nature (Kawabe and Ochi, 1991). During phases of deletion CD3' T cells purified from the spleen of SEB-injected mice exhibit DNA fragmentation provided they are

0'

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1

I

2

3

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4

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5

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6

,

7

.

8

I

9

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, I

0

I IL-1, IL-2, IL-4, tL-6, TNF,IFN, ACTH,GC Death of sensitized animals nsion of VR8 cells

0

1

2

3

4

5

6

7

8

9 1 0

days

Frc. 1. Pleiotropic effects of staphylococcal enterotoxin B (SEB) injection in oiuo. T h e consequences of an intravenous injection of 50-100 pI SEB into adult BALBic mice (Day 0) are show. I n the top of the figure changes in the number of splenic CD4'VP8+ or CD8+ VPS' cells are depicted. In the bottom ofthe figure the chronological development ofcytokine release, death ofu-galactosamine-sensitized animals, hyperexcitability, and anergy of peripheral T cells upon in oitro restimulation with SEB are schematically represented in their chronological development. Moreover, the periods during which VP8' T cells proliferate (assessed b y determination of the incorporation of5-bromo-2'-deoxyuridine) or undergo apoptosis (assessed by measuring DNA fragmentation) are shown. Data are from Kawabe and Ochi (1990,1991), MacDonald et al. (1991,1994), Gonzalo et d.(1993a,b,l994a),Miethke et 01. (l992),Tarazona et al. (19941, and unpublished observations.

222

GUIDO KROEMER

cultured during a short period (>30 min) in vitro (Kawabe and Ochi, 1991; Gonzalo et al., 1994a). I n accord with in uitro experiments discussed above (Section II,A,l), pretreatment of mice with the anti-CD4 mAb H129.19 greatly enhances the sensitivity of CD4' lymph node cells to the SEB-mediated induction of deletion via PCD in vivo (Facchinetti et al., 1992). Superantigens like SEB do not only affect the frequency of reactive T cells. SEB causes an acute cytokine release (Herrmann et al., 1992; Miethke et al., 1992), leading to the secretion of detectable amounts of different cytokines (interleukins-la,-2,-4,-6,-1O,granulocyte/macrophage-colony stimulating factor, tumor necrosis factor, interferon-y) that are detectable 30 min to 4 hr after SEB injection (Gonzalo et al., 1994a). Some of these mediators are produced by macrophages (e.g., interleukins-la and -lo), whereas others are products of T cells (e.g., interleukins-2 and -4, interferon-y) (Bette et al., 1993; Tarazona et al., 1994). SEB also causes the induction of functional antigen unresponsiveness (anergy) among VP8' T cells that survive the deletion phase (Kawabe and Ochi, 1990; Lee and Vitetta, 1992; Baschieri et al., 1993; Perkins et al., 1993). The relation between cytokine production, anergy, and PCD will be discussed under Section IV,D. Peripheral deletion occurs in response to other superantigens besides SEB. Staphylococcal enterotoxin A (SEA) deletes VP3' and VPll' R lymphocytes in vivo (McCormack et al., 1993).Prior expansion of superantigen-reactive T cells is not a prerequisite for clonal deletion since continuous application of low doses of SEA can cause deletion of reactive T cells without prior increase in number (McCormack et al., 1993). As do bacterial superantigen, retrovirus-encoded superantigens provoke peripheral clonal deletion, e.g., Mtv-7 (previously Mls-1") causes peripheral deletion of VP6' cells (Jones et al., 1990; Webb et al., 1990; Desaymard et al., 1993). A single injection of Mtv-7' spleen cells into Mtv-7- Balb/c mice (Day 0) results in changes in the frequency of VP6' cells that follow a similar kinetics as the response of VP8' cells to SEB: rapid expansion of VP6' cells in the spleen and lymph nodes until Day 3, anergy on Day 3, and reduction in VP6' cells until Day 10 (Webb et at., 1990; Dannecker et al., 1991).B cells and CD8' (but not CD4') T cells from Mtv-7' mice efficiently induce peripheral deletion of reactive T cells (CD4Vp6') upon transfer to Mtv-7- recipients, whereas only B cells elicit specific T cell proliferation in uiuo. Deletion of specific T cells without prior proliferation also occurs after injection of low doses of Mtv-7expressing B cells (Waanders et al., 1993). These experiments again suggest that clonal deletion must not be preceded by proliferation of specific T cells.

PHARMACOLOGY OF T CELL APOPTOSIS

223

Instead of superantigens, antibodies specific for TCR epitopes can be taken advantage of to delete a part of the T cell repertoire in uiuo. The monoclonal mouse antibody F23.1, an IgGZa, can be used to delete the same T cell subset as SEB in uiuo. F23.1 recognizes an epitope of the TCR Vp chain that is encoded by all three members of the Vp8 gene family (Vp8.1, Vp8.2, Vp8.3) (Staerz et al., 1985). Intravenous administration of this antibody causes the depletion of Vp8+ T cells that follows a monophasic pattern, i.e., without interruption by a transient phase of expansion as would be the case for SEB. T cell depletion is accompanied by endonucleolytic DNA fragmentation and therefore involves PCD (Table 111). Injection of Fab or F(ab), fragments of this antibody does not induce PCD of VpS+ T cells, indicating that F23.1 eliminates in a n Fc-dependent fashion (Gonzalo et al., l994a). Antibody-dependent cellular cytotoxicity (ADCC) appears to dominate over complement-mediated deletion of Vp8' T cells since (i) these cells die from PCD rather than from necrosis (and complement would induce necrosis); (ii) in uiuo elimination of macrophages partially suppresses the F23. l-mediated depletion of Vp8' T cells; and (iii) F23.1 depletes Vp8' T cells with the same kinetics in A/J mice that are deficient for the C5 complement factor, as in CBA/ Ca mice. In contrast to the SEB, F23.1 fails to activate T cells according to three criteria: (1) it does not induce proliferation of Vp8+ T cells; (ii) it does not stimulate the production of cytokines in uiuo (Gonzalo et al., 1994a); and (iii) it fails to provoke T cell anergy. To confer immunostimulatory properties to this antibody, it is necessary to produce bispecific antibodies that recognize Vp8 plus MHC class I1 (H2 I-E) molecules (Yagi et al., 1994). These data indicate that disparate mechanisms of T cell depletion, activation-induced death in the case of SEB, and macrophage-dependent ADCC in the case of F23.1 account for apoptotic cell death (Table 111). In TCR transgenic mice, peripheral deletion can also be induced by the antigenic peptide that is recognized by the TCR. Female T cells that express a male antigen (H-Y)-specific alp TCR expand during the first 5 days after transfer into an autoantigen-expressing (i.e., male) environment, but most are eliminated between Days 5 and 9 after transfer (Rocha and von Boehmer, 1991). In a similar setting, injection of male cells into female recipients bearing a male antigen (H-Y)specific a / p TCR induces apoptosis of CD8' T cells (Carlow et al., 1992). In mice transgenic for a TCR-specific class I-restricted influenza virus nucleoprotein-derived peptide, one single injection of nominal antigen causes a rapid partial deletion (minimum after 12-24 hr) of CD8+ lymph node cells, followed by their expansion (Mamalaki et al., 1992,1993). In contrast, chronic exposure to the peptide causes a

224

GUIDO KROEMER

profound deletion of CD8’ T cells expressing the transgenic TCR (Mamalaki et al., 1993). From these data it appears that antigenic stimulation causes the deletion of specific T cells. This applies for both superantigens and classical peptidic antigens. Recent evidence suggests that mature human T cells also undergo deletion upon exposure to antigen in uivo. When human thymocytes and peripheral T cells contained in chimeric scid-hu mice are exposed to superantigens (SEB, SEE) in uiuo, they exhibit specific clonal deletion, anergy, and proliferation in uiuo in a way that closely parallels the behavior of murine T cells (Waller et al., 1992). Altogether, it appears that clonal deletion is one of the cardinal mechanisms that shapes the T cell repertoire, in both man and mice.

B. NONANTIGEN-SPECIFIC INDUCTION OF T CELLDEATH In addition to antigen-dependent activation, a number of stimuli can induce T cell apoptosis. This applies to contact-dependent alternative activation pathways, signals perceived through hormone or cytokine receptors, artificial triggering of signal transduction pathways, mild physical damage, and inhibition of intermediate metabolism.

1 . Death Induction by Alternative Activation Pathways As discussed above, stimulation of T cells via the CD2, CD4, and class I molecules primes for TCR-mediated activation death. In determined circumstances activation of T cells via “alternative” (i.e., nonantigenic, non-TCR-mediated) pathways can cause PCD b y itself, i.e., in the absence of TCRICD3 triggering (Table IV). These receptors include CD2, Thy-1, and Fas/Apo-1. Crosslinking of the Fas/Apo-1 antigen, that is expressed on CD4’CD8+ thymocytes (not CD4+CD8- or CD4-CD8’ thymocytes or nonactivated T cells) and activated T and B lymphocytes, especially on memory cells, per se may cause PCD (Trauth et al., 1989; Yonehara et al., 1989). Prior activation of T cells in uitro, e.g., by anti-CD3 or PHA, is necessary to render them susceptible to PCD induction via the Fas/Apo-1 pathway (Miyawaki et al., 1992). Based on sequence homology data, the Fas/Apo-1 antigen belongs to a superfamily of surface receptors that includes the p55 TNF receptor, the low-affinity nerve growth factor receptor, the B cell-specific surface marker CD40, as well as CD27, CD30, 4-1BB, and 0 x 4 0 . Several of these products are involved in the regulation of apoptosis (Itoh et al., 1991). It has been suggested that the Fas antigen is one of the target molecules

TABLE IV INDUCTION OF T CELLDEATH VIA ALTERNATIVESURFACE RECEPTORS ~

Alternative Surface Receptor

Cell Type

Probable Active Principle

Reference

Fasi Apol

Crosslinking Expustiru to Fas ligand

T cell leukemia

Thy1

Crosslinking, showirig syncrgy with CLWTCR signals (Re)activation of T cells via the alternative pathway

Thyrnocytes

Trnuth et d . , 1989; Itoh et a / . , 1981; Owcn-Srhaub et al., 1992; Takahaslii pt ul,, 1994 Nakashinia et ul., 1991

T cell hybridoma CD45RO- thyriiocytes preactivated CD4' m d CDR' human T cells CD4- T cells

Bierer et 01.. 1991; Li et al., 1992; Wesselborg et ol., 199,313; Houlcau e t a / . , 1993 Newel1 et af.,1990

CD4- T cells CD.1' T cell line

Howie et ul., 1994 Danile et uZ., 1993a

CD4' T cell line

Damle et ul., 1993a

T cells

Sambhara and Miller. 1991

Anti-CD2

cu4 CDlla/CD18 CD29/CD49d MHC class I

lndependent crosslinking of the TCR In ciuo injection of anti-CD4 Ligation by ICAM-1 together with anti-atp TCR Ligation b y VCAM-1 together with anti-aip TCR Crosslinking with antibody specific for the a3 domain plus stimulation of CL)J/TCR

Peripheral T cells

226

GUIDO KROEMER

that is triggered during T-T interactions, causing death upon antigenspecific recognition by an effector cell that bears a Fas ligand (Rouvier et al., 1993). Apoptosis of the target cell is Ca2+-independent(Rouvier et al., 1993) and does not involve perforine or granzymes (Suda et al., 1993). The importance ofthe Fas/Apo-1 gene product in the regulation of self-tolerance is underscored by the fact that mutations that reduce its expression ( l p r )or signal-transducing capability (Zpfg) cause severe autoimmune disease. Fas/Apo-1 interacts with a specific ligand, FasL (Suda et al., 1993).A mutation of FasL ( g l d )also causes an autoimmune syndrome (see Section V,A). Another surface molecule that may cause T cell death in the absence of TCR/CD3-mediated stimuli is CD2. Combinations of anti-CD2 antibodies that are mitogenic in certain systems cause death of human T cell lines of different phenotypes as well as of freshly isolated CD8+CD57+T cells (Rouleau et al., 1993; Wesselborg et al., 1993b). According to one report, anti-CD2-induced death is blocked by cyclosporin A and FK 506 (Wesselborg et al., 1993b). Sequential stimulation with a minimum of three anti-CD2 antibodies causes death of up to 60% of nonfractionated human peripheral blood lymphocytes (Rouleau et al., 1994). Human cloned CD4+ and CD8+ T cells and polyclonal T cell lines die from apoptosis when stimulated with mitogenic combinations of anti-CD2 antibodies (Wesselborg et al., 1993b). Similarly, human CD8+CD57+ (not CD8+CD57-) T cells die when exposed to a particular mitogenic pair of anti-CD2 antibodies (Rouleau et al., 1993). The PCD-triggering interaction between CD2 and antiCD2 antibodies could mimic binding of CD2 to its physiological ligands (CD48, CD58). In addition to CD2, Thy-l-specific antibodies can induce T cell apoptosis (Hueber et al., 1994). Mouse thymocytes, especially CD3-CD4+CD8+ and CD3'""CD4+CD8+ cells, die from apoptosis after exposure to certain immobilized anti-Thy-1 antibodies (Hueber et al., 1994). It is noteworthy to remember that the above-mentioned structures, FasIApo-1, Thy-1, and CD2, d o not only cause T cell death but may also function as (co)stimulatorymolecules. Certain monoclonal antibodies directed against Fas/Apo-1 costimulate the anti-CD3-driven proliferation and IL-2 production of T cells (Alderson et al., 1993).Similar costimulatory properties have been reported for Thy-1 (Nakashima et al., 1991). Certain combinations of antibodies specific for the CD2 molecule also induce T cell proliferation and lymphokine production (Meuer et d., 1984). Although the physiological role of antigen-nonspecific death remains to be elucidated, it is very possible that any kind of nonphysiological T cell activation will normally ensue

PHARMACOLOGY OF T CELL APOPTOSIS

PCD to avoid-from the teleological point of view-the nonspecific T cells.

227 expansion of

2 . Death Induced by Soluble Mediators (C ytokines and Hormones) Hormones are soluble mediators that act on the systemic (endocrine) level, whereas cytokines have predominantly local (auto- or paracrine) effects (Kroemer et al., 1993b). Both classes of mediators are constantly regulating the propensity of T cells to undergo apoptosis (Table V). Steroids are the most prominent hormones involved in the regulation of T cell death. It has been known for a long time that thymic cellularity is negatively influenced by glucocorticoids and by sex hormones. Glucocorticoid receptor agonists cause a reduction of thymic cellularity (Claman, 1972) and induce PCD of thymocytes (Cohen and Duke, 1984). On the contrary, bilateral adrenalectomy or medication of the glucocorticoid receptor antagonist RU-38486 cause a rapid increase in thymic size and in the number of splenic T cells (Shortman and Jackson, 1974; Gonzalo et al., 1993a) followed by a PCD-mediated depletion ofthymocytes and splenic T cells (Gonzalo et al., 1993a). Peripheral T cells also respond to glucocorticoids by undergoing apoptosis. The dose of glucocorticoids needed to kill splenic T cells in vitro or in vivo is the same as that required for the induction of thymocyte PCD (Perandones et al., 1993). The glucocorticoid receptor agonist dexamethasone also induces apoptosis of mouse natural killer cells, cytotoxic T lymphocytes (Migliorati et al., 1994),and B220'IgM' pre-B cells (Garvy et al., 1993). Glucocorticoids regulate the antigen-specific deletion of T cells. Instead of exhibiting an expansion of Vp8' T cells on Day 3 after injection of SEB, dexamethasone-treated animals displayed a highly significant rapid deletion of Vp8' T cells that can be easily detected 2 to 3 days after SEB challenge (Gonzalo et nl., 1993a).Similarly, exogenous hydrocortisone enhances the SEB-driven deletion of Vp8' lymph node cells from BALB/c mice and the SEAtriggered elimination of Vpll' cells from C57/B16 animals (LUSSOW et al., 1993).In addition, dexaniethasone enhances the susceptibility ofthymocytes to anti-CD3-driven deletion in vivo (Jondal et al., 1993). Thus, glucocorticoids accentuate the activation-induced death of T cells. Nontheless, these in vivo data are not compatible with in vitro studies suggesting that glucocorticoids antagonize the CD3-triggered death of thymocytes (Iwata et al., 1991). Glucocorticoids are not the only steroids that mediate thymocyte depletion in v i m . Testosterone and estradiol deplete CD4'CD8+ thymocytes after i n vivo injection, although this effect is not mediated via specific receptors expressed on thymic lymphocytes. However, it

TABLE V

INIICCTIUN OF

Substance

'r CEI.1.. DEATH VIA Sl'tAOID

Probable Active Principle

Glucocorticoids

Immediate effect

Physical exercise

Glucocorticoid-mediated stress response Blockade of glucocorticoid receptors (delayed effect) Depletion of endogenous glucocorticoids (delayed effect) Effect on epithelial cells Activation-induced expression of vitamin I> receptors Cnknown

RU-38486 Adrenalectom y Estrogen, testosterone Vitamin D 3 plus phorbol ester 13-cis-retirioic acid Interleukin-2 Withdrawal of IL-2 Interferon-y Tumor necrosis factor-a Lymphotoxin

Unknown Withdrawal of an abligate trophic factor Unknown Mediated by lytic domain of the TNF molecule Unknown

OH

LYMPHOKINE W C E I l O H S Cell Type

Reference

Thymocytes Peripheral T cells Thymocytes in uiuo

Cohen and Duke, 1984; Perandones et al., 1993 Concordet and Ferry, 1993

Peripheral T cells

Gonzalo et ol., 1993a

Peripheral T cells

Gonzalo et ol., 1993a

Thymocytes in uiuo Thymocytes

Hirahara et al., 1994 R. Ramirez et al., unput)lished s u et ol., 1993a

Epstcin-Barr virus-infected T cell lymphoma CD4'CDS' ~ ~ O U Sthymocytes F IL-2-dependent cell lines Malignant T cell lines and human thymocytes Murine thymocytes Thymocytes in uiuo

Migliorati et al., 1993b Duke and Cohen, 1986 Novelli et al., 1994 Hemhdez-Caselles and Stutman, 1993 Hirahara et al., 1994

PHARMACOLOGY OF T CELL APOPTOSIS

229

might involve receptors expressed on epithelial cells (Barr et al., 1982). Castration of male C57/B1/6 mice causes an increase in thymic cellularity without an increase in the percentage of cycling cells (Olsen et al., 1994). These data indicate that sex steroids are involved in the regulation of thymic cellularity. Indeed, sex steroid-mediated thymocyte depletion may underlie thymic involution during puberty, Two classes of vitamins, vitamin A (retinoic acid) and vitamin D3 (24-, 25-dihydroxycolecalciferol), bind to members of the sex steroid superfamily. 13-cis-Retinoic acid induces apoptosis of an EpsteinBarr virus-containing T cell lymphoma expressing high levels of glutathione-S-transferase-.rr (Su et al., l993a). In contrast, retinoids suppress activation-induced PCD in several in uitro and in uivo systems (see below). Vitamin D3 is capable of inducing the death of PMAactivated human T cells (Ramirez et ul., personal communication). The probable role of molecules interacting with the steroid receptor superfamily in the regulation of PCD is also illustrated by the gene nw-77. Nur-77 encodes a member of the steroid receptor superfamily whose ligand is thus far unknown. This orphan steroid receptor is induced by activation of thymocytes with anti-TCR, PMA plus ionomycine, but not dexamethasone (Liu et ul., 1994; Woronicz et al., 1994b). In T cell hybridomas, cyclosporin A and glucocorticoids inhibit PCD and induction of Nur-77 expression by a n t i - C D ~ EThis . suggests a central role of Nur-77 in the regulation of apoptotic cell death (Woronicz et al., 1994a). Whereas Nur-77 mRNA lacks a poly A tail in proliferating cells, cells that are condemned to death bear polyadenylated Nur-77 mRNA. In T cell hybridomas, a dominant-negative Nur-77 mutant or antisense RNA protects against activation-induced cell death (Liu et al., 1994; Woronicz et ul., 1994b). Collectively these data again underline the importance of different types of steroid receptors in PCD regulation. Of course, steroids are not the only class of hormones to intervene in the regulation of apoptosis. For example, growth hormone inhibits the prednisoloneinduced reduction of spleen and thymus cellularity (Franc0 et al., 1990). Regulation of apoptosis by cytokines is more controversial than steroid-mediated control of PCD. Withdrawal of cytokines serving as essential growth factors causes PCD. Lack of IL-2 causes death of IL%dependent cells (Duke and Cohen, 1986). In contrast, IL-2 by itself induces PCD in CD4'CD8+ mouse thymocytes (Migliorati et d., 1993b). Some investigators (Lenardo, 1991; Critchfield et al., 1994) have speculated that mature T cells do not die upon CD3 crosslinking unless they are exposed to a high dose of IL-2 beforehand. However,

230

GUIDO KROEMER

the possible proapoptotic role of IL-2 remains a matter of debate. In the in vitro system, IL-2 can be substituted by other cytokines, such as IL-4, that also prime for deletion (Lenardo, personal communication). Inhibition of IL-2 production in vivo fails to inhibit the superantigendriven deletion of T cells in vivo (Gonzalo et al., 1992; Vanier and Prud’homme, 1992). In addition, administration of exogenous IL-2 in vivo fails to enhance the deletion of VP8’ T cells stimulated by a VP8-specific superantigen in vivo (unpublished observation). Another cytokine reported to positively regulate PCD is interferon-y (IFNy). In a particular murine Thl clone, as well as in human thymocytes, neutralization of IFNy has been reported to inhibit anti-CD3ITCR antibody-triggered PCD (Liu and Janeway, 1990; Groux et al., 1993). However, other studies failed to unravel a critical role of IFNy in activation-induced cell death (Damle et al., 1993a). In synthesis, the impact of two cytokines, IL-2 and IFNy, in the regulation of T cell apoptosis remains to be elucidated. Another cytokine that can induce cell death is tumor necrosis factor (TNF). According to one report (Wang et al., 1994), TNFa mediates the death of CTL induced by TCR crosslinking. Microinjection of antiTNF antibody into CTL as well as inducion of CTL in the presence of antisense T N F oligodeoxynucleotides inhibit anti-CD3-induced cell death, which is consistent with the notion that TNF induced by T cell activation mediates cell death (Wang et aZ., 1994). T N F a can also induce apoptosis of murine thymocytes, but no such effect is seen in freshly isolated peripheral T cells (Hernindez-Caselles and Stutman, 1993). It remains elusive which TNF receptor is involved in mediating cell death and whether endogenous TNF is involved in the regulation of clonal deletion.

3. Arti$cial Triggering of Signal Transduction Pathways To induce cell death, physiological apoptosis-inducing stimuli must trigger intracellular signal transduction pathways. This applies to stimulation of the TCR/CD3 complex, activation of certain alternative pathways, or PCD induction by soluble mediators. Accordingly, a variety of synthetic stimulators of signal transduction pathways cause PCD (Table VI). The second messengers triggered via the TCR/CD3 complex can be mimicked in many in vitro systems by a suitable combination of calcium ionophore and protein kinase C-activating phorbol esters. Accordingly, both agents, calcium ionophore and phorbol ester, induce apoptosis (Kizaki et al., 1989). Treatment of immature thymocytes with ionomycin causes apoptotic cell death (McConkey et al., 1989b).

TABLE VI INDUCTION OF T CELLDEATHBY ARTIFICIALSTIMULATION OF SIGNAL TRANSDUCTION PATHWAYS Substance

Probable Active Principle

Cell Type

Reference

Genistein tyrphostin A23187 12-0-tetradecanoyl 13-acetate (TPA)

Inhibitors of tyrosine phosphorylation Calcium ionophore Phorbol ester (protein kinase C activator) Augmentation of intracellular CAMP

Leukemic cell lines Thymocytes Thymoc ytes

Bergamaschi et a/., 1993 Kizaki et al., 1989 Kizaki et d.,1989

Thymocytes

Suzuki et al., 1991

Elevation of intracellular CAMP Elevation of intracellular CAMP Inhibition of protein kinase C

Thymocytes Thymocytes in ciao Splenic T cells

McConkey e t al., 1990b McConkey et d.,1993 Perandones et al., 1993

Isoproterenol or prostaglandin E in conjunction with TPA Dibutyril CAMP (N-ethyl)-carboxamide-adenosine Staurosporin or H7

232

GUIDO KROEMER

Nonetheless, both ionomycine and phorbol ester are also able to inhibit PCD induction in response to certain stimuli, unravelling the complex regulation of PCD at the level of signal transduction pathways (see below). Interventions on protein kinase cascades can also trigger apoptosis. Thus, treatment of T cells with staurosporine or 1(5-isoquinolinesulfonyl)-2methylpiperazine (H7”),two protein kinase C inhibitors, triggers cell death (Perandones et al., 1993). Another second messenger system with a high potential of apoptosis regulation is cyclic adenosine monophosphate (CAMP).cAMP analogs or agents that elevate CAMPpotentiate the apoptotic response to glucocorticoids and anti-CD3 antibodies (Jondal et al., 1993; McConkey et al., 1993). cAMP and dexamethasone cooperate in the deletion of thymocytes (McConkey et al., 1993). The effect of both of them is counteracted by activation of protein kinase C (McConkey et al., 1992). cAMP also induces cell death in a rat myeloid leukemia cell line (IPC81) (Lanotte et al., 1991). This involves the CAMP-regulated kinase I, since microinjection of the catalytic alpha subunit of CAMP-dependent protein kinase induces PCD (Vintermyr et al., 1993). Whether this kinase is also implicated in CAMP-mediated induction of T cell death remains to be determined. Recent experiments suggest that the regulatory subunit of CAMP-dependent protein kinase Ia, together with its associated kinase activity, are translocated to and interact with the TCR-CD3 complex during T cell activation and capping. Thus, cAMP might favor inactivation via phosphorylation of the TCRfCD3 complex by the CAMP-regulated kinase I (SkHlhegget al., 1994). In this context, cAMP would inhibit TCR/CD3-driven T cell activation. As a matter of fact, cAMP is not a universal inducer of apoptosis. Dibutyryl cAMP retards the apoptosis of IL-3-dependent 32D cells induced by culture in the absence of their growth factor (Berridge et al., 1993).These data, together with the contrasting effect of dibutyryl cAMP on T cell responses in vitro-enhancement of TPA-induced proliferation but inhibition of PHA-driven proliferation (Chakkalath and Jung, 1992)-suggest that the functional outcome of interventions on the cAMP pathway depends very much on other second messenger systems. Thus, the available data argue against the existence of a unique “death pathway” of signal transduction. In contrast, each signal transduction cascade can enhance or inhibit apoptotic cell death. It is the combination of second messenger systems, cell type, differentiation, and activation stage that determines the outcome of each manipulation.

PHARMACOLOGY OF T CELL APOPTOSIS

233

4 . Viral Znfection and Lymphopenia

Enhanced apoptotic turnover of lymphoid cells may cause a severe immunodeficiency. This has become clear from experiments in which the anti-apoptotic bcl-2 protooncogene was eliminated by homologous recombination, leading to the disappearance of the lymphoid system after birth (Nakayama et al., 1993; Veis et aZ., 1993). Expression of the homeobox fusion gene E2A-PBX1 under control of the Ig heavy-chain enhancer causes a reduction of thymocytes and bone marrow B lineage progenitors that is due to an increased cell death (Dedera et al., 1993) (Table VII). Similarly, viral infections can cause immunodeficiencies due to apoptotic decay of lymphoid cells (Table VIII). A number of acute viral infections are accompanied by a reduction in T lymphocyte numbers that may be attributed to an enhanced apoptotic turnover of T cells not compensated for by proliferation. Accordingly, infection of mice with niurine lymphocytic choriomeningitis virus (Razvi and Welsh, 1993) or vaccinia virus (Gonzalo et al., 1994b) is accompanied by apoptosis of peripheral T cells. In both models both CD4+ and CD8+ cells are depleted. In contrast, in human infectious mononucleosis, cell death is restricted to the CD45RO' (activated) populations of CD4' and CD8' cells (Uehara et al., 1992). These cells are deficient for bcl-2 expression compared to CD45RO' cells from healthy controls (Tamaru et al., 1993). Infection with retroviruses causing immunodeficiency is also accompanied b y PCD-mediated depletion of peripheral T cells. An enhanced apoptotic decay is observed in lymphocytes from HIV-infected donors and may contribute to the development of chronic AIDS-associated lymphopenia (Gougeon et al., 1991; Groux et al., 1992; Meyaard et al., 1992). Enhanced apoptotic decay has been observed in both nonstimulated and mitogen-stimulated cells from HIV-infected donors (Groux et al., 1992; Meyaard et al., 1992). It concerns both CD4+ and CD8' T cells (Meyaard et al., 1992), as well as uninfected hematopoietic progenitor (CD34') cells (Re et d.,1993). It is equally observed in macaques infected with simian immunodeficiency virus (del Lano et al., 1993) and murine retroviral models of AIDS (Cohen et al., 1993a); Saha et al., 1994). In contrast, HIV-infected chimpanzees that do not develop AIDS do not exhibit enhanced lymphocyte apoptosis (Gougeon et al., 1993). In summary, virus-induced immunodeficiency is tightly associated with an enhanced apoptotic decay of lymphocytes. Enhanced lymphocyte susceptibility to PCD could negatively affect

TABLE VII GERMLINE-ENCODED ABNORMALSUSCEPTIBILITY OF MOUSELYMPHOCYTES TO UNDERGO APO~TOSIS Cause

to

Mechanisms

bcl-2 null mutation

Failure to express the antiapoptotic protooncogene bcl-2

c-myc transgene (Ep enhancer)

Elevated expression of the oncogene c-myc in lymphocytes

Transgenic homeobox fusion protein E21-PBX1 (Ep enhancer)

Expression of a fusion protein involved in leukemogenesis

lpr and gld mutations

Expansion of a phenotypically abnormal CD2-CD4-CD8alp T cell population

rp W

Functional Consequences

Reference

Progressive loss of T and B lymphocytes from 2 weeks after birth; fulminant lymphoid apoptosis after irradiation or glucocorticoid treatment Enhanced proliferation and elevated apoptotic decay that is blocked by hyperexpression of transgenic bcl2; formation of B cell lymphomas that is accelerated by Bcl-2 Lymphoid hypoplasia, increased thymocyte death, enhanced entrance of CD3+CD4+CD8+and CD3tCD4+CD8- thymocytes into S and G2IM phases; development of CD3+CD4tCD8' lymphomas Spontaneous apoptosis of the expanded T cell population that is inhibited by phorbol ester

Nakayama et d.,1993; Veis et d.,1993

Adams and Cory, 1991; Fanidi et d,1992

Dedera et QZ., 1993

Van Houten and Budd, 1992

TABLE VIII INDUCTION OF T CELLDEATHBY VIRALINFECTION Viral Infection Infectious mononucleosis

t.3

w

w

Cytomegalo virus infection Murine lymphocytic chorionieningitis virus (MCMV) Vaccina virus infection tstl mutant of Moloney murine leukemia virus Infection with chicken anemia virus Human immunodeficiency virus (HIV)

Probable Active Principle

Cell Type

Reference

Patients infected with Epstein-Barr virus Infection of patients Infection of adult mice

CD45RO' T cells

Uehara et o l . , 1902

Peripheral T cells in oico

Meyaard and Miedema, 1994 Razvi and Welsh, 1993

Infection of adult mice Infection of neonatal mice

Peripheral T cells in oiuo Thyniocytes and splenic T cells Thymocytes in uico

Jeurissen et al., 1992

Peripheral T cells

Gougeon et ol., 1991

Infection of newly hatched chicks Various independent mechanisms (see Table 1X)

Gonzalo et al., 1994b Saha et al., 1094

236

GUIDO KROEMER

immune function in a dual fashion. On one hand, it entails a lymphopenia, i.e., a state of immunodeficiency marked by a reduction in the number of lymphocytes. On the other hand, it may compromise immune function, given that antigenic stimulation in uivo will result in deletion rather than productive activation of specific T cells. As to the mechanism of increased apoptosis in HIV infection, several hypotheses have been proposed (Table IX). Although it is clear that the cytopathic effect of HIV infection is mediated by PCD (LaurentCrawford et aZ., 1991), direct infection of lymphocytes cannot explain the massive entrance of cells in apoptosis upon in vitro culture. Activation of CD4+ cells via CD4/gp120 interaction could also contribute to apoptosis (Banda et al., 1992). However, this phenomenon cannot explain the death of CD8+ T cells. Another possible explanation of this finding would be to assume that the direct or indirect (lymphokinemediated) activation of T cells accounts for PCD. In addition, it has been proposed that deprotection of cells from oxidative stress might be involved in HIV-induced PCD (Buttke and Sandstrom, 1994). The HIV-induced increase in apoptotic decay is associated with a reduction in glutathione peroxidase activity that renders cells more sensitive to killing by hydroperoxy fatty acids (Sandstrom et al., 1994). Antioxidants inhibit HIV-associated T lymphocyte apoptosis in uitro (Buttke and Sandstrom, 1994). A further problem that has been addressed by several research groups concerns the question of whether the HIV-driven lymphopenia

HYPOTHETICAL MECHANISMS BY

TABLE IX HIV INDUCEST LYMPHOCYTE Apop~osis

WHICH

Mechanism

Reference ~~~

Cytopathic effect Similarity between Env protein and Fas Interaction of viral gp120 with CD4 Downregulation of manganese superoxide dismutase and catalase leading to deprotection of cells with respect to damage by reactive oxygen species Superantigen-mediated activation of T cell populations defined by their V@ repertoire Nonspecific immune activation

~

Terai et al., 1991; Laurent-Crawford et al., 1991 Zagury et al., 1993 Banda et al., 1992; Oyaizu et QZ., 1993 Sandstrom et aZ., 1994

Imberti et al., 1991 Meyaard and Miedema, 1994

PHARMACOLOGY OF T CELL APOPTOSIS

237

is selective or broadly polyclonal. In fact, in one murine model of AIDS, a retrovirus-encoded superantigen reacts with T cells expressing VpS (Kanagawa et al., 1992). Whereas normal subjects carry a TCR repertoire without major V a or V@ deletions, according to one report (Imberti et al., 1991)HIV-infected individuals show a specific deletion of certain Vp gene products (Vp14 to Vp20) that is independent of the presence of opportunistic infections. Another report shows that HIV preferentially reproduces in Vpl2' T cells and selectively expands such cells in uitro (Laurenceet al., 1992).Studies performed in monozygotic twins discordant for HIV revealed possible perturbations in the frequency of the Vp13 and Vp2l families (Rebai et al., 1994). These data led to the speculation that HIV-encoded superantigens and/or exogenous superantigens might participate in abnormal immune stimulation and T cell depletions. However, a study performed in maternally HIV-infected children failed to detect a selective Vp deletion (Bahadoran et ul., 1993), and thus far the search for HIV-encoded superantigens has failed (Nisini et al., 1994). To avoid an oversimplified view of HIV-induced immunopathology, it has to be stated that the effect of HIV infection on PCD regulation is rather complex. Thus, the HIV-1 Tat protein protects lymphoblastoid, epithelial, and neuronal cell lines fom apoptotic death induced by serum withdrawal (Zauli et at., 1993). This might be relevant to the pathogenesis of HIV-associated neoplasia. Thus, as is true for other viruses, HIV is likely to control both survival and death of its host cells during its life cycle.

5. Apoptosis as

Consequence of Cytotoxic, Physical, or Chemical Damage

ci

Mild physical and chemical damage causes apoptosis rather than necrosis ofthymocytes and mature T cells. Exposure ofcells to temperatures above 40°C (Migliorati et d.,1992), irradiation with y, X, or UV rays (Sellins and Cohen, 1987), induction of DNA breaks by topoisomerase inhibitors (Cotter, 1992; Tepper and Studzinski, 1992), DNAdamaging agents like cis-platin (Evans et al., 1994), culture in the presence of inhibitors of inRNA or protein synthesis (Martin et d., 199O),inhibitors ofthe respiratory chain such as deoxyglucose, inhibitors of purine or pyrimidine synthesis, inhibitors of DNA polymerase activity, and a plethora of further toxic substances induce T cell death by PCD (Table X). Since many of these substances are employed as cytotoxic drugs in the treatment of lymphomas and leukemias, it is reasonable to assume that even cell death induced by rather drastic methods (ionizing irradiation, chemotherapy, and photochemother-

TABLE X INDUCTION OF T CELLDEATHBY PHYSICAL DAMAGE, ANTIMETABOLITES, OR CYTOTOXIC DRUG Substance or Manipulation Cold shock Heat shock Irradiation Aphidicolin Epipodophyllotoxins-like etoposide and teniposide Camptothecin Hydroxyurea 2-Chloro-2'aribino-fluoro2'deoxyadenosine Arabinosylcytosine or 5azacytidine Vincristine Rotenone or antimycin A Oligomycin Methotrexate 6-Nitroso-1,3-benzopyroneor 3nitrosobenzamide Cisplatin Cycloheximide

Probable Active Principle

Cell Type

Reference

Unknown Physical damage DNA damage DNA polymerase inhibitor Inhibitor of DNA Topoisomerase I1 Inhibitor of DNA topoisomerase I Antimetabolite Cytidine analogue

Thymoma cells Thymocytes Thymocytes

Lymphoma PBL

Kruman et al., 1992 Migliorati et al., 1992 Sellins and Cohen, 1987 Cotter, 1992 Tepper and Studzinski, 1992 Cotter, 1992 Johnson et al., 1992 Carson et al., 1992

Cytidine analogues

Thymocytes

Kizaki et al., 1992

Microtubule inhibit01 Mitochondria1 respiratory chain inhibitors Inhibitor of mitochondria1 ATPsynthase Dihydrofolate reductase inhibitor Inhibition of protein poly ADPribose) polymerase DNA-damaging agent Inhibition of protein synthesis

Miyashita and Reed, 1992 Wolvetang et al., 1994 Wolvetang et al., 1994 Miyashita and Reed, 1992 Rice et al., 1992 Proliferating thymocytes Human T cells

Evans et al., 1994 Collins et al., 1991 Martin, 1993b

Actinomycin D D-Galactosamin Tibultyn Photochemotherapy with 8methoxypsoralen + ultraviolet A light Photodynamic therapy sensitized by chloraluminium phtalocyanine Killing of antigen-presenting human T cells by CD4+ cytotoxic T cells Granzyme B-mediated killing of allogeneic target cells Leucyl-leucine methyl ester Injection of F23.1 (anti-VP8) Staphylococcal a-toxin Hydroperoxyeicosa-tetraenoic acid 25-Hydroxycholesterol or 7P, 25dih ydroxycholesterol Extracellular ATP

Inhibition of RNA synthesis Inhibition of RNA synthesis Immunotoxin Combination of chemical and physical damage

Human T cells Periphcral T cells Cortical thymocytes PBL

Martin, 1993b Gorizalu et d., 1993a Raffray et al., 1993 Marks and Fox, 1991

Chemosensitized damage

PBL

Aganval et al., 1991

Cytotoxic effects

Human T cells

Rahelu et al., 1993

Cytotoxic effects

Mouse T cells

Heusel et al., 1994

Conversion into membranolytic metabolites by dipeptidyl peptidase I Partially macrophage-dependent depletion Permeabilization by formation of transmembrane pores Peroxidated lipid acid Oxygenated cholesterol derivatives Unknown

Cytotoxic T cells

Thiele and Lipsky, 1992

Vp8' T cells

Gonzalo et al., l994a

Human T cells

Jonas et a / . , 1994

Human T cell lines Sandstrom et al., 1994 Murine lymphoma cells and Christ et al., 1993 th ymocytes Zheng et al., 1991 Thymocytes

240

CUIDO KROEMER

apy) will usually involve apoptosis rather than necrosis in vivo. This would explain the fact that during anticancer therapy therapeutic episodes are not usually accompanied by local or systemic signs of inflammation, as would be the case for necrotic cell death. Enzymes contained in cytotoxic cell granula may also induce apoptosis. Cytoplasmic granules of cytotoxic T cells and NK cells contain serine proteases (granzymes or fragmentins) which cause apoptosis in the presence of the pore-forming protein perforin (Hayes et al., 1989; Shi et al., 1992a). The most rapidly acting among these enzymes is fragmentin-2 (granzyme B), a unique protease that hydrolyses after asparagine residues and thus shares a unique substrate specificity with interleukin-lp converting enzyme. Granzyme B is necessary for the rapid apoptosis-inductory action of cytotoxic T lymphocytes acting on allogeneic target cells, as shown in mice rendered deficient for granzyme B by homologous recombination (Heusel et al., 1994). Thus, cytotoxic effector T cells kill determined target cell types by inducing apoptosis. In synthesis, molecules contained in cytotoxic cell granules, as well as nonphysiological damage of cells, can induce apoptosis instead of necrosis. 111. Inhibition of T Cell Death

Exposure to the PCD-inducing (proapoptotic) stimulus involves a series of events: signal transduction (in the case of specific receptormediated signals) and mild, still-reversible changes in cellular metabolism. During this phase, before a point of no return has been reached, PCD can still be avoided. Since T cells are continuously exposed to low levels of PCD-inducing stimuli, it has to be postulated that threshold and repair mechanisms counteract apoptosis induction. For example, DNA repair may counteract the induction of PCD at early stages. This is suggested by experiments in which the inhibition of DNA repair by aphidocolin, an inhibitor of DNA polymerase-a, greatly enhances the sensitivity of targets to TNF-induced PCD (Gera et al., 1993). Inhibition of DNA transcription and RNA translation interferes with the induction of cell death in many systems. However, in some cases cell death can be induced and executed even in the absence of RNA and protein synthesis, suggesting that preexisting catabolic cascades only need to be activated. Once a threshold of PCD-inducing signals or cell damage has been reached, the cell is inevitably condemned to death and a metabolic program is executed that causes cell death. This program probably involves several death pathways that are activated in response to any kind of PCD-inducing trigger.

PHARMACOLOGY OF T CELL APOPTOSIS

24 1

The mechanism by which different substances inhibit programmed T lymphocyte death can be rather disparate (reviewed by Kroemer and Martinez-A., 1994; Fig. 2, Table XI). Depending on the level at which such apoptosis-inhibitory (antiapoptotic) substances interfere with apoptosis-inducing (proapoptotic) stimuli, it is possible to distinguish the following possibilities. First, blocking of PCD can be due to direct antagonism, leading to the neutralization of the PCD-inducing agent or the blockade of its receptor. Second, an antiapoptotic compound may be a functional antagonist of a proapoptotic stimulus, functioning as “rescue signal” that prevents otherwise ineludible PCD. This group includes growth factors or contact-dependent signals that function as costimuli and switch an abortive or tolerizing response, induced, e.g., by crosslinking of the antigen receptor, into a productive one. Third, prevention of PCD may be effectuated at the subcellular level by blocking signal transduction pathways that mediate proapoptotic stimuli or by stimulating pathways with a rescue effect. Fourth, the antiapoptotic agent could impede the deployment of the selfdestructive cascade, thereby preventing PCD at a metabolic level that is common to many or all types of apoptosis.

Apoptosisinducing ligand

Level of apoptosis inhibition

I <

phase of commitment point of no return

-

neutralization of the apoptosis-inducing stimulus or blockade of its receptor

ligand Receptorinteraction ’

stimulation- of corece ptors that transmit rescue signals

S e c z d .( messenger cascades

blockade of apoptosisinducing signal transduction or stimulation of rescue pathways

Effector

inhibition of selfdestructive catabolic reactions

preapoptotic phase

no possible intervention Apoptosis

FIG.2. Hypothetical steps of apoptosis. The apoptosis-inducing signal is delivered via specific receptors coupled to signal transduction cascades that, in turn, activate catabolic effector mechanisnrs. During the commitment phase, before the point of no return is attained, apoptosis can be blocked at several different levels. For details and references see text.

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TABLE XI INRIBITION OF LYMPHOCYTE APOPTOSls AT DIFFERENT LEVELS Target Molecule of Inhibition

Level of Inhibition Neutralization

Glucocorticoid receptor Antigedclass I1

Costimulation

Cytokine receptors Costimulator receptors Retinoid receptors IntracelIuIar calcium

Signal transduction

Inhibition of effector phase

Protein kinase C Tyrosin kinases Phosphatases GTP-binding proteins Macromolecular synthesis Endonucleases Chromatin arrangement Free radicals Proteases Poly-(ADP-ribose) polymerase Inhibition of G o 4 1 transition ~

Example for Inhibitors

RU-38486 Neutralizing antibodies masking antigen or class I1 IL-1, IL-2, IL-4, IL-10 anti-CD28, costimulatory cells 9-cis retinoid acid Extra- or intracellular calcium Chelators High dose of phorbol ester, H7 Genistein, tyrphostin Okadaic acid Pertussis toxin Cycloheximide, actinomycin D Zinc, aurintricarboxylic acid Sperniine N-acetylcystein, thioreduxine, glutathione, bcl-2, etc. Specific serine and cysteine protease inhibitors 3-Aminobenzamide Antioncogenes Inhibitors of ~ 3 4 ' ' ' ~

~~~

Note. For details and references consult text.

A. IN VITROINHIBITION OF T CELLDEATH Zn uitro systems have contributed to unraveling the mechanisms of cell death and to developing strategies for the pharmacological inhibition of apoptosis. Given that the manipulations that can be performed in uitro are often too drastic to be applied in vim, I will discuss them separately.

1 . Neutralization of Apoptosis-Znducing Stimuli Antiapoptotic agents may simply neutralize a proapoptotic agent or hinder it from binding to its respective receptor. Thus, antibodies that mask the interaction between superantigens on one hand and the TCR or CD4 of responsive cells on the other hand can be employed to inhibit the clonal deletion of superantigen-reactive T cells in uiuo (Jones et al., 1990; Acha-Orbea et al., 1992). Tripeptide chloromethyl

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ketones inhibit peptide hydrolysis by “fragmentins” in a specific fashion and concomitantly prevent endonucleolysis of cells exposed to these cytotoxic lymphocyte granule serine proteases (Shi et al., 1992a). In addition, the glucocorticoid receptor antagonist RU-38486 (mifepristone, RU486) blocks the glucocorticoid- and CAMP-triggered death of thymocytes in vitro and in vitio (Iseki et nl., 1991; Schwartzman and Cidlowski, 1991; Jondal et nl., 1993).Inhibition ofthe transcription of the TNFa gene or neutralization of its product by a microinjected antibody has been used to block the TCR-driven PCD of cytotoxic T lymphocytes (Wang et at., 1994). On a different level, T cells protect themselves against the PCDinducing effect of hydrogen peroxide by releasing soluble catalase (Sandstrom and Buttke, 1993). Expression of the mouse multiple drug resistance (mdrfl P-glycoprotein in a murine thymoma (W7) variant (MS23) is associated with abnormal resistance to glucocorticoids with both 11- and 17-hydroxyl groups. Such cells accumulate reduced concentrations of drugs including dexaniethasone, and both drug sensitivity and intracellular accumulation can be restored by verapamil (Bour1993). Thus, the proper cell can develop strategies to cope geois et d., with PCD-inducing agents which could contribute to tumorogenesis. 2. Cosigna1s That inactivate Death Programs The antiapoptotic action of certain stimuli can be accommodated in the two-signal model (Bretscher and Cohn, 1970; Schwartz, 1990), according to which lymphocyte activation via the antigen receptor (T cell receptor or surface 1g)-signal l-will entail tolerance by induction of anergy or apoptosis, unless a second, costimulatory signal is provided. Separate delivery of signal 1 or 2, respectively, would cause apoptosis of the T cells, whereas simultaneous stimulation of the antigen receptor and the coreceptor(s) allows for productive T cell activation. In this context, signal 2 inhibits PCU induction via signal 1. Accordingly, the presence of macrophages (that provide cosignals) inhibits PCD induction of murine Th2 (not T h l ) clones responding to antigenic peptide (Wang et nl., 1993). Similarly, monocytes inhibit the SEA-induced death of a human CD4+ clone (Kabelitz and Wesselborg, 1992). Interleukin-1 (IL-1) and phorbol ester that both activate protein kinase C inhibit apoptosis of thymocytes induced by Ca2+ ionophore or anti-CD3 antibody. Ca2+mobilization alone is lethal for thymocytes, whereas costimulation via protein kinase C provides an activation signal (McConkey et al., 1989a,1990). Similarly, both IL-1 and PMA inhibit the thymocyte death in response to agents that elevate CAMP levels (forskoline, an adenylate cyclase activator; prosta-

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glandine E) or mimic CAMPelevations (dibutyryl-CAMP) (McConkey et al., 1990a). A proper combination of ionomycin and PMA inhibits glucocorticoid-induced thymocyte PCD in uitro. Similarly, stimulation with anti-CD3 antibodies together with costimulation via LFA-1 receptors enhances the glucocorticoid resistance of thymocytes (Iwata, 1994). T cells that depend on exogenous growth factor can be rescued from PCD by providing the missing factor. Interestingly, in certain cases, IL-2-dependent cells can be rescued by another growth factor. IL-10 inhibits the apoptotic cell death of human T cells starved from IL-2 (Taga et al., 1993). IL-2 and IL-4 can also rescue T cells from death induced by activation or exogenous glucocorticoids. In human medullary CD1-single positive (CD4+CD8- or CD4-CD8+) thymocytes, IL-2 prevents PCD caused by stimulation with anti-CD3 in uitro (Nieto et al., 1990). In contrast, incubation of total murine thymocytes with IL-2 fails to reduce anti-CD3-triggered DNA fragmentation (Tadakuma et al., 1990). IL-4 inhibits the dexamethasone-induced apoptosis of DN and CD4'SP human thymocytes (Migliorati et al., 1993a). The dexamethasone-induced apoptosis of mouse NK cells or CTL is counteracted by IL-2 and IL-4 (Migliorati et al., 1994). IL-2 impedes the glucocorticoid-induced death of T h l cell lines, whereas IL-4 preferentially rescues Th2 cells (Zubiaga et al., 1992). IL-4 counteracts the IL-2-induced apoptosis of murine CD4+CD8+ thymocytes in uitro (Migliorati et al., 1993b). Anti-H-Y TCR transgenic female mice that are thymectomized exhibit a reduction in the frequency of CD8+ transgene-expressing peripheral T cells after injection of male splenocytes. In this system, transfection of male stimulator cells with IL-2 retards clonal deletion (Kirberg et al., 1993). However, IL-2 does not impede the clonal deletion of thymocytes in uiuo upon contact with peptidic self-antigen (Kroemer et al., 199313)or superantigens encoded b y endogenous retroviruses of the mammalian mammary tumor virus type (Mls) (Kroemer et al., 1991). Moreover, in uitro induction of apoptosis in a human CD4+ cell line by staphylococcal enterotoxin is not modulated by IL-2 and other cytokines (IL-1, IL-4, IFNy, GMCSF, TNFa, TNFP) or neutralizing anti-cytokine antibodies (Damle et al., 199313).Thus, extrathymic clonal deletion induced by superantigens and antigenic peptides obeys different rules. Another cofactor of T cell activation, retinol and its derivatives, might be involved in PCD inhibition. All-trans-retinol, an essential physiological cofactor of human T cell activation (Garbe et al., 1992), blocks the antigen receptor-triggered PCD of thymocytes (Iwata et al., 1992; Yang et al., 1993)in uitro at near-physiological concentrations. It

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also inhibits apoptosis of T cell hybridomas induced by TCR/CD3 ligation or a combination of ionomycin and phorbol ester (Iwata et al., 1992).In contrast, retinol fails to counteract the glucocorticoid-induced PCD of thymocytes in vitro (Iwata et al., 1992). The stereo isomer 9cis-retinoic acid is about 10-fold more potent in PCD inhibition than all-trans-retinoic acid, suggesting that retinoid X receptors mediate the effect of retinol (Yang et al., 1993).

3. Direct Znterventions a t the Level of Signal Transduction Pro- and antiapoptotic stimuli converging on different cellular receptors are integrated by a complex signal transduction machinery that ultimately decides activation of the self-destructive metabolic program causing endonucleolysis and cytolysis. Accordingly, interference with Ca2 mobilization, protein kinases, or phosphatases can abolish apoptosis induction. Protein kinase inhibitors and cyclosporin A prevent the anti-CD3-induced but not the dexamethasone-induced DNA fragmentation in T cell hybridomas (Iseki et al., 1991). In contrast, the Ca2+ ionophore ionomycine inhibits the dexamethasone- but not the anti-CD3-induced DNA fragmentation (Iseki et al., 1991).These interventions unravel distinct signal transduction pathways stimulating PCD. A sustained increase in intracellular calcium concentrations has been considered essential for some apoptotic processes, e.g., lysis of susceptible target cells by cytotoxic T lymphocytes (Allbritton et al., 1988), and glucocorticoid-induced death of thymocytes (Iseki et al., 1991).Although depletion of extracellular Ca2+with EGTA and chelation of intracellular Ca2+with quin-e/AM have initially been reported to inhibit glucocorticoid-induced thymocyte apoptosis (McConkey et al., 1989c),later reports suggest that quin-2/AM inhibits DNA fragmentation but fails to inhibit cytolysis (Iseki et al., 1993). Moreover, exposure of thymocytes to exogenous glucocorticoid does not cause an immediate elevation of intracellular Ca2+that would precede commitment for PCD (Deckers et al., 1993; Iseki et al., 1993). Similarly, EGTA fails to inhibit the glucocorticoid-induced death of T cell hybridomas that, on the contrary, is inhibited by ionomycine (Iseki et al., 1991). Protein kinase C activation is likely to be involved in glucocorticoidinduced thymocyte apoptosis. Accordingly, glucocorticoid induces translocation of the Ca"-independent protein kinase C-E isoenzyme from the cytosolic to the particulate fraction of immature thymocytes but not mature T cells. This process can be inhibited by actinomycin D or cycloheximide (Iwata et al., 1994), indicating the involvement +

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GUIDO KROEMER

of de nouo synthesis of macromolecules. Protein kinase C inhibitors suppress glucocorticoid-induced DNA fragmentation and cytolysis of murine thymocytes (Ojeda et al., 1990; Iwata et al., 1994). In addition, the protein kinase C inhibitor H7 inhibits radiation-induced DNA fragmentation in thymocytes (Ojeda et al., 1992). In contrast, okadaic acid, an inhibitor ofphosphatases 1,2A, and 2B, prevents PCD of B and T lymphoma lines induced by heat treatment or by ionizing radiation exposure, interfering with dephosphorylation of protein(s) that precedes DNA fragmentation (Baxter and Lavin, 1992). Overexpression of the CD45R(O) protein tyrosine phosphatase in the thymus increases the efficiency of TCR-mediated apoptosis and MHC-restricted negative selection in viuo (Ong et al., 1994). Activation of protein kinase C with phorbol ester inhibits apoptosis of MRLIMp-Iprllpr T lymphocytes of the CD4-CD8- phenotype (Van Houten and Budd, 1992). In synthesis, the use of enzymatic inhibitors of phosphokinases and phosphatates suggests the implication of a network of phosphorylating and dephosphorylating enzymes in the regulation of PCD. It remains to be determined which protein kinase C isoenzymes are preferentially involved in signaling death or survival. It is important to note that gross inhibition of signal transduction can only be performed in uitro. The fact that inhibitors of phosphatases and kinases, as well as calcium chelators, act on most cell types in a nonspecific fashion explains why these substances are too toxic to be employed in uiuo. At a more subtle level, treatment of thymocytes or T cells in uiuo with pertussis toxin, an inhibitor of signal transduction via certain GTP-binding proteins, abolishes apoptosis induction via the TCRlCD3 complex (anti-CDE, superantigen) but not via glucocorticoid receptors (Table XII). This applies to superantigen-stimulated mouse T cells as well as to human thymocytes induced to undergo apoptosis by sequential exposure to immobilized a n t i - C D ~ and E soluble anti-alp TCR antibodies (Gonzalo et al., 1994c; Ramirez et al., 1994). In addition, pretreatment with pertussis toxin inhibits the apoptotic cell death of human pre-B leukemia cells dying in response to crosslinking of the IgM and the CD19 receptors, as well as of monocytes activated via the CD69 molecules (Ramirez et al., 1994). In contrast, pertussis toxin fails to prevent the programmed cell death that follows exposure ofcells to the synthetic glucocorticoid dexamethasone (thymocytes, pre-B cells) or to interleukin-4 (monocytes) (Table XII). The capacity of pertussis toxin to suppress activation-induced death is not due to quenching of the activation signal because purified CD4+CD8+thymocytes exposed to pertussis toxin are still capable of

247

PHARMACOLOGY OF T CELL APOPTOSIS

TABLE XI1 EFFECTS OF PERTUSSIS TOXINON APOFTOTIC CELLDEATHOF LYMPHOID CELLS Cell Type ~~

~

Human thymocytes Human pre-B leukemia cells Human monocytes

K562 cells Mouse T cells in uiuo

Effect of Pertussis Toxin on Apoptosis

Apoptosis Inducer ~~

~

Anti-CDSE plus anti-alp TCR dexamethasone Anti-IgM plus anti-CD19 dexamethasone LPS plus anti-CD69 LPS plus 1L-4 Human NK cells Injection of SEB Injection of dexaniethasone

Note. Data from Gonzalo et a / . (1994~)and Raniirez et

(I/.

Inhibition No effect Inhibition No effect Inhibition No effect No effect Inhibition No effect

(1994).

mobilizing Ca2+after alp TCR crosslinking. The apoptosis-inhibitory ef'fect of pertussis toxin depends on the presence of an intact ADP ribosylating moiety. A mutant pertussis toxin molecule that lacks enzymatic activity, but still possesses the membrane translocating activity (Pizza et al., 1989) (Fig. 3), fails to interfere with activation-induced cell death. Another toxin that induces a different spectrum of ADP ribosylation than pertussis toxin is cholera toxin which fails to inhibit PCD. To suppress apoptosis, the intact pertussis holotoxin must be added to cells before the lethal activation step; its addition 30 min after initial activation remains without effect on apoptosis (Ramirez et al., 1994).These experiments unravel a pertussis toxin-sensitive signal transduction event that intervenes during an early step of activationinduced cell death of immune cells.

4 . Inhibition of Catabolic Cascades at the Effector Level Apoptosis involves a defined sequence of catabolic steps that lead to the disintegration of cellular organelles and macromolecules. Early changes that precede internucleosomal DNA fragmentation include telomeric association of chromosomes (Pathak et al., 19941, nucleolar disruption, condensation of heterochromatin into clumps abutting the nuclear membrane (Brown et al., 1993; Cohen et al., 1993b), cleavage of DNA into large (>50 kbp) fragments (Brown et al., 1993), nuclear lamina disassembly (Lazebnik et al., 1993) and an elevated plasma membrane permeability (Ormerod et al., 1993)-The catabolic metabolism that characterizes PCD implies degradation of intracellular proteins, loss of organelle function, and enzymatic fragmentation of nu-

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GUIDO KROEMER

FIG.3. Effect of pertussis toxin mutants on activation-induced cell death. The holotoxin elaborated by Bordetellu pertussis comprises five different subunits, S1 to S5. Within the S1 subunit, pertussis toxin contains the enzymatic activity that ADPribosylates sensitive Gor subunits so that ligand-induced exchange of GDP for GTP on the Ga subunit is blocked. It thus interferes with Gi protein-dependent signal transduction from cell surface receptors. In contrast to the native pertussis toxin molecule, the mutant holotoxin PT9K 129G specifically lacks the ADP-ribosyltransferase activity due to two amino acid substitutions in the S 1 subunit (residues 9 and 129), but retains the T cell mitogenicity, the hemagglutinating activity, and the antigenic determinants of the native molecule (Pizza et al., 1989; Marsili et al., 1992). The antitolerance effect of pertussis toxin depends on ADP-ribosyltransferase activity that resides in the S 1 subunit, whereas its mitogenic and IL-Zinducing potential does not require any enzymatic activity (Kimura et al., 1990; Lobet et al., 1993) and maps to the B oligomer, composed of S2 through S5 (Witvliet et al., 1992; Lobet et al., 1993).

clear DNA by endogenous nucleases. After an initial cutting of D N A at points of interaction with the nuclear scaffold, chromosomal DNA is digested by Ca2+- and Mg2+-dependent DNA endonucleases that cause internucleosomal strand breaks. The regular fashion in which degradation proceeds between nucleosomes gives rise to a laddertype pattern of DNA fragments that are mono- or oligomers of 180-200 base pairs (Cohen, 1991). a. Znterventions on the Cell Cycle. At least some types ofapoptosis are coupled to the cell cycle. Accordingly, activation-induced cell death of T cells appears to be coupled to entrance into S phase, as shown by drugs interfering with cell cycle progression. This has been shown for cells undergoing PCD after TCR/CD3 ligation in vitro (Boehme and Lenardo, 1993), but has not been confirmed for antiCD2-triggered cell death (Rouleau et al., 1994). Induction of PCD by

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glucocorticoids or by a nonactivating anti-TCR antibody (F23.1) in uico is not preceded by entrance into the S phase of cell cycle. This has been shown b y injecting mice with the thymidine analogue 5bromo-2’-desxoxyuridine (BrdUdr)during the phase ofapoptosis induction. Immunohistochemical analysis failed to detect BrdUdr into the nucleus of cells undergoing DNA fragmentation (unpublished observation). In contrast, manipulations that affect cell cycle progression in a more subtle fashion have revealed a role of G, to G, progression in apoptosis induction. Thus, blockade of ~ 3 4 ‘ ~ ‘a~serine-threonine , kinase that controls cell entry into mitosis at the Go-G, checkpoint, using an excess of peptide substrate, renders target cells resistant to apoptosis induction by the lymphocyte granule protease fragmentin-2. A similar effect is obtained by degradation of cdc2 protein in a temperaturesensitive mutant (Shi et al., 1994). Similarly, it has been shown that quiescent Go cells are refractory to cytotoxic T lymhocyte-induced apoptosis. However, transformation of cells with c-myc or infection with herpes simplex virus-1, two manipulations which trigger entry into a G, or GI-like state, render target cells susceptible to apoptosis (Nishioka and Welsh, 1994). This finding probably extends to PCD induced by other stimuli. Inhibition of the action of certain oncogenes by means of antisense oligonucleotides also entails cell cycle blockade and inhibits apoptotic events ligated to progress in the cell cycle (Vaux, 1993).The protooncogene c-myc is necessary for cell cycle progression in the transition between Go and G,. Overexpression of c-myc favors cell death. It has been shown that withdrawal of a mitogenic signal from fibroblasts causes cell cycle arrest during the Go phase concomitant with a reduction in c-myc expression. If c-myc is still expressed, PCD is induced (Evan et nZ., 1992). Bcl-2 inhibits apoptosis induced by c-myc overexpression and synergizes with c-myc in inducing cell proliferation (Bissonnette et al., 1992; Fanidi et al., 1992). Antisense oligonucleotides to the c-myc gene block TCR-mediated (not steroid-induced) PCD of T cell hybridomas (Shi et al., 199213).In pre-B lymphoma cells, however, stabilization of c-myc RNA by means of antisense oligonucleotides hinders cells from undergoing apoptosis following crosslinking of surface IgM (Fischer et d., 1994).Collectively, these data indicate a dual role of c-myc in the regulation of PCD. It has also been shown that antisense oligonucleotides that block the expression of c-fos or c-jun induced by IL-2 or IL-6 deprivation also block apoptosis (Colotta et al., 1992). However, these protooncogene products are important transcription factors (c-fos, c-jun) or cell cycle

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GUIDO KROEMER

regulators (c-myc) relevant to any cell type and therefore it appears improbable that drugs affecting their expression can be used for the inhibition of apoptosis in vivo. Despite these practical considerations, however, it appears that control of Go-G1 transition is critical for the regulation of apoptosis. In certain cases, cells would have to pass beyond this checkpoint of the cell cycle, thus undergoing a kind of “mitotic catastrophe.”

b. Inhibition of the Formation or Action of Free Radicals. Recent studies suggest that PCD is mediated at least in part by free radicals. Accordingly, membrane lipid peroxidation occurs after exposure of 2B4 cells to glucocorticoids well before cell viability is lost, suggesting that peroxidation damage caused by OH’ radicals participates in the metabolic suicide (Hockenbery et al., 1993). Reactive oxygen species cause membrane alterations (lipid peroxidation, activation of phospholipases, and alterations of membrane permeability), DNA damage, and compromise metabolic pathways (Greenspan and Aruoma, 1994). N Acetylcysteine is a well-established thiol antioxidant which, after uptake, deacylation, and conversion to glutathione, functions as both a redox buffer and a reactive oxygen intermediate scavenger. Thus, it reduces reactive oxygen species directly and acts indirectly via increasing the production of glutathione (GSH, an important endogenous antioxidant buffering the intracellular redox state). N-acetylcysteine diminishes the death of cell lines induced by growth factor deprivation (Hockenbery et al., 1993) and inhibits the antigen-driven apoptosis of MBP-specific hybridomas (Sandstrom et al., 1994). Similarly, transfection with the seleno enzyme glutathione peroxidase (GSHPx), that catalyzes the detoxyfying reaction H 2 0 2 + 2 GSH + GSSG + 2 HzO, inhibits apoptotic cell death following IL-3 deprivation of the FL5.12 cell line (Hockenberry et al., 1993). Dimethyl sulfoxide, a membranepermeable hydroxyl radical scavenger, or blockade of hydroxyl radical formation by o-phenanthroline blocks PCD of endothelial cell exposed to endotoxin in vitro (Abello et al., 1994). Thioredoxine, an intracellular thiol reductant, glutathione, a thio antioxidant, and N-(2mercaptoethyl)-1,3-propandiamine, an inhibitor of membrane peroxidation, have also been shown to inhibit PCD (Buttke and Sandstrom, 1994). These data underline the role of reactive oxygen species in PCD. According to several reports (Hockenbery et al., 1993; Kane et ul., 1993), the antiapototic protooncogene blc-2 also inhibits apoptosis on the level of the formation or action of free radicals.

PHARMACOLOGY OF T CELL APOPTOSIS

25 1

c. Inhibition of Endonucleases. A further strategy of apoptosis inhibition aims at the blockade of endonucleases. Bivalent zinc ions inhibit the action of CaZt and Mg2+ endonucleases and thus impede the internucleosomal DNA fragmentation into oligomers of 180-200 base pairs that in most cases accompany PCD (Duke et al., 1983). However, suppression of the ladder-type endonucleolysis does not prevent PCD in all systems. Thymocytes exposed to dexamethasone will die exhibiting key morphological features ofapoptosis (e.g., heterochromatin condensation) even in the presence of zinc or CaZ+chelators at concentrations that completely inhibit internucleosomal DNA fragmentation (Cohen et al., 1992a; Iseki et al., 1993). This dissociation between apoptosis morphology and DNA fragmentation has been confirmed in isolated liver nuclei treated with Ca2* and Mg2+ in the presence of Zn2+ (Sun et n l . , 1994). Zinc fails to inhibit the initial cleavage of DNA in larger fragments of approximately 700,300 and 500 kilobase pairs, suggesting that enzymes other than the zinc-inhibitable Ca2+/Mg2'-dependent endonucleases are sufficient for PCD execution (Brown et al., 1993). Aurin tricarboxylic acid, an inhibitor of endonuclease activation, has been reported to prevent PCD o f T cells in response to glucocorticoid and anti-CD3 (McConkey et al., 1989b). It has also been shown to inhibit DNA fragmentation and apoptosis of activated T cell hybridomas (Shi et al., 1990; Vukmanovic and Zamoyska, 1991; Mogil et al., 1994). In contrast, aurin tricarboxylic acid inhibits DNA fragmentation, but not cell death induced by heat shock, in a T cell hybridoma (Mogil et al., 1994). Similarly, T cell hybridomas exposed to aurin tricarboxylate doses that completely block internucleosomal endonucleolysis still exhibit mitochondria1 failure after anti-CD3 stimulation (Vukmanovic and Zamoyska, 1991). These data shed doubts on the possibility of inhibiting PCD with ATA. Further substances that inhibit DNA fragmentation without suppressing cytolysis are enediynes (Nicolao et al., 1993). Hence, at least four agents interfering with DNA fragmentation do not inhibit cell death in a durable fashion: zinc, calcium chelators, aurin tricarboxylic acid, and enediynes.

d . Protease Inhibitors. Experiments involving protease inhibitors suggest that enzymatic proteolysis is also involved in regulated cell death. The serine protease inhibitors N-tosyl-L-phenylalanylchloromethyl ketone and N-tosyl-L-lysilchlormethyl ketone inhibit proteolysis and endonucleolysis in human promyelocytic leukemic

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HL-60 cells exposed to the topoisomerase I inhibitor camptothecin, as well as in thymocytes treated with the glucocorticoid agonist prednisolone (Bruno et al., 1992). Three chemically distinct serine, but not cysteine, protease inhibitors prevent the dexamethasone- or teniposide VM-26-induced internuclosomal DNA fragmentation of thymocytes, cell shrinkage, and nuclear collapse, though fail to affect the highmolecular-weight DNA cleavage (>50 kbp) and loss of cell viability (Weaver et al., 1993). These serine protease inhibitors do not inhibit the TCR-triggered PCD of the murine T cell hybridoma 2B4. However, in this cell line, substances that specifically inhibit cysteine poteases (trans-epoxysuccinyl-~-leucylamido-(4-guanidineo)butane = E-64 and leupeptin) or calpain (acetyl-leucyl-leucyl-normethional) prevent anti-CD3- and anti-Thyl- but not dexamethasone-induced PCD (Sarin et aZ., 1993). Thus, different proteases may be speculated to participate in different types of PCD, serine proteases in glucocorticoid-driven cell death, and cystein proteases in activation-induced death. However, it appears improbable that the sole inhibition of these proteases is sufficient to confer long-term protection against cell death.

e . Inhibition of Other Metabolic Processes Associated with Apoptosis. The polyamine spermine that modifies chromatine arrangement inhibits the GC and Ca2+ionophore-induced DNA fragmentation and PCD of thymocytes, whereas depletion of intracellular spermine with methylglyoxal-bis-(guanylhydrazone)induces spontaneous D N A fragmentation. Other polyamines, such as putrescine and spermidine, have no effect in this system (Brune et al., 1991). Treatment of yirradiated lymphocytes with ubiquitin sequence-specific antisense oligonucleotides reduces the frequency of PCD, giving rise to the speculation that ubiquinilation of nuclear proteins might be involved in chromatin disorganization and oligonucleosomal DNA fragmentation (Delic et al., 1993). It remains to be determined whether spermine and inhibition of ubiquinilation confer long-term protection against cell death. The aromatic amide 3-aminobenzamide, an inhibitor of nuclear poly(ADP-ribose) polymerase, prevents the calcium-induced activation of endonucleases in rat liver nuclei (Jones et al., 1989a), inhibits the TNF-induced cytolysis of U937 cells (Wright et al., 1992), and interferes with K562 target cell killing by human natural effector NK cells, not lymphokine-activated killer cells (Monti et al., 1994). However, 3aminobenzamide does not block the enodnucleolysis that accompanies the death ofCTL targets or the glucocorticoid-triggered death ofthymo-

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253

cytes: it merely interferes with cytolysis (Redegeld et al., 1992; Hoshino et al., 1993). It has been suggested that activation of poly-(ADPribose) polymerase subsequent to DNA fragmentation in an attempt to repair DNA causes an excessive consumption of NAD and ATP, thus provoking an energy deficiency which contributes to membrane disruption (Redegeld et al., 1992). However, the fact that experimental blockade of poly-(ADP-ribose) polymerase by 6-nitro-l,2-benzopyrone and 3-nitrosobenzamide augments the activity of nuclear endonucleases in human leukemic cell lines (Rice et al., 1992) argues against the possibility that this enzyme could serve as a molecular target for PCD inhibition. A further candidate for inhibiting apoptosis at the effector level is tissue transglutaminase. Dexamethasone-stimulated thymocytes, as well as lymphoid cells undergoing PCD, express mRNA encoding this enzyme and an elevated catalytic activity (Piacentini, 1994). Since transfection with antisense complementarity DNA in antisense orientation inhibits apoptosis in neuroblastoma cell lines, this molecule might also be a target for PCD inhibition in lymphocytes. B. IN Vrvo INHIBITION OF T CELLDEATH As will be discussed in this section, T cell apoptosis can be prevented by in uiuo manipulations. On one hand, apoptosis-inhibitory oncotransgenes can be targeted to lymphoid cells by means of tissuespecific promoters. In view of the progress of gene therapy, this approach may lay ground for future interventions on lymphoid PCD by somatic gene manipulations. On the other hand, a number of pharmacological agents have been successfully employed to inhibit T cell apoptosis in uiuo (Table XIII).

1 . Manipulation of Apoptosis-Regulatory Protooncogenes a. Bcl-2. Bcl-2 is a protooncogene that specifically regulates the survival of cells from the lymphoid system. As shown in mice deficient for hcl-2 expression, this protooncogene is dispensable for the maturation of lymphocytes until the second postnatal week of life, although its expression is a conditio sine quu non for the survival of peripheral lymphocytes later on (Nakayama et al., 1993; Veis et ul., 1993). BcZ-2 is a member of a family of genes that is involved in PCD regulation. Bcl-x, a bcl-%related gene, gives rise to two different transcripts arising from alternative splicing. Whereas the larger product Bcl-x, inhibits cell death, as does Bcl-2, the smaller product of the bcl-x gene, Bclxs, inhibits the survival of growth factor-deprived cells (Boise et aZ., 1993). The proportion between PCD-preventing (Bcl-2, Bcl-x,) and

TABLE XI11 MANIPULATIONOF T CELLA~op~osrs IN VIVO IMMUNOPHARMACOLOGICAL

ta

cn 4

Agent that Causes T Lymphocyte Depletion

Inhibitor of PCD RU-38486

Dexamethasone (DEW

All-trans retinol

Competes with DEX for glucocorticoid receptor occupancy and neutralizes biological effects of DEX No effect

Pertussis toxin

N o effect

SEB Early Phase (12-24 hr)

SEB Late Phase (4-10 days)

Impedes early deletion when administered simultaneously with SEB

No effect on deletion when applied from Day 4

Conzalo et al., 1993a

Postpones early deletion by 12 hr in spleen, lymph nodes, and thymus Inhibits deletion of

Partial inhibition of deletion when administered from Day 4

Gonzalo et al., 1994a

Partially inhibits deletion

Conzalo et al., 1994c

Reference

CD4+VP8' (not CD8+) spleen cells and \7@' thymocytes

t o

01 01

Linoniide

Impedes PCD of peripheral T cells (not th ymocytes)

Cyclosporin A

N o effect

Aurintricarboxylic acid N-acetylcysteine

No effect Partial inhibition of deletion

Partially inhibits deletion of peripheral T cells when applied 3 days before SEB N o effect

Inhibition of thymocyte depletion nd

of CD4' and CD8' spleen and lymph node cells when administered together with SEB; no effect when injected on Day 4 Partially inhibits deletion of peripheral T cells when applied before SEB injection No effect or slight enhancement of deletion, when SEB is administered repeatedly nd

Gonzalo et al., 1992; Vanier and Prud'homme, 1992 Heeg et al., 1993 Mogil et nl., 1994

nd

Zamzami et al., 1994

Gonzalo et al., 1994b

256

GUIDO KROEMER

PCD-inducing (Bcl-xs, Bax) members of the Bcl-2 family may determine the fate of lymphocytes (Boise et al., 1993; Oltvai et al., 1993). The mutual inhibition of both classes of members of the Bcl-2 family involves heterodimerization (e.g.,between Bcl-2 and Bax).The molecular excess of one or the other substance would then determine the propensity to undergo PCD (Oltvai et al., 1993). The bcl-2 protooncogene is expressed in a tissue localization that is suggestive for its role in cell survival regulation (Hockenbery et al., 1991; LeBrun et al., 1993).Within the T lymphoid system, bcl-2 mRNA is expressed at a high level in immature CD4-8- thymocytes, as well as mature CD4+8- cells, whereas CD3'OWCD4+CD8+thymocytes, the fraction of thymocytes that is subject to selection processes, downregulate the bcl-2 mRNA (Tao et al., 1994). Moreover, it is expressed to a higher degree in naive quiescent T cells than in activated cells with a memory phenotype (Salmon et al., 1994). Similarly, Bcl-2 is highly expressed in pro-B cells and mature B cells but not in pre-B or in nascent bone marrow B cells (Merino et al., 1994). The Bcl-2 protein localizes to different intracellular membranes, namely the outer mitochondrial membranes (Hockenbery et al., 1990), the endoplasmatic reticulum, and the nuclear membranes (Chen-Levy and Cleary, 1990; Monaghan et al., 1992). Nevertheless, deletion of a C-terminal sequence determining the membrane localization of Bcl-2 does not abrogate its antiapoptoticpotential (Hockenbery e t al., 1993).Bcl-2 hyperexpression reduces PCD in different cell types and in response to rather divergent stimuli including radiation, mild hyperthermia, growth factor withdrawal, glucocorticoids, c-myc, and overexpression of the interleukin-lp-converting enzyme (Vaux et al., 1988; McDonnell et al., 1989; Miura et al., 1993; Nufiez et al., 1990; Strasser et al., 1990,1991a; Cuende et al., 1993). Similarly, it protects cells against oxidative stress induced by t-butyl-hydroperoxide (Zhongetal., 1993),H202,and menadione, a quinone compound that undergoes redox cycles intracellularly and causes superoxide radical formation (Hockenbery et al., 1993). In another experimental system, bcl-2 has been reported to inhibit the generation of reactive oxygen species induced by glutathion depletion (Kane et al., 1993). Overexpression of transfected Bcl-2 into cells reduces lipid peroxidation in the 2B4 murine T cell hybridoma exposed to glucocorticoids (Hockenbery et al., 1993), suggesting that the Bcl2-mediated inhibition of cell death might involve an antioxidant pathway. Bcl-2 might also influence signal transduction pathways. This possibility is favored by the finding that Bcl-2 protein associates with the human ras-related GTP-binding protein R-ras p23 (FernandezSarabia and Bischoff, 1993).

PHARMACOLOGY OF T CELL APOPTOSIS

257

Overexpression ofthe protooncogene bcl-2 partially protects lymphocytes against nonspecific induction of apoptosis (i.e., PCD triggered by growth factor deprivation, glucocorticoids, ionizing radiation, heat shock, anti-CD3q ionomycine, and phorbol ester) (Sentman et al., 1991; Strasser et al., 1991a,l994; Cuende et al., 1993).Although expression of transgenic Bcl-2 in lymphoid cells causes an increase in lymphoid organ cellularity, it does not cause a complete tumor-like deregulation of the lymphoid system. This could be related to the fact that Bcl-2 fails to inhibit the engulfment of senescent cells by macrophages, as shown for neutrophils overexpressing Bcl-2 (Lagasse and Weissman, 1994). Bcl-2 expression rescues B but not T cell development of scid mice, and thus overcomes the need of positive selection in the B cell system (Strasser et al., 1994a).In contrast, in mice expressing Bcl-2 in the thymus, self-antigen-driven clonal deletion is near to intact (Sentman et al., 1991; Strasser et al., 1991a; Tao et al., 1994). Nonetheless, Bcl-2 reduces the efficiency of intrathymic deletion in the male peptide-specific model (Strasser et al., 1994b; Tao et al., 1994). Thy-1-induced thymocyte apoptosis is also relatively resistant to Bcl-2 (Hueber et al., 1994), suggesting that activation-induced cell death, even if it is mediated via alternative pathways, is only moderately influenced by Bcl-2. WEHI-231 cells with a pre-B cell phenotype transfected with bcl2 are as susceptible to PCD induction via crosslinking of sIgM as vector-transfected control cells, although they are rendered comparatively resistant to death by heat shock (Cuende et ul., 1993).Similarly, certain WEHI-231 sublines that are relatively resistant to the induction of anti-IgM-mediated apoptosis still express high Bcl-2 levels (Gottschalk et ul., 1994), indicating a lack of correlation between resistance to activation-induced death and Bcl-2 expression. In mice carrying transgenes encoding Bcl-2, an anti-hen egg lysozyme (HEL) antibody and/or a membrane-bound form of mHEL, Bcl-2 inhibits clonal deletion of self-reactive B cells. This was shown by reconstituting host mice expressing HEL with bone marrow cells of anti-HEL antibody transgenic mice bearing the bcl-2 transgene. However, it fails to interfere with a predeletional step of tolerance induction that causes a developmental arrest at the stage of immature B cells (B220'"", IgM'"", HSA'"Rh)(Hart1eyet al., 1993). In contrast to the above data that do suggest an influence of Bcl-2 on clonal deletion of nascent bone marrow B cells, another group has reported that Bcl-2 only inhibits clonal deletion of mature self-reactive CD5+, as well as CD5-, lymphocytes in the peritoneum, but not that of immature cells in the bone marrow. This has been tested in double-transgenic mice that

258

GUIDO KROEMER

express the bcl-2 gene as well as an immunoglobulin gene encoding an anti-erythrocyte antibody (Nisitani et al., 1993) Altogether, these data suggest that in R cells, as well as in T cells, the apoptotic pathway responsible for negative selection is relatively resistant to Bcl-2.

b. p53. p53 is another protooncogene that plays an important role in PCD regulation. p53 overexpression in certain cell lines causes apoptosis (Shaw et al., 1992; Yonish-Rouach et al., 1991), whereas inactivation of p53 by genetic mutations (Hollstein et al., 1991) or binding of its activation moiety to the oncoprotein MDM2 (Oliner et al., 1993) correlates with progression through the S phase of the cell cycle and oncogenic transformation. Since Bcl-2 inhibits p53-induced apoptosis, p53 controls an event upstream of the Bcl-2 pathway. It is conceivable that p53 functions as a control of DNA integrity before replication. In dividing cells, low-dose irradiation and chemical DNA damage cause a massive p53 nuclear accumulation concomitant with a G, cell cycle arrest that may be mediated by the tumor growth suppressor gene. WAFl/CIPl (Eldeiry et al., 1994; Fritsche et al., 1993; Kuerbitz et al., 1992). Thus, p53 might cause apoptosis if DNA damage is too important to be repaired. I n contrast, in the case of limited damage p53 prolongs the G , phase during which DNA repair can be performed (Lane, 1992). In accord with this hypothesis, induction of apoptosis by agents causing DNA damage (y-irradiation, exposure to DNA topoisomerase I1 inhibitors) is dependent on p53 expression, as demonstrated in mice rendered p53 deficient by the homologous recombination technique (Clarke et al., 1993; Lowe et al., 1993). On the contrary, apoptosis induced via other pathways not primarily targeted to chromosomal DNA, e.g., PCD induced by glucocorticoids, anti-CD3, or stimulation with ionomycin plus phorbol ester, does not require p53 expression (Clarke et al., 1993; Lowe et al., 1993). c. Pim-1. Recently, it has been demonstrated that expression of pim-1 oncotransgene under the transcriptional control of the immunoglobulin heavy-chain gene enhancer reduces the susceptibility of thymocytes to undergo PCD in response to glucocorticoids in vivo (Moroy et al., 1993). This suggests that, in addition to bcl-2 and p53, further oncogenes intervene in the regulation of programmed lymphoid cell death. 2 . lnterventions on Steroid Receptors Recent experimental evidence indicates that glucocorticoid receptor occupancy determines whether antigenic stimulation will cause clonal

PHARMACOLOGY O F T CELL APOPTOSIS

259

expansion or deletion in uiuo (Wick et al., 1993). Injection of the superantigen SEB, that reacts with T cells expressing products of the T cell receptor VP8 locus, causes an immediate, transient increase in the serum concentration of corticosterone that is maximal 90 min postinjection and returns to control values within a period of 6 hr (Gonzalo et al., 1993a). Apparently, this glucocorticoid boost is essential for the apoptosis-mediated deletion of SEB-reactive (i.e., VP8') cells in the thymus or in the spleen that can be observed as early as 12 hr after SEB administration. The acute depletion in VP8'CD4+ or VP8+CD8+splenocytes and VP8'CD3' thymocytes is glucocorticoid dependent, since it is fully abolished by coadministration of saturating amounts of the glucocorticoid receptor antagonist RU-38486 (Gonzalo et al., 1993a,1994a).These results have been confirmed by experiments in which endogenous cortisone synthesis in the adrenal gland was prevented by DL-aminoglethimide pretreatment (MacDonald et al., 1993).Moreover, RU-38486 inhibits the peptide-mediated deletion of T cells in transgenic mice bearing a peptide-specific class 11-restricted TCR ( Jondal, personal communication). RU-38486 also impedes the deletion of CD4'CD8' thymocytes caused by administration of the a n t i - C D ~ Eantibody 2C 11 or 5'-(N-ethyl)-carboxamido-adenosine,an adenosine deaminase-resistant adenosine analogue that binds A2-type receptors and thereby activates adenylic cyclase and causes CAMP formation (Jondal et al., 1993; McConkey et al., 1993). As discussed above (Section II7B,2),coadministration of high amounts of exogenous glucocorticoid dexamethasone or hydrocortisone aggravates the SEBor anti-CD3~-induceddeletion of relevant target cells in uiuo (Gonzalo et al., 1993a; Jondal et al., 1993; Lussow et al., 1993).Consequently, interaction of glucocorticoid with its receptor is essential for antigendriven deletion of T cells in uiuo, revealing a cooperative interaction between two proapoptotic pathways in thymocytes and peripheral lymphocytes: that promoted by glucocorticoid and that triggered by antigen. The level of glucocorticoids present during an immune response determines whether antigen-specific T cells will undergo deletion or, on the contrary, mount a productive immune response. This underscores the importance of defects in glucocorticoid-mediated immune regulation that contribute to the development of autoaggression in several animal models of spontaneous autoimmune disease (Wick et al., 1993). All-trans-retinol is another agent targeted to the steroid receptor family. It reduces the SEB-induced depletion of VP8' thymocytes and splenocytes in uiuo. In contrast, retinol fails to reduce the glucocorticoid-induced apoptosis of thymocytes and peripheral T cells in uiuo (Gonzalo et al., 1994a). Therefore, at least two pathways are involved

260

GUIDO KROEMER

in SEB-driven deletion: one that depends on the presence of endogenous GC and another that can be blocked by retinol. These data provide a hypothetical explanation why vitamin A (retinol) deficiency causes an immunodeficiency (Semba et al., 1993). Lack of retinol availability would enhance the propensity of T cells to undergo apoptosis.

3. Pertussis Toxin Suggests G Protein-Mediated Regulation As discussed above (Section II,A,2, Fig. l), SEB has multiplepleiotropic effects on the immune system in uiuo, causing a cytokine syndrome and complex changes in the frequency of VPS' T cells. Intravenous injection of this bacterial superantigen causes a transient activation and expansion of SEB-reactive VPS' T cells, as well as a specific downregulation of the immune response through partial deletion of superantigen-reactive T cells and anergy of surviving T cells. Coadministration of as little as 0.5 p g pertussis toxin together with SEB abolishes most of the negative SEB effects (deletion, anergy), though it does not affect T cell activation and proliferation. Pertussis toxin abrogates the SEB-driven deletion of VP8+CD4' (not VPS'CDS' ) splenocytes that is observed early (12-24 hr) after SEB injection. Moreover, it antagonizes the late ( 1 4 days) deletion of VPS'CD4' and VPS'CDS' peripheral T cells that follows a transient expansion of such cells. This phenomenon is associated with a significant reduction in apoptosis and endonucleolysis and is not due to a compensatory increase in proliferation of SEB-reactive T cells as determined by means of a combined fluorometric analysis of cell cycle and DNA alterations associated with PCD. In addition, simultaneous injection of pertussis toxin and SEB fully abolishes the incapacity (anergy) of VP8' T cells to proliferate upon TCR crosslinking in vitro. All these effects are also observed in thymectomized animals, thus excluding the possibility that pertussis toxin might act by enhancing the maturation and export of thymic T cells to the periphery. The capacity of pertussis toxin to impede clonal deletion and anergy induction depends critically on its ADP-ribosyltransferase activity because a nonenzymatic pertussis toxin mutant fails to act in this biological system. Pertussis toxin selectively antagonizes or impedes the delivery of negative signals to T cells stimulated by superantigens without interfering with the transmission of stimulatory signals (Gonzalo et al., 1 9 9 4 ~ )These . data strongly suggest the involvement of pertussis toxin-sensitive Gi proteins in the regulation of T lymphocyte PCD. In contrast to pertussis toxin, cholera toxin, another ADP-ribosylating toxin, has inhibitory effects on murine T cell activation. This effect is mediated by the B subunit alone and does not involve elevations in

PHARMACOLOGY OF T CELL APOPTOSIS

26 1

CAMP or changes in the phosphatidyl inositol second messenger system (Woogen et al., 1993). Moreover, cholera toxin (not the B subunit) causes a covalent modification of the CD3 6-chain (Haack et al., 1993). It remains to be determined which downstream regulatory events are involved in the antiapoptotic effect of pertussis toxin. ADPribosylation of G(i)proteins catalyzed by pertussis toxin causes downregulation of protein kinase C (Winitz et al., 1994), but the relevance of this effect on PCD regulation is still elusive.

4 . Cyclosporin A Cyclosporin A is capable of antagonizing PCD induction in some experimental systems. Cyclosporin A selectively inhibits the activation (antigenic peptide)-induced cell death of a T cell hybridoma, though it does not affect apoptosis induced by glucocorticoids (Zacharchuk et al., 1990). In human mature T cells or single positive (CD4+CD8or CD4-8+) medullary thymocytes, cyclosporin A inhibits cell death induced by stimulation with anti-CD3 in the absence of accessory cells. In this experimental system, the cyclosporin A effect can be abolished by addition of exogenous IFNy (Groux et al., 1993), suggesting that cyclosporin A acts by inhibiting the production of the proapoptotic cytokine IFNy. In mice expressing a transgenic male peptide (H-Y antigen)-specific a//3 TCR, cyclosporin A has been reported to inhibit intrathymic positive selection (in female mice) and to delay negative selection in the male (i.e., autoantigen-expressing) thymus (Urdahl et al., 1994). Thus, it causes the accumulation of a low amount of mature CD4-CD8' thymocytes expressing the malespecific TCR in the male thymus (Urdahl et uZ., 1994). It has been reported that cyclosporin A perturbs intrathymic T cell development, interfering with the differentiation of immature CD3-CD4+CD8+thymocytes into mature CD3+CD4+CD8- or CD3+CD4-CD8+ T cells (Gao et al., 1988; Kosugi et al., 1989), possibly by damaging stromal cells of the thymus. In mice and rats, cyclosporin A causes a disturbance of thymocyte maturation and emigration, resulting in the export of double positive (DP, CD4+CD8+)T cells into lymph nodes (Fischer et al., 1991; Prud'homme et al., 1991; Zadeh and Goldschneider, 1993). In an i n vitro model of clonal selection, cyclosporin A prevents antigen-induced deletion of a T cell clone grown on a monolayer of H-2-matched thymic epithelial cells (Kosaka et ul., 1990). In T cell hybridomas, the capacity of cyclosporin A to prevent anti-CD3induced apoptosis has been attributed to an inhibition of calcineurin activity (Frumen et al., 1992). Accordingly, at concentrations of cyclosporin A and the related drug F K 506 that inhibit negative selec-

262

GUIDO KROEMER

tion in vivo, a decrease in calcineurin phosphatase activity can be observed in thymocyte lysates (Bierer et al., 1993). Altogether, these data suggest that cyclosporin A inhibits negative selection by four different but nonexclusive mechanisms: (i) by suppressing the production of cytokines like IFNy that are necessary for PCD induction; (ii) by disruption of thymic architecture; (iii) by favoring the export of immature thymocytes to the periphery; and (iv) b y blocking signal transduction at the level of calcineurin. Nonetheless, it would be simplistic to assume that cyclosporin A is a universal blocker of negative T cell selection. In an in vivo model of superantigen-induced peripheral T cell deletion, continuous treatment with cyclosporin A does not inhibit deletion and, on the contrary, enhances the elimination of superantigen-reactive peripheral T cells (Gonzalo et al., 1992; Vanier and Prud’homme, 1992). In contrast, cyclosporin A may abolish the induction of T cell anergy (Vanier and Prud’homme, 1992). This latter finding has not been confirmed by Hermann Wagner’s group (Heeg et al., 1993) who reports that SEBdriven deletion, as well as anergy, are cyclosporin A resistant. In synthesis, cyclosporin A appears to be incapable of inhibiting the deletion of mature T cells, although it partially inhibits the deletion of thymocytes in uivo.

5. Inhibitors of Catabolic Metabolism As discussed in Section III.A.4, multiple effector pathways participate in the final steps of apoptosis, which explains the difficulty to inhibit PCD at late steps of metabolic suicide. This could explain the rather unsatisfactory protection against death conferred by substances that act on late stages of the apoptotic process. Aurin tricarboxylic acid (ATA),a blocker of endonuclease activation, has been reported to inhibit apoptosis of thymocytes in vivo (Mogil et al., 1994). It impedes the loss of thymic cellularity induced by injection of an anti-CD3 antibody and counteracts the SEB-triggered deletion of Vp8’ thymocytes (Mogil et al., 1994). However, it has to be critically noted that ATA does not block the glucocorticoid-induced death of thymocytes and splenic T cells in vivo. At doses at which ATA does block DNA fragmentation, it fails to inhibit an early step of the apoptotic cascade, namely loss in mitochondria1 potential (unpublished observation). Although it delays the loss of cell viability, it does not provide actual protection against dexamethasone-induced T cell death. Another antiapoptotic agent that can be administered in vivo is N acetylcysteine (NAC), a potent antioxidant. To obtain protection

PHARMACOLOGY OF T CELL APOPTOSIS

263

against dexamethasone-induced thymocyte and splenic T cell depletion, the dose of NAC that has to be administered in uiuo is so high (21 g/kg per day) that NAC per se has nonspecific toxic effects and induces lymphopenia (unpublished observation). Thus, it appears that NAC is not suitable for the inhibition of T cells apoptosis in uiuo.

6. Linomide Linomide (a quinoline-3-carboxamide) is an immunostimulatory agent with an unknown mode of action that, by virtue of its anticancer effects, is undergoing clinical trials (Bengtsson et al., 1992). Linomide inhibits the endonucleolysis and depletion of peripheral T lymphocytes (not thymocytes) exposed to different apoptosis-inducing stimuli in uiuo (Gonzalo et al., 1994b). Linomide effectively inhibits the deletion of VP8+CD4+and VP8’CDS’ splenic T lymphocytes in BALB/ c inice injected with the VP8-stimulatory superantigen SEB (Gonzalo et al., 1994b). This effect is observed both during the early (12-18 hr) SEB-driven phase of deletion (D’Adamio et al., 1993)as well as during the second wave ofdeletion (>4 days postinjection) (Kawabe and Ochi, 1991) that follows a phase of clonal expansion and concerns cells that have undergone at least one round of division (Gonzalo et aZ., 1994a). In both phases, linomide reduces the amount of DNA fragmentation to about half of controls treated with SEB only. Linomide specifically affects deletion, but does not interfere with the SEB-driven clonal expansion or induction of anergy (Gonzalo et al., 1994b). Linomide is also effective in preventing the deletion of splenic CD4+ and CD8’ cells induced by “mega” doses of dexamethasone (1mg per mouse). Twenty-four hours after administration ofdexamethasone, splenic cellularity is reduced by about 70%, unless linomide is provided, in which case the reduction is lower than 20% (Fig. 4). Consequently, linomide is likely to interfere with a rather distant event of the apoptotic cascade, as it blocks the glucocorticoid- and the superantigen-triggered PCD of peripheral T cells (Table XIII). In contrast, linomide fails to rescue CD4+CD8+ thymocytes from the effect of exogenous glucocorticoids and does not increase the frequency of T cells that are clonally deleted in the thymus due to their unwarranted reactivity with self-superantigens (Mls) (Gonzalo et al., 1994b). In uiuo administration of linomide also reduces PCD of CD4-CD8- cells (Van Houten and Budd, 1992) recovered from the hyperplastic lymph nodes of MRL/Mp-lpr/Zpr mice (Gonzalo et ul,, unpublished data). These data indicate that linomide is a universal inhibitor of apoptosis in peripheral (not thymic) T lymphocytes.

264

GUIDO KROEMER 150 I h

-/

m~mw

c

spleen

CI

control

DEX

DEX +

DEX

+

linomide linomide 100 m f l g 300 mg/kg

FIG.4. Effect of linomide on the dexarnethasone (DEX)-induced depletion of T cells in uiuo. Depletion of thymic or splenic mononuclear cells was assessed 18 hr after intraperitoneal injection of dexarnethasone (1 rng/animal).Pretreatment with linomide (100 or 300 rng/kg body wt per day during 3 days) greatly reduces the sensitivity of splenic T cells to apoptosis induction.

The molecular mode of action by which linomide inhibits lymphocyte apoptosis and stimulates immune responses (see below) is unknown. It appears that linomide blocks an early event of PCD. In a model of dexamethasone-induced splenic T cell death, it interferes with the zinc-resistant DNA fragmentation into high-molecular-weight fragments (>50 kbp) and abolishes early apoptotic changes in cellular morphology (Zamzami et al., 1994). In addition, linomide interferes with an early functional change affecting cells committed to PCD. Before T cells demonstrate oligonucleosomal ladder-type DNA fragmentation, they exhibit a loss of the mitochondrial potential. This loss in potential can be assessed by a simple cytofluorometric method using a dye (3,3'-dihexyloxacabocyanineiodide (DiOC,(S)) that is incorporated into cells depending on their mitochondrial potential (Petit et al., 1990). Following injection of dexamethasone or SEB, splenic T cells never exhibit DNA fragmentation after ex uiuo isolation, although these cells do exhibit a loss in DiOC6(3) incorporation (Zamzami et al., 1994). Only after a short period of in uitro culture at 37°C do splenic T cells (>60 min) exhibit DNA fragmentation and a loss in chromosomal material (Kawabe and Ochi, 1991). Linomide inhibits the loss in mitochondrial potential and thus inhibits a relatively early event in the apoptotic cascade. Linomide does not lead to a downregulation of glucocorticoid receptors, nor does it inhibit all biological effects of glucocorticoids. Gluco-

PHARMACOLOGY OF T CELL APOPTOSIS

265

corticoids have a high anti-inflammatory potential, as well as an important immunosuppressive effect that is caused in part by the physical elimination of lymphocytes. Comedication of linomide abolishes the dexamethasone-induced depletion of splenocytes but leaves intact its anti-inflammatory effect in a model of local inflammation (foot pad swelling and popliteal lymph node assay). Moreover, linomide does not interfere with the glucocorticoid-mediated inhibition of IL-2 production in uiuo (Zamzami et d.,1994). In synthesis, linoniide inhibits an early stage of the apoptotic cascade without exhibiting a general inhibitory effect on glucocorticoid function. IV. Theoretical Insights Gained by Apoptosis Modulation

The pharmacological induction and inhibition of T lymphocyte apoptosis provides a methodological procedure for the elucidation of the (patho)physiology of PCD regulation. Thus, the manipulation of cell death has contributed to several conceptual advances: the multiplicity of pathways leading to cell death, the complex regulation of apoptosis, and the existence of redundant effector pathways that guarantee that a cell that is convicted to death will actually die. Moreover, pharmacological modulation of PCD has allowed for the mechanistic dissociation between different mechanisms of immune tolerance. A. MULTIPLEPATHWAYS INVOLVED IN APOFTOSIS REGULATION A plethora of different stimuli are capable of inducing apoptotic T cell death (Tables 11, IV-VI, VIII, and X). In view of the high number of' conditions causing PCD, several manipulations with limited antiapoptotic effects have been employed to discriminate between different pathways involved in the genesis of apoptosis. Agents that inhibit apoptosis allow for the establishment of dichotomies between resistant and susceptible types of cell death. As will be described briefly in this section, the spectrum ofactivity of different PCD-inhibitory manipulations is rather disparate, thus underlining the multiplicity of pathways involved in apoptosis regulation. Cohen (1992a) attempted a classification of different types of PCD depending on whether cycloheximide, an inhibitor of protein synthesis, would influence the induction of apoptosis (Table XIV). Conditions in which inhibition of DNA transcription or RNA translation impede the activation of the suicidal metabolic program would require induction mechanisms, whereas the induction of apoptosis by inhibitors

TABLE XIV CLASSIFICATION OF PROMOFTOTIC STIMULI ACCORDINGTO THEIRSENSITIVITY TO CYCLOHEXIMIDE Type Induction (inhibitable by cycloheximide)

Release Transduction (not inhibitable by cycloheximide)

Stimulus

Cell Type

Reference

Anti-CD3 SEB Glucocorticoids Glucocorticoids Epidophylotoxins Irradiation Growth factor withdrawal Cycloheximide or Actinomycine D Cytotoxic granules Tumor necrosis factor Anti-Thy1 antibody UVB light Heat shock Cold shock K+-specific ionophores

Thymocytes Thymocytes Thymocytes Mature T cell lines Thymocytes Thymocytes Many cell types

Smith et al., 1989 D’Adamio et ol., 1992 Cohen and Duke, 1984 Zubiaga et al., 1992 Walker et al., 1991 Sellins and Cohen, 1987

Peripheral T cells

Martin et al., 1990

Target cells Thymocytes Thymocytes Peripheral T cells Thymocytes Thymoma Lymphoid cell lines

Shi et al., 1992 Hernandez-Caselles and Stutman, 1993 Hueber et al., 1994 Bazar and Deeg, 1992 Sellins and Cohen, 1991 Kruman et al., 1992 Ojcius et al., 1991

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267

of macromolecular synthesis would represent a release mechanism (Martin et al., 1990). Induction mechanisms include the death ofthymocytes in response to anti-CD3 (Smith et al., 1989a), glucocorticoid (Cohen and Duke, 1984), irradiation (Sellins and Cohen, 1987),as well as the death of WEHI-231 cells stimulated with anti-IgM (Benhamou et nl., 1990).Transduction pathways, not influenced by cycloheximide, would dominate in target cells attacked by cytotoxic lymphocytes (Shi et al., 1992a) or cells exposed to TNF (Wright et ul., 1992), Kt-specific ionophores (Ojcius et al., 1991), or hyperthermic shock (Sellins and Cohen, 1991). However, the criterium whether cycloheximide actually inhibits cell death or not cannot be employed to distinguish between “active” cell death and other types of apoptosis. First, cycloheximide inevitably induces apoptosis by itself (Martin, 1993b), given that it is a rather rude biochemical tool, and therefore at most temporarily postpones the apoptotic process. This applies to other substances that perturb intermediate metabolism, such as the RNA synthesis inhibitor actinomycine D (Cotter et al., 1992), indicating that apoptosis can occur in immune cells without recourse to macromolecular synthesis. Second, in certain cases, when cycloheximide itself does not postpone apoptosis, other substances block the apoptotic machinery, indicating the existence of an active contribution of the dying cell to the death process. Thus, blockade of the cell cycle regulatory kinase ~ 3 4 “ren~‘ ders target cells resistant to apoptosis induction by the lymphocyte granule protease fragmentin-2 (Shi et al., 1994) although this type of cell death is resistant to inhibition of protein synthesis. In summary, the utility of cycloheximide or related inhibitors of macromolecular synthesis to unravel the physiology of PCD has to be questioned. Another molecule that has been employed to discern various pathways of apoptosis induction is cyclosporin A. The deletion of CD4+CD8+thymocytes induced by anti-TCR antibodies, and not that by glucocorticoids or anti-Thy-1 antibodies, is blocked by cyclosporin A, thus allowing to differentiate between “activation-dependent” cell death and other effects not depending on antigenic stimulation (Zacharchuk et al., 1991; Hueber et al., 1994). However, cyclosporin A also exerts rather “antigen-nonspecific” effects, rendering target cells resistant to cytotoxic T cells (Hudnall, 1991) and inhibiting anti-CD2induced death of peripheral T cells (Wesselborg et al., 1993b). The antiapoptotic ef‘fect that cyclosporin A is exerting in some experimental systems (e.g., T cell hybridomas) is mimicked by the immunosuppressive macrolide FK-506, which in turn is antagonized by rapamycine (Staruch et al., 1991).

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Alternatively, a classification of different types of apoptosis may take advantage of the fact that overexpression of the protooncogene bcl-2 protects lymphocytes against nonspecific induction of apoptosis (i.e., PCD triggered by growth factor deprivation, glucocorticoids, ionizing radiation, heat shock, anti-CD3~,ionomycine, and phorbol ester) but largely fails to inhibit the induction of apoptosis via stimuli directed to the antigen receptor, e.g., by self-antigen to thymocytes or by antiIgM antibodies to WEHI-231 cells (Sentman et al., 1991; Strasser et al., 1991a; Cuende et al., 1993). Nonetheless, this difference is not absolute given that bcl-2-transgenic mice exhibit an accumulation of self-reactive thymocytes or B lymphocytes (Siege1 et al., 1992; Strasser et al., 1991a,b71994b).Thus, the utility of distinguishing between bcl2-resistant and bcl-2-sensitive pathways of PCD remains elusive. However, the existence of a Bcl-2-salvable and another (relatively) Bcl-2resistant pathway of PCD is suggested by the fact that Bcl-2 prevents apoptosis of target cells of cytotoxic T cells from growth factor deprivation, but fails to protect from PCD induced by cytotoxic T cells (Vaux et al., 1992). Bcl-2 also fails to inhibit the death induced by mitochondria1 respiratory chain inhibitors (Wolvetang et al., 1994) and tumor necrosis factor (Vanhaesebroeck et al., 1993). Although the spectrum of antiapoptotic action of Bcl-2 is broad, certain types of PCD are clearly resistant to Bcl-2 hyperexpression. Manipulation of another oncogene involved in the regulation of some types of apoptosis allows for the establishment of another dichotomy than that dictated by bcl-2. Germline disruption of the p53 oncogene reveals that homozygous p53 null thymocytes are resistant to induction of apoptosis by radiation and by DNA-damaging agents (the topoisomerase I1 inhibitor etoposide, 5-fluoruracil, adriamycin), but retain normal sensitivity to glucocorticoids and to stimulation with ionomycin plus phorbol ester (Clarke et al., 1993; Lowe et al., 1993). These experiments clearly illustrate the multiplicity of apoptotic pathways that may or may not depend on macromolecular synthesis, cyclosporin A-sensitive signals, bcl-2, or p53 expression in different combinations. From these data a complex picture of PCD regulation emerges.

B. COMPLEX REGULATIONOF APOPTOSIS Similar to mitosis, PCD is regulated in a complex, polyfaceted, cell type- and differentiation stage-dependent regulation, and constitutes the final outcome of multiple signal transduction pathways. This is illustrated by the fact that the same substance can either favor or prevent apoptosis.

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Thus, IL-2 inhibits the glucocorticoid-induced death of certain T cell lines and thymocyte subsets (Nieto and L6pez-Rivas, 1989), but is thought to program alp and y16 T cells to activation-induced death ( Janssen et al., 1991; Lenardo, 1991) and to induce PCD in mouse thymocytes (Migliorati et al., 1993b). An ambiguous role of cytokines in apoptosis regulation has also documented for IFNy and IL-4. IL4 inhibits the PCD of glucocorticoid-stimulated Th2 cell lines and cytotoxic T lymphocytes (Migliorati et al., 1994; Zubiaga et al., 1992) and rescues pre-B cells from activation-induced death (Baixeras et al., 1993), but induces apoptosis of lipopolysaccharide-activated monocytes (Mangan et al., 1992). IFNy mediates PCD in some experiments (Groux et al., 1993; Liu and Janeway, 1990), but is a growth factor for certain cell lines. Retinol induces apoptosis ofa T cell lymphoma (Su et al., 1993a), but inhibits anti-CD3-induced death of T cell hybridomas (Iwata et al., 1992). Similarly, artificial inducers of signal transduction pathways, such as ionomycin or phorbol esters, can both inhibit and induce apoptosis (Tables VI and XI),depending on the experimental design, thus unravelling a dual role of intracellular calcium and protein kinase C in PCD regulation. Even the protein synthesis inhibitor cycloheximide has a dual effect on PCD (see above, Table XIV). Inhibitors of DNA topoisomerase type I1 generally induce apoptosis (Tepper and Studzinski, 1992) but inhibit the TNF-induced apoptotic death (Nishioka and Welsh, 1992). Further proof that apoptosis regulation is indeed an extremely complex process comes from experiments showing that two stimuli that per se induce PCD, when combined, may succeed in preventing apoptosis. This has been documented for thymocytes in which the proapoptotic stimuli anti-CD3 and heat shock on one hand and glucocorticoids on the other hand are mutually antagonistic iiz vitro (Iwata, 1991,1994; Migliorati et al., 1992). Thus, it may be postulated that there is no single signal transduction cascade that will always induce cell death. Rather, it appears that cell survival is the outcome of a (labile) equilibrium state. When entropy increases above a threshold level, loss of this equilibrium entails apoptotic cell death. C. REDUNDANT EFFECTORPATHWAYS INVOLVED IN APOPTOTIC CELLDEGRADATION The frustrated attempts to inhibit apoptosis by blocking catabolic processes suggest that several cascades of organelle disassembly and macromolecular disintegration are cooperating in PCD. Thus, inhibition of one single effector mechanism, e.g., endonucleases (by zinc

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ions or aurintricarboxylic acid), serine, or cysteine proteases, autophagosome formation, or formation of oxygen radicals, is rather inefficient in inhibiting apoptosis (Kroemer and Martiez-A., 1994). From the teleological point of view it makes sense that PCD does not involve just one single death mechanism but several independent ones, thereby reducing the vulnerability to single-locus mutations affecting the regulation or execution of PCD that would have deleterious effects for the individual, e.g., via oncogenic transformation. A further example for redundant regulation of cellular longevity is provided by transgenic mice expressing the antiapoptotic protooncogene bcl-2 in neutrophils. Although these cells become comparatively resistant to apoptosis, senescent neutrophils are still recognized and eliminated by macrophages. This is probably the reason why the transgenic animals exhibit normal in uiuo turnover of neutrophilic granulocytes (Lagasse and Weissman, 1994). If these data could be extrapolated to the T cell system, this would explain why transgeneenforced overexpression of the bcl-2 gene by itself is not sufficient to cause the development of lymphomas (Korsmeyer, 1992) and that additional mutations have to accumulate to allow for tumor development. In this light, it appears improbable that universal “killer genes” that would be involved in several different types of cell death in an obligate fashion (Schwartz and Osborne, 1993)will be identified in the future. It appears more likely that, at most, simultaneous silencing of several killer genes would affect the effector phase of apoptosis. D. INSIGHTS INTO THE PHYSIOLOGY OF LYMPHOCYTE TURNOVER AND CLONAL DELETION Pharmacological modulation of T cell death has provided important insights into the (patho)physiology of lymphocyte turnover. Apoptosis is hardly detectable on freshly isolated lymphocytes. Even after injection of drugs with a high proapoptotic potential, such as SEB or dexamethasone, apoptotic morphology and ladder-type DNA fragmentation cannot be detected on freshly ex uiuo isolated peripheral T cells, unless such cells are cultured during a short period (30 min to several hours) (Kawabe and Ochi, 1991; Carlow et al., 1992; Gonzalo et al., 1993a). In this system, massive internucleosomal DNA fragmentation, a rather late step in PCD (Brown et al., 1993), appears to be an exclusive in uitro phenomenon. To detect the breakdown of DNA into lowmolecular-weight fragments in freshly isolated T cells, sensitive PCRbased techniques are required (D’Adamio et al., 1993). A similar finding has been reported for human circulating lymphocytes. Although

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a great number of anticancer drugs induce PCD of proliferating cells in uitro, few if any apoptotic cancer cells can be detected ex uiuo after in uiuo treatment. Although glucocorticoid analogs and the topoisomerase inhibitor etoposide induce apoptosis of acute lymphoblastic and myeloblastic leukemias in uitro and cause a strong reduction of leukemia cell counts in uiuo, apoptotic cells are not found in circulation (Matsubara et al., 1994). This is related to the fact that apoptotic cells are undergoing changes in the physicochemistry of their membrane (loss in electric charge, modifications in the composition of the glycocalix, flipping out of phosphatidylserine residues normally only found on the inner sheath of the lipid bilayer, etc.) allowing for their recognition and removal by the phagocytic cells in uiuo well before the morphological and biochemical changes normally associated with in uitro apoptosis would occur (Savill et al., 1993). In this sense, typical apoptotic death wouId constitute a default pathway only occurring in the absence of adjacent cells endowed with phagocytic capacity, as is the case during in uitro culture of isolated cells. Further insight gained from PCD inhibition concerns the role of deletion in the maintenance of immune tolerance. It has long been unknown whether apoptotic deletion and functional nonresponsiveness (anergy) ofT cells would result from a qualitatively or quantitatively different stimulation (reviwed by Kroemer et al., 1992). Thus, several authors have speculated that anergy would result from tolerization processes that are quantitatively insufficient to induce immediate deletion (Sprent and Webb, 1992; Fulcher and Basten, 1994). Anergic T cells would simply be situated in the antechamber of death. However, this possibility has been ruled out by pharmacological manipulations of SEB-induced immune tolerance. SEB induces both deletion and anergy of surviving Vp8' T cells (Section II,A,2). RU-38486, retinol, and linomide are antiapoptotic agents that only affect SEBinduced deletion but leave intact SEB-induced anergy. In contrast, cyclosporin A, exogenous IL-2, and cycloheximide only block the induction of anergy, but do not affect the reduction of Vj38' T cells in this system (Table XV). In synthesis, qualitatively different pathways determine whether T cells will be eliminated in a irreversible fashion or rather survive and become anergic. A further question concerns the role of cytokines in peripheral clonal deletion. As discussed above, SEB induces the production of a whole series of different cytokines, some of which inhibit or induce PCD: IL2, IFNy, IL-4, IL-10, and TNF. To determine whether SEB-induced cytokines might participate in the regulation of cell death, a number of substances that modulate in uiuo cytokine secretion, namely

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TABLE XV QUALITATIVELY DIFFERENTIAL REGULATIONOF SUPERANTIGEN-INDUCED DELETION AND ANERCY Inhibition of SEB-Induced Immunomodulator RU-38486 (10 mg ip 2 x per day) Retinol (5 mg ip every 2 days) Linomide (10 mg per os per day) Pertussis toxin (1 p g iv together with SEB) Cyclosporin A (500 p g ip per day) Interleukin-2 PEG (1.5 x lo5 every 2 days) Cycloheximide (1 mg ip together with SEB)

Deletion

Anergy

+

-

Gonzalo et al., 1993a

-

Gonzalo et al., 1994a Gonzalo et al., 1994b

+ + + -

-

+ + + +

Reference

Gonzalo et al., 1994c Gonzalo et al., 1992; Vanier and Prud’homme, 1992 Unpublished observation Yuh et al., 1993

cyclosporin A, dexamethasone, and chlorpromazine, have been employed in uiuo. After comparing the effect of these drugs on cytokine production and SEB-driven deletion (Table XVI), it appears that these cytokines, including TNFa, are not rate-limiting factors of deletion or T cell survival in viuo. V. Functional Consequences of Apoptosis Modulation The pharmacological modulation of cell death either aims at correcting diseases linked to an abnormal resistance of T cells to apoptosis or, on the contrary, at reducing their unwarranted apoptotic decay (Table I). In this section, I will evoke the therapeutical consequences as well as the undesired side effects of interventions on T cell apoptosis.

A. DEPLETION OF T CELLLYMPHOMA OR LEUKEMIA CELLSAND DELETION OF AUTOREACTIVE T CELLS 1. Treatment of T Cell Lymphomas or Leukemias

As a general rule, it appears that tumorogenesis involves deregulated proliferation, abnormal resistance to PCD, or a combination of both. Therefore, at least in some cases, induction of tumor cell apoptosis

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273

TABLE XVI DISSOCIATION OF CYTOKINE PRODUCTION AND ACTIVATION-INDUCED CELLDEATH Substance

Effects on Cytokine Serum Levels Induced by SEB

Strong inhibition of IL-2 and I F N y production, suppression of T N F secretion Dexamethasone Complete inhibition of IL-2, IFNy, and T N F production Chlorpromazine Strong inhibition of 1L-la, IL-2, IFNy, IL-4, T N F , GM-CSF; amplification of IL-10 production Linoniide Moderate inhibition of T N F production, no effect on IL-2, I F N y , IL-4, and IL-10 secretion

Cyclosporin A

Effects on SEB-Driven Deletion of VPS' Peripheral T Cells No effect or enhancement of deletion Enhanced deletion of T cells No effect on deletion

Inhibition of deletion

Note. Data from Gonzalo et uI. (lt)Y2,1993a.b,19Y4a,b), Tarazona el ul. (1Y94), arid unpublished obszrvations.

constitutes an etiological treatment of tumor development. As discussed under Section II.B.5, apoptotic cell death of lymphoma or leukemia cells can be induced by irradiation, as well as by different types ofchemotherapy. High energy irradiation and conventional chemotherapy, however, do not only affect tumor cells but also damage and eliminate normal cells. A novel strategy for the treatment of lymphoproliferative syndromes and malignomas consists of developing antibodies that signal death to lympoid tumor cells. One example for this type ofapproach is provided by monoclonal antibodies against the Fad Apo-1 molecule. Anti-Apo-1 induces apoptosis of adult T cell leukemia cells (ATL) isolated ex vivo from patients with ATL (Debatin et ul., 1993).In the same sense, anti-idiotypic antibodies capable of causing Ig signal transduction may be used for the induction of apoptosis in B cell leukemia cell (Vuist et ul., 1994). It remains to be determined whether TCR-specific reagents (antibodies, superantigens) may also be employed for the selective elimination of monoclonal T cell malignancies.

2. Treutment of Autoimmune Diseuse and Lymphoproliferation Autoimmune diseases are another class of pathologies that are caused by the persistence of T cells that ought to be eliminated by PCD. In several animal models of spontaneous autoimmune disease,

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genetic defects causing an abnormal resistance of peripheral T cells to PCD have been identified. This applies both to models of systemic lupus erythematosus (strains homozygous for the lpr,lpr"g, or gld mutations) and to an animal model of type I diabetes mellitus (nonobese diabetic, NOD mouse), as listed in Table XVII. The lpr mutation renders the Fas gene inoperative ( Watanabe-Fukunaga et al., 1992; Wu et al., 1993); the gld mutation affect the Fas ligand (Takahashi et al., 1994); and one of the diabetes susceptibility loci of the NOD strain maps to the bcl-2 gene (Garchon et al., 1994). In all three models, the susceptibility of peripheral T cells to undergo apoptosis is reduced (Russel and Wang, 1993; Scott et al., 1993; Garchon et al., 1994; Leijon et al., 1994). In the case of the lpr mutation, it has been clearly shown that deficient expression of the Fas gene in the T cell compartment, and not in other cell types such as B cells, accounts for the development of autoimmune disease. Expression of the normal Fas gene under the control of the T cell-specific CD2 promoter is sufficient to impede autoimmune disease development in MRLIMp-lprI1pr mice (Wu et al., 1994). The data obtained in this latter lupus-prone strain may be extrapolated to human systemic lupus erythematosus (SLE). Patients with SLE overexpress a variant of Fas that lacks the transmembrane exon, giving rise to a soluble molecule. Mice injected with such a soluble Fas molecule display autoimmune features, indicating that this alteration could indeed be involved in the pathogenic cascade (Cheng et al., 1994). The phenotypically abnormal lymphocyte population that expands in mice homozygous for the lpr mutation contains an elevated percentage of cells expressing products ofthe Vp8.2' and VP8.3' genes (Singer and Theofilopoulos, 1990; Herron et al., 1993).Accordingly, semispecific modulators targeted to members ofthe Vp8 family have a prophylactic effect on lupus development. In female MRLIMp-ZprIlpr mice, injection of the monoclonal IgG2a antibody F23.1 (specific for VpS.1, VpS.2, and Vp8.3) every 2 days (100 pg) starting from 8 weeks of age until 5 days prior to necropsy (at 8 months of age) causes a depletion of Vp8' and "double negative" (CD4-CD8-) cells in the thymus, lymph nodes, and spleen. Paralleling the reduction of the lpr-related splenomegaly and lymphadenopathy F23.1 attenuates the autoimmune manifestation (proteinuria, arthritis, cutaneous ulcers, glomerulonephritis), suppresses the surge in rheumatoid and anti-nuclear (antiDNA) antibodies, and augments the mean life span of MRLIMp-lprl lpr mice (M. de Alborin et al., 1992). Similarly, several groups have demonstrated the potential beneficial effect of the bacterial superanti-

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gen SEB, another substance targeted to Vp8’ T cells (Kim et al., 1991; Gonzalo et al., 1994d). Despite the Fas defect (that must concern all lymphocytes), T cells exhibiting a “normal” CD4’CD8- or CD4-CD8’ phenotype are susceptible to the induction of clonal deletion by the SEB. As in normal mice, intravenous SEB injection to 2- or 6-month-old female MRL/Mp-lpr/lpr mice causes a transient expansion of SEB-reactive Vp8+T cells, followed by a deletion of this subset (Kim e t d.,1991; Herron et al., 1993).However, the SEB-driven reduction in CD4+CD8-V08+cells is somehow mitigated compared to l p r l + or + / + controls, and the frequency of abnormal VP8’CD4-CD8cells is not modulated at all (Herron et ul., 1993; Gonzalo et al., 1994d). Whereas CD4-CD8- T cells are completely resistant to SEB-mediated deletion in uiuo, their precursors appear susceptible to SEB-induced deletion. A single injection of SEB prior to the surge of phenotypically abnormal CD4-CD8- T cells in peripheral lymphoid organs, at 2 months of age, is sufficient to cause a stable long-term (6 months) deletion of DN cells (Gonzalo et al., 1994d). This is accompanied b y a significant amelioration of autoimmune parameters. The mechanism of “memory” determining this long-term SEB response remains to be elucidated. The possibility of preventing autoaggression by means of TCR Vpspecific antibodies is not restricted to the MRL/Mp-lprllpr strain. Antibodies specific for product of the Vp8 gene family have been successfully employed in the prophylactic treatment of experimental autoimmune encephalitis (Acha-Orbea et al., 1988; Urban et al., 1988; Zaller et al., 1990), spontaneous diabetes arising in NOD mice (Fukuda e t al., 1989), and type I1 collagen-induced arthritis (Osman et al., 1993). Thus, antibodies specific for determined Vp families may be used in the prophylaxis of autoimmune diseases mediated by a restricted TCR repertoire. A different approach has been tested in mice transgenic for a Va2.3/ VpS.2 TCR specific for myelin basic protein that develops a spontaneous multiple sclerosis-like autoimmune diseases (Goverman et al., 1993).In such transgenic mice, multiple intravenous injections of high doses of the nominal antigenic peptide induce the deletion of peripheral T cells and impede the development of the disease (Critchfield et al., 1994). These data point to the possibility to induce apoptotic depletion of autoaggressive T cells by three different (semi-)specific approaches: TCR-specific antibodies, cross-reactive superantigens, and high doses of antigenic peptide.

DEFECTSIN Cause I p r mutation

(recessive) 1\3

4

m

ZpFg mutation

(recessive)

gld mutation (recessive) B ~ 1 - (dominant) 2 ~ ~ ~

THE

TABLE XVII CLONAL DELETION OF SELF-REACTIVE T CELLS I N

THE

MOUSE

Mechanisms

Functional Consequences

Reference

Insertion of a retrotransposon in the second intron of the Fas gene, causing abnormal transcription and splicing of the Fas mRNA and greatly reduced expression of the Fas proteins

Expansion of CD4-CD8- alp T cells. Generalized autoimmune disease with glomerulonephritis, arthritis, and arteriitis; reduced life span of mice; enhanced resistance of peripheral T cells to spontaneous or induced apoptosis; expression of transgenic Fas under the control of the CD2 promoter (i.e., in mature T cells and precursors) is sufficient to correct defect Allelic to Zpr mutation

Watanabe-Fukunaga e t al., 1992; Adacha e t al., 1993; Wu e t al., 1993,1994

Same manifestations as lpr, but nonallelic to l p r and Z p f g mutations Correlation with elevated IgG levels, periinsulitis, sialitis and enhances

Takahashi et a[., 1994

Point mutation that alters the cytoplasmic tail of the Fas protein, thus abolishing signal transduction via Fas Point mutation in the C-terminal region abolishing Fas-mediated triggering of cell death RFLP in bcl-2 gene specific for the NOD mouse

Watanabe-Fukunaga et al., 1992

Garchon et al., 1994; Leijon et al., 1994

like span of peripheral T cells in tiitro

bcl-2 transgene

Transgene-enforced hyperexpression of the anti-apoptotic bcl-2 protooncogene

nude (recessive)

Thymic aplasia

Neonatal thymectomy

Acquired athymia

Cyclosporin A treatment

Inhibition of TCR-mediated signal transduction? Suppression of the production of the PCD-inducing cytokine IFNr? Disruption of thymic architecture? Export of immature T cells? Disruption of thymic architecture?

to

-l -l

Graft-versus-host disease

Accumulation of self-superantigen- or self-peptide-specific thymocytes; glomerulonephritis in certain genetic backgrounds Accumulation of T cells that normally are deleted in the thymus; severe immunodeficiency and susceptibility to develop autoimmune lesions after IL-2 treatment Accumulation of T cells that are deleted in euthymic controls, spontaneous organ-specific autoimmune symptoms in susceptible strains Increase in the frequency of autoreactive T cells in the periphery; autoimmune disease after neonatal treatment in some strains

Strasser et al., 1991b, 19940

Defective MIS”-driven deletion of host thymocytes in the thymus

Hollander et al.. 1994

Fry et al., 1989; Kroemer et al., 1991

Smith, H. et al., 1989; Kojima and Prehn, 1981

Gao et al., 1988; Kosugi et al., 1989; Fruman et al., 1992; Groux et al., 1993; Zadeh and Goldschneider, 1993

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B. IMMUNOSTIMULATORY EFFECTSOF APOFTOSIS-INHIBITORY DRUGS-PREVENTION OF IMMUNODEFICIENCY AND INDUCTION OF AUTOIMMUNE DISEASES

1 . lmmunostimulation Pharmacological treatments that enhance the probability of lymphocytes to survive antigen-mediated stimulation can be expected to have immunostimulatory effects in viuo. Thus, they enhance desired (antitumoral and antiviral), as well as undesired (autoaggressive), immune responses in uiuo. Linomide might exert at least some of its immunostimulatory effects by virtue of its PCD-blocking potential: potentiation of antitumor immunity (Kalland, 1986),enhancement of delayedtype hypersensitivity reactions (Stblhandske and Kalland, 1986), acceleration of cardiac allograft rejections (Wanders et al., 1989), and aggravation of collagen type 11-induced arthritis (Kleinau et al., 1989), as well as of sialadenitis of MRL/Mp-lpr/lpr mice (Jonsson et al., 1988). Similarly, it is tempting to relate the capacity of retinol to break neonatal allotransplantation tolerance (Malkovsky et al., 1985) with its antiapoptotic properties (Iwata et al., 1992). By preventing PCD of peripheral lymphocytes undergoing stimulation by (neo)self-antigens, linomide or retinol would impede the downregulation of immune reactions against allo- and autoantigens.

2 . Autoimmune Side Effects of Antiapoptotic Drugs The fact that linomide per se has a rather low autoimmune potential may be attributed to the fact that this substance only inhibits PCD of peripheral T cells, and not that of thymocytes. Moreover, the effect of linomide is restricted to deletional tolerance in the sense that this drug does not reverse superantigen-induced anergy in uiuo (Gonzalo et al., 199413). The limited proautoimmune potential of retinol may also be related to the fact that it has no effect on anergy and only a limited antiapoptotic potential. In contrast, two further substances that can be employed in uiuo have relatively strong proautoimmune side effects: pertussis toxin and cyclosporin A. The capacity of pertussis toxin to subvert key mechanisms involved in the establishment of immunological tolerance (deletion and anergy) might explain its immunostimulatory and proautoimmune potential. Pertussis toxin is a potent adjuvant (Wilson et al., 1993),accelerates the development of spontaneous autoimmunity (Goverman et al., 1993), and elicits organ-specific autoimmune diseases when coadministered together with the relevant autoantigen (Broekhuyse et al., 1992; Goverman et al., 1993).

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The capacity of cyclosporin A to perturb clonal selection in the thymus (Jenkins et al., 1988; Urdahl et al., 1994)is likely to be involved in its autoimmune side effect. Irradiated hosts transplanted with syngenic bone marrow and then treated with, and withdrawn from, cyclosporin A develop a “syngenic graft-versus-host reaction” that bears all clinical hallmarks of allogenic graft-versus-host disease, including erythroderma, dermatitis, and alopecia (Marcos et al., 1986; Jones et nl., 1989b). As is known from animal models, this syndrome only develops in individuals that bear an intact thymus after discontinuation of cyclosporin A therapy (Sorokin et al., 1986). However, abolition of clonal deletion is not the only mechanism by which cyclosporin A could cause autoimmune lesions. Cyclosporin A abolishes anergy induction in some in uiuo systems (Vanier and Prud’homme, 1992). Moreover, a lack of suppression appears to be involved in cyclosporin A-induced autoimmune disease (Nucy et al., 1993). 3. Prevention of Immunodeficiency Although PCD inhibition favors the development of autoimmune diseases, it also has positive effects on immune function. Inhibition of apoptosis may be taken advantage of for the prevention of acquired immunodeficiencies. Of course this is trivial in the case of vitamin A (retinol) deficiency, that causes an immunodeficiency (Semba et al., 1993), and can be easily treated by supplementation with retinol derivates that inhibit T cell apoptosis. PCD-inhibitory agents could also be useful in the prevention of virus-induced lymphopenias. Linomide prevents the loss in peripheral CD4’ and CD8+ lymphocytes caused by infection with vaccinia virus and simultaneously reduces the degree of endonucleolysis observed in purified CD4+ and CD8’ T cells from virus-infected Balb/c mice (Gonzalo et al., 1994b). Similarly, attempts to prevent lymphocyte apoptosis from HIV-infected persons have been performed in uitro. Cyclosporin A, anti-CD28 antibodies (Groux et al., 1992), fibroblast-derived cytokines (Pandolfi et al., 1993), as well as antioxidants, such as N-acetylcysteine (Buttke and Sandstrom, 1994), catalase, vitamin E, or 2-mercaptoethanol (Sandstrom et al., 1993), have been shown to inhibit lymphocyte death from HIV carriers in vitro. It remains to be determined whether these or related substances can be successfully employed to inhibit lymphocyte PCD in uiuo and whether this will be beneficial for the immune function of patients infected with HIV.

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4 . Experimental Treatment of Septic Shock Inhibition of PCD might have a positive effect in another pathology: septic shock. Bacterial products involved in septic shock (endo- and exotoxins), as well as lymphokines induced by such products (TNF, TNFy, Section II,B,2), are known to induce PCD. This has been extensively discussed for superantigenic exotoxins (Section II,A,2) that induce PCD by activating T cells via the TCR, as well as nonsuperantigenic staphylococcal a-toxin that permeabilizes cell membranes (Jonas et al., 1994). Similarly, endotoxin (lipopolysaccharide) induces PCD of monocytes (Mangan and Wahl, 1991) and endothelial cells (Abello et al., 1994).A number of substances that augment the sensitivity of mice to endotoxin or superantigen-mediated septic shock per se induce T cell apoptosis (Table XVIII), whereas others that inhibit lethal septic shock, such as linomide, inhibit apoptosis. Further studies will be needed to determine the putative contribution of massive leukocyte PCD to the pathogenesis of multiorgan failure and disseminated intravascular coagulation typical for septic shock. VI. Conclusions

T lymphocyte apoptosis can be induced by precise combinations of signals delivered through the TCR, alternative activation pathways, cytokine, and hormone receptors. In addition, perturbation of cellular metabolism by viruses, toxins, antimetabolites, or mild physical damage causes PCD. The complex regulation of PCD and cell survival involves particular combinations of signals rather than specific pro- and antiapoptotic pathways. This explains that a number of extracellular stimuli, as well as artificial inducers and blockers of signal transduction, may both enhance and diminish the proclivity of lymphocytes to undergo PCD. Agents capable of inducing or impeding apoptosis help elucidate the complex regulation of programmed T cell death. PCDinducing and -inhibitory manipulations have contributed to the unraveling of the relation between clonal deletion, anergy, and immune tolerance. Moreover, they have unraveled the multiple pathways involved in the induction and execution of PCD. Antiapoptotic effects can b e effectuated at four different levels: (i) interception of a stimulus causing PCD, (ii) functional antagonism to an otherwise PCD-inducing trigger, (iii) interference with signal transduction cascades, and (iv) blockade of catabolic enzymes participating in cellular suicide. These four levels reflect a hierarchy of specificity, toxicity, and effectivity. Most of the agents that in-

TABLE XVIII PUTATIVE CORRELATION BETWEEN LEUKOCYTE APOPTOSIS A N D LETHALSEFTIC SHOCK Substance

Effect on Septic Shock

Effect on Leukocyte Apoptosis

Reference

D-Galactosamine

Pertussis toxin

N o effect on SEB- or LPS-induced shock

Linomide

Inhibits septic shock

Rapid apoptosis of peripheral T cells Apoptosis of peripheral T cells Inhibits activation- hut not dexaniethasone-mediated T cell apoptosis Inhibits activation- but not glucocorticoid-induced T celldeath Inhibits dexamethasone and SEB-induced T cell death

Gonzalo et al., 1993a

RU-38486 Retinol

Enhances sensibility to both SEB- and LPS-induced septic shock Enhances sensibility to SEB- or LPS-induced shock N o effect on SEB- or LPS-induced shock

Gonzalo et al., 1993a Gonzalo et d.,1994a Gonzalo et d., 1994c Gonzalo et al., 1993h, 1994b

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hibit PCD at the first two levels have selective effects and counteract death only in response to determined stimuli. In contrast, drugs blocking late events in the apoptotic machinery are highly nonspecific. Compounds that antagonize the first events following exposure to PCD-inducing stimuli have a lower spectrum of side effects, while substances targeted to distal events tend to be too toxic to acquire therapeutic value in vivo. Neutralization of apoptotic signals, as well as their interception at the level of signal transduction, is highly effective in PCD inhibition in response to the specific apoptosis-inducing stimulus to which they are targeted. In contrast, substances that inhibit catabolic events at the effector level of apoptosis tend to be comparatively ineffective and in many cases only offer a delay in death rather than long-term protection against death. Drugs inducing lymphocyte PCD can be employed in the therapy of lymphoid tumors and autoimmune diseases mediated by T lymphocytes that have failed to be deleted. Indeed, most cytostatic and immunosuppressive substances induce apoptosis of T cells, T cell precursors, or T cell-derived tumor cells. Idiotype- or antigen-specific protocols of PCD induction may be particularly successful in the treatment of autoimmune diseases mediated by a restricted TCR repertoire. In contrast, antiapoptotic drugs might be employed to counteract the increased apoptotic decay of lymphocytes after viral infection. Moreover, they exert immunostimulatory effects by impeding the deletion andlor augmenting the longevity of lymphocytes stimulated b y modified self-products (e.g., tumor antigens) or exogenous antigens. As an unwarranted side effect they can aggravate preexisting autoimmune diseases or provoke autoaggression ex novo. Drugs that inhibit lymphocyte apoptosis and that have been employed in vivo include cyclosporin A that, although predominantly immunosuppressive, in determined circumstances induces an autoimmune syndrome: alltrans-retinol, that is capable of breaking neonatal tolerance; linomide, that has a wide range of immunostimulatory effects; and pertussis toxin, that has a strong proautoimmune and immunostimulatory capacity. Whereas the effect of cyclosporin, retinol, and pertussis toxin is restricted to T cells activated via the antigen receptor, linomide has a broader antiapoptotic spectrum and inhibits both antigen- and glucocorticoid-induced lymphocyte PCD. Cyclosporin A acts on thymocytes, linomide on peripheral T cells, and both retinol and pertussis toxin on both populations. The fact that linomide only subverts deletion of peripheral T cells explains why this drug combines strong inimunostimulatory effects with a reduced autoimmune diseaseinducing potential.

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Future studies will elucidate the pathophysiology of T lymphocyte apoptosis, thus aiding to extend the list of pro- and antiapoptotic molecular devices that combine therapeutic efficiency with specificity for T lymphocytes. It will be particularly interesting to apply apoptosisinhibitory drugs to the treatment of slowly progressive diseases caused by an enhanced apoptotic decay of T lymphocytes such as AIDS. ACKNOWLEDGMENTS

I am indebted to Anna Senik and Charles Auffray for constant support. Moreover, I thank Naoufal Zamzami, Maria Castedo, Philippe Marchetti (Villejuif, France), and Rafael Rainirez (Cbrdoba, Spain) for allowing me to cite their unpublished work and Drs. Terje Kalland and Gunnar Hedlund for helpful suggestions. This work was partially supported by Centre National d e la Recherche Scientifique (CNRS), Association pour la Recherche sur le Cancer (ARC), Fondation Nationale pour la Recherche Medicale (FNRM), Institut National d e la Sante et d e la Recherche Medicale (INSERM), and Leo Fountlation.

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ADVANCES IN IMMUNOLOGY, VOL. 58

lntraepithelial lymphocytes and the Immune System GEK-KEE SIM Basel Institute for Immunology, Grsnzacherrtrasse 487, Basel CH4005 Switzerland

I. Introduction

11. T Cell Receptor Expression at Different Anatomical Sites 111. CD4 and CD8 on Intraepithelial Lymphocytes

IV. Origin of IEL V. Selection VI. Homing VII. Antigens and Antigen Recognition VIII. Functional Attributes IX. Concluding Remarks References

297 300 306 308 31 1 316 318 324 330 33 1

I. Introduction

Lymphocytes are not only present in circulation and in typical lymphoid organs, such as the thymus, spleen, lymph nodes, and Peyer’s patches, but they are also frequently found in the epidermis and in the epithelia of nonlymphoid organs (e.g., the gastrointestinal, respiratory, and urogenital tract). This article examines the rapidly expanding literature on the origin, phenotype, antigen receptor repertoire, specificity, and function of lymphocytes in various epithelial tissues. The mechanisms underlying the localization of these lymphocytes to specific anatomical sites, the selection of special repertoires in different tissues, and the contribution of these factors to organspecific immunity will be discussed. A. THECONCEPTS OF EPITHELIAL IMMUNITY AND MUCOSALIMMUNITY

For decades, immunology has addressed problems of lymphocyte diversity, repertoire selection, function, and cellular interactions exclusively through events which occur in either the primary (fetal liver, bone marrow, and thymus) or the secondary (spleen and lymph nodes) lymphoid organs. It was considered that self- and foreign antigens in these organs were sufficient for the immune system to discriminate 297 Copyright D 1995 by Academic Press, Inc. All rights of reproduction in any form re5ewed.

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self from non-self and mount efficient immune responses as needed. Effector cells activated in an immune response could either migrate to the site of inflammation or release soluble antigen-specific products, such as antibodies, into circulation. Lymphocytes present in the epithelial layers of various nonlymphoid organs in normal animals were not well characterized and were thought to be indistinguishable from those of the systemic pool. It is now evident that intraepithelial lymphocytes are mainly of the T lineage. The study of intraepithelial lymphocyte subsets, particularly the structure of their antigen receptors, indicates that clonotype dominance among epithelial T cells can be tissue specific. This key finding led to the recent burst of interest in “epithelial immunity.” There are many reasons to consider epithelial and mucosal immunity as a group of distinct and complex functions of the immune system and to assume that such functions have been selected in phylogeny under powerful pressures. For many pathogens, the mucosa offers an easy and apparently undefended gate of entry. At the same time, the mucosal surface itself offers an excellent milieu for the growth of various pathogens. The local production and concentration of IgA antibody on mucosal surfaces are part of an important albeit not wellunderstood defense mechanism against pathogens. The role of IgA in mucosal defense has recently been reviewed (1). Besides viruses, some pathogens with a pronounced tropism for epithelia have developed the ability to live and multiply in the intracellular environment, out of the reach of antibodies. To combat these, the immune system could, in principle, develop two distinct mechanisms. First, it could induce a series of intracellular events which would lead to the destruction of the intruder within. Second, it could develop mechanisms which would specifically detect the invaded cells and eliminate them by a cytotoxic mechanism. These tasks of local defense are not easy. Often, an intraepithelial immune response fails to eliminate the aggressor before doing some self-damage. In such cases, typical forms of local defense, such as granuloma formation, are more effective in containing the invader and preventing the spreading of the pathogens to adjacent healthy tissues. As we will see, there are strong indications that resident lymphocytes participate in the formation of such organized defense structures.

ARE MAINLYT CELLS B. INTRAEPITHELIAL LYMPHOCYTES Intestinal intraepithelial lymphocytes (i-IEL) are probably the most extensively investigated set of intraepithelial lymphocytes (IEL). More than a century ago, there were already indications of the exis-

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tence of resident lymphocytes among the epithelial cells of the intestines (2). Characterization of these cells by immunofluorescence performed on tissue sections showed that practically all the lymphocytes interspersed between the villous epithelial cells of the murine intestine were T cells (3,4). Extensive immunohistochemical studies confirmed that immunoglobulin-expressing B lymphocytes were rarely found within the epithelial layer, but were present in the lamina propria (5). In addition, most of the intraepithelial lymphocytes purified from the intestines of humans, mice, and rats express surface markers such as Thy-1 and CD8, which are phenotypic markers normally associated with T cells (6,7). These early observations paved the way for further characterization of the T cells in this important anatomical compartment. Historically, the occurrence of various lymphoproliferative cutaneous diseases and cutaneous lymphomas has fostered the belief that lymphocytes are a normal constituent of the skin, although direct evidence in support of this has been obtained only very recently. In 1983, two groups of investigators discovered a new population of cells in the epidermis of the mouse. These cells are dendritic in morphology, but are positive for the Thy-1 antigen (8,9). These dendritic epidermal cells, termed dEC, express Ly-5 but not other common T cell markers such as Ly-1, -2, or -3. Their identity remained obscured, until reagents against the T cell antigen receptor complexes became available, and they were identified as y6 T cells (10-13). Lymphocytes in normal human skin were also examined (14). Despite the differences in morphology, frequency, as well as the type of T cell receptors they expressed in the two species, in both humans and mice, the lymphoid cells in the skin are exclusively T cells. It is estimated that there are about 5 million T cells in the skin of a mouse, and around 4 billion in that of an adult individual (15,16). The cellular components of the lung reside within a very narrow interstitium amongst an extensive network of extracellular matrix consisting primarily of collagen. In sections of normal human lungs, a few lymphocytes can be seen, interspersed between the other cellular occupants. However, the number of resident pulmonary lymphocytes in an individual is high when the total volume of the interstitium is taken into account. After lung tissues have been digested with enzymes to allow better access of the resident cells, the number of lymphocytes recovered from an individual (0.4-1 x 10") is comparable to the total number in the peripheral blood (17). In comparison, the number of lymphocytes recovered in bronchoalveolar lavage is only a tenth of what was recovered from the resident population. Similar preparations,

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also obtained after removal of intravascular blood by perfusion, yielded comparable cell number per gram oftissue from mice and other rodents (18).In all these preparations, 70-80% of the lymphocytes turned out to b e T cells. Now that a large panel of monoclonal antibodies directed against T cells is available, particularly those against the cup or y6 T cell receptor (TCR) and the TCR-associated CD3 complex, the predominance of T lymphocytes among other IELs has become easy to document. Thus, IEL from the tongue and uterus have also been shown to be mainly T lymphocytes (19,20). These immunological reagents, employed in conjunction with biochemical and genetic approaches, have yielded extensive information on the repertoire of T cell receptors in various epithelial tissues. Moreover, in the mouse, they have also provided valuable information on the origin of different sublineages of IELs at different locations. There are striking differences between the a/3 and the y6 T cells with respect to their tissue distribution, receptor expression, specificities, and function. Since many of these differences have been revealed by studies on IEL, they will be addressed in this article. Since information on many aspects of I E L in other species is not available, only data pertaining to human and mice will be discussed in detailed. II. T Cell Receptor Expression at Different Anatomical Sites

In general, T lymphocytes develop primarily in the thymus. These cells normally express clonally distributed diverse antigen receptors known as TCR. Each T cell receptor consists of a heterodimer of two polypeptide chains, either cup or y6, in association with the CD3 polypeptide complex (21-23). The a/3 and y6 polypeptides are encoded by genes of the TCRa, -& -y, and -6 loci. As is the case for immunoglobulin genes, V and J or V, D, and J gene segments present in the germline configurations undergo somatic rearrangement during lymphocyte development to generate functional T cell receptor genes. The potential for combinatorial diversity is enormous, and a large repertoire of T cell receptors can be produced (24). Theoretically, the number of possible variants is estimated to be between 1O'O and 1015. The vast majority of T cells in circulation and in the lymphoid organs express the cup TCR, usually in conjunction with either the CD4 or the CD8 coreceptor, depending or whether they recognize peptide antigens in the context of class I1 or class I MHC molecules. The less frequent population of T cells expresses the y6 TCR, usually in the absence of CD4 or CD8 (25-28).

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The structure and organization of the murine and human T cell receptor a, p, y, and 6 loci have been extensively reviewed (22,23,29). Although there are fewer V and J segments in the TCRy and -6 loci compared to the TCRa and p loci, there is extensive junctional diversity in the antigen receptors of thymic y6 T cells in adults. The paucity of the y and 6 germline encoded elements is easily compensated for by 6 TCR rearrangements which employ both D61 and D62 segments in the same rearrangement in all three reading frames. In addition, N region nontemplated random nucleotide addition can occur at each of the three junctions. Thus, the potential repertoire of both y6 and ap T cells is enormous (23). It is therefore rather surprising to find that in the mouse, IEL in several epithelial tissues are T cells which bear mainly invariant, monomorphic y6 TCR (see Table I). At other locations, although the diversity of the TCR repertoire is high, there is evidence for preferential usage of specific Vy genes (22,30,31).At the moment, there are good indications that a developmentally regulated program of Vy gene rearrangement is at least partially responsible for these observations (30-32). In humans, both cup and y6 T cells are present in IEL, but ap T cells predominate in most normal epithelia that harbor lymphocytes (14,33). Invariant human y6 TCR have not been described, but oligoclonal expansion of y6 T cells seems to be frequent in pathological conditions (see Sections VII and VIII). In addition, now that more data are available, oligoclonal expansion of Cup T cells appears to be increasingiy common. A. INVARIANT y6 T CELLRECEPTORS OF MURINEIEL I. The S k i n

The Thy-l+ dEC found in the skin of all normal mouse strains are in intimate contact with keratinocytes and differ from Langerhans cells in their lack of class I1 expression (8,Y). They were first demonstrated to be y6 T cells by immunoprecipitation of skin-derived T cell clones using antibodies to CD3 and TCRy and -6 chains (10,12,13). In sharp contrast to the diverse y6 and ap TCR present on T cells in lymphoid tissues (22,23), practically all dEC (or s-IEL for skin intraepithelial lymphocytes) express identical TCR which consist ofVy5Jyl in associaBoth y- and 6-chains exhibit typition with V6lD62J62 chains (34,35). cal “fetal” type rearrangements (36) in that there is essentially no N region diversity present at the VJ or VDJ junctions of the rearranged y and 6 genes. In addition, the same TCR is expressed on dEC from all mouse strains tested regardless of their genetic background. This suggests that antigen recognition b y dEC is not restricted b y polymor-

TABLE I y6 T CELLRECEP~ORS OF MURINEINTRAEPITHELIAL LYMPHOCYTES ~

Tissue Skin

TCRy VY~JY~

Tongue

VYSJY~

Vagina and uterus

Vy6Jyl

Lung

VY~JY~

TcRG V6 1D62J62

V61D62J62 V61J62Di32 V6lJ62D62

V Y ~ J1Y V65 V66, 4, 7 Intestine

VY~JY~

V64, 5, 6, 7

Diversity

Strain Variation

None None

None observed None observed

None None None None None None Limited High Limited High High High High

None observed None observed None observed None observed None observed None observed BALB GXYS high C57BL/6 GXYS low Invarant delta in BALB C57BL/6, C3H, AJ, DBAR None observed V64 high in IE'-positive mice

Status in Nude Mice Absent Absent Some highly diversified VylV66 Absent Absent Absent Absent Absent Absent No difference No difference No difference No difference No difference No difference

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phic MHC structures. These unusual observations have led to questions regarding a TCR-based mechanism for tissue-specific homing, a stringent selection process based on the recognition of a limited number ofantigens by an invariant TCR structure, and a specialized biological function for these cells.

2 . The Tongue and Female Reproductive Tract A systematic immunohistochemical survey of various murine epithelial tissues using monoclonal antibodies which detect all crp TCR or y6 TCR has led to the identification of T cells which reside in the tongue, esophagus, vagina, and uterus (15,19).ap and y6 T cells are both present in the reproductive tract (r-IEL). crp T cells are mainly found in the subepithelial areas, while y6 T cells are most prevalent in the stratified squamous epithelium of the vagina and among the columnar epithelium of the uterus. The y6 T cells in these locations, like the dEC, exhibit a practically homogenous TCR repertoire. In this case, the predominant TCR consist of a Vy6Jyl chain which pairs with a V6lD62J62chain. As is the case with dEC, there is no N region diversity present in any of the joining regions (19), and no variation in TCR composition between mouse strains has been detected. It is intriguing that the &chain is identical to the one expressed in dEC. In addition, there are nine amino acids at the VJ junction ofthis Vy6Jy1 chain which are identical to those at the VJ junction ofthe d E C Vy5Jyl chain ( ~ 4 3 0 )The . striking similarity of these canonical y6 TCR may indicate a selection for similarity of specificity or function of these cells despite their differential accumulation at different locations. In analogous studies, the y6 T cells associated with the epithelium of the tongue were shown to be another homogenous set of y6 T cells. Surprisingly, IEL from the tongue express y6 TCR which are identical to those found on the y6 T cells of the reproductive epithelia (19). 3. The Lung The kinetics of colonization of the murine lung by lymphocytes show that, at birth, there are more y6 than crp T cells in the lung (37), indicating early homing ofy6 T cells to this site. However, the situation reverses rather rapidly such that in the adult, y6 T cells constitute only 8% of the resident pulmonary lymphocytes (37,38). It appears that by Day 20 after conception and continuing into early life, the resident pulmonary T cells (RPL) of the y6 lineage consist primarily of cells which express an invariant TCR of the Vy6JyllV61D62J62 type, which is identical to that found in the tongue and the female reproductive tract (37). With increasing age, the Predominant Vy6

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T cells are gradually replaced by Vy4, along with the concurrent acquisition of a highly diversified TCR repertoire. Nonetheless, T cells which bear the canonical Vy6VS1 TCR are still present, and can be activated upon in situ immunization with mycobacterial antigens administered in aerosols (39). It is of interest to note that in the first 2 weeks of life, the majority of Vy4 genes expressed in the lungs carry the invariant fetal type y gene rearrangement marked by the GYS junction (37,40). In older mice, they are replaced by y genes with limited junctional diversity characterized by the GxYS junctions. In addition, there is a strainspecific expansion for the BALB invariant delta chain (BID) in mice of the BALB but not of the C57BL/6 background (41). This selection operates on cells bearing a specific VSSDSZJSl rearrangement similar to the other invariant y- and S-chains described above by the marked lack of N region diversity. It is dependent on strain-specific background genes but not on polymorphic MHC-encoded determinants (41). Thus, it appears that a common feature among all T cells bearing the invariant yS TCR described so far is their apparent lack of dependency on polymorphic MHC determinants during maturation. B. DIVERSIFIED ap

AND

yS

T CELLRECEPTORS I. In Mice

In contrast to the T cells which bear invariant yS TCR, i-IEL from young mice generally consist predominantly of yS T cells which express a highly diversified TCR repertoire (15,42-46). However, similar to the s-IEL and r-IEL described above, i-IEL also observe the rule of preferential Vy gene usage. Vy7 is the predominant Vy gene utilized in i-IEL, primarily in combination with V64, although a few other VS genes (VS5, -6, and -7) are also used to a lesser extent (35,46,47). Unlike the s-IEL and r-IEL, the i-IEL exercise extensive usage of N region diversity for both y- and S-chains. In the light of recent documentations of stem cell differences (48), i-IEL are likely to be derived from T precursors which differ from those that give rise to yS T cells bearing invariant TCR. An extensive survey of i-IEL from different strains of mice of different ages indicates that the proportion of TCR ap+ i-IEL can vary from 10 to 80%, depending on the age of the animal, the microbial flora of the gut, and the genetic background of the mice (46,4952). I n general, the fraction of a@ T cells increases with age, paralleling the increase in immunological experience. There is no evidence for selective Vp gene usage, and no indication of a gut-specific ap TCR

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repertoire in normal mice. Overall, the TCR aP repertoire of i-IEL resembles that found in the peripheral lymphoid organs, except that the TCRap'CD8a'p- subset contains self-reactive Vp clones that are normally deleted in the periphery by recognition of self-superantigen (53). In older mice, in contrast to newborn mice, the prevailing set of T cells among resident pulmonary lymphocytes expresses the ap TCR (37).The overall repertoire of these T cells appears to be similar to that found in the lymphoid organs, at least at the level of VP gene usage (54). The y6 T cells at large are Vy4+, and among several V6 transcripts present, V66 appears to be most abundant (41). The junctional diversity associated with V66 is high, exemplifying the full potential of the 6 locus in the combined usage of both D6 gene segments in combination with multiple N region insertions. In mice of the BALB background, the GxYS type of convergent amino acid sequence motif is most prominent. However, this is not the case in C57BL/6, where a more diverse sample of N regions has been documented (40).The predominance of V66 rather than V65 distinguishes this set of y6 T cells from those of the lymphoid organs. 2. In Human

IEL in human also consist mainly of T lymphocytes, but in general,

up T cells are more abundant (33,55,56). Recent analysis of the TCRP

chains expressed by i-IEL revealed a marked skewing toward the expression of one or several V region genes in individual donors, indicating that human ap TCR+ i-IEL are oligoclonal (57-59). The majority of i-IEL appear to be derived from the expansion of a few T cell clones, but these clones can utilize a number of different V/3 genes. The predominant VP genes are frequently expressed among i-IEL of different individuals. In one analysis, PCR cloning and sequence analysis of the predominant Vp family in two individuals revealed an identical VDJ sequence in 13 out of 21 clones obtained from one donor, and a different repeated sequence in 18 out of 27 clones examined in the second donor (59). These data suggest that the preferential usage of certain Vps in i-IEL is due to an oligoclonal T cell expansion which may reflect the response ofthese T cells to a restricted set of as yet undefined antigens present in the gut. Among human intestinal intraepithelial lymphocytes, the level of y6 T cells is significantly higher than that found in the blood and in the lamina propria. Thirteen to thirty-seven percent of i-IEL have been reported to express the y6 TCR (55,56). There is no evidence of preferential Vy gene usage among normal human y6 TCR+ i-IEL as there is in mice, but there is a marked increased in V61 ex?ression

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instead (55).In contrast, the circulating y6 T cells in peripheral blood show a preponderance of V62 TCR. The reasons for the predominance of Vy7 in murine i-IEL and that of V61 in human i-IEL may not be different (see below). Although some studies showed that in most epithelia in humans, the level of y6 T cells is not significantly greater than that in the peripheral blood (14,33); it should be noted that in general, the representation o f 7 6 T cells in the blood and in the lymphoid organs of humans is approximately 10 times higher than that in mice (28,60). The greatest difference between human and murine IEL probably lies in the s-IEL population, where the uniform presence of y6 T cells in mice is contrasted with the fewer but clearly detectable human epidermal T cells. Nonetheless, about 4 billion T cells are present in the skin of a normal adult, compared to 5 million in a mouse (15,16). These human epidermal T cells are not dendritic in morphogy, and are primarily of the crp T lineage (14,61). The oligoclonal amplification of both ap and y6 IEL in association with various diseases suggests that the human I E L repertoire has been at least partially shaped by antigen exposure.

111. CD4 and CD8 on lntraepithelial lymphocytes

The differentiation antigens CD4 and CD8, which are coexpressed on immature thymocytes (CD4'CD8+ double positive), are found on distinct subsets of mature ap T cells in the periphery. The expression of these antigens on mature T cells generally correlates with helper T cell function or cytotoxic T cell activity, respectively. A small fraction of peripheral CD3' T cells are CD4-CD8-, and the majority of these double negative T cells express the y6 TCR. TCRaPfCD4-CD8T cells can also be found in the periphery, but at a very low frequency. Murine dendritic epidermal T cells which bear the yS TCR are all Thy-1 'CD4-CD8- (8-lo), while human skin epidermal T cells are predominantly CD8' (61).Among murine resident pulmonary T cells, the majority of y6 T cells are also CD4-CD8-. Most of the ap T cells are either CD4+ or CD8+. Surprisingly, CD4-CD8- TCR-ap' cells, which normally constitute less than 1% of T cells in the peripheral lymphoid organs, make up 10% of murine RPL (38). At present, it is not clear whether this is a reflection of selection driven by special antigens typical for this particular microenvironment or whether it is the result of a preferential homing of a particular subset of T cells.

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The majority of intestinal IEL in all species examined so far are CD8' (6,55,62). In addition, in the same animal, some i-IEL are Thy-1+, while others are Thy-1- (63).Thy-1 expression in i-IEL appears to be associated with cellular activation. In humans, the predominant ap i-IEL are mainly CD8+ (55,56), while the y8 i-IEL consist of both CD4-CD8- and CD8+ subsets. In mice, ap i-IEL may be CD4+ or CD8+.In addition, about 5% of IEL are CD4+CD8+double positive ap T cells. The representation of these double positive cells varies among different inbred strains. Unlike their thymic counterpart, most of the double positive i-IEL express high levels of TCR and a CD8aa homodimer rather than a C D 8 4 heterodimer (45,50). Currently, it is not certain whether these CD4+CD8+i-IEL are differentiation intermediates similar to double positive thymocytes, or whether they belong to cells of a functionally distinct subset. The observation that iIEL in athymic nude mice express CD8aa and not CD8ap led to the conclusion that the CD8aa homodimers are present on T cells which mature extrathymically, while thymus-derived T cells express the ap form ofCD8 (64).However, in adult thymectomized, fetal liver reconstituted radiation chimeras, CD8a+P+i-IEL can also be generated in the absence of the thymus (6566).Using the aa form of CD8 as a marker, it appears that most y8 T cells and at least some ap T cells in the intestinal epithelium of normal mice are of extrathymic origin. Moreover, i-IEL can now be divided into a number of phenotypically distinct subsets for functional analysis (Table 11).The significance of the evolutionarily conserved dominance of CD8-positive T cells in the intestine remains to be elucidated.

TABLE I1 EXPRESSION OF CD4, C D 8 a , A N D CD8p ON MURINEINTESTINAL INTRAEPTITHELIAL LYMPHOCYTES

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IV. Origin of IEL

A. DEVELOPMENTALLY PROGRAMMED TCR GENEREARRANGEMENT AND

STEMCELLSWITCHING

During the early stages of murine thymus development, T cell receptor genes are rearranged and expressed in an ordered fashion, resulting in the appearance of y6 T cells before crp T cells (27,67). Within the y and 6 loci, the V genes also rearranged in a defined sequence, and overlapping waves of y6 T cells expressing distinct Vy genes are generated according to a preset developmental program (28,30,68,69). The first set of y6 T cells appears on Day 14 of gestation. These cells carry the invariant Vy5/V61 receptors found on the dendritic epidermal sIEL (68). Vy6IVGl-positive cells appear next, expressing the canonical TCR that predominate in the tongue, uterus, reproductive tracts, and the lungs of young mice. Transcripts for these two Vy genes are only found in the fetal thymus and are not detectable after birth (69,70).In the late fetal and adult thymus, cells expressing other y6 TCR begin to appear. These TCR consist mainly of Vy4, some Vyl, Vy2, and Vy7, in conjunction with several V6s (15,30).The productive y6 TCR rearrangements in the two sets of early thymocytes are characterized by the complete lack of nontemplated (N) nucleotides. These types of rearrangements result from a lack of terminal transferase activity in the T precursors and are known as “fetal” type rearrangement (36,71-73). In contrast to the fetal thymocytes, the junctional sequences of the TCR rearrangements in the adult thymus, particularly in the 6 rearrangements, are much more diverse than those generated in the fetal period. The relationship between the early appearing y6 T cells with invariant TCR and the late-appearing y6 T cells with diverse TCR is not clear. One could ask whether T cells generated early in ontogeny are endowed with special homing receptors for epithelial tissues, or whether the T cell receptor itself is a homing receptor. Although the early fetal thymocytes share the same TCR with the s-IEL and r-IEL, it does not prove that the lifelong population of these epithelial T cells is derived from a single wave of fetal thymocytes that seed the periphery only once early in ontogeny. Evidence of a specific precursor committed to the generation of y6 fetal clonotypes was first obtained from thymic transplant experiments. Transplantation of fetal and not adult thymic lobes into athymic nude mice resulted in the appearance of s-IEL which expressed the canonical Vy5V61 TCR previously absent in the nude mice (74). Such cells were not detected if the fetal thymi were depleted of Vy5 T cells in utero before

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transfer. These data established the fetal thymic origin of s-IEL and raised the possibility that differences in the T precursors between the fetus and the adult animal prevented the development of Vy5 s-IEL in the y6 T-depleted fetal thymus transplant. Subsequently, thymic repopulation experiments performed on deoxyguanosine-treated fetal thymic lobes revealed that Vy5 d E C can only be generated from fetal stem cells (from fetal liver) and not from adult bone marrow-derived stem cells (48). In addition, the fetal thymic environment is also essential, since intrathymic injection of fetal stem cells into adult thymi failed to generate Vy5 dEC. Thus, both the appropriate microenvironment and the right type of stem cells are critical. These data illustrate the complexity of the regulatory process invoked in the production of a set of intraepithelial T cells. Developmentally ordered TCR rearrangement is not unique to murine thymocytes. In human, early TCR rearrangements result in the appearance of the V62 subset, while the V61 set of rearrangements occurs later (75,76). Val-positive T cells are the most frequent y6 T cells found in normal human intestine, while the V62 subset has been shown to undergo age-related extrathymic expansion that results in its predominance among adult human peripheral blood leukocytes (77). It is noteworthy that in both human and mice, the preferred y and 6 genes expressed in i-IEL (Vy7 for mice and V61 for human) are TCR genes that are expressed in the thymus only after birth. This could reflect some special requirements for the expression of these genes, as are discussed in the next section.

B. THYMIC AND EXTRATHYMIC CONTRIBUTIONS Athymic nude mice have a small number of T cells in the periphery, and this number increases with age. It was thought that although the majority of T cells mature in the thymus, there is a less efficient pathway for T cell maturation outside of the thymus. Until recently, this issue was a matter of some controversy (78). An alternate view was that T cells in nude mice were not generated outside of the thymus but were products of an inefficient differentiation process that took place in an abnormal thymic rudiment. Detail characterization of T cell phenotypes showed that while thymic-derived CD8+ T cells were Lyt-2+Lyt-3+(i.e., CDSa+CD8P+),CD8 T cells from nude mice were Lyt-2+Lyt-3- (CD8a+CD8P-) (79). Consequently, at least for some CD8' T cells, CD8afCD8P- could be a marker which distinguished between T cells that mature in the thymus and those that are generated outside of the thymus.

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GEK-KEE SIM

From the discussion in the previous section, it is apparent that the normal differentiation of s-IEL requires a thymus at a very specific time in development. Indeed, although Thy-l+ dendritic cells are present in the skin of nude mice, these cells are mainly CD3- and do not express TCR (80). However, in older nude mice, a few CD3’ T cells are present in the epidermis (81).These cells differ from the s-IEL of normal mice in their Vy gene expression (Table I ) and in exhibiting a high degree of junctional diversity. The second set of early fetal thymocytes, represented by the Vy6VS1 T cells which colonize the uterus, is also absent in nude mice ( 1 5 ~ 9 ) . It is also absent among resident pulmonary lymphocytes isolated from newborn nude mice (37).Its generation is therefore most likely dependent on the fetal thymus. In contrast, the invariant delta chain BID, which is found in Day 17 fetal thymus and not in the adult thymus, is present in nude mice (41,82). It should b e noted that although BID is a fetal-type rearrangement, it employs V, D, and J segments which are most frequently found in “adult” thymic rearrangements. Unlike other canonical fetal y and S rearrangements, it does not have any nucleotide deletion of the germline coding sequence. Thus, by Day 17, fetal T precursors which do not express terminal transferase have acquired the ability to generate restricted sets of yS T cells, both in the fetal thymus and also extrathymically. It is not clear whether this is due to a change in the inducibility of the T precursors to rearrange a different set of V y and 6 genes or to different sets of stem cells that emigrated from the fetal liver. Recent studies utilizing CD8a’P- expression as a marker for extrathymically generated CD8 T cells showed that in normal mice the majority of i-IEL are actually of extrathymic origin, despite the presence of the thymus (Table 11) (45,50). This approach is particularly significant in the case of i-IEL, since the majority of T cells at this location are CD8 positive. Analysis of radiation chimeras obtained b y fetal liver reconstitution of thymectomized hosts led to the conclusion that both y8 and aP i-IEL could be generated extrathymically (65,66). It is of interest to note that in mice, the predominant Vy gene in i-IEL (Vy7) is known to rearrange late in the thymus (28).A comparable situation is present in humans, where the VS1 subset which predominates in the intestine is also known to rearrange late. It is conceivable that iIEL are generated from stem cells which are already precommitted to rearrange Vy7, regardless of where rearrangement occurs. Alternatively, it could reflect the homing of pre-T cells which have already rearranged their TCR prior to their arrival at the respective organs, and that the thymic epithelium and the intestinal epithelium merely

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31 1

provide the microenvironment needed for further maturation. At present, these possibilities cannot be excluded. The evidence presented so far indicates that the factors which influence the generation of thymic-dependent y6 T cell repertoires specific for various epithelial lymphocytes included differences in stem cells, maturation environment, and targeted Vy6 gene rearrangement. Similar factors could operate extrathymically in generating i-IEL. The stem cells could be predisposed to rearranging their TCR genes efficiently outside of the thymus, targeting a specific V gene for that purpose through the differential expression of a distinct set of DNA binding factors. This specific set of factors could be preferentially induced by the interaction of the T precursor with factors present in an organspecific epithelial environment. Indeed, for such T cells, the intestinal environment would play a role similar to that of the fetal thymic environment in the case of s-IEL. This possibility is supported by evidence indicating that resident pulmonary lymphocytes and intestinal intraepithelial lymphocytes can be generated in sitv (37,79).The presence of lymphoid precursors isolated from the lungs has been demonstrated in reconstitution experiments using scid mice as recipients. In addition, transcripts for the recombination activation genes, RAG-1 and RAG2, which are active in immature lymphocytes but not in mature T cells, have been detected in RPL and i-IEL preparations which are devoid of TCR+ cells (37,79).Their presence implies an ongoing process of receptor gene rearrangement in these populations which is consistent with the notion that these cells mature locally. Overall, it is clear that both ap and y6 IEL can be generated inside and outside of the thymus. But it is not certain whether the products of both pathways are functionally equivalent. V. Selection

A. THYMIC SELECTION

For a@T cells, negative and positive selection are key events which determine whether an ap TCR-bearing thymocyte is to die or to exit the thymus as a functionally mature T cell (83).The outcome is ultimately a consequence of the avidity of the TCR for the available peptide/ MHC complexes in the thymus (84,85). Accordingly, ap IEL that are thymic derived appear to observe these rules, as illustrated b y the TCR@+CDSa+@+i-IEL.In mice, among i-IEL of this phenotype, which could be of thymic origin, specific sets of self-reactive Vps have been deleted (53,65). In the anti-HY a@ TCR transgenic system, T

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GEK-KEE SIM

cells with this phenotype follow the same selection pattern as their counterparts in the peripheral lymphoid organs (66).There are some uncertainties concerning the thymic origin of the CD8a+P+i-IEL7 since they can also be regenerated in fetal liver reconstituted, irradiated, adult thymectomized hosts (65,66). The role of thymic selection in the generation of the yS TCR repertoire is a much more complex issue. This is largely due to our lack of understanding of the antigen-recognition properties of y6 T cells in general, and our limited knowledge of their maturation process. Cumulative data obtained in the past few years have provided increasing evidence against the need for thymic selection in the development of the majority of y6 T cells. First of all, in mice of the BALB background, the &chain ofVS5-positive resident pulmonary T cells consists predominantly of a BALB invariant delta (BID).This is not the case in C57BL/ 6, but not because of negative selection, since expansion of these cells occurs in F1 (C57BL/6 x BALB/c) mice (41). This difference is not due to H-2 encoded genes. BALB.B mice, which bear the same H-2 haplotype as C57BL/6 mice, behave like BALB/c rather than C57BL/ 6. This was the first indication that polymorphic MHC determinants were not involved in the antigen-recognition process of at least some y6 T cells. Moreover, the levels of BID expression among C57BL/6 and BALB/c thymocytes are the same, implying that there is no selection in the thymus for these cells (82). One could argue that in both C57BL/ 6 and BALB/c mice, these cells have been positively selected by different sets of ligands in the thymus by low-affinity interactions with the TCR (84,85). Later, outside of the thymus, antigen(s) present only in mice of the BALB background is responsible for the observed expansion. Even if this were the case, one should note that this putative thymic positive selection process could b e completely disposed of, and it would have no effect on the strain-specific peripheral expansion of these cells. Indeed, the same strain-specific expansion is also found in BALB/c nude mice but not in C57BL/6 nude mice, negating a need for positive selection in the thymus for their rescue from programmed cell death (41). An analogous situation was seen in the GxYS family of y6 T cells, which express T cell receptors consisting of Vy4 chains of identical length but differing at only one amino acid at the V-J junction (40). BID is present in the fetal thymus, while GxYS is found in the adult thymus (40,41,82). In the light of previous discussions on stem cells and microenvironment changes that can occur in ontogeny, it appears that in these cases, the thymus merely provides a breeding ground and has no apparent influence on the high and low phenotypic expression of these T cells observed in the periphery. This is the case,

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despite the fact that these two sets of y6 T cells are generated in the thymus at two different windows in development. Data from MHC class I-deficient mice obtained by creating mutants harboring nonfunctional p-2 microglobulin genes showed no detectable difference in y6 T cell expression between homozygous class Inegative mutants and their phenotypically normal heterozygous litter mates (86).A survey of class I1 negative mutants also reveals no appreciable effects on y6 T cell expression (87). In both cases, epithelial tissues and lymphoid organs were examined. The type of y6 TCR and the number of y6 T cells found in various tissues appear normal. These data do not rule out the absolute requirement for thymic selection based on the recognition of some yet unidentified ligands, but they strongly suggest that the rules for y6 T cell selection differ from those of crP T cells in at least one major aspect: that neither the polymorphic nor the monomorphic T cell-defined epitopes of MIHC molecules play a crucial role in the maturation process of y6 T cells. However, the issue is far more complex, since some data for positive and negative selection have been obtained from studies of mice transgenic for the y6 TCR of two class Ib reactive hybridomas, KN6 and G8, derived from the thymus and spleen, respectively. KN6, a C57BLi6 thymocyte-derived hybridoma, recognizes the T22 (TL”) gene product expressed in H-2” but not in H-2d mice (88). In H-2“ transgenic mice which expressed the y and 6 TCR genes of KN6, transgenic y6 T cells were twice as frequent in the thymus and fivefold more abundant in the spleen compared to H-2” transgenic mice. Unlike transgenic y6 T cells from H-Zd mice, cells bearing the KN6 TCR from H-2b mice were unreactive to TL“ products. It was concluded that T cells expressing the transgenic receptors were rendered anergic in the self-reactive TLb background. G8 is derived from BALB/c nude mice, and it also recognizes a T L region encoded protein which is expressed in H-2“ and not in H-2d mice (89). In this case, cells expressing the y6 TCR transgenes were present in the thymus and spleen of H-2d’d mice, but not in H-ebidmice. These data suggest that the mechanisms of self-tolerance in y6 T cells are similar to those employed by ap T cells, i.e., deletion and anergy induction. Evidence for a requirement for positive selection in these systems was obtained by mating these TCR transgenic mice to the /3-2m-negative mutant mice. In TCR transgenic p-2m-negative homozygous mice, y6 T cells bearing the transgenic receptors were observed at high frequencies in the thymus of both H-2b and H-2d mice, but failed to exit to the lymphoid organs or react to H-2” spleen cells (90,91). These findings indicate that the maturation of transgenic y6 T cells was blocked in P-2m-negative mice

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GEK-KEE SIM

because these mice did not express P-2m-associated class I and class I-like molecules, among which were the antigens that positively select these T cells. The special role of the thymus in the generation of the invariant Vy5Jyl and Vy6Jyl subsets has been discussed earlier. It was thought that this developmental dependency on the thymus, coupled with the fact that all the productive rearrangements were identical, was likely to reflected a TCR-mediated positive selection (34,36,92). From a heterogeneous population of rearrangements, a homogenous repertoire would be generated as a consequence of a stringent selection process. An alternate explanation is that differentiation factors, further imposed upon the observed ordered sequence of Vy gene rearrangements, could create some inherent bias in the fine specificity of the recombination machinery such that a particular rearrangement of the targeted V gene predominates regardless of the functionality of its expressed product (30,93). Two different sets of recent experiments show that a bias recombination mechanism is indeed in effect (32,94). In TCRG knockout mice which have deleted the CG gene, no functional y8 T cell receptors are produced. Nonetheless, pre-T cells in these mice still perform VyJy gene rearrangements. Early y gene rearrangements in these mutants are identical to those generated in wild-type mice in that the invariant Vy5Jyl dEC-type rearrangements are still predominant (94).In a different experimental system, a rearranging substrate, consisting of the germline Vy4, Vy5, andVy6 fragment, was placed in front ofthe JylCyl fragment. Frameshift mutations were introduced into the coding sequence of the Vy genes which rendered them nonfunctional. Transgenic mice were generated using this construct, and the junctional sequence ofthe rearranged mutant Vy5 genes in newborn thymocytes was determined (32). Again, the prevalent Vy5 rearrangement consisted of the invariant Vy5 junctions found in s-IEL. These data emphasize the role of developmental programming rather than selection in the generation of yG IEL. Obviously, the idea that T cells can mature in the thymus without undergoing selection as part of the maturation process is a novel concept which is surprising to many at a time when a detailed picture for positive and negative thymic selection of crP T cells is rapidly emerging (84,85).However, results of earlier experiments using the immunosuppressive drug cyclosporin A (CsA) already suggested that there is a major difference in the thymic maturation process of y8 and ap T cells (Y5,96). Treatment of adult mice with CsA blocked the differentiation of CD4+CD8+thymocytes into single positive CD4+ and CD8' crp T

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cells, but did not affect the maturation of yS T cells. Since CsA interferes with proteins involved in T cell activation (971, it presumably acts on a$ thymocytes at the stage of positive selection. The data imply that either there is a lack of positive selection for most y6 thymocytes or a different set of activation signals is received by y6 and ap T cells in the process of positive selection. One can consider several hypotheses which will accommodate all the data obtained to date. Yet, until a greater understanding of the nature of yS TCR antigen recognition is attained, the general role of the thymus in the selection of yS T cells remains only speculative.

B. EXTRATHYMIC SELECTION Thymic-independent selection of yS T cell receptors expressing the BID and GxYS motifs has been discussed in the previous section (40,41,82).The strain-specific expansion of these yS TCR in nude mice follows the same pattern as that in euthymic mice. Thus, part of the y6 T cell repertoire, whether generated in the thymus or not, is selected by non-MHC self-antigens in extrathymic environments. Strainspecific extrathymic selection of yS i-IEL has aIso been reported (98). In H-2kmice, 50-70% ofyS i-IELare V64+,while only 30%express VS4 in H-2"mice. Further analysis of i-IEL from congenic and recombinant inbred mice has implicated I-E molecules in this selection. Despite this, class II-negative mutant mice still express a substantial level of VS4-positive i-IEL (87). In TL"-specific y6 TCR transgenic mice, deletion of self-reactive G8 yS T cells occurred in the thymus and spleen of H-2b mice (89). However, these self-reactive yS T cells were present in the intestine of the same mice (99). Compared to cells derived from H-2d mice, cells from H-2' mice appeared to be anergic. In addition, their number decreased in time. These cells were most likely of extrathymic origin, since most i-IEL were generated extrathymically, and these anergic cells also expressed CD8ar+P- molecules. These data led to the conclusion that tolerance of self-reactive intraepithelial yS T cells could be achieved through functional T cell inactivation, and that this form of selection could occur extrathymically. Analysis of the VP repertoire of i-IEL revealed that self-Mls-la reactive V p subsets were deleted in the CD8a+P+fraction, just like in the lymph nodes and spleen. These self-reactive cells, however, were present in the CD8a'P- fraction (53).Since CD8a+P- i-IEL originated extrathymically, it was concluded that while thymic-derived i-IEL (CD8atP+) underwent clonal deletion in the thymus, self-reactive iIEL generated outside of the thymus did not undergo negative selec-

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tion (53,100).This is apparently not the case. Fetal liver reconstitution studies performed on adult thymectomized radiation chimeras showed that TCRaP+ CD8afPt i-IEL were also generated in these thymectomized hosts in the absence of the thymus (65). Since self-reactive iIEL of the CD8atp+ phenotype are deleted in the appropriate thymectomized hosts, it follows that negative selection does occur in the intestinal environment, by clonal deletion, among extrathymically generated T cells. Moreover, functional analysis of TCRaP i-IEL subsets revealed that while both CD8atPt and CD8atP- aP T cells respond to stimulation with Con A, only cells expressing the CD8atPt heterodimer can proliferate in response to TCR crosslinking. Accordingly, all TCRaP+ i-IEL which are CD8a’P- are either nonfunctional or require special activation signals which distinguished them from “normal” aP T cells (65). The second set of experiments investigating the role of extrathymic selection on aP T cells generated independently of the thymus was performed using TCR transgenic mice expressing a receptor specific for the male antigen H-Y in the context of H-2b (66). Adult thymectomized radiation chimeras, similar to those described above, were used and similar experiments were performed. Negative selection of the transgenic T cells was observed in H-2b male mice, strengthening the conclusion that negative selection of self-reactive i-IEL are thymus independent. In addition, in these experiments, an elevated number of extrathymically generated CD8a’Pt transgenic T cells was present in H-2Db but not in H-2Dd female, indicating that extrathymic positive selection of the anti-HY receptor also occurs in the genetic background previously shown to be permissive for thymic-positive selection (66). These data, as illustrated by the extrathymically generated CD8atp+ T cells (65,66),indicate that intraepithelial T cells which mature extrathymically undergo selection as part of their maturation process, and that this selection occurs outside of the thymus. VI. Homing

The striking correlation between Vy gene usage and the tissue distribution of y6 T cells (Table I ) prompted earlier considerations that the y6 TCR might serve as tissue-specific homing receptors. y6 T cells, displaying specific sets of TCR, could be selectively retained upon recognition of tissue-specific ligands. This idea was discarded after the y6 TCR transgenic mice were generated. In mice transgenic for the invariant Vy5 d E C T cell receptor, the majority of intestinal intraepithelial lymphocytes expressed the dEC TCR rather than their normal

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Vy7 TCR repertoire (101). Likewise, in mice transgenic for the TLbspecific Vy4V65 KN6 TCR, the s-IEL and i-IEL expressed the transgenic K N 6 receptor. Similar observations have been made for other transgenic mice which expressed either a different Vy4 or a Vy6 transgenic TCR (89; Sim and Olsson, unpublished observations). For the strictly thymic-dependent invariant Vy5V61 s-IEL and Vy6V61 r-IEL, it is not clear how they migrate to their respective homes after maturing in the fetal thymus. One possibility is that different organs are receptive to colonization by lymphocytes at different stages of ontogeny, and the sequential generation of different subsets of y6 T cells coincides with the different timing oftissue receptiveness. This would eliminate the need for special homing receptors. This appears unlikely, in the light of the observation that transplantation of fetal thymus into adult nude mice resulted in the specific colonization of the skin by Vy5V61 dEC cells (74). Rather, in such a highly regulated developmental program which targets distinct Vy genes for rearrangements at defined periods of ontogeny (32,94), it is likely that there will be a coordinated expression between the y and 6 TCR genes and specific homing receptors on the sublineages of y6 T cells. In the case of extrathymically generated T cells, there is evidence for in situ generation of these T cells (37,79). In this case, the question is not how IEL arrived at their particular locations, but what keeps them where they are. One postulate is that IEL which home to a particular tissue express adhesion molecules specific for that tissue, and these molecules facilitate their tropism for that tissue. Intestinal intraepithelial lymphocytes injected into scid mice preferentially repopulate the intestinal epithelium and not other tissues (102). In contrast, the intestinal epithelium ofscid mice cannot be efficiently repopulated with lymphocytes isolated from the thymus or Peyer’s patches. Data obtained from parabionts using congenic T cell markers showed that T cells in the general circulatory system do not colonize the gut epithelium but do enter the Peyer’s patches and the lamina propria (65).Taken together, these results support the notion of tissue-specific homing receptors,” which are most likely to be distinct pairs of adhesion molecules (103). The human niucosal lymphocyte integrin aEP7, defined by the monoclonal antibody HML-1, is expressed on practically all i-IEL but only on 2-6% of peripheral blood lymphocytes which are of the memory phenotype (104-106). Since HML-1 specifically blocks the binding of i-IEL to epithelial cells (107), it is currently considered to be the intestinal homing receptor of human lymphocytes (103). A similar integrin in mice, aM290P7, defined by the monoclonal antibody M290 “

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(108,109),could b e the counterpart of aE in man. The ligand of aEP7 has not yet been identified. How and when these specific integrins are turned on has yet to be determined. There are some indications that TGFp enhances the expression of aEP7 (104). This is especially interesting, considering that TGFp has been shown to induce B cells to switch to the IgA subclass-the predominant immunoglobulin isotype found at mucosae. A putative skin homing receptor has also been recently identified on human T cells of the CD45RO memory phenotype. Cutaneous lymphocytes can be differentiated from other lymphoid populations by their preferential expression of the cutaneous lymphocyte associate antigen CLA (110). This antigen has recently been shown to act as the skin-homing receptor for human T lymphocytes by binding to Eselectin at sites of chronic skin inflammation (1 11,112). However, the proportion of CLA+ lymphocytes in circulation is rather high (16%), and it has not been excluded that CLA might target T cells to the epidermis of other inflamed organs. VII. Antigens and Antigen Recognition Most aP T cells recognize antigens presented by MHC molecules, but it does not appear that y6 T cells behave in the same manner. Overall, antigen-specific MHC-restricted reactivities are the exception rather than the rule for y6 T cells, and the nature of the antigens recognized by y6 T cells still awaits clarification. Since antigen presentation is crucial for T cell responses, it is probable that different cell types residing at various epithelial locations may serve as specialized antigen-presenting cells, employing perhaps even nonconventional antigen-presenting molecules. The antigens may be self or foreign, and they may be tissue specific, produced b y either pathogens with a tropism for that tissue, or local cells suffering various forms of stress. There is much to learn about how epithelial surveillance is accomplished, particularly with respect to the contribution of self- and nonself-antigens in this process.

ap T CELLREACTIVITIES The specificities of ap T cells among IEL have not been extensively A.

addressed in the murine system. In human, T cell clones derived from diseased lesions have been analyzed. Under these circumstances their reactivities tend to be specific for the disease-causing agents (see Section VIII). The CD4 and CD8 subsets appear to exhibit the same pattern of antigen recognition as their systemic counterparts. It is also likely that in the few normal situations examined, the available IEL

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repertoire in humans reflects prior exposure to antigens. This is suggested by the fact that the majority of TCRa/3+ IEL in human skin and intestine are of the CD45RO memory type (57,61).Hence, T cell lines generated from IEL may provide some insights into the prevalent reactivities of T cells residing in various epithelia. Using this approach, it was first demonstrated that i-IEL undergo oligoclonal expansion (58), most likely as a consequence of some as yet undetermined antigenic stimulation. The tendency for oligoclonal expansion of certain V/3 subsets in human i-IEL has now been substantiated by additional studies (58,59,113). The most striking observation to emerge from the first report was the documentation that lines and clones derived from iIEL exhibit CD1-specific cytotoxicity, the dominant clone in this case being specific for C D l c (58). The CD1 locus on human chromosome 1 encodes for at least three molecules which are structurally similar to class I molecules encoded by the MHC on chromosome 6. More recently, it was demonstrated that the product of C D l b can present Mycobacteria tuberculosis antigens to human CD4-8- a/3 T cells in a fashion analogous to MHC class II-restricted antigen presentation (114). Taken together, these data suggest that i-IEL may recognize gene products of the CD1 family in mounting cell-mediated immunity against microbial pathogens present in the intestinal environment. CD4-8- a/3T cells have recently been the focus of several investigations, converging at the conclusion that at least in PBL, they undergo rather stringent selective expansion of a limited number of clonotypes ( 1 15,116).The limited usage of particular TCRs by multiple donors indicates that these cells recognize a limited spectrum of antigens, most likely presented by molecules with limited polymorphism. CD4-CD8- a/3 T cells have also been shown to recognize mycobacterial antigens presented on C D l molecules (114). As discussed earlier, in murine resident pulmonary lymphocytes, there is an unusually high representation of CD4-CD8- a/3 T cells (38).These double negative a/3 T cells are reactive to mycobacteria antigens but their reactivity is not restricted by H-2-encoded molecules (117). The accumulation of this special subset of T cells in the pulmonary environment could be an indication of a local expansion of antimicrobial immunity, reflecting a major antigen specificity of the T cells in this organ. B. yi3 T CELLREACTIVITIES 1 . Recognition of Self-Antigens

The highly restricted tissue localization and the absence of clonal diversity in the yS TCR of murine s-IEL (Vy5') and r-IEL (Vy6+) suggests that the biological function of these cells may be different

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from those that express diversified ap and y6 TCR. Specifically, it has been proposed that since these cells exist in a restricted environment, they are not selected to react to a variety of antigens, but rather, to monitor the integrity of the epithelial tissue in which they reside. This could be achieved by reacting to stress-induced self-antigens present on the cells that surround them (34,118). The reactivity of dEC cells provided some evidence in support of this hypothesis. Dendritic epidermal T cells in mice are found in tight contact with keratinocytes, raising the possibility that a keratinocyte antigen serves as the ligand for the dEC TCR. Both freshly isolated and cultured keratinocytes, but not other antigen-presenting cells, were able to stimulate freshly isolated dEC cells to produce IL-2 and to proliferate in vitro (119). The response was specific for the dEC TCR but no MHC restriction was demonstrable. It was proposed that the d E C ligand was only expressed by keratinocytes under abnormal circumstances such as cellular stress. Cells expressing the canonical VyGVS1 T cell receptors characteristic of r-IEL TCR did not react to keratinocyte despite the fact that both s-IEL and r-IEL use identical &chains and have identical CDR3 amino acid residues spanning the VJ junction of the y chains (19,34).This led to the speculation that both TCR might recognize the same self-antigen in association with different tissue-specific antigen-presenting molecules. The dEC ligand has yet to be identified, but it appears to be a protease-sensitive peptide found only in the tryptic digest of keratinocytes and not of other cell types (120). Understanding how the closely related monomorphic Vy5V61 and VyGVS1 T cells recognize their respective ligands should be helpful for our understanding of yS T cell reactivity. As mentioned earlier, the expansion of T cell clonotypes bearing the BID is driven b y a gene product that is polymorphic between the C57BL/6 and mice of the BALB background. This is not influence by polymorphism in the classical MHC region (41). Recently, detailed genetic studies using recombinant inbred strains of mice in conjunction with a strain distribution survey strongly indicate that the selfligand to which BID react is an endogenous retroviral antigen (121). It is known that different endogenous retroviral sequences can become activated in different cell types as a consequence of local infection, direct cellular injury, or activation. The case of BID suggests that some y6 T cells might recognize self-ligands which result from cellular assaults, rather than recognizing actual antigens produced by pathogens. The reactivity of other IEL to other self-antigens as well as heatshock proteins will be considered below.

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2. Recognition of Mycobacterial Antigens and Heat-Shock Proteins Conventional MHC-restricted CD4+ or CD8’ a@T cells specific for mycobacterial antigens are an integral part of the T cell repertoire (122,123). It is therefore surprising to find a preferential expansion of y6 T cells in the early phases ofhuman skin infection by Mycobacteriua leprea (124).y6 T cells isolated from tuberculoid leprosy lesions proliferated in vitro to M . Zeprae cell wall antigens and tuberculin purified protein derivative (PPD), but not to recombinant heat-shock proteins of mycobacteria. In contrast, T cells isolated from immunologically unresponsive lesions produced by the same pathogen did not show any evidence of y6 T cell expansion, but consisted predominantly of a@ T cells. Mice exposed to aerosols containing M . tuberculosis antigens (PPD) responded b y an expansion of resident y6 T cells in the lungs (38). Such T cells can be propagated in vitro with sonicated mycobacteria extract. In addition, cells stressed by heat shock can further stimulate the proliferation of in vivo primed y6 T cells (39).These studies suggest that there is a link between the mycobacteria ligand and the cellular ligand in stressed cells which activate y6 T cells. Mycobacteria reactivity has turned out to be a major reactivity of not just a@T cells but also y6 T cells in both mice and humans (38,124-127), but the nature of the antigenic component that the two types of T cells react to seems different. One human study initiated using PBL from tuberculin test negative donors showed that practically every other clonable y6 T cell in the blood responded to killed M . tuberculosis, but very few reacted to PPD or HSP65 (128). Some human y6 T cell clones reactive to mycobacteria antigens have been shown to also react to HSPs, to Burkitt lymphomas such as the Daudi cell line which is @-2mnegative, and to bacteria ranging from Escherchia coli to Listeria (61,126,129,130). In addition, the recognition of Daudi can be inhibited by anti-HSP50 antibodies (131).The common feature among these observations is that all mycobacteria reactive human y6 T cells, regardless of their other specificity, express Vy9V62 TCR which consist of highly diversified junctional sequences (132-134). This type of reactivity is strongly suggestive of superantigen recognition. The nature of the mycobacterial “superantigen” and its mode of action are yet to be clarified. Fractionation of mycobacterial antigen preparations showed that the major y6 T cell stimulatory components were present in a low-molecular-weight fraction (2-10 kDa), were protease resistant, and exhibited lectin-binding activity (135,136).

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These nonpeptide antigens have been further characterized as a family of related phosphorylated compounds in the molecular weight range of 500-600 Da (137). One of these antigens is a 5’-triphosphorylated thymidine containing compound, and its T cell stimulatory effect has been shown to be specific for human y6 T cells bearing the Vy9V62 receptors. The unexpected finding that the major stimulatory mycobacterial antigen for human y8 T cells is a nucleotide conjugate is particularly intriguing, since it is in line with the general evidence of stressinduced antigens as targets of y6 T cell reactivity in immunity. In mice, a series of hybridomas predominantly expressing the VylV66 TCR are autoreactive, but the nature of the autoantigen is unclear. Studies from one group of investigators showed that hybridomas obtained by fusing newborn thymocytes to BW5147 produced IL2 spontaneously, and that the production can be inhibited by antiCD3 antibody (138). IL-2 production in these hybridomas can also be augmented by the addition of PPD, recombinant mycobacteria HSP65, or peptide 180-196 of HSP65 (127). These reactivities did not appear to be MHC dependent. The TCR of these hybridomas consisted predominantly of Vyl and V66 and are highly diversified at the CDR3 region. In studies performed by another group, the reactivities of a set of autoreactive VylV66-positive hybridomas derived from three different sources of T cells were compared: s-IEL, newborn thymocytes, and adult spleens (139-141). The spontaneous production of lyinphokine in all cases was inhibited by anti-CD3 antibody and also by antibody to the vitronectin receptor. The later studies failed to demonstrate reactivity to PPD, to recombinant HSP65, or to the HSP65 180-196 peptide despite the fact that the same type of V gene usage was verified, and all hybridomas under investigations were autoreactive. There are two issues that needed to be clarified since these data were all obtained from hybridomas. First of all, are freshly isolated VylVS6-positive cells autoreactive? Second, do they express vitronectin receptors? This information is necessary for evaluating the nature of the endogenous ligand of T cells that express VylV66.

3. Superantigens In general, superantigens are viral and bacterial products (142,143). A mycobacteria-derived superantigen which stimulates human Vy9V62 T cells has been discussed above (137).It would be informative to know whether the same purified compound is also stimulatory for murine y6 T cells and, if so, to determine the target V region(s) of the reactive TCR.

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Various staphylococcal enterotoxins can act as superantigens for different Vp T cell subsets. Human Vy9-bearing cells can lyse target cells coated with SEA but not SEB or SEC (144). This specific lysis is dependent on the expression of MHC class I1 antigen on the targets, but is not haplotype specific. In this respect, the action of SEA on TCR expressing Vy9 is reminiscent of its action on cyp T cells expressing the target Vp (142). There is, however, one distinction. While SEA can stimulate both cytotoxicity and proliferation of ap T cells (145), it can only cause Vy9 T cells to kill but not to proliferate. It is currently not clear whether this difference is due to distinct pathways of signal transduction between these two cell types, to possible differences in accessory molecules, or to a difference in the structure of the antigen/ T cell receptor complex.

4 . Classical and Nonclassical MHC Antigens Murine and human y6 T cell clones that react to MHC class I and class I1 antigens are rare (31).The vast majority of human y6 T cell clones activated in limiting dilution cultures were not specific for the HLA antigens of the stimulating cells (146), and a large number of murine hybridomas tested do not exhibit alloreactivity at the frequency expected for ap T cells. There is only one demonstrated case of y6 T cell reactivity to a peptide antigen presented by MHC molecule (147). In this report, three y6 T cell clones isolated from the synovial fluid of a rheumatoid arthritis patient by repeated in uitro stimulation with mycobacteria tuberculosis antigen were found to be reactive to the same tetanus toxin peptide presented by a nonpolymorphic MHC class I1 molecule, HLA-DRw53. In the case of a murine hybridoma specific for the synthetic polymer GT presented by Qa-lb, the sequence of the peptide has yet to be established (148). One might consider that in general, unlike cyp T cells, y6 T cell receptors are not structured or selected to react to classical class I and class I1 antigens. Several cases of y6 T cell reactivity to nonclassical class I antigens encoded in the murine TL region have been described. The KN6 and G8 clones mentioned earlier are good examples (88,89). In addition, y6 T cells specific for CD1 have also been found (114). It appears that y6 T cells are equally impartial to classical MHC antigens and nonclassical MHC molecules. Recent evidence suggests that yS T cell recognition of MHC molecules, conventional or otherwise, is different from the recognition of peptide/MHC complex by a/3 T cells (149). Studies on the class l b (TL”)-specific clone G8, and the I E k =specific clone LBK5, indicate

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that antigen processing and peptide loading are not required in order to activate either of these clones. Moreover, peptides apparently do not affect their specificities, and amino acid substitutions in the H-2 molecules which affect aP T cell recognition do not have any influence on these yS T cells. These data are consistent with the rare alloreactivity found in yS T cells and the curiously broad cross-reactivity observed in their recognition of MHC (146,150). They could be interpreted as suggesting that y6 T cell receptors recognize antigens in a fashion similar to immunoglobulin (149). VIII. Functional Attributes

A. EFFECTOR POTENTIAL OF IEL Intraepithelial lymphocytes are capable of a wide range of effector functions known to T cells. T cells isolated from the intestines of humans and mice are predominantly CD8+ and are cytolytic when activated (44,55,58,151). The majority of murine i-IEL are TCRyS' and express the CD8aa homodimers, but the absence of CD8p on these cells apparently does not impair their cytolytic function (45,50,64). These cells acquire Thy-1 antigen and lytic capacities following in vivo exposure to microbial organisms (63). For example, adaptation of germ-free animals to conventional housing resulted in the development of previously absent lytic activities. The natural ligands of cytolytic i-IEL are not known. However, human CD4-CD8aP-TCR+i-IEL lines may have a biased cytotoxicity for C D 1 molecules

(58).

In an early study, i-IEL from normal mice and mice infected with the gut nematode Trichinella spiralis were compared for their ability to produce T cell-derived lymphokines in response to in vitro stimulation with Con A or with specific worm antigens (152). Compared to splenic T cells, Con A-stimulated i-IEL produced minimal amounts of IL-2 and intermediate levels of IFNy, IL-3, and GM-CSF. i-IEL isolated from mice infected with T . spiralis produced high levels of IL-3 and GM-CSF when challenged in vitro with Trichinelladerived antigens, while naive i-IEL did not respond to this stimulus. Thus, antigen-specific T cells could arise in the intestinal epithelium during gut infection. Antigen-specific IEL can usually be elicited in immune responses to infection and cellular abnormalities, but a wide range of antigen specificities is attributed to a@ T cells, while yS T cells appear to react to a more narrow range of agents. In pathological conditions, resident pulmonary lymphocytes and cutaneous T cells are also able to synthesize a large array of lymphokines (153-156).

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A more extensive analysis of the types of lymphokines which normal murine i-IEL were capable of producing showed that both TCRaP and TCRy6 i-IEL could produce IL-2, IL-3, and IL-6, but not IL-4 or IL-5. However, the levels of IL-2 and IL-3 produced by y6 T cells were much lower compared to aP T cells. In addition, other lymphokines, such as INFy, TNFa, and TGFP, were also detected (157,158). When murine TCRaP+ i-IEL isolated from mice orally immunized with sheep red blood cells were tested for lymphokine production b y enzyme-linked immunospot assays, both T H 1 and T,2 cells were revealed, and the frequency of IL-4 and IL-5-producing cells was higher than that of IL-2 and INFy producers (159). These studies illustrate that TCRaP+ i-IEL are likely to possess the same range of helper activities known to systemic T cells, and that the priming conditions are key factors which determine their functional differentiation pathway (160-163). It is possible that the correct priming conditions have not yet been established for TCRy6+ IEL, but to date there is little evidence that y6 T cells can also be subdivided into TH1and TH2subsets. Moreover, TCRaP-deficient mice which have normal y6 T cell compartments do not exhibit normal T-dependent antibody responses (164), suggesting that circulating y6 T cells do not function as helper T cells for B cells. However, since y6+ i-IEL in the intestine produced as much IL-6 and TGFP as cup+ i-IEL (157),and these two cytokines are implicated in IgA production (165,166), it remains to be determined whether y6+ i-IEL can facilitate the differentiation of IgAsecreting B cells. Activation-linked expression of Fc receptors (FcR) on y6 TCR' IEL may be a potential mechanism for expanding the range of antigen recognition of y6 T cells, particularly in mice, where the V gene usage is highly tissue specific. y6 T cell lines isolated from murine skin were found to express Fc receptor (167). Resting y6 T cells from the spleen and intestine of normal mice did not express FcR until activated with anti-CD3 antibody (168). Under these circumstances, practically all splenic y6 T cells and a large fraction ofthe intestinal y6 IEL expressed high levels of IgM and IgA FcR and low levels of IgG FcR. Additionally, high levels of IgA and IgM Fc receptors were also found on activated y6 TCR cells in hepatic granulomas of schistosome-infected mice (168).The significance ofthese observations has yet to b e directly demonstrated. Early studies showed that murine CD8+ intraepithelial lymphocytes proliferated in response to Con A and IL-2 at a lower frequency compared to that of the splenic CD8+ population (1 in 500 vs 1 in 8, respectively) (169).More recently, the in vitro proliferative potential +

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of murine i-IEL was further investigated by stimulating these cells with anti-CD3 or anti-TCR antibody. In agreement with the Con A stimulation data, these agents also elicited minimal proliferative responses even in the presence of accessory cells (102,170). This lack of proliferation could not be overcome by stimulating in the presence of T cell-dependent cytokines or phorbol ester. Moreover, unlike splenic T cells, stimulation of i-IEL failed to induce the expression of IL-2 receptors. Since these i-IEL could lyse target cells in antiCD3 or anti-TCR antibody redirected assays, but would not respond to proliferative signals delivered through the same channels, they appeared to be functionally distinct from other peripheral cup or yS T cells which generally responded well to proliferative signals mediated through the CD3-TCR complex. In contrast to i-IEL, resident pulmonary lymphocytes and dendritic epidermal T cells of the skin proliferate readily under stimulation with mitogens, calcium ionophors, or signals delivered through the TCR complex when augmented with lymphokines (38,171,172). It is possible that the majority of murine i-IEL represent the end product of a differentiation process, frozen in place to perform their limited function as eliminators of undesirable companion cells in the epithelia. Alternatively, there might be an inherent mechanism which prevent the proliferation of i-IEL in their tight epithelial environment, except under very strictly defined conditions. The bimodal distribution of CD2 on i-IEL, and the correlation of CD2 expression with proliferative potential, is consistent with the later view (173). The multifaceted functions of i-IEL in particular are in agreement with their high degree of phenotypic heterogeneity (Table 11).

B. IMMUNITY TO INFECTIONS AND DISEASES The participation of IEL in immunity to infections and diseases has recently been further examined in some instances, following the discovery of T cells which bear the y8 rather than the ap T cell receptors. These cases will be discussed briefly. These two types of T cells might complement each other in immunity, and their differential activities might b e revealed by their independent kinetics of response to infection, the type of effector function they exhibit, and the type of diseases with which they are preferentially associated.

1. In the Skin In skin lesions of patients with cutaneous infections, such as leprosy and American cutaneous leishmaniasis, there is a local accumulation of y8 T cells (124,174). The majority of the y8 T cells in the dermal

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granulomas of leishmania lesions are V61 or V62 positive, but there are more Val-positive T cells present in the epidermis. In addition, there is a striking preferential usage of the JSl gene segment in combination with either of the V6s (175). Within a given lesion, the y6 T cells show limited junctional diversity and are essentially oligoclonal. This is in marked contrast to the highly diversified TCR displayed by y6 T cells in the blood of the same individuals. A similar pattern of y8 TCR gene segment usage and oligoclonal expansion was also seen in leprosy lesions (175,176). These observations are consistent with the possibility that y6 T cells responding to different infections in the same tissue may recognize a limited set of nominal antigens shared between distinct pathogens and/or a limited set of common antigens expressed by the hosts. The strongest evidence for y6 T cell involvement in immunity to these infections lies in the fact that y6 T cell expansion is only observed in the reversal lesions where bacteria clearance occurs and not in the lepromatous sites. The ap T cell responses in leprosy lesions are heavily skewed toward expression of members of the Vp6 gene family (177). Moreover, analysis of the deduced amino acid sequences in these Vp chains shows that there are conserved amino acid residues and amino acid motifs in the CDR3 region. The bias forVp6 family could be due to a superantigen type of selection in the immune response to M . leprae. From this Vp6 subset, certain ap clonotypes that recognized mycobacterial antigens in a conventional manner are preferentially expanded, thus contributing to the above observation. A parallel can be drawn in the T cell responses to M . Zeprae between the expanded y6 and ap T cell receptor (175-177). In both cases, there are oligoclonal expansions of TCR which employ highly restricted sets of gene segments. Given the apparent difference in the nature of the antigen-recognition process between ap and y6 T cells (discussed under Section VII), it would be informative to define the molecular nature of the ligands that are specific for ap and y6 T cell clones isolated from such lesions. Functional subsets of epidermal T cells involved in these infections have also been delineated by analyzing patterns of lymphokines produced by T cells which accumulated in these lesions. In situ hybridization of tissue sections obtained from individuals with leprosy showed that in reversal reactions there is a much higher accumulation of mRNA for INFy and serine esterase compared to the lepromatous tissues (178).The accumulation of TH1INFy-producing cells, together with cytotoxic cells in tissues from reversal reactions, is in line with the ability to clear the bacilli and generate tissue damage in these locations.

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In cutaneous leishmaniasis lesions there is also a skewing of the TCR Vp representation in both CD4+ and CD8+ T cells compared to the repertoire of the respective subsets in the blood of the same donor (179). The overrepresented Vps in the CD4+ and CD8+ subsets differ, and the cytokines expressed by these subsets are also different. The CD4+ subset is characterized by IFNy expression, and the CD8+ subset is characterized by IL-4 and IL-10 expression. I n the mouse, resistance to Leishmania is associated with a T H 1 cytokine profiIe (IL2 and IFNy), whereas susceptibility to infection is associated with the production of TH2cytokines (IL-4, IL-5, and IL-10) (153,156,180,181). The data on human indicate that both T H 1 and TH2subsets of T cells can also be elicited in the skin in the course of an immune response, and that the local balance of T,1 and TH2cytokines at the skin lesions is likely to determine the outcome of the infection (182). In support of this, IL-2 and INFy mRNA are prevelent in localized cutaneous leishmaniasis, while in the destructive mucocutaneous form of leishmaniasis, IL-4 mRNA is more abundant.

2. In the Lungs Sarcoidosis is a granulomatous disease of unknown etiology, characterized by the accumulation of large numbers of T lymphocytes at diseased locations such as the lung. An expansion of Vy9+ T cells has been observed in some patients. The TCR junctional regions of Vy9 and VS2 genes expressed in the lungs of these patients were rather limited compared with that of normal individuals (183). This limited diversity of TCR junctional regions among some individuals with sarcoidosis suggests an oligocIona1 T cell response to specific antigenic stimuli. Taken together with the observation of y6 T cell expansion in cutaneous granulomas described above, it is tempting to speculate that yS T cells play a specific role in granuloma formation in diseases and infections, where such structures are beneficial for the host’s defense. In mice undergoing a primary influenza virus infection ap T cells accumulated early in the inflamed lungs, while substantial numbers of yS T cells appeared much later. Upon secondary challenge, yS T cells now responded sooner: within a few days after infection (184,185). The response of such “memory” yS T cells suggests that they actively participate in the immune process. These yS T cells are not constitutively cytotoxic when recovered directly from the site of infection, while lytic effectors are present among the aP T cell population (186). Cytokine mRNA analysis showed that among the TCRap+ subsets, transcripts for IFNy and TNFp were common in the CD8+ population,

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whereas mRNA for IL-4 and IL-10 was much more prevalent in CD4+

T cells. Among yS T cells recovered from the inflammatory exudate,

mRNA for IL-2, IL-4, and IFNy was most abundant (154). At present, there is clear evidence for the participation of both arp and yS T cells in clearing influenza virus infections in the lungs, but the details of how the different types of T cells cooperate to achieve this are still missing.

3. In the Intestines The proportion of intraepithelial T cells which express the y6 TCR increases significantly in the intestines of patients with coeliac disease (187-189). This increase in the number of y6 T cells can be correlated with genetic markers for coeliac disease susceptibility: DR3, DQA, and DQB (190). Expansion ofyS Tcells in the gut epithelium ofhealthy individuals which carry the susceptibility markers has also been observed, leading to the suggestion that an increased frequency of yS T cells might be necessary for the development of the typical lesions of coeliac disease. A large fraction of these TCRyS+ cells expressed the VS1JS1-encoded epitope revealed by monoclonal antibody delta TCS 1 (187). However, detailed phenotypic analysis of a large number of yS T cell clones isolated from coeliac patients indicated that these V a l + yS T cells were derived from a phenotypically heterogeneous population, the majority of which exhibit cytolytic activities (189). Although coeliac disease is activated by gluten exposure, reactivity of these clones to gluten has not been demonstrated. It is of interest to note that V a l JSl yS T cells are also prominent in human cutaneous lesions (see above). Thus, one cannot rule out the possibility that these VSlJSl y6 T cells are actually activated by some common sets of selfantigens, perhaps a superantigen, produced during tissue destruction. Most murine i-IEL are of the y6 T cell lineage and tend to be cytolytic when activated. However, when mice were infected orally with virulent Listeria monocytogenes at doses which caused bacterial invasion through the intestinal epithelia, both ap and yti i-IEL from these mice expressed high cytolytic activities in antibody-redirected killer assays, but failed to lyse target cells pulsed with listerial antigens (191). In contrast, IFNy secretion was induced by both anti-TCR antibody and by target cells pulsed with listerial antigens. These findings suggest that L. monocytogenes induced IFNy secretion by y6 i-IEL from mice suffering from intestinal L. monocytogenes infection and provide further evidence for a role of IFNy-secreting IEL in local resistance against infection (152,191). +

+

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IX. Concluding Remarks

In recent years, the surging interest in intraepithelial lymphocytes has resulted in the accumulation of a large volume of knowledge, providing a solid foundation for advancing our quest for understanding the role of IEL in immunity. At present, key efforts are still oriented toward elucidating the basic rules of the game. There are many questions to be answered. What governs the peculiar nonstochastic generation of diversity dominant among intraepithelial TCRy6 lymphocytes? What is the mechanism for the tissue-specific distribution and peripheral clonal expansion of such cells? What is the effective antigen specificity and biological function ofthe various intraepithelial lymphocytes in local immunity? How do the different subsets of IEL orchestrate their efforts to achieve host defense? So far, the effector functions of y6 T cells seem to complement those of a@T cells (Section VIII). It is conceivable that once activated, y6 T cells will accomplish their mission of host defense in a similar fashion as their a@counterparts. But y6 T cells are far from being a redundancy. There is now convincing evidence that ap and y6 T cells are not activated b y the same means (Section VII). First and foremost, the nature of the antigens and the antigen-recognition requirements of y6 T cells appear to have little overlap with those of ap T cells; while a/3 T cells recognize peptide antigens bound in the groves of MHC molecules, it is practically impossible to find an antigen-specific, MHC-restricted y8 T cell. Rather, it appears that they might recognize antigens in ways analogous to immunoglobulin. Their preference for microbial antigens, and the ease with which they are activated by this category of antigens, could be an indication that although both a@ and y6 T cells respond to microbial invasions, y6 T cells recognize a different subset of these antigens, without the limitation conferred by MHC presentation. Second, there are now several reports that the TCR-associated CD3 complex can be different for $3 and y8 T cells. In CD35-negative mice, a@T cells are no longer generated while y6 T cells mature normally (192-194). These y6 T cells are able to assemble their TCR/CD3 complex using the y subunit of FcR in place of CD35. It has also been shown that in normal mice, this alternate form of CD3 complex is used by some y6 T cells (195). The implications of these findings are obvious, but the actual functional effects of these differences must await further verifications. Third, wherever it has been measured, the kinetics of response during an infection seem to differ for ap and y6 T cells (196-198). +

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Thus, it appears likely that TCRys+ T cells act in concert with TCRaP' T cells to provide immune surveillance, complementing each other functionally through their differential TCR recognition and activation processes. In the light of current data, one might consider yS T cells as a hybrid between classical T and B lymphocytes, mediating cellular immunity through the effector functions of aP T cells, while recognizing antigens without MHC presentation. In this case, the signals that are involved in translating antigen recognition into cellular activation have yet to be deciphered. ACKNOWLEDGMENTS

1 thank K. Canipbell and L. du Pasquier for reading the manuscript and A. Augustin for critical discussions. The Basel Institute for Immunology was founded and is supported by F. Hoffiiiann-La Roche Ltd., Basel, Switzerland.

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ADVANCES

IN IMMUNOLOGY,VOL. 58

leukocyte Migration and Adhesion BEAT A. IMHOF' and DOMlNlQUE DUNONt 'Basel Institute for Immunology, Basel CH4005, Switzerland; and tUnivenite Pierre el Marie Curie, CNRS-URA 1135, Paris, France

I. 11. 111. IV. V. VI. VII. VIII.

IX. X. XI.

XII. XIII. XIV.

Introduction Leukocyte Migration Lymphocyte and Endothelial Cell Adhesion Molecules Selectins Selectin Ligands Integrins Immunoglobulin Superfhnily Molecules Other Molecules Involved in LeukocyteEndothelial Adhesion Chemotactic Molecules Involved in Integrin Activation Th e Model of Leukocyte-Endothelial Cell Recognition: An Adhesion Cascade Molecular Basis of Specific Homing of Leukocytes: Combinatorial Diversity in Leukocyte-Endothelial Cell Recognition Recruitment of Lymphocytes to Specific Organs Recruitment of Leukocytes during Inflammation Outlook References

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1. Introduction

Leukocytes are the principal actors in the body's defense system against invading microorganisms (1,2).This defense system has a nonspecific branch consisting of granulocytes and macrophages and a specific branch of lymphocytes. Granulocytes (neutrophils, eosinophils, and basophils) release cytotoxic compounds from their intracellular granules to their local environment when they encounter microorganisms. This random destruction happens rapidly but it may also harm healthy tissue of the body. Macrophages, the other class of defense cells from the nonspecific immune system, can ingest and destroy microorganisms by phagocytosis or, in a similar way to granulocytes, by the secretion of cytotoxic compounds. However, macrophages can also act more specifically by collaborating with lymphocytes and their products. 345 Copyright 0 1995 by Academic Press, Inc. All rights of reproductmn in any form reserved.

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The lymphoid system comprises the cellular components responsible for antigen-specific immune defense. B lymphocytes produce antibodies that bind to foreign organisms and facilitate their destruction, either by activating the complement system which in turn can perforate the membrane, or by “opsonizing” the microorganism, i.e., trigger phagocytosis due to receptors for antibodies and the macrophage surface. T lymphocytes act mainly by cell-to-cell contact. One subpopulation of T lymphocytes recognizes and kills cells which bear foreign antigen (e.g., after virus infection); the second subpopulation helps to modulate the activity of other hemopoietic cells in the immune response or helps to multiply effector cells. All of these leukocytes patrol the body by circulating through the blood and lymphatic system ensuring a continuous surveillance which is a prerequisite for efficient defense (3). Upon tissue damage and inflammation, leukocytes are recruited from the blood to sites of injury, and this trafficking displays exquisite specificity (4-6). For instance, neutrophils selectively enter sites of acute inflammation or tissue damage, while eosinophils extravasate into sites of allergic reactions and parasitic infestations. The migration of lymphocytes is even more selective and includes a complex pattern of recirculation that relates to differentiation and activation (see below). This review will present a general model of adhesion between leukocytes and endothelial cells, the molecules that are involved, and it will emphasize how the homing specificity of lymphocyte subsets to different lymphoid organs is ensured, and how leukocyte migration to sites of inflammation is regulated. II. leukocyte Migration

To efficiently protect the body from infectious organisms, the cells of the immune system circulate as nonadherent cells in the blood and lymph, and migrate as adherent cells into tissues when necessary. Rapid transition between adherent and nonadherent states is the key to the dual functions of immune surveillance and responsiveness. Circulating lymphocytes in the blood have first to adhere to and then to cross the endothelial lining in order to enter the various lymphoid tissues which are involved in recirculation. One exception is the spleen, where small penicillar arterioles may terminate open ended in the parenchyma, allowing continuous unhampered access of blood leukocytes ( 7 ) .Another characteristic of lymphocyte migration to the spleen is related to the absence of lymph vessels in this organ. In all other secondary lymphoid organs, a high percentage of lymphocyte

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extravasation happens in a histologically distinctive postcapillary venule, the high endothelial venules (HEV) (8).“High endothelial cells,” in contrast to other vascular endothelial cells, have a typical cuboidal morphology. Most recirculating lymphocytes selectively bind to the endothelium of HEV, but they almost ignore normal vascular endothelium (9,lO).This is in strong contrast to the situation in inflamed sites. Under these conditions, the damaged tissue appears to trigger new adhesion properties of the adjacent endothelium, resulting in local extravasation of leukocytes which includes many lymphocytes.

A. LYMPHOCYTES In adult mammals the bone marrow contains the precursors for B and T lymphocytes (11). B cell precursors remain in the bone marrow to complete their differentiation in situ, whereas T cell precursors migrate to the thymus and differentiate there. Lymphocytes produced in these primary lymphoid organs are called naive because they have not yet encountered the molecules which their antigen receptors recognize (12). These naive lymphocytes are exported to the periphery and localize preferentially in organized secondary lymphoid organs, including the lymph nodes, Peyer’s patches, and spleen (9). In early life, naive T cells appear to show no preference in their extensive migration to these different lymphoid organs (13,14).Their migratory properties appear to be determined ontogenically as a function of their class, and they probably traffic continuously between the different secondary lymphoid organs using lymph and blood circulation systems until they die, or they respond to their cognate antigen which is accumulated and presented in secondary lymphoid tissues (compare Fig. 1). Following antigen stimulation with the appropriate secondary signals, naive T cells (CD45RA’) transform into effector/activated T cells (15).These cells then transform into memory cells (CD45RO+),becoming smaller and losing some of the markers associated with acute activation. The question whether or not CD45RO+memory cells revert back to CD45RA’ cells remains highly contentious (16,17).At present, it is uncertain whether long-lived memory cells actually exist, as there is good evidence that they depend on continuous antigenic stimulation (12,16,18).This is an important consideration for concepts of lymphocyte homing because with this model memory cells need to be continuously reactivated, which has a bearing on migration patterns. Memory and effector lyniphocytes are exported back to the blood and display migratory properties different from naive cells. They migrate to nonlymphoid tissues at a low rate and continuously recirculate from the blood by taking a route through the draining afferent lymph vessels

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FIG.1. Pattern of lymphocyte circulation. Lymphocytes patrol the body ensuring continuous immune surveillance. In order to migrate from one compartment to another they use transportation provided by the circulatory system. Extravasation is organ specific. T h e figure illustrates the tissue-specific recirculation established in sheep.

of the organ-specific lymph node, the efferent lymph vessel, and finally the thoracic duct back into the blood. Once activated at a specific site of the body, memory cells migrate remarkably selectively to the tissues which were originally involved in the foreign antigen exposure (Fig. 2). Evidence that lymphocytes migrate nonrandomly was first proved by the distinctive migration pattern of bladeffector cells. Blast cells were found to localize to the gut and various other tissues and not to peripheral lymph nodes. In sheep, lymphocytes draining from the gut were labeled with fluorescein isothiocyanate (FITC). The T cells that preferentially migrated back to the gut were small, memory CD45RO' cells ( 1 5 ~ 9 ) Isolated . intraepithelial lymphocytes of the gut exclusively home to the gut or to gut-associated tissues such as Peyer's patches. In addition, murine activated T cell clones, when injected

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intravenously, failed to recirculate through lymphoid tissues. In pigs, blast cells in the lung migrated differently compared with blood or spleen blast cells (20). Similarly, various human T cell lines derived from different tissues showed selective binding to mucosal, synovial, or lymph node HEVs (21). In contrast with this specific migratory behavior, inflammation augments the influx of lymphocytes into tissues but at the same time reduces the selectivity that normally governs homing.

FIG.2. Lymphocyte homing to peripheral tissues and lymph nodes. T h e illustration shows memory lymphocytes which home to the gut (dark gray cells) or the skin (white). These cells subsequently enter regional afferent lymphatic vessels which direct the cells to a draining lymph node. In contrast, naive lymphocytes home by afferent postcapillary high endothelial venules ( H E V )into lymph nodes (black cells). In noninflamrnatory tissue, these naive cells make u p to 90% ofcirculating lymphocytes. After homing, both cell types leave the lymph node by the efferent lymphatics and recirculate via the thoracic duct into the blood.

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The primary naive response takes time to develop because a novel antigen must drain to local lymph node and be scanned by thousands of naive T cells before a specific interaction between an antigenpresenting cell and a T cell is achieved. In contrast, the secondary memory response is faster and it can be argued that the tissue-selective homing of memory T cells assists in the speed of this secondary response, enhancing the efficiency of the immune surveillance to those tissues where the antigen initially entered the body. The specific memory T cells are preferentially located at the point of entry of the antigen which would lead to an inflammation reaction almost immediately. This rapid memory response is also probably assisted by the status of memory T cells which might need only minimal secondary signals from antigen-presenting cells to be activated. Moreover, antigen which drains to local lymph nodes induces the entry of many more memory T cells into the node, leading to the focusing of memorytype T cells to the sites of antigen deposition, i.e., to the lymph nodes which drain the "infected" tissue and to the tissue itself. Analysis of B cell homing has been limited; naive B cells are IgM+IgD+and constitute the great majority of the B cells that recirculate between blood and lymphoid tissues (22). Injection of fluorescentlabeled B and T lymphocytes in several mammalian species indicated that spleen absorbed a maximum number of labeled cells as early as 1 or 2 hr after injection, while lymph nodes did so about 18 hr afterwards (23). Up to 40% of the injected lymphocytes was recovered 1hr later from the spleen. Quantitatively, the spleen is thus the predominant organ in lymphocyte recirculation, surpassing the total cell number circulating through all the lymph nodes. In man, the circulating blood contains about 10" lymphocytes, which have a blood transit time of 25 2 6 min, resulting in an exchange of 48 times per day (about 5 x 10" lymphocytes). The daily recovery from the thoracic duct in man is only about 3 x 10" and in rat 2 x lo9(23,24),which is equivalent to about 6 or 7%of the daily emigration which reflects the lymphocyte circulation through lymph nodes, tonsils, and Peyer's patches. Although there is a bias in their migration routes, naive and memory lymphocytes collected in about equal numbers in the thoracic duct of the rat when observed for 7 days (25). B. OTHERLEUKOCYTES Granulocytes are the most abundant leukocytes with the neutrophils constituting up to 70% of all circulating white blood cells. They are the first cells which arrive at sites of tissue inflammation. In contrast

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to lymphocytes they are short lived and do not recirculate. They are produced in the bone marrow during hemopoiesis and circulate as mature cells in the blood for several hours before they enter the tissue. The survival time within the tissue is then limited to several days. Prerequisite for tissue entry are molecular changes on the surface of blood vascular endothelial cells that signal inflammatory reactions or injury. This leads to adhesion and extravasation of circulating granulocytes. The adhesive interaction is spacially specific and occurs primarily in postcapillary venules. The magnitude of the response and the temporal characteristics vary with the nature of the inflammatory stimulus. It can be rapidly induced and rapidly decreased or it can be sustained long term over hours. This indicates that the molecules which are involved are different or differentially expressed or regulated (5,26,27, and see below). 111. lymphocyte and Endothelial Cell Adhesion Molecules

The recirculation and homing patterns of leukocytes were established in uiuo using reinjection of isolated cells after fluorescence or radiolabeling them (28). In 1976, Stamper and Woodruff described an elegant technique which involved the in uitro binding of viable leukocytes on the HEVs contained within frozen tissue sections of target organs (29) (Fig. 3). Antibodies obtained from animals either immunized against the leukocytes or the endothelial cells were selected for their ability to inhibit the binding of leukocytes with the HEVs in tissue sections (30). The successful antibodies were then used to characterize and purify the adhesion molecules and later clone the corresponding gene (31,32).Although this assay is rudimentary, it has yielded a large harvest of adhesion molecules. These have been subsequently identified to belong to the selectins, the integrins, the immunoglobulin superfamily, or to a group consisting of highly glycos ylated molecules. IV. Selectins

The selectins designated L-, P-, and E-selectin (33) are a family of adhesive receptors found on leukocytes (L) (31,32,34,35),platelets and endothelial cells (P) (36,37), or endothelial cells alone (E) (37,38). These receptors belong to a family because they share a common mosaic structure consisting of an aminoteminal C-type lectin (sugarbinding) domain, a single epidermal growth factor (EGF)-like domain, several short consensus repeats (SCR) similar to those found in regula-

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FIG.3. Photomicrograph of lymphocytes which bind to a frozen section of a peripheral lymph node. Lymphocytes were isolated from peripheral blood and allowed to bind to freshly frozen sections of peripheral lymph nodes. Nonadherent lymphocytes were eliminated by washing. Bound cells are cytostained darker than surrounding cells. Note that the adherent lymphocytes line along a high endothelial venule. Magnification is SOOX.

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tory proteins that bind complement, a transmembrane domain, and a short C-terminal cytoplasmic domain (Fig. 4). The level of sequence homology among the lectin and EGF-like domains with the actual molecules is 60-70%, while the homology amongst the SCR domains falls to about 40%. The main difference between these three molecules is the number of SCR: 2, 8/Y, and 6 for L-, P-, and E-selectin, respectively. The location of selectin genes on mouse and human chromosome 1 is consistent with a close evolutionary relationship to complement-receptor genes which are positioned on the same chromosome in both species (39). Selectins are known for their binding to carbohydrate ligands via the lectin domain. In fact, anionic carbohydrates are able to block in vitro adhesion of lymphocytes to lymph node HEVs and it was shown that the tetrasaccharides, sialyl Lewis X and sialyl Lewis A (sLex, sLea), or the sulfated forms thereof, have a ligand activity for all three selectins (Figs. 5 and 6) (40,41). Such sugars can be positioned on and presented by proteins to the selectins (see below). The lectin-binding domain for these carbohydrates on the selectins has been mapped by mutating single amino acids, and the correct protein conformation seems to depend on a bound Ca2+cation (42,43). A. L-SELECTIN

L-selectin is expressed by all leukocytes except by activated, memory lymphocytes (35,44,45). It was originally described by Gallatin, Weissman, and Butcher in 1983 as a lymphocyte “homing receptor” involved in the initial attachment of lymphocytes to HEVs in lymph nodes (30). Although it was initially convenient to think in terms of a single function for L-selectin, its widespread distribution on all classes of leukocytes has made this view untenable. In fact, L-selectin contributes to both lymphocyte and neutrophil entry into inflammatory sites (44,46-49). Antibodies to L-selectin block homing of lymphocytes into lymph nodes and solubilized L-selectin-IgG chimeric molecules block leukocyte migration to peripheral lymph nodes and to inflamed peritoneum of mice (50,51). L-selectin expression by leukocytes can be modulated. Cell activation by chemokines (see below) or by tumor promoter (e.g., phorbol myristate acetate, PMA), downregulates Lselectin expression on the plasma membrane by shedding (52). Thus, a soluble form of L-selectin can be found in plasma (53,54), and such soluble L-selectins can be detected in serum of animals with experimental autoimmune uveitis before any other signs of the disease. IFNa is the only immunomodulatory cytokine so far which increases the cell-surface density of L-selectin and this correlates with increased

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B. P-SELECTIN P-selectin has emerged as a versatile receptor on endothelial cells and platelets for neutrophils and macrophages (55).On the cell surface

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of the endothelial cells P-selectin initiates the earliest phase of leukocyte recruitment into inflammatory sites (56-58). Expression of Pselectin on activated platelets is important in the recruitment of leukocytes to thrombi and in the induction of fibrin production during hemostasis. Visualization of adhesion of flowing leukocytes to immobilized, activated platelets showed “rolling” of these leukocytes on the platelet layer (59). This was inhibited by antibodies against P-selectin. It has also been found that activated platelets induce superoxide anion production by monocytes and neutrophils through P-selectin interactions, whereas resting platelets do not (60).The production of superoxide was blocked by antibodies against P-selectin, sLex, or soluble P-selectin. In contrast to L-selectin in which only the lectin domain is functional, P-selectin functions with its lectin and with the EGF-like domain (61). The latter may serve as a cooperative ligand-binding site unique to P-selectin, and this may explain why P-selectin can mediate

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PSGL-1 MAdCAM-1

FIG.6. Outline of the structure of highly glycosylated selectin ligands. MAdC h 1,GlyCAM-1, and CD34 are L-selectin ligands. PSGL-1 is a ligand for P- and E-selectin. MAdCAM-1 binds with high efficiency to a4@7 integrins. The cytoplasmic protein part, C-terminus, is indicated as COOH, disulfide bridges are indicated with S-S. Cys indicates protein regions with high cystein content. Molecular domains with homologies to ICAM-1, VCAM-1, mucin, or immunoglobulin A, are indicated.

intercellular adhesion even at low expression levels. Upon platelet or endothelial cell activation, P-selectin becomes phosphorylated within seconds on cytoplasmic tyrosine, threonine, and serine residues, and the molecule is translocated to the plasma cell membrane (62,63).The phosphotyrosine and phosphothreonine disappear after a few minutes, whereas phosphoserine remains stable. This regulation may influence the function of P-selectin itself or may transduce signals into the cells. P-selectin is constitutively expressed and stored in Weibel-Palade bodies, and it can be translocated to the plasma membrane within minutes upon cell induction by histamine, thrombin, the complement factor C59, oxygen radicals, or various neuropeptides (37,64-66). The increase of P-selectin expression on activated cells is temporarily regulated because the molecule is rapidly internalized to vesicles from where it may recycle (63).However, unlike other recirculating receptors (e.g., LDL receptor) a large proportion of P-selectin is targeted to

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and degraded in lysosomes due to a short cytoplasmic amino-acid domain and/or acylation 011 Cys766(63). Deletion of 10 cytoplasmic amino acids increases the half-life of P-selectin from 2.3 to 9.5 hr. Rapid degradation is of ultimate importance for upregulation of cellsurface P-selectin expression only after a secretory stimulus. It is conceivable that failure of rapid degradation would lead to the accumulation of P-selectin on the cell surface and result in chronic arrest of circulating leukocytes by vascular endothelium, i.e., chronic inflanimation. In addition to the upregulation of intracellular transport, P-selectin can also be regulated transcriptionally upon cell activation by the cytokine tumor necrosis factor-a (TNFa) (37,671.The P-selectin genepromoter region contains putative binding sites for ETS and NFKB/ re1 families, a GATA motif, and a sequence related to the GT-IIC element of the SV40 enhancer. Some of these regions have been shown to be involved in cytokine-dependent gene activation (68). In mouse endothelial cells, a maximal amount of P-selectin was produced after 2 or 3 hr of TNFa incubation (37). Interestingly, brain endothelial cells do not have preformed P-selectin in their Weibel-Palade bodies but it can be newly synthesized by TNFa induction (67). C. E-SELECTIN E-selectin is probably the most specific, inducible endothelial cellsurface molecule which is involved in the adhesion of neutrophils, monocytes, and T cell subsets to inflammatory but not to normal tissues (38,69). Similar to L- and P-selectin, E-selectin can bind sLex and sLea with its lectin domain, and this binding is enhanced when these sugars are coupled to proteins (70,71). However, the lectin domain of E-selectin can bind to some ligands which do not bear sLex (see below). Similarly to cell-surface P-selectin, E-selectin is rapidly downregulated by internalization and degradation in lysosomes; however, the intracellnlar targeting mechanism of E-selectin is still unknown (63,72). Expression of E-selectin of endothelial cells is transcriptionally upregulated by the inflammatory cytokines interleukin 1 (IL-1) or TNFa, by neuropeptides (37,65,73),as well as by bacterial endotoxin such as lipopolysaccharide (LPS) (74). Upon endothelial cell stimulation, newly synthesized E-selectin is detected after 3-6 hr and decreases to basal levels after 48 hr (37,75).Endothelial E-selectin upregulation can also be obtained by the two cations Ni2+ and Co2+;both are known contact sensitizers which can lead to contact hypersensitivity (76).Cytokine-induced E-selectin expression can be hampered by

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the incubation of endothelial cells with transforming growth factorp (TGFP) or interleukin 4 (IL-4), and both together lead to an additive effect (77).Interestingly, TGFP is produced by endothelial cells themselves in an inactive form and has to be processed by other adjacent cell types (78).In comparison to TGFP and IL-4, antioxidant reagents have a more restricted effect: they inhibit TNFa but not IL-1 or LPSinduced E-selectin expression (79). These reagents can act on NFKB-like DNA binding proteins and block the NF-KB-like enhancer element present in the human E-selectin gene-promoter region (79). This would indicate that E-selectin may also be transcriptionally regulated b y oxidative stress: for example, in atherosclerosis.

V. Selectin Ligands

A. CARBOHYDRATES L-selectin ligand binding activity was completely abrogated when the cells were treated with benzyl GalNAc which blocks O-linked (mucin-type) sugar synthesis (80).This suggests that the tetrasacharides sLex and sLea are presented to selectins by proteins (Fig. 6) (70,81,82).In addition, lipid-bound sugars may also become valuable candidates as selectin ligands. For instance, HNK-1 is an antibody raised against human natural killer cells (83). It recognizes sulfated glycosphingolipids and sulfoglucuronyl-containing neolactosylceramides (SGNL lipids) which are present in the nervous system and in vascular endothelial cells. It has recently been found that these SGNLs are ligands for L- and P-, but not E-selectin. The binding is different from the sLex-selectin interaction and it is Ca2+ independent, i.e., the glycolipid may bind to an alternative region of the lectin domain in the selectins. In fact, the better-known selectin ligands belong to a recently described family of adhesion molecules which are of the mucin type (84-86). A significant percentage of their molecular mass is composed of O-linked, sulfated, carbohydrate side chains. These highly glycosylated proteins bear the tetrasacharide, sLex, sLea, or their sulfatated forms which have ligand activity for all three selectins (87). Consequently, although selectin ligands have been identified using one selectin, most of them may bind to two or to all three selectins (87,88). B. L-SELECTIN LICANDS By using an L-selectin-IgG fusion protein, two glycoproteins expressed on endothelial cells have recently been isolated and cloned:

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GlyCAM-1 and CD34 (Fig. 6) (89,90). Both serve as a platform for the Ca2+-dependentpresentation of carbohydrate ligands to L-selectin. A third receptor, MAdCAM-1, exhibits a dual function since it also binds a 4 integrins (91-93).

1. GlyCAM-1 GlyCAM-1 is expressed mainly in HEVs of peripheral lymph nodes (89) and this is consistent with its function as a specific vascular adhesion molecule (addressin) for lymphocyte homing to the lymph nodes. However, its role as a vascular addressin is controversial because it is a secretory molecule which does not contain a transmembrane domain (89,94),but part of it may be associated with the endothelial cell surface (A. Ager, personal communication). GlyCAM-1 can also bind to Eselectin, and it is likely that the soluble molecule acts as an inhibitory modulator ofcell adhesion (88,95).Components ofthe afferent lymphatics (antigens?) may regulate GlyCAM-1 expression since suppression of lymphatic circulation in lymph nodes by ligation of the afferent vessel results in a complete loss of GlyCAM-1 mRNA expression and lymphocyte adhesion to HEV (96)probably because of a loss of antigen challenge. 2 . CD34 The vascular sialomucin CD34, another L-selectin ligand, is expressed on a diverse range of blood vessels as well as on hemopoietic progenitors as a transmembrane cell-surface protein (90,97). On the luminal side of small vascular endothelium CD34 is localized on interdigitating processes and in vitro it has been found enriched on villi and sprouting processes (98).Thus, this L-selectin ligand is expressed and presented to circulating leukocytes by subcellular regions which are most exposed to the blood flow. The L-selectin ligand activity of CD34 depends predominantly on appropriate glycosylation and sulfation and this has significant consequences for in vitro research on this molecule. L-selectin-bearing leukocytes do not bind to CD34 expressed by the endothelial cell line b.End.3 or by nonendothelial cells after gene transfection (90). Thus, the post-translational modification of CD34 is cell specific. It has been speculated that the accurately glycosylated or sulfated molecule mediates lymphocyte homing to lymph nodes and serves as an L-selectin ligand for lymphocytes and neutrophils to sites in the periphery (85). Similar to E- or P-selectin, the expression of CD34 by endothelial cells can be regulated by cytokines. However, the effect of IL-1, INFy, and TNFa on CD34 expression is reciprocal to the selectins. CD34

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is downregulated from endothelial cells at the same inflammatory cytokine concentrations which created an upregulation of the selectins, VCAM-1 or ICAM-1 (see below) (98,99). Such regulation has been observed in patients with skin lesions resulting from graft-versus-host disease. In skin regions with massive lymphocyte infiltration, endothelial CD34 expression was low and E-selectin increased (99). Hence, CD34 is likely to be involved in the circulation of leukocytes under normal, but not under inflammatory, conditions.

3. MAdCAM - 1 The mucosal addressin MAdCAM-1 is a transmembrane protein which exhibits a complex structure leading to a dual function: it combines three Ig domains and a mucin-like region between Ig domain 2 and 3 (91-93). This molecule is expressed mainly on HEV of Peyer’s patches and on venules in small intestinal lamina propria, on the marginal sinus of the spleen, and on HEV of embryonic lymph nodes (100).It is interesting to note that MAdCAM-1 exhibits an IgA-like Ig domain which could be correlated with its expression in the digestive tract area where most ofthe IgA antibodies are secreted. Similar regulating elements in the gene-promoter regions of IgA and MAdCAM-1 may be responsible for this gut-specific regulation. The function of MAdCAM-1 as an endothelial cell adhesion molecule was directly demonstrated b y the ability of the purified receptor to mediate adhesion of normal lymphocytes and cell lines (101).MAdCAM-1 serves as a ligand of L-selectin and a4p7 integrin, both involved in lymphocyte homing to Peyer’s patches (91-93,102). The a 4 p l integrin binds MAdCAM-1 with only very low affinity. C. P-SELECTIN LIGANDS Two P-selectin ligands have been characterized so far: the recently cloned molecule P-selectin glycoprotein ligand-1 (PSGL-1) (Fig. 6) and a 120-kDa ligand (103-105). Surprisingly, L-selectin might also be a ligand for P-selectin and E-selectin (106): first, because the rolling of leukocytes on a platelet layer could be inhibited by antibodies directed against P-selectin or E-selectin (106,107); and second, since L-selectin can bear sialylated carbohydrates which serve as ligands for P-selectin and E-selectin.

I . PSGL-f Recently, PSGL-1, a P-selectin ligand, was isolated by screening for transfected cDNA that translated ligand activity (103).The screening was performed with COS cells cotransfected with 3/4-fucosyltransferase which allowed the correct glycosylation of the protein

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necessary for P-selectin recognition. PSGL-1 is a transmembrane sialomucin of 110 kDa associated in dimers by a disulfide bridge and it is expressed on various leukocytes and perhaps other cells.

2 . 120-kDa P-Selectin Ligand McEver and co-workers isolated human P-selectin and used this as a probe in Western blotting to identify a ligand expressed only on myeloid cells. It was a 120-kDa (reduced form, 250-kDa nonreduced) sialoprotein and its binding to P-selectin affinity columns was Ca2+ dependent (104).The ligand contained the sLex motif, and a-2-3 linked sialic acids which are most probably O-linked (105). However, the amount of sLex contained in the 120-kDa ligand was less than 1% of the total cell membrane-bound sLex. The 120-kDa protein may serve as a specific selectin ligand because the sugar side chains form a clustered patch of sialylated fucosylated O-linked oligosaccharides (105). Albeit likely, the similarity of this protein to PSGL-1 awaits further clarification. D. E-SELECTIN LICANDS The identified ligands of this selectin have not yet been cloned; they form a series of antigens found on cutaneous leukocytes: the cutaneous leukocyte antigens (CLA) (108-110), a 250-kDa receptor, and the sialyl stage-specific embryonic antigen (SSEA-1) (111).

1 . CLA One family of ligands for E-selectin is known as CLA and can be recognized by the anticarbohydrate antibody HECA 452 (108).The molecules are highly sialylated and they are found in a population of memory lymphocytes in inflammatory lesions of the skin. Since E-selectin is expressed on the venular endothelium in these inflammatory sites, a role for E-selectin in the recruitment of these “skin homing” lymphocytes is suggested (112). However, the antibody also recognizes other glycoproteins located on HEV of lymphoid tissues and on HEV-like blood vessels in inflammatory tissues (109).

2 . 250-kDa E-Selectin Ligand Another type of cell-specific E-selectin ligand has been found on ruminant y6 T cell receptor (TCR)-type lymphocytes (113).E-selectin induction by TNFa in skin results in recruitment of y6 cells to this organ. The binding of this single-chain molecule (MW 250-kDa) to E-selectin was not inhibitable by antibodies to CLA nor to sLex; however, it was cation dependent and could be blocked with EDTA.

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3. SSEA-1

The sialyl SSEA-1 is expressed on natural killer cells and absent on resting T and B lymphocytes (111).However, concanavalin A activates lymphocytes and leads to sialyl SSEA-1 expression. All sialyl SSEAl-positive cells were able to bind to E-selectin present on endothelial cells. E. MONOSPECIFIC SELECTINLICANDS An important finding in this field is the recent identification of ligands which interact specifically with one selectin. Vestweber and co-workers used mouse recombinant P- or E-selectin IgG fusion proteins and applied them for affinity purification of mouse and human leukocyte ligands (95,114). P-selectin bound specifically to a 160-kDa and E-selectin to a 150-kDa glycoprotein (ESL-1). Both ligands are monospecific, i.e., they do not bind to the other selectin respectively and they are Ca2+dependent. For both ligands the functional carbohydrate side chains are N-linked as N-glycosidase F destroys the selectinbinding activity. The 160-kDa P-selectin ligand is sensitive for O-sialoproteases, i.e., contains O-linked sialic acids in its selectinbinding domain. The E-selectin ligand does not contain these 0linked sugars. Interestingly, the same group also identified a 130 and a 230-kDa protein which binds to both P- and E-selectin (95). In contrast to the monospecific ligands, the binding activity was not affected by N-glycosidase F, and signals in affinity isolation experiments were 10-15 times weaker. It has been speculated that these mouse proteins may be related if not identical to human PSGL-1 or the 120-kDa P-selectin ligand (95,103,104). They may belong to a family of sialic acid containing O-glycosylated mucins which would also include GlyCAM-1 and CD34. Their binding site on the selectin’s lectin domain may even differ from that of the monospecific ligands. VI. lntegrins

Integrins are adhesion molecules that are involved in many biological processes including embryonic development, maintenance of tissue integrity, and leukocyte homing (115-1 17). They are heterodimeric proteins consisting of noncovalently associated a and /3 subunits generally of 150 and 100-kDa, respectively (Fig. 7, Tables I and 11). At present, 15 a- and 8 @-chainsare known and at least 21 different heterodimers have been found. Leukocyte populations can express 13 different integrins from the existing repertoire and 6 of them are

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p subunit

Extracellular domain

FIG.7 . Outline of the structure of integrins and the combinations of a-and p-chains. Disrilfide bridges are indicated with S-S; the cation-binding sites are shown by the Ca symbol, and C means a cystein-rich domain.

important in the leukocyte-endothelial interaction: they belong to the subfamilies. Both a and p subunits are transmembrane glycoproteins and several cytoplasmic domains of the integrin subunit interact indirectly with cytoskeletal actin filaments by the proteins talin or a-actinin. At the N-terminus of the a subunit are seven homologous tandemly repeated a2,aL,aM, domains (numbered from I to VII) (1 18).Some a subunits, al, a E ,and ax,exhibit an inserted domain, or I domain, located between repeated domains I1 and 111, whereas the extracellular domains of other a subunits are composed of heavy and light chains joined by a single disulfide bond (119).The last three of four repeated domains are thought to contain EF hand-type-like domains that bind the divalent cations Ca2+or Mg2+(119,120).They are essential for integrin function, and the nature of the cation can affect the affinity and specificity for ligands (120-123). Ligand specificity of p l and p2 integrins depends mostly on the associated a-chain. For example, a5pl is a receptor for fibronectin (RGD domain), a6pl for laminin, and a4pl for fibronectin (CS-1 domain) and VCAM-1. In many cells there is a retention of an intracellular pool of immature p l integrin chains associated with the chaperon calnexin (124).Cell-surface expression of mature heterodimers is then regulated by biosynthesis, assembly, and transportation of the a-chain. The level of a-chain expression can be regulated by the

PI, &, and p7 integrin

TABLE I BIOCHEMICAL CHARACTERISTICS OF INTECRINSUBUNITS ~

Name of Subunit

CD Classification (Human)

Size (kDa) (Nonreduced/Reduced)’

CD49a CD49b CD49c CD49d

200/210 160/165 150/135; 30 140/150; 40 180l80; 70 155/135; 20 150/130; 30+31 120/100; 30 160/140; 25 140/ 150

CD49e CD49f ff7

a8

(Chicken)

ff9 QL

~~

~~~~

~~~~~~~

CDlla

nd/ 180

Alternative Splicing (Cytoplasmic Part)

+

I Domain

+ +

SS Bridge

+ +

+ + +

+

+ -t

“M ax

CDllb CD1 l c

“E “IIb

a”

PI

Pz P3 P4

P5 PS

P7

Px

W

0,

in

a

+ + +

nd/170 nd/150 170/150; 25

CD41 CD51

145/120; 25 1501125; 25

CD29 CD18 CD61 CD49f CD51 CD51 CD49d CD51

120/130 90/95 95/115 200/205 97/110 100-110/? 105/120 95/97

Size of human niolecules except for a,(rat) and (I* (chicken). nd, not done.

c

+

+ c

+

+ (two types)

TABLE I1 THEINTECFUN FAMILY OF ADHESION RECEFTORS AND THEIRLICANDS Name of Receptor

P1Integrins alp1 a91

QBl

ad1

ad31 a7P1

Synonyms

Ligands"

VLA-1 VLA-2, GPIa-Ha, ECMRII VLA-3, VCA-2, ECMRI VLA4, LPAM-2 VLA-5, FNR, GPIc-IIa, ECMRVI VLAS, EA-1 VLA-7

Coll (1, IV), LN Coll (I, IV), LN, FN? Ep, LN, Nd/En, FN, Coll ( I ) FN, VCAM-1 FN LN, X? LN ? ? FN

%Pl 4

1

ffS1

Pe Integrins a1P2

4%

LFA-1 Mac-1, CR3 ~150-95

ICAM-1, -2, -3 CBbi, Factor X, Fb, ICAM-I Fb, C3bi?

Binding Sequences in Ligandb

DCEA RGD EILDV RGD

RGD

p3 Integrins a11bP3 4

3

p7 Integrins aEPi

ff97

Other @ ad34 4

5

a"P8

J

GPIIb-IIIa VNR

Fb, FN, vWF, VN Fb, FN, vWF, VN, Tsp, LN Osp, Bspl

M290 IEL, HML-I LPAM-1

E-Cad FN alt., VCAM-1, MAdCAM

GPIc-IcBP, TSP-180, A9, EA-1

LN VN FN ?

RGD

EILDV

RGD RGD

" Abbreviations for ligands: Coll, collagen (subtypes); LN, laminin; NdlEn, nidogenlentactin; Ep, epiligrin; FN, fibronectin; F N alt., fibronectin containing the IIlCS region; Fb, fibrinogen, vWF, von Willebrand factor; VN, vitronectin; Tsp, thrombospondin; Osp, osteopontin; Bspl, bone sialoprotein1; C3bi, inactivated form of C3b component of complement; Factor X,coagulation factor X; MAdCAM, mucosal addressin recognized by MECA-367 mAb; E Cad, E cadherin. Minimal binding sites recognized by integrins are indicated by their amino-acid sequences.

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addition of inflammatory cytokines, such as TNFa, and this regulates the amount of cell-surface expression of a particular integrin (125,126). One of the most important mechanisms of integrin function is the rapid transition from a nonadhesive, low-affinity state to a transient high-affinity state (115). It is likely that the increased adhesiveness of integrins is due to a conformational change caused by activation; this was suggested because certain mAbs react with p l or p2 integrins only after activation and others can activate integrins after binding (121,127-129). A motif conserved in all cytoplasmic domains of achains close to the membrane, the GFFKR motif, appears to be particularly important in affinity modulations (130,131). When this motif was deleted from the cytoplasmic domain of aL, the receptor switched into a high-affinity state (132). Results from studies of a2,a4,and truncation suggest that sequences on the C-terminal side of the GFFKR motif may mediate physiological activation of PI integrins (131).In contrast, LFA-1 is constitutively active when it is expressed in COS cells, and partial deletions of the aLcytoplasmic domain appear to have no effect on adhesion to ICAM-l(l33).In addition, truncation of p cytoplasmic domains also results in reduction of cell adhesion mediated by integrins (132,134). Thus, the inside-out signaling pathways probably involve both a and p cytoplasmic domains, resulting in changes in conformation of the heterodimeric integrin. Although tyrosine, threonine, and serine are conserved in integrin cytoplasmic domains, detailed studies on a6Ap1and a d Bhave so far failed to identify a significant role for phosphorylation in activation (133-137). This controllable adhesiveness of integrins provides a versatile mechanism for the arrest and tight adhesion of circulating leukocytes on vascular endothelium rapidly followed b y intermediate adhesion during transendothelial migration and finally deadhesion at extravasation (5,138,139). Different modes of cell-cell interaction can induce a higher affinity; for instance, interaction of T lymphocytes with the vascular endothelium through the adhesion molecule CD31 transduces activating signals into T cells which in turn activate integrins (140,141). Similar signals are also obtained upon interaction of the 16 different chemokines with their 7 transmembrane receptors (see below and Table 111) (142,143). Thus, the ubiquitous expression in many tissues of most integrins does not preclude constitutive activity of these adhesion molecules. In addition to integrin affinity modulation regulated by signals generated from the inside of the cells (inside-out signaling), studies indicate that integrin themselves can transmit signals into the cells (outsidein signaling) (130,144). Ligation of integrin receptors may initiate a

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TABLE 111 CYTOKINES INVOLVEDIN SIGNALING Cytokine a-Chemokines IL-8 (NAP-1) IP-10 ENA-78 MIP-2aP (GRO-a,-p,-y,MGSA) NAP-2 P-Chemokines MIP-la (LD-78) MIP-lp (ACT-2) RANTES MCP-1,-2,-3 Others

SF (HGF)

C5a Formyl peptides

Target Neutrophils, basophils, T cells Monocytes, T cells Neutrophils Neutrophils Neutrophils Monocytes, T cells Monocytes, T cells Monocytes, eosinophils, T cells Monocytes, basophils, T cells T cells Neutrophils Neutrophils

Note. 1L-8, interleukin 8; NAP-I, neutrophil attractantiactivation protein-I; 1P-10, y-interferon-induced peptide; ENA-78, epithelium-derived neutrophil attractant 78; MIP-2ap, niacrophage inflammatory protein ap; GRO-a,-p,-y, growth regulating protein a, p, y; MGSA, melanoma growth-stimulating activity; RANTES, regulated on activation, normal T-cell expressed and secreted; MCP1,-2,-3,monocyte chemotactic proteins 1, 2, 3; SF. scatter factor; HGF, hepato-

cyte growth factor; C5a, fifth complement component a. Names in parentheses indicate other names of the same or highly homologous cytokines.

variety of cellular responses including differentiation, proliferation, differential gene expression, cytoskeletal assembly, migration, and gel contraction (115). For instance, signaling through lymphocyte functional antigen-1 (LFA-1) integrin in T cells regulates activation and proliferation (145),and signaling via a6 integrins regulates the activity of chemotactic receptors (146). Among the earliest molecular events are tyrosine phosphorylation, activation of a PKC-type pathway leading to cytosolic alkalinization, and calcium fluxes (130).Outside-in signaling through integrins is different to signaling mediated by classical receptors since integrin cytoplasmic domains possess neither kinase nor phosphatase activities. Upon ligand interaction, integrin cytoplasmic domains interact with tyrosine kinases, leading to signal transduction. The use ofmAbs or purified immobilized ligands to crosslink specific integrins led to the detection of three subsequent cellular events: alkalinization, protein phosphorylation, and calcium fluxes. Clustering of LFA-1 integrin with anti-a, antibody resulted in the release of

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intracellular Ca2+and an increase in intracellular pH (147,148). Ligation of VLA-4 on T cells stimulates tyrosine phosphorylation of a 105kDa protein, indicating that engagement of VLA-4 on T cells activates tyrosine kinase activity (149). Indeed, specific tyrosine kinases, i.e., the focal adhesion kinase p ~ 1 2 5 ~ are * ~ ,concentrated in adhesion plaque and are phosphorylated in response to cell attachment to extracellular matrix components (150,151).

A. p 2 INTECRINS

The in uiuo function of a set of integrins was found by analysis of immunocompromised patients with congenital leukocyte adhesion deficiency (LAD I). This disease was characterized by the absence of stable adhesion between leukocytes and endothelium (152). Three different integrins sharing the p2-chain, LFA-1, Mac-1, and p150,95, were found to be missing in these LAD I patients because the gene coding for the p2 subunit was mutated. 1 . LFA-1 LFA-1 is present on the cell surface of most leukocytes and interacts with the first Ig domain of the ICAMs, members ofthe immunoglobulin superfamily (see below). Several domains of the LFA-1 a-chain seem to be responsible for this interaction. Monoclonal antibodies which bind to the inserted integrin domain affected ICAM-1 binding (5). Recombinant fragments of a,-chain which contained the putative cation-binding domains V and VI bind directly to ICAM-1 (118). In addition to leukocyte-endothelium interaction (see below), LFA-1 participates in many other cell-cell interactions such as T cell activation by antigen-presenting cells or the killing of virus-infected cells b y cytotoxic T lymphocytes (153). 2. Mac-1

Mac-1, found predominantly on granulocytes and macrophages, is also a receptor for ICAM-1 but does not bind to ICAM-2 and -3. In addition, it can bind to the C3bi component of complement and fibrinogen. Thus, Mac-1 is a receptor involved in recruiting myeloid cells to inflammatory sites.

B. pl INTEGRINS A second set of integrins combines the pl-chain with variable a subunits (a1 to a9). They are called the very late antigens (VLA) because the first ones to be identified (VLA-1 and VLA-2) were only expressed at a late stage after T cell activation. With two exceptions

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they are expressed in most tissues and their ligands are molecules of the extracellular matrix. 1. a4pl

The a 4 p l (VLA-4) integrin is mainly expressed on leukocytes and binds VCAM-1 on endothelial cells, as well as fibronectin, a component of the extracellular matrix. The a4-chain can be expressed on cell surfaces as a 180 or a 150-kDa form; the latter can also appear as a cleaved configuration with 80 and 70-kDa chain fragments (154,155). These forms may have consequences on the differential activation of this integrin by cellular signals. VLA-4IVCAM-1 interaction is probably the most important adhesion pair involved in leukocyte attachment to the endothelium at inflammatory sites and also in lymphocyte differentiation (156-158). Together with a 2 p l and a5pl it is responsible for the spreading of leukocytes which adhere to endothelium (128). For leukocyte transendothelial migration a 4 p l seems to be the major player since blocking of this integrin blocks extravasation (159). 2. a6pl An interesting function is also assigned to the a6pl integrin: it is expressed by endothelial cells and promotes homing of T cell progenitors to the thymus (160-163). In addition, this molecule is involved in leukocyte homing in normal, noninflammatory tissue (127; Ruiz et al., submitted for publication). It is thought that a6 integrin expressed on the luminal side of the endothelium recognizes a ligand on lymphocytes which is different from laminin. Occupancy o f a 6 by this receptor induces signal transduction into the endothelial cells and the activation of a novel adhesion molecule involved in homing. C. p7 INTEGRINS 1. a4p7

a4p7 heterodiiner is expressed on a subset of lymphocytes which colonize the gut and gut-associated lymphoid tissues (164-167). The a4p7 recognizes the niucosal endothelial ligand MAdCAM-1 (see above) and mediates lymphocyte homing to Peyer’s patches. However, this integrin, formerly named LPAM-1, can also bind very efficiently to VCAM-1 and fibronectin (92,102,167). In contrast to MAdCAM-1, which is involved in lymphocyte recirculation under normal conditions, interactions with VCAM-1 may only become important in inflammatory reactions.

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IMHOF AND DUNON

2. aEP7 This integrin, also called a I E d 7or ( ~ " ~ $ 7 , is involved in the interaction of lymphocytes with the intestinal epithelium (168-171). It appears on lymphocytes only after their appearance in the gut and can be upregulated on lymphocytes in vitro by TGFP, a cytokine which is present at a relatively high concentration in the gut area (170,172,173). Recently it has been described that the heterotypic adhesive interactions between epithelial cells and intraepithelial lymphocytes are mediated by E-Cadherin, the ligand of aEP7integrin (334). VII. Immunoglobulin Superfamily Molecules

The immunoglobulin (Ig) superfamily encompasses a large group of molecules with multiple immunoglobulin-like domains (Fig. 8). Each domain is usually encoded by a discrete exon and consists of a

VCAM-1.7NCAM1.6 CD31

CAM1

ICAM-3

in V-CAM 1 6

2 COOH

cow

COOH

COOH

c2:

5

C2f

6

COGH

FIG.8. Th e general structure ofthe immunoglobulin superfamily molecules involved in leukocyte-endothelial interaction. T h e cytoplasmic protein part, C-terminus, is indicated as COOH, and protein-bound sugar side chains are indicated as circles (lacking of this symbol indicates that glycosylation is not yet determined). C2 stands for constant immunoglobulin domain 2. Disulfide bridges are indicated with S-S.

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primary sequence of 60-70 amino acids with a disulfide bridge spanning 50-70 residues; several other conserved residues are involved in establishing a tertiary structure referred to as an antibody fold. Five members of this family are involved in leukocyte-endothelial cell interaction: ICAM-1 (CD56a), ICAM-2 (CD56b), ICAM-3 (CD56c), VCAM-1 (CD108), and PECAM-1 (CD31) (174-179). Four of them, ICAM-1, ICAM-2, VCAM-1, and CD31, serve as endothelial ligands for leukocytes.

A. ICAMs ICAM-1 and ICAM-2 are products of distinct but homologous genes containing five and two Ig domains, respectively (174,175,180). They were both initially identified by their ability to interact with LFA-1 integrin (175,181,182). ICAM-1 binds LFA-1 by the first domain and has also been found to bind to Mac-1 integrin by a distinct site in its third Ig domain (183,184). The two Ig domains of ICAM-2 are homologous to the two aminoterminal domains of ICAM-1 and the first domain also binds LFA-1. In addition, ICAM-1 is a ligand for the major group of human rhinovirus serotypes (184-186), and Plasmodiumfulcipurunz-infected erythrocytes also use ICAM-1 as an endothelial cell receptor by a binding site which partly overlaps the LFA-1binding domain (187,188).Finally, ICAM-1 interacts with the leukosialin CD43 expressed on T lymphocytes as well as on monocytes, neutrophils, platelets, and some B cells, although the significance of this binding in adhesion processes of normal leukocytes is yet to be established (189-191). The numerous ligands of ICAM-1, including its use by viruses or parasites, indicate that this adhesion molecule plays a central role in immune cell interactions. ICAM-1 is weakly expressed on resting endothelium, but its expression increases strongly after several hours of stimulation by IL-1, TNF, or interferon-y (192-196). In contrast, ICAM-2 is constitutively expressed at a high level on resting endothelial cells and its expression is not augmented by activation (197,198); interestingly, the affinity of ICAM-2 for LFA-1 seems to be weaker than that of ICAM-1(199,200). This stresses the responsibility of ICAM-2 for constitutive low transendothelial leukocyte trafEc, whereas de aovo expression of ICAM-1 regulates the main inflammatory traffic, as demonstrated in ICAM-l-deficient mice (201). On leukocytes, the ICAM molecules differ strikingly by their expression: ICAM-1 is widely expressed in an inducible manner, ICAM-2 is absent on these cells, and ICAM-3, a recently discovered five Ig domain molecule, is strongly expressed on resting lymphocytes and monocytes ( 179,198-200). All three ICAMs contribute to antigen-

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specific interactions so that inhibition with mAbs to all three is required to completely block LFA-l-dependent antigen-specific T cell responses (199).Their differential expression suggests that they play different roles in T cell responses. ICAM-1 may act through its association with the multichain high-affinity IL-2 receptor (202), and ICAM3, which is expressed on resting T cells (200), might be important in the initiation of immune responses. Although ICAM-3 has never been found on endothelial cells, it could be involved in lymphocyte-endothelium interaction as it might competitively inhibit LFA-1 binding to ICAM-1 (200).

B. VCAM-1 The vascular cell adhesion molecule VCAM-1 was originally identified as a cytokine-inducible adhesion molecule on human endothelial cells, mediating the binding to leukocytes and melanoma cells (178,203,204). VCAM-1 is a ligand for the a 4 p l integrin (VLA-4) and binds weakly to a4P7 (102,205-207). A single VCAM-1 gene gives rise through alternative splicing to distinct isoforms (204,208-21 1). The major form of VCAM-1 in humans contains seven Ig domains of which domains 1-3 are homologous to domains 4-6 (204). VLA-4 integrin binds VCAM-1 through the first and the fourth Ig domain (208,212). A splicing variant lacking domain 4 has been described but it is only a minor form of VCAM-1, whose biological significance as well as specific binding properties are not yet known (208). A glycolipid-anchored VCAM-1 isoform has recently been cloned in the mouse which contains the first three Ig domains and is glycosylated differently (210,211,213). In addition, a soluble 95-1 10-kDa form of VCAM-1 has been purified from the supernatant of human cultured endothelial cells as well as from blood of patients suffering from rheumatoid arthritis and SLE (214,215). VCAM-1 is absent on resting endothelial cells, but the cells respond to IL-1 and TNF by upregulating the expression of VCAM-1, with maximal activity reached by 6-12 hr (216,217). Interestingly, IL-4 acts on endothelial cells to increase the expression of VCAM-1, but not E-selectin or ICAM-1 (216). VCAM-1 expression is regulated at the transcriptional level and analysis of5' flanking sequences in the human VCAM-1 gene has revealed the presence of two NFkB sites as well as other functional elements (213,218). These statements suggest that VCAM-1, as well as ICAM-1, are regulators of lymphocyte extravasation at sites of inflammation. VCAM-1 is also expressed in several nonvascular cell types, including populations of dendritic cells found in lymph nodes and skin, bone marrow stromal cells, and synovial cells in inflammed joints (219,220). At these locations it is involved

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in the adhesion of lymphocyte precursors to bone marrow stromal cells, the binding of B cells to lymph node follicular dendritic cells, and VCAM-l/VLA-4interaction acts as a costimulator for T cell proliferation (156,221). Together with a7pl integrin, VCAM-1 and VLA-4 also play a role in myogenesis since both molecules are expressed on immature muscle cells, and antibodies directed against these molecules block secondary myotube formation (222).

C. CD31 PECAM-l/CD31 is a six domain molecule which mediates both leukocyte and platelet/endothelial cell adhesion and transendothelial migration (177,223-228). CD31 is expressed on platelets and on most leukocytes and is constitutively present on endothelial linings in uiuo. CD31 mediates adhesion through homophilic interaction; however, a heparin-binding consensus sequence (LKREKN) on domain 2 may mediate heterophilic interaction with cell-surface or extracellular matrix proteoglycans (229-231). It appears that Ig domain 6 may also be important in CD31-mediated heterophilic interactions, as the epitopes for two adhesion-blocking antibodies map in this domain (232).A more striking property of CD31 is its ability to activate pl and p2 integrins by ligand-induced signaling, conferring to CD31 a critical role in the regulation of leukocyte adhesion to endothelium (140,141).Phosphorylation of the serine residues in the CD31 cytoplasmic domain may be one ofthe activation events detected after platelet treatment with PMA or thrombin (230). In addition, when endothelial cells come into contact with each other to form a cobblestone-like monolayer, CD31 redistributes to the cell border and is thought to participate in the endothelial cell-endothelial cell interaction that limits vascular permeability (233). The ability of CD31 to move to cell-cell borders suggests a requirement for cytoskeletal interaction. Indeed, partial deletions of the CD31 cytoplasmic domain perturbed cell-cell border localization of CD31 and cell agregation (234).Moreover, some of these deletions modify CD31mediated binding from a heterophilic to a homophilic process (234; Piali et al., in preparation). In fact, PCR analysis revealed several transcripts which were alternatively spliced in the cytoplasmic region leading to CD31 isoforms which could exhibit different binding specificities (235).

D. L1 The L1 cell adhesion molecule is a 200-kDa transmembrane glycoprotein that comprises six Ig domains. It appears to mediate homophilic Ll-Ll or heterophilic L1-NCAM binding at the surface of adjacent

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IMHOF AND DUNON

cells of the nervous system in a cation-independent way (236,237). Recently, L1 expression has also been found on lymphoid cells, which bound to noninduced endothelioma cells, and this interaction was blocked by anti-L1 antibodies (238). In contrast to the nervous system the binding was Ca2+,Mg2+,and temperature dependent. The binding must be heterophilic since the endothelioma cells did not express L1. VIII. Other Molecules Involved in Leukocyte-Endothelial Adhesion

A. CD44 The CD44 proteoglycan is a widely expressed cell-surface protein (239,240). Many isoforms of CD44 are generated by alternative splicing from a single gene, containing 19 or 20 exons, located on chromosome 11 in humans and chromosome 2 in mice (241,242). Five CD44 isoforms have been encountered in leukocytes. CD44 mediates cell adhesion mainly by its binding to hyaluronic acid (HA) but it can also interact with the extracellular matrix molecules collagen, laminin, and fibronectin (241,243-245). The binding to extracellular matrix molecules was observed in vitro with only some CD44 isoforms and after covalent addition of chondroitin sulfate (244). CD44 purified from the placenta, however, binds fibronectin or collagen I only poorly (239). Whether the interaction of chondroitin sulfate moieties occurs in vivo and whether this interaction is important in mediating lymphocyte homing is uncertain. Some anti-CD44 antibodies were found to induce CD44 shedding from the cell surface (246). The size of the shedded molecule corresponded to the soluble form of CD44 found in human serum. In contrast, while binding of CD44-expressing cells to HA occurs at low efficiency, crosslinking of CD44 by mAbs which recognized other epitopes strongly increased this binding (247). CD44 transfected into Jurkat T cells did not bind to HA but this function was achieved after activation by TPA (248). Such activation was not possible with CD44 mutants with cytoplasmic tail deletions. This suggests that the activity of CD44 can be regulated similarly to the versatile function of integrins and that the cytoplasmic tail of CD44 is critical for binding of HA to the CD44 extracellular domain (248). However, this binding should not require CD44 cytoplasmic interaction with cytoskeleton but could involve new protein synthesis as observed in the inducible binding of human lymphocytes to HA via CD44 (249). CD44 was thought to participate in lymphocyte binding to HEVs, mainly in Peyer's patches, and to activated endothelial cells; however,

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the data are too controversial to provide definite proof. Whereas binding of lymphocytes to cultured endothelial cells and to stromal cell lines involved HA in certain conditions, the binding of lymphocytes on frozen sections did not (241,250,251). Thus, this in uitro cell-cell binding might involve at least two CD44 ligands, including HA, and one of them could b e a sulfated proteoglycan (252). I n uiuo injection ofCD44 mAb or Fab fragments has no effect on extravasation oflymphocytes into lymphoid organs during normal trafficking in the mouse (247). On the contrary, antibody-induced shedding of CD44 from lymphocyte membranes resulted in inhibition of edema and leukocyte infiltration at a site of cutaneous delayed-type hypersensitivity 24 hr after challenge (253). These results indicate that CD44 is not necessary for normal leukocyte circulation but is required for leukocyte extravasation into an inflammatory site involving nonlymphoid tissue. CD44 is also involved in the bone marrow stroma interactions and maturation of lymphoid precursors (239,254). In the mouse, CD44 expression by prothymocytes in bone marrow and injections of CD44 antibodies have suggested that CD44 plays a role in thymus homing (255,319-322). Finally, CD44 can also modulate T cell responses. CD44 has recently been shown to interact with a chondroitin sulfate form of invariant chain, a nonpolymorphic glycoprotein that associates with MHC class I1 molecules (256). This interaction can stimulate class 11-dependent allogeneic and mitogenic T cell responses. A putative function of CD44, when expressed on endothelial cells, is its capacity to bind and present chemokines to those leukocytes which are in contact with the endothelium (257). Chemokines are defined as chemotactic cytokines and have a characteristic glycosaminoglycan binding site (Table 111). Thus, these chemokines can be carried by CD44 through its heparan sulfate or chondroitin sulfate side chains as demonstrated for MIP-1P. Signaling by chemokines leads to integrin activation and tight endothelial adhesion of the leukocytes (see below).

B. VAP-1 AND L-VAP-2 Vascular adhesion protein-1 (VAP-1)is an endothelial adhesion molecule that is strongly expressed in venules of most human lymphoid organs except mucosa-associated tissue (258). Low expression can also be found by endothelium in brain, skin, kidney, liver, and heart. In these organs it is highly upregulated at the site of chronic inflammation, e.g., such as bowel diseases or dermatoses (259). Anti-VAP-1 monoclonal antibody blocked adhesion of lymphocytes to HEV in frozen

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IMHOF AND DUNON

tissue sections. The antibody precipitated a 90-kDa glycoprotein and its N-terminal sequencing revealed no significant homology to other adhesion molecules. Also defined by a mAb, the lymphocyte-vascular adhesion protein2 (L-VAP-2)has a molecular mass of 70-kDa. L-VAP-2 is constitutively expressed on human umbilical vein cells and the anti-L-VAP-2 antibody inhibits lymphocyte binding to these cells (260). This mAb stains a subpopulation of venules in lymphoid and nonlymphoid tissues as well as a few HEV in lymphoid tissues. L-VAP-2 is also expressed on 20% of peripheral blood lymphocytes, preferentially on B cells and CD8' T cells. IX. Chemotactic Molecules involved in lntegrin Activation

A. CHEMOKINES The proinflammatory chemokines belong to a family of 16 homologous members (Table 111).They induce changes in cell shape, release of intracellularily stored enzymes, formation ofbioactive lipids, respiratory burst, and, most importantly, the activation of integrins and chemotactic migration (142,143,261,262). The chemokines, also called intercrines, are small chemotactic peptides with a MW of 8-10 kDa produced by a variety ofcell types. They have four cysteines conserved in all members of the family; they are called a-chemokines when the first two cysteins are interrupted by one amino acid (C-X-C) and p-chemokines when they are together (C-C) (Table 111). a-Chemokines attract and activate neutrophils, p-chemokines act on monocytes, eosinophils, and basophils, and both types can react with subsets of lymphocytes. For example, the a-chemokine IL-8 has been shown to attract memory T cells and natural killer cells although, later on, the effect on T cells has been questioned (263-266). The p-chemokines RANTES and MCP-1 attract memory T cells, whereas MIP-la and -p lead to naive T cell migration (257,267-270). Chemokines have a heparin-binding domain which is utilized for the immobilization on and presentation of the chemokine by endothelial proteoglycans to circulating leukocytes (257,271). The actual binding sites for cellular chemokine receptors and proteoglycans do not overlap, and they are even located on opposite sides of the molecule (271). The large family of cytokine receptors for CC and CXC chemokines have relatively high affinities ranging from pico- to nanomolar (142). For some of these receptors the genes have been cloned. They are expressed as polypeptides with molecular weights of approximately

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40kDa and they have sequence homologies of25 to 80%. The predicted structure includes hairpins with seven putative transmembrane domains of 20-25 amino acids, typical for G protein-coupled receptors (272,273). Chemokine receptors cross-react to a certain extent with the different members of the chemokine family, but they d o not react with other chemoattractants such as f-Met-Leu-Phe, C5a, LTB4, PAF, and possibly hepatocyte growth factor/scatter factor (142). Most ligandbound receptors are endocytosed within 10 min and the receptors are recycled (142). The chemotactic response is of short duration because the receptors are rapidly desensitized and this occurs particularly at high ligand concentrations (274,275). The pathway by which chemokines activate integrin activity mainly includes the heterotrimeric guanine nucleotide-binding proteins (G proteins) linked to the receptors (276,277).Ligand hintling is coiipled to transduction into an intracellular signal. This begins with the activation of the heterotrimeric G protein which includes an a-, p-, and ychain. It promotes the exchange of GDP, which is bound to the a subunit, by GTP and the subsequent dissociation of the a-GTP complex from the yi3 heterodimer. The GTP-bound a subunit and the free py subunit can then interact with effector proteins, such as phospholipases, and this in turn can lead to the cleavage of phosphatidylinositol into diacylglycerol and inositol-1,4,5-triphosphate, activators of protein kinase C and endoplasmatic Ca’+ channels, respectively (276). Termination of the signal occurs when the GTP bound to the a subunit of the G protein is hydrolyzed to GDP SO that the a unit reassociates with the y6-chain and inactivates the G protein. The signal transduction process of chemokine receptors can be uncoupled by Bordetella pertussis toxin which irreversibly inactivates the a-chain by ADPribosylation; thus these G proteins are of the G, type (276,278).

B. OTHEHCHEMOTACTIC MOLECULESATTRACTINGLYMPHOCYTES 1 , Hepatocyte Growth Factor (HGF) The chemokine family is not unique in its ability to trigger T cell adhesion and chemotaxis. Recently, HGF, or scatter factor, was able to induce chemotactic migration on memory T cells (279). H G F belongs to a multigene family which encodes a 90-kDa protein with a structure and sequence homologous to plasminogen and other enzymes involved in blood clotting (280,281). The primary translation prodiict is an inactive precursor which is proteolytically processed into an active heterodimer. Like the chemokines, HGF also has a heparin-binding domain by which it may be arrested on endothelial

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proteoglycans and presented to approaching leukocytes. Indeed, HGF can be found associated with vascular endothelium, and HGF staining was increased in endothelium in inflamed tissues (279). The only high-affinity HGF receptor described so far is the protooncogene c-Met (282). It is a transmembrane receptor tyrosine kinase composed of a 50-kDa a- and a 145-kDa p-chain expressed mainly on epithelial cells (283). HGF binding to c-Met causes epithelial cell growth, differentiation, and scatter. Although HGF induced chemotaxis on T cells, it was not possible to identify the c-Met receptor on these cells (279). Thus, the lymphoid system may use an alternative receptor in order to more specifically regulate cell traffic.

2. p2- Microglo bulin The small ll-kDa polypeptide p2-microglobulin (P2m) is the common small subunit of MHC class I antigens and as a free soluble molecule it is chemotactic for pro-T cells (284-287). This chemotactic activity was found with plasma pzm from rat and human and with recombinant mouse P2m, suggesting that no additional maturation of this protein is necessary for the chemotactic activity (284). Rat bone marrow cells migrating toward P2m were resting cells and could acquire T cell markers in coculture experiments with thymic stroma. In the chicken embryo &m attracts bone marrow cells which colonize the thymus (287). During early embryogenesis, peaks of P2m RNA transcripts and of free &m protein synthesis were only detected in the thymus.

MOLECULESATTRACTINGMYELOIDCELLS C. OTHERCHEMOTACTIC

f . C5a The complement product C5a is chemotactic for neutrophils, eosinophils, and macrophages, but not for lymphocytes (288). It is a 74 aminoacid peptide which is found in serum of animals treated with immune complexes or endotoxin. Similar to the chemokines, the C5a receptors also belong to the type of molecules which span the plasma membrane seven times (142,289). On myeloid cells, these receptors, together with receptors for f-Met-Leu-Phe, are the most abundant with 100-200,000 sites per cell. In comparison, chemokine receptors are expressed at a density of20-40,000 sites per cell and platelet-activating factors (PAF) or leukotriene B, (LTB,) (see below) receptors have less than 10,000 sites (290).

LEUKOCYTE MIGRATION A N D ADHESION

38 1

2 . Bacterial Formyl Peptides Native, bacterial-derived formylated peptides can be simulated by the tripeptide f-Met-Leu-Phe. They provoke activation and chemotactic migration of neutrophils but show no reaction with lymphocytes. The peptides also bind to specific G protein-coupled receptors which are expressed as allelic forms of a polymorphic gene (291).

3. PAF Platelets produce a group of acetyl-alkylglycerol ether analogs of phosphatidylcholine called PAF (288). PAF causes platelet aggregation and is a potent chemoattractant for neutrophils, eosinophils, and macrophages, but not for lymphocytes (26).PAF is produced by mast cells, basophils, and endothelial cells and the former two cell types secrete the functionally active lipid. The PAF receptor also has a seven transmembrane structure as that described for chemokines, but it is monospecific.

4 . LTBJ Leukotriene B, is a chemotactic lipid with similar characteristics as PAF (288). It is mainly chemotactic for neutrophils and also has no effect on lymphocytes. LTB, is produced by activated mast cells via the lipoxygenase pathway of arachidonic acid metabolism. T h e LTB, receptor resembles those of PAF (292). X. The Model of Leukocyte-Endothelial Cell Recognition: An Adhesion Cascade

The various adhesion molecules presented above were identified in static binding assays. In “reality,” leukocytes are transported by the blood stream and collide with endothelial cells under shear stress. Based on in uiuo observations, Butcher proposed a multistep model of leukocyte binding to endothelium (4).In the same year, Lawrence and Springer built an in vitro chamber that allowed microscopic analysis of adhesion under different dynamic flow conditions similar to those found in capillary venules (293). They showed that selectinmediated interactions are weak and neutrophils rolled along but were not arrested on lipid surfaces coated with P- or E-selectin (Fig. 9). However, rolling cells on bilayers containing P-selectin and ICAM-1 could be totally arrested when the integrin LFA-1 was activated by adding a chemokine (293).Thus, in blood vessels the selectins initiate the first leukocyte contact with the endothelium and this leads to

-

382

Control

IMHOF A N D D U N O N

a

-

f

Selectin + ICAM-1

fintegrin activation factor

FIG.9. Illustration of leukocytes rolling in a flow chamber as described by Lawrence and Springer (see text). Control: the cells flow through a chamber without any coating of adhesion molecules. dE-selectin: the chamber is now coated with either of the two selectins (squares). The flowing cells adhere loosely by glycosylated ligands and start to roll along the coated support. Selectin + ICAM-1: the chamber is now coated with either of the selectins (squares) and with the immunoglobulin superfamily molecule ICAM-1. Before activation the cells roll along the selectin; upon activation (e.g., by chemokines) the cells adhere tightly by the activated integrins on ICAM-l-coated support.

leukocyte rolling. Endothelial contact promotes activation of leukocyte integrins b y chemokines, which are secreted or presented by the endothelium or other adjacent cells. Induced adhesion by activated integrins is tight and stops rolling (Fig. 9). This important breakthrough in the understanding of the cell adhesion mechanism leads to a fourstep model: (i)establishing tenuous adhesion or tethering, (ii) delivery of a triggering signal that activates leukocyte integrin function, (iii) establishing strong adhesion to the endothelium, and (iv) subsequent migration to endothelial junctions and transmigration into the surrounding tissue (Figs. 9 and 11) (4,5,41,56,138,139,293,335).

A. TETHERING / ROLLING Under normal circumstances the slowest flow rates in the circulatory tree occur in the postcapillary venules. This area would therefore be expected to be the location for leukocyte interaction with the endothehum under normal conditions. At sites of inflammation, the blood

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flow rate is reduced due to vessel dilatation, which offers increasing opportunities for a passing leukocyte to collide with the endothelium. Tethering refers to the establishment of loose and somewhat transient adhesion between leukocytes and endothelium. Because of the shear forces by the blood stream, this results in the rolling of leukocytes along the endothelium. It is now clear that selectins and their ligands participate in the tethering of all leukocytes. In addition, the large number of selectin ligands suggests that some cells may simultaneously express two or more of these molecules which could lead to a precise tuning of this step. Some of these ligands could be involved in real tethering, whereas others are involved in the rolling of leukocytes. Tethering is important because it allows a short-term cell-cell contact that takes long enough for the preparation of the second step in the adhesion cascade to occur, i.e., the triggering. B. TRIGGERING During this step, lymphocytes respond to ligands on the endothelial cell surface by signaling, which in turn wilI activate strong adhesion in probably less than seconds. The objective of this triggering is to turn on the adhesive function of integrins. Although circulating leukocytes express substantial amounts of different integrins on the cell surface, they are not functionally active. As already mentioned, triggered integrin activation on leukocytes can be mediated by chemokines and other chemotactic signaling molecules. Chemokines are heparinbinding molecules which upon secretion can become associated with cell-surface proteoglycans of the endothelial cells. One prominent proteoglycan is the adhesion molecule CD44 which has been shown to bind the p-chemokine MIP-10 (294). In this manner the chemokine is then available for presentation to the rolling naive T lymphocytes. These cells then respond to the chemokine contact with a 4 p l integrin activation followed by tight cell adhesion. Triggering of rolling leukocytes can also be achieved by the Ig superfamily adhesion molecule CD31. CD31 is present on all endothelia, monocytes, neutrophils, and on some of the T lymphocyte populations. Occupancy of CD31 on leukocytes, possibly by homotypic ligand interaction (Fig. 11; Tables IV and V), leads to signal transduction followed by integrin activation (138).

C. STRONG ADHESION Integrins, once their function is induced, undoubtedly play a major role as the main force or “glue” that sticks leukocytes to endothelium. It is this strong adhesion that can rapidly bring flowing T cells to a

TABLE IV ADHESION MOLECULESIN LEUKOCYTE-ENDOTHELIUM INTERACTION Name Selectins L-selectin

Expression Pattern

Cellular Ligands

ECM Ligands"

Adhesion Step

All leukocytes

GlyCAM, CD34, MadCAM-1

Tethering

P-selectin

Platelets, inflamed endothelium

Tethering

E-selectin

Inflamed endothelium

PSCL-1, 120-kDa SLex bearing protein 150-kDa SLex bearing protein

Tethering

Integrins alp1 (VLA-1)

T cell subsets

Collagen, LN

a 2 p l (VLA-2)

T cell subsets

Collagen, LN?

a3pl (VLA-3)

Resting T cells

Collagen, LN?

a 4 p l (VLA-4)

Resting T cells

FN, tsp VCAM-I

a 5 p l (VLA-5)

Resting T cells

a 6 p l (VLA-6)

Resting T cells, endothelium

?

FN, tsp LN, kalinin

Implicated In: Lymphocyte homing to lymph node Leukocyte homing to inflammation sites Lymphocyte/myeloid cell homing to inflammation sites Lymphocyte/myeloid cell homing to inflammation sites

Lymphocyte/ECM interaction LymphocyteIECM interaction Lymphocyte/ECM interaction Lymphocyte/ECM interaction Strong adhesion Leukocyte homing to inflamed tissues Lymphocyte/ECM interaction Strong adhesion? Pro-T cell homing to thymus Lymphocyte/ECM interaction

Strong adhesion? Lymphocyte homing to Pryer’s patches Strong adhesion’? Gut IELiintestinal epithelium interaction Strong adhesion General role in leukocyte extravasation Strong adhesion Homing to inflamed tissues

a4p7

Lymphocytes

MadCAM-1, VCAM-1

aIELP7

Mucosal T cells

E-Cadherin

aLP2 (LFA-1)

Leukocyte subsets

ICAMs

a&2 (Mac-1)

Leukocyte subsets

ICAM-1

Fibrinogen, C3bi

aXp2 (p150/95) avp3

Leukocyte subsets Leukocyte subsets

?

Fibrinogen VN, FN, fibrinogen

Leuko., inflammed endothelium

LFA-1, Mac-1

Strong adhesion

ICAM-2

Endothelium

LFA-1

Strong adhesion

ICAM-3 VCAM-I

Resting T cells Inflammed endothelium Endothelium, naive T cells, platelets, monocytes, neutrophils

LFA-1 ~ 4 P (VLA-4) l

Strong adhesion

CD31, GAG

Triggering

Ig superfamily molecules ICAM-1 c)

m

in

CD31

General role in leukocyte extravasation General role in leukocyte extravasation Leukocyte homing to inflammed tissues T cell homing to lymph node?

(continued )

TABLE IV (Continued ) Name Highly glycosylated molecules CD44 GlyCAM-1 CD34

Expression Pattern

Leukocytes Lymph node and lung endothelium Endothelium

Cellular Ligands

ECM Ligands'

Adhesion Step

L-selectin

? Tethering

Many functions Homing to lymph node

L-selectin

Tethering

a4p.7 L-selectin

? Tethering

Homing to inflammed tissues Homing to lymph nodes? Lymphocyte homing to gut

?

HA, collagen, F N

0

m

m

MAdCAM-1

a

Mucosal endothelium

Implicated In:

LN, laminin; FN, fibronectin; tsp, thrombospondin; C3bi. complement subunit; VN, vitronectin; HA, hyaluronic acid.

TABLE V THEDIFFERENT “ORGAN-SPECIFIC” ADHESION STEPS Peripheral Lymph Node HEV Tethering Lymphocyte Endothelial cell

w co

4

Triggering Lymphocyte

Endothelial cell Strong adhesion Lymphocyte Endothelial cell

L-selectin I CD34, GIyCAM-I? Ga,-coupled receptors 1 chemoattractant?

ad2

1

ICAM-I-:!

? I CD312

Peyer’s Patch HEV

Gut

L-selectin I MAdCAM-1, CD34(?) ?

Ga,-coupled receptors I chemoattractant?

1 CD31?

aJPi

a d 2

I MAdCAM-1

1

ICAM-1,-2

Skin

L-selectin I MAdCAM-1, CD34 (P) Ga,-coupled receptors I MCP-l? HGF? MIP-l?

CD31IGAG

I CD31?

ff&

1 MAdCAM-1

CLA

I E-selectin Ga,-coupled receptors 1

MCP-l? HGF? MIP-l? a&

I MAdCAM-1

CD31/GAG

I CDGl?

PLP2

I ICAM- 1,-2(?)

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IMHOF AND DUNON

halt. The predominant pathways are mediated by the integrin adhesion molecule pairs VLA-4/VCAM-1, LFA-l/ICAM-l, LFA-l/ICAM-2, and Mac-1IICAM-1 (see Table IV). In addition, the a4P7IMAdCAM-1 pair plays a role in specific lymphocyte homing to Peyer’s patches.

D. LEUKOCYTE-ENDOTHELIAL TRANSMIGRATION Transendothelial migration is a rapid event; once a leukocyte sticks to the luminal side of the endothelium it takes only a few minutes to reach the subendothelial basal membrane. The transmigration is a oneway trafficking event as the leukocytes are trapped by the extracellular matrix of the basal membrane (159). The process of transmigration starts with locomotion of adherent leukocytes toward the endothelial cell-cell junctions. While moving forward the cell steadily forms new adhesion contacts at the migration front and reduces adhesion at the “back.” Overall, cell migration requires adhesion which is not too strong, otherwise it would lead to immobilization. Several mechanisms may contribute to modulation of the adhesion force. The first is the transience of augmented integrin function, probably because the signals given to leukocytes by interactions with CD31 or chemokines decrease rapidly (225). The second mechanism is shedding; for instance, L-selectin is shed from the cell surface as a consequence of leukocyte activation, and this may help in releasing a stationary cell to migrate (44). Significant numbers of soluble adhesion molecules are found in the blood, and they can reduce the leukocyte adhesion strength by blocking adhesion ligands (53,295). Similar to L-selectin, soluble forms of E-selectin and ICAM1are produced by proteolytic cleavage from endothelial cells (38,296). In contrast, soluble forms of P-selectin are directly generated by alternatively spliced transcripts missing the transmembrane region (297,298), and soluble forms of VCAM-1 are produced by alternative splicing leading to glycolipid-anchored molecules possibly followed by cleavage of the lipid (211,213). The GlyCAM-1 molecule does not contain a transmembrane domain and it can be found in a soluble form (89,94). These soluble molecules, in addition to the migration modulating effect, may also inhibit inopportune arrest of leukocytes to endothelia at inappropriate sites (295). In uitro migration assays have demonstrated that antibodies against the integrins a4P1, a5P1, LFA-1, the proteoglycan CD44, and the signaling adhesion molecule CD31, inhibit leukocyte migration through endothelial monolayers (159,225,227,299). The precise role of these molecules in the process of transmigration is not yet known. However, T cells harvested during or immediately after transendothelial migration from in uitro cultures can adhere with high affinity to

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the extracellular matrix molecule fibronectin; circulating T cells do not adhere. It appears that the two integrins, a401 and a501, are primarily involved in this function (225,227). Engagement of a4Dl integrin with VCAM-1 can induce the expression of a 72kDa gelatinase on the surface of T cells which may facilitate T cell migration into perivascular tissue (337).The role of LFA-1 is less clear, but LFA-1deficient T cell clones also showed decreased transmigratory capacity (299). XI. Molecular Basis of Specific Homing of leukocytes: Combinatorial Diversity in leukocyte-Endothelial Cell Recognition

As already mentioned, research was initially focused on the identification of adhesion receptors which ensured homing of a specific leukocyte subset, i.e., naive T cells or neutrophils which home to a specific organ, or adhesion receptors from which a tissue-specific expression was found. As the adhesion molecules were identified, it became clear that cell-specific receptors, with the exception of GlyCAM-1 and MAdCAM-1, may not exist. For instance, while both L-selectin and CD31 are expressed on naive lymphocytes and may contribute to the specific movement ofnaive cells into lymph nodes, they are also widely expressed by myeloid cells. This illustrates that the rules are not so simple. The question then arises as to how specificity can be achieved (some combinations of chemokines, adhesion molecules, and their ligands which may lead to tissue-specific homing are illustrated in Table V and Fig. 11). First, there are structural variations within adhesion molecules which are not yet fully understood. VCAM-1, CD31, P-selectin, and some integrin subunits exhibit alternative splicing of their transcripts which probably leads to proteins of different specificity or affinity. The selectin ligands can be differentially glycosylated depending on the cell type expressed, and this also modifies the adhesion properties. Second, the multiple-step model allows combinations of different adhesion molecules, chemokines, and their receptors which generate extended diversity of specific leukocyte-endothelial interactions. One ligand pair is used in a limited number of leukocyte-endothelial cell interactions, each of them being defined by the leukocyte cell type and the origin of the endothelial cell. The advantage of this model is that relatively few ligand-receptor pairs are needed for tissue-specific leukocyte homing. Even less individual molecules are needed in as much as one adhesion receptor can have several different ligands. Admitting that each step requires one ligand-receptor pair, 425 different specific homing possibilities can maximally be obtained with 27

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IMHOF AND DUNON

pairs in a three-step model (5 selectin-ligand pairs x 17 chemokines or CD31 x 5 integrin-ligand pairs = 425 and 5 + 17 + 5 = 27) (4). Consequently, inflammatory tissue which recruits leukocytes from blood with low selectivity requires expression of very few different adhesion molecules and chemokines to be fully efficient. Another advantage of the multiple-step model is that it avoids simultaneous contacts of the different ligand pairs needed in specific leukocyte-endothelium adhesion. Such simultaneous contacts could require the formation of multimolecular complexes and they could decrease the efficiency of specific leukocyte-endothelium recognition. The probability that simultaneous bimolecular reactions occur is lower than the occurance of a series of independent bimolecular reactions. Indeed, in the multistep model, tethering, triggering, strong adhesion, and transmigration occur sequentially. Although some of these steps may overlap at a given time throughout the whole homing process, single bimolecular reactions mainly take place. XII. Recruitment of lymphocytes to Specific Organs

A. PERIPHERAL LYMPHNODEHOMING For lymphocytes there are two independent routes of entering the peripheral lymph nodes (PLN) from the blood (Fig. 2). T cells can enter directly through the HEV barrier or they can enter the peripheral tissue through flat endothelium and circulate by the afferent lymphatics into the lymph node parenchyme. It has been suggested that it is mostly naive T cells that take the first, and activated or memory cells that take the second pathway (9). Analysis of lymphocyte emigration from the blood through HEVs to PLN provided the first basis to propose a sequential step model of lymphocyte adhesion to HEV (300). Two experiments confirmed this hypothesis: first, mAbs to L-selectin almost completely blocked emigration of lymphocytes from the blood into PLN (30,301).In agreement with this finding, naive T cells which home mainly to PLN are L-selectin positive, whereas effector/activated T cells and a large proportion of memory CD45ROf are Lselectin negative (302). Lymphocyte from L-selectin deficient mice did not bind to peripheral lymph node HEV and these mice had a severe reduction in the number of lymphocytes localized to peripheral lymph nodes (336).Accordingly, the L-selectin receptors GlyCAM-1 and CD34 are expressed on PLN HEVs (85,89,90). Second, mAbs to the integrin LFA-1 also markedly reduced or almost completely abolished lymphocyte migration into PLN (303),although LFA-1 on blood lymphocytes is in a low-affinity state and requires activation for

LEUKOCYTE MIGRATION AND ADHESION

39 1

binding to ICAM-1 and ICAM-2 (198,304).Activation of LFA-1 occurs during the rolling step which is mediated by the L-selectin and involves signaling by G proteins. Inhibition of lymphocyte emigration to PLN by pertussis toxin suggests that G protein-coupled receptors of the aiclass are required for lymphocyte emigration, i.e., for the strong adhesion step (278,300). The molecules which activate LFA-1 by these G protein-coupled receptors, while the cells are in contact with HEVs, are not known. Although L-selectin can transduce signals into cells by protein kinases it is not coupled to G proteins (Rosato et ul., personal communication) (305). It is more likely that CD31 or unidentified chemokine and chemokine receptors are responsible for this activation.

B. HOMINGTO GUT-ASSOCIATED TISSUES The most organized lymphoid structures in the wall of the gut are Peyer’s patches; they are specialized for sampling antigen from the gut lumen and presenting it to lymphocytes. Gut lymphocytes are also found scattered in the lamina propria, underlying the digestive epithelium, and in the epithelium layer. Anti-L-selectin mAbs, as well as mAbs directed against MAdCAM-1, and the a4 or p7 integrin subunits block around 50% of lymphocyte emigration from blood to Peyer’s patches and to the intestine proper (49,301,306,307). The fact that anti-MAdCAM-1, a4, and 0 7 integrin antibodies have no effect on lymphocyte recirculation to PLN enhances the role of L-selectin/MAdCAM-1 and a4p7IMAdCAM-1 interactions in lymphocyte homing to the area of the gut (100,301,306,308). L-selectin and a4p7 integrins are preferentially expressed on naive and on a subset of memory T cells, respectively. This suggests that the L-selectin/MAdCAM-1 interaction may occur preferentially between naive T cells and Peyer’s patches HEV, and gut endothelia and a4P7IMAdCAM-1 interaction occurs between memory T cells and gut endothelia (15,19,306, 309). MAbs to LFA-1 inhibit T cell recirculation to Peyer’s patches but they have no effect on recirculation to the remainder of the gut (Fig. 2) (303). G protein-coupled receptors are also involved here in a similar way as already seen for the naive T cell homing to PLN (278). Lymphocytes roll along Peyer’s patches for a few seconds before they arrest and emigrate. Prior treatment of lymphocytes with pertussis toxin prolongs the rolling of lymphocytes on HEV indefinetely so that the lymphocytes pass out of the Peyer’s patch without being arrested. Activation of blood lymphocyte integrins is required for the last step of arrest on HEV before the lymphocytes can emigrate into the lymphoid compartment of Peyer’s patches. A subpopulation of

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IMHOF AND DUNON

gut lymphocytes penetrates the basement membrane of the intestinal epithelium and localizes in between enterocytes; these intraepithelial lymphocytes (IELs) express the (YEP7 integrin (170).Inhibition of IEL binding to intestinal epithelial cells by anti-a, antibodies indicates that the intestinal epithelial cells express a ligand of a& integrin. In fact recently the epithelial adhesion molecule E-Cadherin has been found to bind to (YEP7 integrin (334). The aE&+ T cell subset, which is of the memory type, represents 2-6% of peripheral blood lymphocytes and these cells are CLA-1 and L-selectin negative (310,311). TGFP, produced in intestine, induces expression of a& on intraepithelial lymphocytes (312). The cytokine TGFP also induces IgM+ B lymphocytes to undergo IgA class switching; note that the predominant Ig secreted in the mucosa is of the IgA class and that the gut addressin MAdCAM-1 contains an IgA-like domain (93,313). The cytokine TGFP in the gut may transform “homeless” lymphocytes into gut-specific lymphocytes which can recirculate to the gut. Such “address imprinting” may also be found with natural killer cells which are found in the lamina propria (314). C. SKINHOMING

A major entry of pathogens, in addition to the gut, is the skin. The T cells that localize and migrate through the skin are almost exclusively

of the memory phenotype, but unlike gut memory cells, skin memory T cells express the carbohydrate-bearing protein, CLA, which can bind to E-selectin (Fig. 2) (110). Indeed, a CLA+ memory T cell subset binds to E-selectin (108).E-selectin is induced on dermal endothelial cells in delayed-type hypersensitivity and in chronically inflamed skin (315).In addition, Fab fragments of anti-E-selectin antibodies inhibit the recruitment of lymphocytes at the site of delayed hypersensitivity. Thus, the interaction between E-selectin and CLA may contribute to T lymphocyte skin tropism. The strong expression of VLA-4 on CLA+’ memory T cells and inhibition of skin homing by Fab fragments of anti-VCAM-1 mAb suggest that the gluing/strong adhesion step is ensured by the VLA-4/VCAM-1 interaction. However, the involvement of the LFA-l/ICAM-2 interaction in this step cannot be excluded. Recently, it has been suggested that extravasation of activated T cells in the skin may adhere by a3pl integrin to the ECM component epiligrin (316). However, the precise mechanism by which T cells penetrate the basement membrane and accumulate in the epidermis is unknown. Furthermore, it is speculated that intradermal migration of E-cadherin-expressing T cells may interact by this adhesion molecule with keratinocytes (317).

LEUKOCYTE MIGRATION A N D ADHESION

393

D. PRO-TCELLHOMING TO THE THYMUS The pro-T cell line FTFl, isolated from fetal thymus, was shown to bind to frozen sections of thymus and liver from newborn mice as well as to an embryonic endothelial cell line (160,318). This property of pro-T cells appeared to be restricted to vessels in hemopoietic tissues. EA-1, a mAb directed against a6 integrins (a&, and ad4), was found to block the binding of pro-T cells to thymus-derived endothelium on frozen sections. Anti-LFA-1 antibody did not inhibit the binding of pro-T cells to thymic endothelium, but it slightly increased the inhibitory effect of EA-1 mAb suggesting that LFA-1 plays an accessory role in the endothelium binding of pro-T cells mediated by a6 integrins. Although ad1integrin is a laminin receptor (117), the EA-1 mAb does not inhibit binding o f a d 1 +cells to laminin, which suggests the existence of a novel ligand for a6 integrins on pro-T cells (161). Recently, it has been found that a6 integrin occupancy by pro-T cell ligands can also lead to signaling in endothelial cells. As a consequence, a further adhesion molecule is activated and this leads to tight adhesion of pro-T cells to the endothelium (Naquet, personal communication). Thus, the study of thymus homing may lead to the discovery ofa new dynamic cell adhesion mechanism which may not need chemokines and functions by activation of the endothelial target cell. A role in thymus colonization by pro-T cells has also been ascribed to the hemopoietic standard form of CD44 (240). Anti-CD44 antibodies inhibit homing of fluorescently labeled bone marrow cells to the thymus, and CD44 is expressed on bone marrow cells able to repopulate the thymus of irradiated mice at long term (319-322). Finally, L-selectin might also belong to this group of pro-T cell homing molecules since it is expressed on bone marrow cells, presumably on pro-T cells, and on the most immature thymocytes (323). The role of chemotatic factors in thymus homing seems to be restricted to the migration of T cell progenitors from the vascular endothelium through the perithymic mesenchyme to the thymic epithelium (324). Cheniotactic peptides of thymic origin have been partially purified from mammals and avian embryos (325,326). To date, the bestcharacterized chemotactic molecule involved in thymus homing during embryogenesis is P2m (287). However, the thymus colonization in P,ni-deficient mice is normal (327), which indicates that other molecular entities with chemotactic properties must exist. Pro-T cell migration from the perivascular space toward the thymic epithelium requires extracellular matrix proteins as anchoring points. In the presence of thymic chemotactic factors, quail hematopoietic precursors were able

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IMHOF AND DUNON

to transverse a human amniotic basement membrane (328).The inhibition of this process by fibronectin-specific antibodies or b y synthetic peptides containing RGDS, a cell-binding sequence of fibronectin, suggests that T cell precursors interact with fibronectin during this migration. It has also been shown that laminin-specific antibodies inhibit this invasive process. XIII. Recruitment of leukocytes during Inflammation

Leukocytes adhere poorly to resting endothelial cells. During inflammation the phenotype of the endothelium is dramatically modified (Fig. 10). This change results from the contact of endothelial cells with “alarm” cytokines such as IL-1, TNF, or IFNy. Both IL-1 and T N F are mainly produced by macrophages stimulated by microbial products, whereas IFNy is released by natural killer cells and T cells that have encountered their specific antigen. These different inflammatory cytokines induce cell-surface expression of adhesion molecules on endothelia. The first which appears is P-selectin; it is continuously synthesized by endothelial cells on a low level and stored in Weibel-Palade bodies, but it is translocated to the plasma membrane within seconds upon stimulation with these alarm cytokines, as well as with thrombin or histamine. Only the alarm cytokines induce Pselectin synthesis by endothelial cells. Cell-surface P-selectin allows the tetheringholling of leukocytes at the site of tissue injury. E-selectin is predominantly found in cutaneous inflammatory sites after induction of its biosynthesis by IL-1 or TNF, but its maximal expression can only be reached after 4 to 6 hr of endothelial stimulation. E-selectin promotes tethering of neutrophils, monocytes, and lymphocytes.

-

..-.. ...,.*.... 111,11111,*1111,111

-0

1

2

4

0

24

40

P-Selectin E-Selectin I-CAM-1 V-CAM-1

72

Endothelial Cell Activation by Cytokines (hours)

FIG.10. Temporal expression of endothelial adhesion molecules induced by inflammatory reactions.

LEUKOCYTE MIGRATION AND ADHESION

395

Within minutes after stimulation with cytokines, endothelial cells produce chemokines, leukotriene B4, and PAF; all are proinflammatory agents which can activate integrins on rolling leukocytes. As already mentioned in detail above, activated leukocyte integrins bind to the Ig superfamily molecules ICAM-1 and VCAM-1. Both are poorly expressed on resting endothelial cells but their expression is dramatically increased within a few hours by the same alarm cytokines (Fig. 10). The increase of permeability is also a characteristic component of inflammation. Chemoattractants aIone will promote little leukocyte influx in the absence of vascular permeability increase. The ability of CD31 to redistribute to the cell border when coming into contact with endothelial cells suggests that it participates in the endothelial cell-endothelial cell interactions that limit vascular permeability. In this regard, mediators, such as thrombin and histamine, that act on endothelium to cause cellular retraction and to increase permeability, may do so through an effect on CD31 (329). In summary, the production of inflammatory molecules and induction of a few adhesion molecules, such as P- and E-selectins, ICAM1, and VCAM-1, should be sufficient to massively recruit leukocytes to inflammation sites. This is confirmed in P-selectin and ICAM-1 knockout mice which exhibit major defects in inflammatory responses (201,330). This relatively simple induction appears to be autocatalytic since stimulated leukocytes themselves secrete proinflammatory molecules. One way to limit the inflammation process and its autocatalytic pathway is by the shedding ofadhesion molecules from the cell surface (ICAM-1, E-selectin) and by the production of alternatively spliced soluble adhesion molecules (P-selectin, VCAM-1). They reduce the efficiency of leukocyte homing which finally has a moderating effect on inflammation.

XIV. Outlook

A conceptual leap in the field of leukocyte migration and homing was the description of adhesion as a multistep process (Fig. 11 and Table V). An important part of' this process is rolling and integrin activation of leukocytes by chemokines or CD31. New mechanisms arise which also include the activation of adhesion molecules located on endothelial cells under noninflammatory conditions. Inflammation is a vital and dramatic event, and it is probably for this reason that the repertoire of inflammatory adhesion molecules is larger than that for normal cell trafficking. The molecular dissection of leukocyte homing leads to the development of new therapeutic drugs which block adhe-

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IMHOF AND D U N O N

1. Rolling

2. Triggering

3.Strong Adhesion

4. Migration

FIG.11. Illustration ofthe interaction ofa leukocyte with the endothelium. The four steps are rolling or tethering, triggering, strong adhesion, and migration.

sion in autoimmune diseases and chronic inflammation. It may also shed light on the metastasis process as it exhibits the same steps: blood transportation of cells, organ-specific recognition, extravasation, and an invasive process. The ability of some cells to metastasize could be due to the abnormal regulation of expression of adhesion molecules which are known to participate in leukocyte homing, as is the case for VCAM-1, ICAM-1, CD44, and a6pl integrins, and many other molecules (161,331-333). ACKNOWLEDGMENTS The authors thank Nicole Schoepflin and Jerome Aarden for artwork, Hans Spalinger and Beatrice Pfeiffer for photography, and Drs. Luca Piali, Dietmar Vestweber, and Charles Mackay for critical reading and improving of the manuscript. D. D. is partially supported by the Association pour la Recherche contre le Cancer (ARC). The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche & Co. Ltd, Switzerland.

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306. Hamann, A., Andrew, D. P., Jablonski-Westrich, D., Holzmann, B., and Butcher, E. C. (1994). Role of a4-integrins in lymphocyte homing to mucosal tissues in vivo. J. ltnmunol., 152, 3282-3293. 307. Picker, L. J., and Butcher, E. C. (1992). Physiological and molecular mechanisms of lymphocyte homing. Annu. Reo. Zmmunol. 10, 561-591. 308. Issekutz, T. B. (1991). Inhibition of in vivo migration to inflammation and homing to lymphoid tissues by the TA-2 monoclonal antibody. A likely role for VLA-4 in vivo. 1. Zmmutiol. 147, 4178-4184. 309. Mackay, C. R., Marston, W., and Dudler, L. (1992). Altered patterns of T cell migration through lymph nodes and skin following antigen challenge. Eur. J. lnimunol. 22,2205-2210. 310. Cerf-Bensussan, N., Jarry, A., Brousse, N., Lisowska-Grospierre, B., Guy-Grand, D., and Griscelli, C. (1987). A monoclonal antibody (HML-1) defining a novel molecule present on human intestinal lyniphocytes. Eur. J. lmmunol. 17, 1279-1285. 311. Picker, L. J., Terstappen, L. W., Rott, L. S., Streeter, P. R., Stein, H., and Butcher, E. C. (1990). Differential expression of homing-associated adhesion molecules by T cell subsets in man. J. lmmunol. 145, 3247-3255. 312. Parker, C. M., Cepek, K. L., Russell, G. J., Shaw, S. K., Posnett, D. N., Schwarting, R., and Brenner, M. B. (1992). A family of 87 integrins on human mucosal lymphocytes. Proc. Natl. Acad. Sci. USA 89, 1924-1928. 313. CoKman, R. L., Lebman, D. A., and Shrader, B. (1989).Transforming growth factor p induces IgA production by lipopolysaccharide-stimulated murine B lymphocytes. J. E x p . Med. 170, 1039-1044. 313. Van Tol, E. A. F., Verspaget, H. W., Pena, S., Elzo Kraemer, C. V., and Lamers, C. B. H. W. (1992). The CD56 adhesion molecules is the major determinant for detecting non-major histocompatibility complex-restricted cytotoxic mononuclear cells from the intestinal lamina propria. Eur. J. Immunol. 22, 23-29. 315. Cotran, R. S., Gimbrone, M. J., Bevilacqua, M. P., Mendrick, D. L., and Pober, J . S. (1986). lnduction and detection of a human endothelial activation antigen in vivo. J. Exp. Med. 164, 661-666. 316. Wayner, E. A,, Gil, S. G., Murphy, G. F., Wilke, M. S., and Carter, W. G. (1993). Epiligrin, a component of epithelial basement membranes, is an adhesive ligand for a 3 p l positive T lymphocytes. J. Cell. Biol. 121, 1141-1152. 317. Wayner, E. A,, Hoffstrom, B., and Pittelkow, M. R. (1994). Cooperative role of a381 and E-cadherin in mediating T lymphocyte adhesion to keratinocytes. Submitted for publication. 318. Ruiz, P., Dunon, D., Hesse, B., and Inihof, B. A. (1991). T lymphocyte precursors adhere to thymic endothelium. I n “Lymphocyte Reaction and in Vivo Immunology” (B. A. lmhof, S. Berrih-Aknin, and S. Ezine, Eds.), pp. 953-957. Dekker, New York. 319. O’Neill, H. C. (1987). Isolation of a thymus homing Iyt-2-,L3T4- T-cell line from mouse spleen. Cell. lmmunol. 109, 222-230. 320. O’Neill, H. C. (1989). Antibody which defines a subset of bone marrow cells that can migrate to the thymus. Immunology 68, 59-65. 321. O’Neill, H. C., Ni, K., and O’Neill, T. J. (1992). Lymphoid precursor cell lines have capacity to migrate to multiple lymphoid sites. Immunology 76, 631635. 322. Horst, E., Meijer, C. J. L. M., Duijvestjin, A. M., Hartwig, N., Van der Harten, H . J , , and Pals, S. (1990). The ontogeny of human lymphocyte recirculation: High

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endothelial cell antigen (HECA-452) and CD44 homing receptor expression in the development of the immune system. Eur. J . lmmunol. 20, 1483-1489. Terstappen, L. W. M. M., Huang, S., and Picker, L. J. (1992). Flow cytometric assessment of human T-cell differentiation in thymus and bone marrow. Blood 79,666-677. Le Douarin, N. M. (1978). Ontogeny of hematopoietic organs studied in avian embryo interspecificchimeras.In “Differentiation ofNorma1and Neoplastic Hematopoietic Cells” (B. Clakson, P. A. Marks, and J . E. Till, Eds.), Vol. 5, pp. 5-31. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Champion, S., Imhof, B. A,, Savagner, P., and Thiery, J. P. (1986). The embryonic thymus produces chemotactic peptides involved in the homing of hemopoietic precursors. Cell 44, 781-790. Ben Slimane, S., Houillier, F., Tucker, G . , and Thiery, J. P. (1983). In vitro migration of avian hemoietic cells to the thymus. Cell. D# 13, 1-24. Zijlstra, M., Bix, M., Simister, N. E., Loring, J . M., Raulet, D. H., and Jaenisch, R. (1990). p2-microglobulin deficient mice lack CD4-CD8’ cytolytic cells. Nature 344,742-746. Savagner, P., Imhof, B. A., Yamada, K. M., and Thiery, J. P. (1986). Homing of hemopoietic precursor cells tothe embryonic thymus: Characterization of an invasive mechanism induced by chemotactic peptides. J . Cell Biol. 103, 2715-2727. Anderson, A. O., and Shaw, S. (1994). Lymphocyte trafficking. In “Clinical Immunology” (R. R. Rich, T. A. Fleisher, B. D. Schwartz, W. T. Shearer, and W. Strober, Eds.), in press. Mayadas, T. N., Johnson, R. C., Rayburn, H., Hynes, R. O., and Wagner, D. D. (1993). Leukocyte rolling and extravasation are severely compromized in P selectin deficient mice. Cell 74,541-554. Rice, G. E., and Bevilacqua, M. P. (1989). An inducible endothelial cell surface glycoprotein mediates melanoma adhesion. Science 246, 1303-1306. Johnson, J. P. (1991). Cell adhesion molecules of the immunoglobulin supergene family and their role in malignant transformation and progression to metastatic disease. Cancer Metastasis Reu. 10, 11-22. Gunthert, U., Hofmann, M., Rudy, W., Reber, S., Zoller, M., Haussmann, I., Matzku, s.,Wenzel, A., Ponta, H., and Herrlich, P. (1991).A new variantofglycoprotein CD44 confers metastatic potential to rat carcinoma cells. Cell 65, 13-24. Cepek, K. L., Shaw, S. K., Parker, C. M., Russel, G . J., Morrow, J. S., Rimm, D. L. and Brenner, M. B. (1994). Adhesion between epithelial cells and T lymphocytes mediated by E-cadherin and an integrin a&. Nature, in press. Lawrence, M. B., Bainton, D. F. and Springer, T. A. (1994). Neutrophil tethering to and rolling on E-selectin are separable by requirement for L-selectin. Immunity, 1, 137-145. Arbones, M. L., Ord, D. C., Ley, K., Ratech, H., Maynard-Curry, C., Otten, G., Capon, D. J., and Tedder, T. F. (1994). Lymphocyte homing and leukocyte rolling and migration are impaired in L-selectin-deficient mice. Immunity, 1,247-260. Romanic, A. M., and Madti, J. A. (1994). The induction of 72-kD gelatinase in T cells upon adhesion to endothelial cells is VCAM-1 dependent. J . Cell Biol., 125, 1165-1 178.

ADVANCES IN IMMUNOLOGY, VOL. 58

Gene Transfer as Cancer Therapy GLENN DRANOFF' AND RICHARD C. MULLlGANt 'Dona-Farber Cancer Institute ond Harvard Medical School, Baston, Massachusetts 021 15; ond

t Whitehead Institute for Biomedical Reseorch ond Department of Biology, Massochusetts Institute of Technology, Cambridge, Mossochusetts 02142

I. 11. 111. IV. V. VI. VII.

Introduction Tumor Antigens Gene-Transfer Techniques Genetic Modification of Tumor Cells Antigen-Based Vaccination Strategies Adoptive Imniunotherapy Reduction to Practice References

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1. Introduction

The development of recombinant DNA technologies has generated remarkable insight into the mechanisms underlying carcinogenesis. The recognition that specific mutations in cellular oncogenes result in transformation (12) has catalyzed dramatic advances in the study of gene regulation, signal transduction, cell cycle control, differentiation, and metastasis. While the impact of this knowledge on the management of patients with cancer has been only modest to date, these new paradigms are likely to stimulate substantial improvements in cancer diagnosis and therapy. The identification of genes critical to the initiation and maintenance of neoplasia, for example, provides a powerful framework for devising genetic-based diagnostic tests to identify cancer earlier in its natural history, when existing therapies are more efficacious (197). Cancer-related genes also offer new prospects for developing rationally designed chemotherapeutic agents, such as farnesyltransferase inhibitors to subvert mutant ras proteins (114), and specific antagonists of angiogenesis factors (95). The development of high-efficiency gene-transfer systems has sparked optimism that gene therapy for cancer will become feasible as well. Gene therapy, broadly defined as the introduction of genetic material iqto a patient's tissues with the intent of achieving therapeutic 417 Copyright D 1995 by Academic Press, Inc. All rights olreproduction in any lorm reserved.

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benefit, offers perhaps the most direct application of recombinant DNA technology to patient management (147).Several clinical trials involving gene transfer in cancer patients have already been launched worldwide. Most of these experiments involve manipulating the host’s immune response to cancer. While immunologic approaches to cancer therapy have been explored with oscillating enthusiasm throughout this century, recent studies employing gene transfer in murine tumor models have generated renewed optimism for this strategy. In this article, we discuss the advances in tumor immunology which have fostered the current excitement, highlight critical features of highefficiency gene-transfer systems relevant to these studies, and speculate on the contributions that gene transfer is likely to make toward improving our understanding of the host-tumor relationship and creating more effective cancer treatments. II. Tumor Antigens

Acritical assumption underlying experimentation in tumor immunology is that an effective antigen-specific immune response can be generated against cancer. Historically, the difficulty in identifying tumorspecific antigens has perhaps been the strongest factor contributing to the recurring skepticism of immunologic measures for combatting cancer (190). Studies of tumor transplantation in mice at the turn of the century, for example, initially stimulated great excitement when tumors were rejected by apparently identical littermates. However, more detailed analysis revealed that the targets of such attack were not tumor-specific antigens per se, but rather the major histocompatibility (MHC) antigens (75,184).While this work thus provided the foundation for principles of allograft transplantation (133), inbred mouse strains (200), and MHC-restricted antigen presentation ( 1 12), tumor immunology suffered a significant setback. More convincing evidence for the existence of tumor-specific antigens emerged from the classic vaccination experiments of Prehn and Main (163).These investigators and others demonstrated that in syngeneic murine model systems, tumor-specific immunity could sometimes be established by virtue of inactivating parental tumor cells through either irradiation or surgical excision (4,56,57,80,170).Mice successfully vaccinated b y these maneuvers were able to reject subsequent challenge of viable parental tumor, in contrast to naive animals which succumbed to this challenge. An intriguing characteristic of this vaccination was its exquisite specificity (10). Immunologic protection was not cross-reactive to distinct tumors, even those which had been gener-

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ated in the same animal by carcinogen treatment. Klein and co-workers extended this work by showing that the autochthonous host was also capable of generating a specific antitumor immune response (111). These vaccination/challenge experiments were used to classify murine tumors on the basis of revealed immunogenicity. A striking finding from this analysis was that tumors shown to be immunogenic almost always had been induced by identifiable carcinogens (4,110,117,198), including chemicals (typically methy lcholanthrene), ultraviolet irradiation, and oncogenic viruses (such as SV40, adenovirus, or polyoma). These tumors could be distinguished from “spontaneous” tumors which had originated in the absence of obvious mutagens and were usually nonimniunogenic in vaccination/challenge experiments. The occasional spontaneous tumor which was immunogenic in this assay had been tested only after prolonged passage in culture, and so its relationship to the original tumor was somewhat unclear. This sharp division in tumor immunogenicity highlighted underlying concerns regarding the suitability of these model systems for predicting the immune response in cancer patients (84). Although remarkably little compelling data were ever marshalled to support the view, some maintained that human tumors were inore closely related to the spontaneous nonimmunogenic murine tumors, in contrast to those associated with carcinogens (85).Ironically, while Hewitt’s exhaustive survey significantly dampened enthusiasm for the concept of tumor-specific immunity, a recent reexamination ofhis work raises the intriguing possibility that the nonimmunogenic tumors did indeed induce an immune response evident upon rechallenge, albeit one of stimulation, rather than inhibition of tumor growth (164). Boon and colleagues repudiated the notion that even nonimmunogenic spontaneous tumors were incapable of generating a protective immune response by the application of chemical mutagenesis techniques (14,230,231).In these studies, rare tumor variants (turn- clones) obtained following in uitro selection in N-niethyl-N’-nitrosoguanidine were, unlike the parental lines, incapable of growing in syngeneic hosts. Some ofthese variants possessed the additional striking property that following their in viuo rejection, they induced protection not only against subsequent challenge of the mutant clone, but also against the unmanipulated parental line. The clear implication of these results was that the parental tumor possessed antigenic targets potentially recognizable by the immune system, but to which an effective response previously had not been generated. These vaccination studies collectively suggested that irnmunogenicity was a property intrinsic to most, if not all, experimental tumors

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and shifted a central question for tumor immunology away from whether tumor antigens existed at all to their biochemical and genetic characterization. Initial strategies for identifying these targets involved application of monoclonal antibody technology. While much was learned about the cell surface of both malignant and normal cells with this approach, with few notable exceptions, such as the idiotype of B cell malignancies (21,119), humoral detection techniques failed to reveal unique tumor-associated proteins. The most intriguing results of this analysis have instead been the identification of tumorassociated alterations in carbohydrate epitopes, including aberrant post-translational modifications of glycoproteins and quantitative changes in cell membrane glycolipids (82). In contrast, improved understanding of the molecular mechanisms underlying antigen processing for T cell recognition dramatically accelerated the search for putative tumor antigens. The notion that peptide fragments derived from both intracellular and exogenous proteins could be presented in the grooves of MHC class I and I1 molecules (71,219)expanded the pool of potential tumor antigens and provided an attractive explanation for the previous limitations of monoclonal antibody-based strategies. Boon and colleagues employed a genetic screen using activation of tumor-specific T cell clones to identify several candidate antigens from both murine and human cancers (15,18,45,196,228,229).Some of these proteins are novel gene products which are either aberrantly expressed in tumors (P1A and MAGE-1) or mutated versions of their normal counterparts (P91A and P198), whereas others appear to be normal cellular proteins (tyrosinase). Activation of tumor-specific T cell clones is also critical to current efforts aimed at purifying and sequencing antigenic peptides eluted from tumor MHC molecules (61,204,205) and heat-shock proteins (225). The latter have been shown, likely on the basis of their chaperone function for peptides, to be associated with the tumor-rejection antigens of the methylcholanthrene-induced murine sarcomas (201). A tumor-specific T cell clone derived from a patient with carcinoma of the pancreas has also been shown to react with the mucin MUC-1 through an otherwise cryptic epitope of the protein core revealed by abnormal glycosylation in the tumor (7,100,101). An additional strategy for identifying tumor antigens has been to determine whether known oncogenic proteins can be recognized by T cells. In uitro responses to ras (60,68,104,158,199), BCR-ABL (33), p53 (88,207,240), PML-RAR (64), and neu (47) were demonstrated in murine and human systems following stimulation with antigenpresenting cells that had been either peptide pulsed or infected with

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a retrovirus expressing the candidate gene. Related studies have also shown in vitro T cell responses to viral oncoproteins, including the products of the human papillomavirus E7 gene (29,54)as well as several Epstein-Barr virus genes (148).A particularly interesting variation on this approach combined the use of cell lines defective in the presentation of endogenous antigens with knowledge of the defined amino acid motifs for peptides eluted from M H C molecules to identify candidate target peptides within the mutant proteins (54,88,207). This impressive array of potential antigens convincingly demonstrates that the T cell repertoire can recognize tumor-associated proteins, but it also highlights the central issue that these proteins nonetheless fail to provoke a response sufficient to prevent tumor progression in vivo. Although elucidation of the mechanisms of this escape will require considerable investigation, the identification of candidate antigens represents a significant contribution to this effort. Quantitative assessments of the precursor frequency and affinity of T cells directed at these proteins in cancer patients and tumor-bearing animals (19,39) will provide important information relevant to exploring potential mechanisms of peripheral tolerance to cancer, including anergy, suppression, clonal deletion, and ignorance (153). Measurement of antigen-specific T cell responses will also be central to evaluating the efficacy of novel strategies to augment host antitumor immunity. While enthusiasm accompanying the identification of candidate tumor-specific T cell antigens is certainly justified, a word of caution must be invoked emphasizing that it is still unclear whether these proteins represent the appropriate targets for immunotherapy of cancer. An assessment of the importance of putative antigens will need to consider several factors, such as the function ofthe candidate protein in the maintenance of the transformed state (which is significantly related to the consequences of selecting for antigen-loss variants), the efficiency by which the intact protein is processed and incorporated into the relevant M H C molecules, and the affinity of the T cell receptor for the target antigen (42,137,138,169).Ultimately, the development of successful immunotherapies will be required to accurately evaluate the potential of candidate antigens to serve as tumor-rejection antigens. A number of strategies for the immunotherapy of cancer are under active investigation. These include various manipulations designed to enhance the immunogenicity of tumor cells, tumor antigen-based vaccination schemes, in vitro expansion and adoptive transfer of tumorspecific T cells, systemic administration of recombinant cytokines, and tumor-reactive monoclonal antibodies conjugated to various toxins (154,221,232). High-efficiency gene-transfer systems are likely to in-

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fluence most profoundly those efforts involving genetic modification of tumor cells or cells involved in different stages of the antitumor immune response. With these applications as a framework for discussion, we highlight relevant features of current gene-transfer systems. Ill. Gene-Transfer Techniques

Strategies for the gene therapy of cancer can be broadly classified according to the site where genetic modification of target cells takes place (146). In one scheme, designated the ex vivo approach, target cells are removed from the patient, established in culture, transduced with an appropriate vector, and returned to the patient. In addition to the gene-transfer technique, this strategy relies on the ability to efficiently harvest the relevant target cells, to manipulate them in culture without inducing significant alterations oftheir biologic properties, and to transplant them in such a fashion that they perform their intended function in vivo. An alternate approach to gene therapy involves methods which accomplish gene transfer in vivo. These maneuvers require the ability to modify a sufficient number of target cells to achieve therapeutic benefit and to target the genetic modification to the relevant tissue. This latter scheme offers a number of potential advantages over the ex vivo approach, including the need for fewer manipulations of target cells and the simpler administration of therapy. Some gene-transfer systems, however, require replication of the target cell for successful transduction, and in these cases, limited understanding of the physiologic signals regulating cell division in vivo presents significant constraints on effective implementation. In addition to the site of gene transfer, strategies for gene therapy can also be usefully classified according to the duration of expression of transduced gene product. For applications in which short-term expression is sufficient, integration of the transferred DNA sequences into the target cell genome is not required, and the transduced cells need not be long lived. Liposomes, synthetic conjugates, naked DNA, and some viral vectors are appropriate for these studies. In contrast, for those applications in which persistent expression is desired, integration of the transferred sequences into the genome of stem cells (which have the unique property of self-renewal as well as commitment) is critical. Retroviral and adeno-associated viral vectors are necessary for these efforts. The high-efficiency gene-transfer systems developed for short-term expression studies differ in important ways regarding their suitability for in vivo versus ex vivo applications. While varying the composition

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of lipids in a liposome particle may afford a modest degree of selectivity in targeting particular cell types (35,130),the development of synthetic conjugates is much more promising in this context (239). By coupling a cell-surface protein through a polylysine linkage to naked DNA, the investigator in principle can limit the uptake of these constructs to target cells expressing the relevant receptor. Most developed in this regard are particles linking transferrin (for efficient uptake by cells) to either inactivated adenovirus or fusogenic peptides derived from influenza virus to reduce degradation in acidic endosomes following internalization (38,40,41,233,234). The ability of these conjugates to target specifically in uiuo the appropriate tissues on the basis of receptor expression, however, still remains to be determined. The administration of naked DNA in uiuo by either direct injection or through a gene gun has proven surprisingly effective for vaccination efforts and potentially allows the facile delivery of large numbers of different sequences (62,226,235). Replication-defective viral vectors offer powerful approaches to transient transfection both in uitro and in uiuo. Most advanced in this context are adenovirus-based systems which deliver extremely high titers of viral particles and high-level expression, at least temporarily (11).The pioneering studies of Shenk demonstrated that El-region gene products could be supplied in trans by a packaging cell, allowing for the production of defective vectors with genes of interest replacing the E l region (103).While there is some concern that viruses lacking E1A may nonetheless undergo low-level replication in uivo (167,192), potentially allowing transfer of genetic material from the infected target cells, the major limitation of this system appears to be the immunologic response against adenoviral products retained in the vector. Studies to determine which proteins are the most frequent targets for cytotoxic T lymphocytes and antibodies and the consequences of deleting these genes from the vector are important issues for further study. Herpes virus vectors are also under active investigation (69,129), but the complexity and limited understanding of the herpes virus genome pose significant difficulties for effective usage of these systems. Although replication-defective vectors have recently been constructed (70), there is evidence that these viruses can still be toxic to target cells (102). Vaccinia vectors also have clear applications in transient expression studies (141),albeit lysis of the infected cell is an inevitable outcome with current systems. For strategies in which persistent expression is desired, retroviral vectors currently are the system of choice (these vectors are also useful for short-term expression studies.) Most vectors are based on the Molo-

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ney murine leukemia virus. The development of packaging cells which provide the viral gag, pol, and env genes in trans has allowed the construction of stable high-titer production systems which do not generate replication-competent virus (44,128). The generation of similar packaging lines using 293 cells, a highly transfectable line, further allows the production of high titer virus without the need to isolate stable cell lines (159). While a detailed analysis of vector design is beyond the scope of this review, recent vectors which utilize the viral long terminal repeat (LTR) to drive transcription of both the fulllength message encapsulated into viral particles as well as the spliced transcript encoding the inserted gene have proven highly versatile for studies requiring high-efficiency gene transfer without the need for selection (51).Current areas of interest in vector design include modifications of the LTR to increase expression in hematopoietic cells and the inclusion of elements which allow for controlled expression. While the retrovirus offers considerable advantages for gene-transfer investigations, a number of factors may ultimately limit its clinical usefulness, particularly for strategies involving in uiuo gene transfer. Most relevant in this context is the requirement for target cell replication in order for proviral integration to occur. Recent studies suggest that factors present during mitosis play critical roles in this process (176). HIV represents an important exception to this rule, however, and efforts to define the relevant elements of the HIV genome and to incorporate them into existing vectors are underway (121,238). The lability of the amphotropic viral particle has also hindered efforts to date at concentrating virus, although pseudotyping with the envelope protein of vesicular stomatitis virus has demonstrated some promise (20).While the risk of insertional mutagenesis with retroviral-mediated gene transfer remains difficult to quantify, it is clear that replicationcompetent virus can pose a significant safety concern. Thymic lymphomas with multiple retroviral integrations developed in primates receiving transplants of bone marrow previously exposed in culture to stocks of defective retrovirus which had been spiked with known replicationcompetent virus (48). Adeno-associated virus (AAV), a parvovirus requiring adenovirus to complete its life cycle, has attracted significant attention as a vector system by virtue of its selective integration into a region of chromosome 19 and the notion that the virus can integrate into resting cells (185,222). The relevant area of chromosome 19, however, has been shown to be near a breakpoint commonly observed in chronic lymphocytic leukemias (132),rendering unclear the advantages of this targeted insertion. Furthermore, vectors in which the viral genome has been

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replaced with the gene under study appear to lose this site specificity. Although of great importance scientifically, little convincing data have yet been proffered regarding the ability of AAV to integrate, as opposed to infect, nonreplicating cells. This array of high-efficiency gene-transfer systems presents a powerful armementarium for strategies aimed at enhancing the immune response to Cancer. Application of these techniques to both murine and human tumor systems is increasing at an exponential pace. We now discuss selected areas of these investigations which appear particularly promising. IV. Genetic Modification of Tumor Cells

The use of tumor cells as therapeutic vaccines against cancer has been explored throughout this century. Although a few convincing responses have been observed (139),most regressions have been only partial and of limited duration. Many attempts to enhance the immunogenicity of these vaccines have yielded only modest gains (155). These manipulations have included the addition of Bacitlus Calrnette-Guerin (BCG) or Corynebacteriurn paruurn to inactivated tumor cells, infection of tumor cells with Vaccinia or Newcastle virus, preparation of tumor cell lysates, chemical modification of tumor cells, and the coadministration of allogeneic cell lines. The development of more potent strategies has been hindered by the limited understanding of which properties of tumor cells and which immunologic mechanisms are critical to successful vaccination. The cloning of cytokine genes has profoundly influenced efforts to elucidate the cellular and molecular basis of vaccination. These small glycoproteins play central roles in regulating the activities of the hematopoietic and lymphoid elements involved in immunologic responses (2). Delineating the precise function of these molecules in uivo is complicated, however, by the apparent redundancy and pleiotropy of cytokine activities as well as by the complex interactions among these molecules. The availability of recombinant proteins in pharmacologic quantities and the ability to manipulate these products at the genetic level has allowed the testing in an empiric fashion of the effects of these molecules on the host response to cancer. These investigations have yielded remarkable insights relevant not only to the development of more effective cancer treatments, but also to the improved understanding of cytokine biology. Forni and associates pioneered the application of recombinant cytokine to the site of an existing tumor mass to induce a local antitumor

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response (59). These studies indicated that injections of IL-2 stimulated the influx of macrophages, neutrophils, eosinophils, natural killer cells, and lymphocytes which led to the rejection of the growing tumor and, in some cases, to protection against subsequent challenge of live tumor (58). Many other cytokines were tested in this assay, although IL-2 proved most potent. An informative extension of this strategy involved the injection of cytokine into the draining lymph node rather than to the site of the primary tumor. Interestingly, IL-4 and IL-1 demonstrated much greater activity when administered in this way compared to peritumoral injection (17). Collectively, these studies indicated that defects in cytokine production either locally at the site of a growing tumor or in the draining lymph node were related to tumor progression, and suggested that these defects might be amenable to therapeutic intervention. A technical advance in this approach, allowing superior delivery of cytokine to the site of tumor, was the genetic modification of tumor cells to express cytokine genes. The experiments of Tepper and coworkers (214) convincingly demonstrated that tumor cells engineered to secrete a particular cytokine could profoundly influence the host antitumor response in ways previously not revealed by local injections of recombinant cytokine. Although initially intended to provide a continuous supply of systemic IL-4, plasmacytoma cells expressing this gene product surprisingly were rejected by syngeneic hosts. Macrophages and eosinophils were abundant at the transfected tumor site, and subsequent work provided some evidence that eosinophils were essential for tumor rejection (215). Tumor cells expressing IL-4 were also eliminated by immunocompromised mice, leading these investigators to conclude that the effect represented an inflammatory, rather than immune, response. An important characteristic of this system was the achievement of a threshold amount of IL-4 production, with lower levels stimulating only partial protection. While sufficient local IL-4 secretion resulted in the rejection of unmodified tumor cells admixed with the transfected cells, parental tumor implanted at distant sites was unaffected. The immunologic response to tumors engineered to express particular cytokine genes was first suggested by several studies examining the effects of tumor cells secreting IL-2 (53,65)or interferon-y (52,66,236). Tumor cells were rejected by CD8-positive lymphocytes and natural killer cells when IL-2 was synthesized, and by natural killer cells, T cells, and macrophages when interferon-y was produced. Rejection of transfected cells in some cases also conferred protection against subsequent challenge of parental cells. One group argued that the

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function of IL-2 in this system was to bypass, in the tumor bearing host, a defective helper arm of the immune system (53).More detailed analysis of this concept has made its acceptance problematic, however, as an extensive histologic study of the rejection of an IL-2-transfected sarcoma revealed a striking absence of infiltrating lymphocytes at the site of the tumor (5).In contrast, significant paracortical enlargement was present in the draining lymph node, suggesting that this was the important site of T cell activation, These discrepancies conjure up the recurring controversy regarding the relative importance of peripheral versus central mechanis-s of immunologic sensitization (6,206). A flurry of studies examining the effects of engineering tumor cells to express a large number of different cytokine genes followed these initial reports. One useful way to classify these experiments is according to whether rejection of the transfected tumor cells occurred. Several genes proved capable of inducing this rejection, at least in some model systems. In addition to IL-2, IL-4, and interferon-? already discussed, these included tumor necrosis factor (TNF) (3,13, 213), IL-1 (50),IL-3 (165), IL-6 (145,161), IL-7 (1,86,131), IL-12 (208), granulocyte-colony stimulating factor (34,203) JE (177), and IP-10 (124). The effector cells varied with the gene introduced, but included macrophages, eosinophils, neutrophils, natural killer cells, and CD4and CD8-positive lymphocytes. In contrast to these molecules, other cytokines did not stimulate rejection of the genetically modified cells. These included granulocyte-macrophage-colony stimulating factor (GM-CSF) (51),macrophage-colony stimulating factor (49), IL-5 (118), IL-9 (227), and transforming growth factor+ (27,218). It remains unclear what characteristics of the tumor cells and what mechanisms of the host response are critical to the rejection or growth of the cytokine transfected tumor cells. Clarification of these issues is an important area for further study and will benefit efforts directed not only at modifying tumor cells, but also at optimizing the systemic administration of recombinant cytokines. Most relevant to cancer vaccination strategies are the genetic manipulations of tumor cells which lead to protection against subsequent challenge of wild-type tumor. In addition to the studies mentioned earlier using IL-2 and interferon-? expressingcells, subsequent investigations demonstrated apparently similar immunostimulatory properties oftumor cells engineered to secrete IL-1(50), IL-4 (73),IL-6 (145, 161), IL-7 (1,131), IL-12 (208), T N F (3),and GM-CSF (51).Although this long list of molecules hints at another illustration of redundancy in cytokine function, it is necessary to emphasize that most of these studies have not properly evaluated the ability of these molecules

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to enhance antitumor immunity. As previously discussed, the classic studies of Prehn and Main convincingly illustrated that the immunogenicity of some murine tumors could be revealed by vaccination with irradiated tumor cells or by surgical excision of a growing tumor mass (163,170). Yet many of the current gene-transfer studies only compare the level of protection accompanying the rejection of live, cytokinesecreting cells to that of either naive hosts or animals inoculated with live, nontransfected cells. Many of the tumors examined in these studies were considered by the investigators to be either non- or poorly immunogenic based on the ability of a small number of wild-type tumor cells to grow progressively in naive hosts. Our own studies, however, indicated that some of the models used to show the activity of IL-2 (CT-26 colon adenocarcinoma, CMS-5 fibrosarcoma), interferon-y (CMS-5, C1300 neuroblastoma), IL-4 (RENCA renal adenocarcinoma), and TNF (WP-4 fibrosarcoma) were significantly immunogenic following irradiation (51). In particular, we found that ifcomparable numbers of irradiated, wild-type vaccinating cells and live, wild-type challenge cells were used as reported in the experiments involving live, genetically modified tumor cells, equivalent levels of systemic immunity could be achieved. Other workers confirmed these results in additional model systems (87). This inherent immunogenicity markedly complicates the assessment of the immunostimulatory properties of these molecules. In such systems, it is necessary to perform careful titrations of genetically modified cells and wild-type irradiated cells to properly evaluate the magnitude of enhancement of systemic immunity. Ideally, genetically modified cells should be irradiated too, ensuring equivalent amounts of vaccinating cells and tumor antigens. In models in which parental cells have significant vaccinating ability, the absolute amount of tumor antigen is a dominant variable, and very small changes in the numbers of vaccinating cells (as little as twofold variations) can give rise to profound differences in the efficiency of protection, depending on the precise conditions of the assay (51).This finding is especially relevant to those experiments which compare live, genetically modified tumor cells with irradiated, nontransfected tumor cells. Many live, genetically modified cells persist and likely replicate in the host for some time before they are rejected, resulting in a comparatively larger tumor antigen inoculum relative to that presented b y equal starting numbers of irradiated, nontransfected cells. The appropriate control for this persistence is complicated, however, because the mechanism of cell destruction could be critical for vaccination. Maneuvers which kill wild-type cells by virtue of “suicide genes,” such as thymidine kinase

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(16) or cytosine deaminase (144) (together with systemic infusion of the appropriate antimetabolite), may not reproduce the same effects as inoculating irradiated parental cells. Furthermore, hyperimmunization schedules which typically consist of administering irradiated, wild-type tumor cells on a weekly basis also are unlikely to model the kinetics and magnitude of antigen exposure following injection of live, genetically modified cells. While the resolution of these difficulties is somewhat uncertain, it is nonetheless critical to bear in mind when analyzing those experiments in which live and irradiated cells are compared. In addition to establishing the proper baseline value of immunogenicity, experiments evaluating the efficacy of genetically modified tumor cells as immunogens should also compare the activity of several different gene products within the same tumor model in order to determine the rank order of potency. The development of versatile retroviral vector systems has allowed us to perform such an analysis in several different tumor systems (51). In the B16 melanoma model, for example, of10 different cytokines and other potential immunomodulators tested, GM-CSF was by far the most potent molecule for augmenting antitumor immunity, with IL-4 and IL-6 demonstrating weaker, though detectable, activities. Of note in our analysis, we were unable to reveal activity of IL-2, interferon-y, or tumor necrosis factor. Probably relevant to this difficulty is the fact that irradiated, parental B16 cells generate little, if any, significant immunity, in contrast to those model systems studied earlier with these genes. An intriguing property of the immunostimulation generated by GM-CSF was that inactivation ofthe GM-CSF-secreting cells was required, as live cells grew progressively and killed the host from toxicities associated with high systemic levels of the cytokine. Inactivation of the GM-CSF-expressing cells could be accomplished either by the coexpression of IL-2 (cells secreting IL-2 alone were rejected but did not stimulate significant immunity; live cells secreting both molecules were rejected) or b y irradiation (which did not adversely affect cytokine secretion). The immunostimulatory properties of GM-CSF could also be demonstrated in the immunogenic systems previously studied by others after appropriate conditions were established to measure true enhancement over the level achieved b y irradiated, parental cells alone. One concern in comparing different studies of genetically modified tumor cells is that the amount of cytokine produced by different vector systems may be an important determinant ofthe ability to detect immunostimulation. Tumor cells engineered to secrete cytokines by our retroviral vectors synthesized comparable amounts of cytokine as had

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been reported by others. Furthermore, we tested serial 10-fold dilutions of these cytokines over a 10,000-fold range without revealing new antitumor properties at lower doses (unpublished results). While admittedly this survey could miss activities evident over only a very narrow range of cytokine levels, achieving such fine control would pose substantial difficulties in the clinical application of these studies. A second issue to consider in comparing investigations of different model systems is whether clones or populations of tumor cells were used for vaccination. The very high efficiency of gene transfer achieved with our retroviral vectors obviates the requirement for selection of transduced cells, and allows the testing of tumor populations as immunogens (51).Most other studies, in contrast, examined the properties of highly selected clones generated by a variety of less efficient genetransfer techniques. Significant variation in the vaccination efficacy of different clones is apparent in at least some of these experiments (25),and it remains unclear how frequently bias is introduced by the selective reporting of results attained with the most “potent” clones. The classic immunization studies in murine tumor models illustrated that these tumors were often antigenically heterogeneous (lo), so considerable caution should be applied to the interpretation of studies which examine only small numbers of clones, as both positive and negative influences of clonal variation (due only to properties intrinsic to the cells) could alter the immunostimulation attributed to a given gene product. Our finding that GM-CSF was the most potent molecule for inducing antitumor immunity was surprising, since most previous studies of the cytokine had concentrated on its activities as a hematopoietic growth factor (134). The strength of immunostimulation effect was also noteworthy, as BCG and C. parvum were ineffective in several of the systems in which GM-CSF-expressing tumor cells were highly active (186, unpublished results). To begin to elucidate the mechanisms underlying this enhancement, a histologic analysis of relevant vaccinating and challenge sites in the B16 melanoma model was performed (51). At the injection site of irradiated, GM-CSF-expressing tumor cells, an abundant influx of macrophages, eosinophils, and lymphocytes was evident, in contrast to the site of irradiated, parental cells where only a scant lymphocytic infiltrate was present. Unfortunately, because of the lack of reliable antibodies, we were unable to assess morphologically the participation of epidermal Langerhan cells and dermal dendritic cells in this response. (In humans, however, subcutaneous administration of recombinant GM-CSF increases the number and activity of these cells (106)which are readily identified by mono-

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clonal antibodies directed against C D l a or S100.) The draining lymph node of animals vaccinated with GM-CSF-transduced tumor cells displayed dramatic enhancement of the paracortical T cell areas and, to a lesser extent, follicular hyperplasia, as opposed to the relatively unstimulated lymph node of animals vaccinated with irradiated, parental cells. The paracortical T cell area is the site to which the antigenpresenting cells of the epidermis and dermis migrate after stimulation (125),and its enlargement in successfully vaccinated animals suggests that it participates in T cell priming. Finally, the challenge site of GMCSF vaccinated animals was characterized by abundant eosinophils, macrophages, and lymphocytes, in contrast to the only occasional lymphocyte evident in animals vaccinated with wild-type irradiated cells. The recritment of eosinophils was especially intriguing, suggesting the evolution of a TH2-like response, rather than the T H 1 delayedtype hypersensitivity reaction which might have been expected (140). Our findings in the B16 system of the weak but detectable activity of IL-4 and the lack of activity of interferon-? were consistent with this observation (63,191). The role of GM-CSF in promoting TH2like resposes in vivo and the relative efficacy of different helper T cell subsets in antitumor immunity are important areas for future studies. The involvement of T lymphocytes in the GM-CSF-stimulated response was confirmed in several ways. By administering neutralizing monoclonal antibodies to a series of animals, we showed that both CD4- and CD8-positive lymphocytes were essential for vaccination at both the priming and the effector phases, whereas natural killer cells were irrelevant (51). CD4-dependent proliferation and tumorspecific CD8 blockable killing also were evident in preparations of draining lymph node and spleen. Since the B16 melanome line studied is MHC class 11 negative, and uninducible with interferon-? treatment, the requirement for CD4-positive T cells strongly suggests that the relevant antigen-presenting cells in this response are derived from the host. The abundant macrophages and (presumably) dendritic cells present at the vaccinating site are attractive candidates, as GM-CSF can increase their numbers from hematopoietic progenitors and augment their function as antigen-presenting cells (24,93,202). Collectively, these results imply that irradiated GM-CSF-expressing tumor cells initiate and amplify an antitumor immune response at the level of antigen-presenting cells, thereby leading to the activation of CD4and CD8-positive lymphocytes. This strategy for tumor vaccination seems preferable (and more potent) to earlier attempts aimed at bypassing a putative defective helper arm (53).

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In addition to vaccination studies in the naive host, several groups have investigated models of preexisting tumor, applying genetically modified cells as therapy. In this work, GM-CSF (51),IL-2 (36), IL-6 (161), interferon-y (162), and IL-4 (although this study failed to assess the efficacy of wild-type irradiated cells) (73) were effective, albeit only against small tumor burdens. This latter finding has provoked much controversy. Some expressed concern that the inability to eradicate large tumor burdens implies that the potency of immunostimulation is only modest, that the effector cells are unable to traffic effectively to bulky, abnormally vascularized tumor masses, and that the growing tumor mass induces a state of peripheral tolerance (216). Indeed, it has been reported that the T cell receptor proteins of tumorbearing animals display evidence of dysfunction (136).While all of these reservations certainly justify careful contemplation, it should also be considered that the lack of efficacy against bulk disease in these systems is simply due to kinetics; the growth rate of these tumors may be sufficiently high so as to preclude the opportunity for developing an adequate immune response. Ultimately, resolution of this vexing issue will require clinical studies in cancer patients with established metastatic disease. An additional point relevant to the analysis of therapy studies in murine tumor models is that injection of cytokine transfected tumor cells result in the delivery of cytokine both locally and systemically. In this regard, recombinant IL-2, IL-4, IL-6, TNF, and interferon-y (but not GM-CSF) all demonstrate some antitumor activity when administered systemically to tumor-bearing hosts (122,142,157). This efficacy likely reflects the augmentation of natural immune effector mechanisms (74),rather than those of antigen-specific immunity. Current gene-transfer experiments have not included controls which adequately model this systemic release (delivery of equivalent amounts of cytokine by transfected fibroblasts implanted at a site distant from irradiated, wild-type tumor), rendering it difficult to evaluate the relative contributions of natural versus specific immunity to the therapeutic effect. Studies which examine the interactions of systemic cytokine administration and vaccination with cytokine transfected tumor cells will be of great interest. One apparent exception to the general inability of transfected tumor cells to cure substantial burdens of preexisting tumor is the system employing antisense transcripts to insulin-like growth factor-1 (IGF-1). In rat glioma and murine embryonal carcinoma models, the transfection of tumors with constructs expressing antisense IGF-1 results in an impressive CD8-positive T cell response capable ofeliminat-

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ing not only the genetically modified tumor cells, but also an established, bulky, nontransfected tumor (223,224).It will be instructive to test the efficacy of cytokine transfected cells in these systems as well, for unique properties of these tumor models may be associated with a more general ability to eradicate larger tumor burdens with vaccination strategies. An intriguing feature of the IGF-1 antisense experiments is that the immunostimulatory effect is restricted to the transfected cells, as the coinjection of irradiated wild-type tumor cells of a second type does not generate protection against subsequent challenge of the second tumor. Apparently, the phenotypic changes associated with inhibition of IGF-1 expression in the transfected cells lead to a potent T cell response (which does not seem to require priming) against the tumor. More detailed studies of the mechanisms involved and the relevant target antigens are dearly warranted. In addition to engineering tumor cells to express cytokines, investigators are introducing other genes of immunologic importance in order to provoke an antitumor response. One approach involves the liposomalmediated delivery into a growing tumor mass of D N A encoding an allogeneic MHC class I molecule. Reminiscent of earlier studies which employed the coinjection of allogeneic (or xenogeneic) and autologous tumor cells (96,98,237),this work indicated that syngeneic tumor rejection could occur as a by-product of the alloreactive response (YO, 149,160).The development of immunity to subsequent challenge with parental tumor was also observed. Unfortunately, it is difficult to evaluate the potency of the immunostimulation from the data presented in this study, as systemic immunity can also be generated in this model with irradiated, wild-type tumor cells alone. It also seems likely that the enhancement of immunity associated with this approach involves the local production of cytokines. Many groups are attempting to endow tumor cells with the properties of an antigen-presenting cell by introducing selected molecules. The first of these studies examined the characteristics of tumor cells expressing high levels of syngeneic MHC class I molecules by virtue of gene transfer (89,127,211).These tumor cells were rejected by syngeneic hosts which then became immunized against low levels of subsequent parental tumor challenge. Of considerable interest was the finding that MHC class I expression sometimes resulted in a complex phenotype, with paradoxical enhancement of metastasis formation, perhaps related to decreased recognition of the transduced tumor by natural killer cells (72,109).The introduction of MHC class I1 molecules in some systems has also resulted in tumor rejection and systemic immunity (156). An intriguing variation of this approach was to

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transfect the gene encoding HSP 65 into tumor cells with the intent of enhancing loading of antigenic peptides into tumor MHC molecules; tumor rejection and subsequent immunity developed in the one model examined (123).The effects of engineering tumor cells to produce the costimulatory molecule B7-1 have also been reported in several studies (9,30,220).While B7-1 expression has sometimes led to the rejection of tumor cells by syngeneic hosts, this has been limited to those tumors shown to be significantly immunogenic in irradiatiodchallenge experiments (31). Although protection against subsequent parental tumor challenge has accompanied the elimination of B7-1 transfected cells, the magnitude of immunostimulation has been difficult to assess. Live B7-1-expressing tumor cells persist for a significant period in the host before rejection, complicating, as discussed earlier, comparison with the vaccinating ability of an equivalent burden of wild-type tumor cells. A different approach for the genetic modification of tumor cells involves the in vivo delivery of the herpes simplex thymidine kinase gene by either retroviral (43)- or adenoviral-mediated gene transfer (32).While considerable uncertainty persists regarding the ability of retroviral vectors to infect a growing tumor mass in vivo, this is not the case for adenoviral vectors which can accomplish this efficiently. The systemic administration of ganciclovir following infection selectively kills thymidine kinase-expressing cells. This strategy has demonstrated striking antitumor activity in some rodent models which in fact appears to exceed that expected from the number of tumor cells transduced, raising important questions regarding the underlying mechanisms. Although much has been emphasized regarding the biochemical basis of this “bystander effect,” it is likely that an immunologic component contributes to the rejection in normal hosts. Indeed, a study of hepatic metastases treated in this fashion revealed a significant influx into the tumor sites of CD4- and CD8-positive lymphocytes and macrophages (23). In this context, it would be of great interest to compare the efficacy of thymidine kinase-induced tumor regression with that achieved by injecting appropriate numbers of wild-type irradiated cells into the growing mass. Many of the strategies using gene transfer to enhance tumor immunogenicity have either recently entered or will shortly enter clinical trials in cancer patients. Those efforts involving the ex vivo modification of tumor cells should seek to minimize the number and complexity of in uitro manipulations in order to best maintain antigenic heterogeneity. Although a number of candidate tumor antigens have been identified and are guiding the selection of some tumor cell lines as vaccines (67),

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it is worth reemphasizing that the appropriate targets for immunotherapy still remain to be defined. Given this uncertainty, vaccination with autologous tumor cells would seem to maximize the opportunity of developing a clinically meaningful response. We have recently demonstrated in this regard the ability to efficiently infect with a retroviral vector short-term primary explants of a variety of human tumors (97). The administration of irradiated tumor cells, which are unable to replicate but remain metabolically active, should also be emphasized, as this reduces the risk of inoculating tumor cells potentially rendered more virulent by virtue of insertional mutagenesis or in vitro manipu1,'3t'1011. V. Antigen-Based Vaccination Strategies

The identification of candidate tumor antigens immediately suggested their potential use as immunogens for vaccination studies. Both peptide and full-length protein formulations are being tested. Free peptides in general have been poor inducers of cytotoxic T cell responses in uiuo, stimulating considerable effort to improve methods for peptide vaccination. Limited understanding of the mechanisms underlying the deficiencies of free peptide administration, however, has made this effort somewhat empirical. Among the most exciting approaches are the incorporation into the vaccine of lipid moieties, either as liposomes or lipopeptide conjugates (46,83,150,166,187,209), and generic helper T cell epitopes (194). A particularly novel strategy emphasizes the use of recombinant p2-microglobulin to stabilize the ternary complex of MHC class I, p2-microglobulin, and peptide molecules (175). Soluble proteins in a variety of new adjuvants suitable for use in humans show great promise for generating potent cellular and humoral responses as well. In this context, the activity of a fusion protein engineered to express both the idiotype of a B cell lymphoma and GM-CSF is noteworthy (212). The potency of linking soluble proteins to several different types of particles has stimulated interest in specific antigen-presenting cells specialized in processing exogenous, particulate antigens for presentation to cytotoxic lymphocytes (22,115,173,174). The critical role of professional antigen-presenting cells in vaccination has inspired efforts to manipulate these cells directly so as to achieve a more potent immunologic response. Dendritic cells and activated B cells are currently the most attractive targets for these investigations, as several studies in vitro and in vivo suggest that they are particularly effective in stimulating both CD4- and CD8-positive

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lymphocytes (91,92,94,99,105,107,113,116,120,126,135,189).Methods have recently been developed to generate large numbers of dendritic cells from hematopoietic progenitors (24,93) and thus will allow the careful testing of the functional properties of these cells in a number of vaccination strategies. Indeed, splenic dendritic cells pulsed with peptide have been shown to stimulate significant cytotoxic T cell responses against HIV and p53 in mice (151,210,240). Langerhans cells and dendritic cells incubated with tumor fragments can also prime an antitumor immune response following inoculation into syngeneic hosts (55,76). An interesting recent study fused tumor cells with activated B cells to generate a specific antitumor immune response, presumably by exploiting the specialized antigen-presenting capabilities of the B cells (81). The application of gene-transfer techniques is likely to impact dramatically on efforts to use antigen-presenting cells for the augmentation of antitumor immunity. Studies of influenza virus presentation by splenic dendritic cells demonstrated that infection of these cells was markedly superior in stimulating a CTL response in comparison to incubating the cells with inactivated virus (152). Introduction of DNA into dendritic cells via liposomes resulted in successful antigen presentation in uitro to CD4- and CD8-positive lymphocytes (150). Retroviral-mediated gene transfer of Epstein-Barr virus immortalized B cells with a gene encoding the PML-RAR protein was effective in generating antigen-specific CD4-positive lymphocytes from the peripheral blood of healthy donors (64). Investigations of the efficacy in uiuo of retrovirally transduced dendritic cells and B cells expressing candidate tumor antigens are currently under way in our laboratory. In addition to studies examining professional antigen-presenting cells as immunogens in uiuo, efforts to investigate the potential of a variety of other cell types are also warranted. I n this regard, fibroblasts expressing the human papillomavirus E7 gene generated a potent immune response upon transplantation into syngeneic hosts which led to protection against subsequent challenge with E 7 expressing tumor cells (29). Although fibroblasts themselves are unlikely to serve as the relevant antigen-presenting cells in this approach, they nonetheless can function as a reservoir for release of antigen, which is then processed by host professional antigen-presenting cells. Their use in this fashion is similar to tumor cell-based vaccinations, and coexpression of genes like GM-CSF should enhance their immunostimulatory properties. Keratinocytes, endothelial cells, and myocytes should be similarly examined. The injection of naked DNA in uiuo has also been shown to engender strong CTL responses, particularly against HIV,

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influenza, and carcinoembryonic antigen (37,172,226,235). While the precise mechanism of priming in these cases is uncertain, processing of either the naked DNA or the expressed protein by host professional antigen-presenting cells seems likely. Antigen-based vaccination schemes offer perhaps the most direct test of the potency of potential tumor antigens in cancer immunotherapy. It is important in this regard to critically compare the efficacy of antigen and tumor cell-based strategies. While it is tempting to speculate that manipulations using professional antigen-presenting cells will prove superior, considerable uncertainty persists. Little is currently known about the optimal antitumor effector populations and their relative stimulation by different vaccination strategies. Ultimately, the combination of tumor cell and antigen-based vaccines may prove most potent. VI. Adoptive lmmunotherapy

The in vitro expansion and subsequent infusion of tumor-specific T cells into tumor-bearing hosts has proven efficacious in a variety of murine tumor models (28,77,78). Both CD4- and CD8-positive T cells have activity, apparently independent of the MHC status ofthe tumor, suggesting that multiple antitumor effector mechanisms can be recruited. Tumor-reactive T cells have been generated from several sources in these studies, including spleen, peripheral blood, and the site of growing tumor. Administration of tumor infiltrating lymphocytes (TILs) in conjunction with recombinant IL-2 has demonstrated clear efficacy in some patients with melanoma and renal cell carcinoma (180,181,217). Adoptive transfer of lymphokine-activated killer cells, the heterogeneous population of antitumor effectors obtained by culture of peripheral blood mononuclear cells in high doses of IL-2, in conjunction with systemic administration of high doses of recombinant IL-2 has also shown activity in some tumor models and patients, although the relative importance of the infused cells versus the IL-2 remains unclear (79,143,178,179,183). Gene-transfer techniques are likely to have many important applications in optimizing adoptive immunotherapy efforts. Indeed, the first clinical trial of gene transfer in humans involved the marking by retroviral-mediated gene transfer of TIL cells (182). This study revealed that TILs were rapidly cleared from the circulation and only inefficiently localized to the tumor after reinfusion. These results highlighted the important issue of whether direct cytolytic activity of these cells was relevant to the observed antitumor effects; current interest

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in this area has shifted to the patterns of cytokines released (8,188). Because TILs do traffic, albeit inefficiently to tumor sites, more recent investigations have sought to infect these cells with viruses expressing cytokine genes. Efficient transduction of TILs has proved technically difficult, although efforts to improve the gene transfer and then characterize the efficacy of this approach in model systems are clearly warranted. One of the fundamental issues in strategies employing TILs is whether the T cells present at the site of a growing tumor mass are the most appropriate for ex uiuo expansion and adoptive transfer. It is of considerable interest that E 1A-specific T cell clones (generated from spleen) can eradicate bulky E1A-expressing tumors in mouse models (108)and that cytomegalovirus (CMV)-specific human T cell clones (generated from peripheral blood) can reduce the incidence of CMV pneumonia in bone marrow transplant recipients (171). Improved antigen and tumor cell-based vaccination strategies may lead to the generation of more potent TILs. In this context, tumors expressing IL-2, IL-3, or interferon-y have generated TILs with enhanced cytolytic activity in uitro (165,168,193). Studies examining the TILs stimulated by GM-CSF-expressing tumor cells are under way. Lymphocytes harvested from the draining lymph node after vaccination represent an additional attractive target. This approach has already demonstrated some activity in mice vaccinated with irradiated tumor cells admixed with C. paruurn and is currently being tested in patients (26,195). T cells primed and expanded in uitro by professional antigenpresenting cells should also be evaluated. VII. Reduction to Practice

The convergence of multiple areas of research in tumor immunology has generated considerable enthusiasm that immunologic approaches to cancer treatment will prove successful. The identification of tumor antigens, the cloning of cytokine genes, improved understanding of antigen-presenting cell function, and more detailed information regarding T cell activities have provided tumor inimunology with a more solid foundation than at any time before. The diversity of gene-transfer systems is well suited to broad application in the many approaches to cancer immunotherapy. These systems have already impacted dramatically on studies involving the genetic modification of tumor cells and will likely figure prominently in efforts to modify antigen-presenting cells and tumor-reactive T cells. Clinical studies already under way using gene-transfer techniques

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will begin to define the potential efficacy of these strategies in patients. These trials early on should consider immunologic interventions within the broader context of other cancer treatments as well, for potential synergies with chemotherapy, radiation therapy, systemic cytokines, angiogenesis inhibitors, and surgery are all ripe areas for study. If activity of one of the immunologic approaches can be established, the need to make this treatment more widely available will catalyze the search for simplifying ways of accomplishing gene transfer. 111 uiuo strategies which employ liposomes, synthetic conjugates, or adenoviral vectors for the delivery of genes to growing tumor masses i n situ are very attractive in this regard. Immunologic interventions, however, may critically depend on characteristics of the local microenvironment-blood flow, stromal architecture, abundance and activation of hematopoietic and lymphoid elements, and cellular trafficking patterns-which are heavily influenced by the progressing tumor. The ectopic transplanation of ex uiuo manipulated cells into a tumor-free microenvironment may represent an important element in the development o f a more effective antitumor response. Careful studies in model systems will be necessary to properly assess these competing concerns. Ultimately, treatments which combine the most favorable features of ex uiuo and in uiuo strategies may prove most efficacious and practical. Gene-transfer technology is likely to change the care of cancer patients and in so doing help reveal the intricacies of the host-tumor relationship.

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233. Wagner, E., Zatloukal, K., Cotten, M., Kirlappos, H., Mechtler, K., Curiel, D. T., and Birnsteil, M. L. (1992). Coupling of adenovirus to transferrin-polylysine/DNA complexes greatly enhances receptor-mediated gene delivery and expression of transfected genes. Proc. Natl. Acad. Sci. USA 89, 6099-6103. 234. Wagner, E., Plank, C., Zatloukal, K., Cotten, M., and Birnstiel, M. L. (1992). Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin-polylysine-DNA complexes: Toward a synthetic virus-like gene-transfer vehicle. Proc. Natl. Acad. Sci. USA 89, 7934-7938. 235. Wang, B., Ugen, K. E., Srikantan, V., Agadjanyan, M. G., Dang, K., Refaeli, Y., Sato, A. I., Boyer, J., Williams, W. V., and Weiner, D. B. (1993). Gene inoculation generates immune responses against human immunodeficiency virus type 1. Proc. Natl. Acad. Sci. USA 90,4156-4160. 236. Watanabe, Y.,Kuribayashi, K., Miyatake, S., Nishihara, K., Nakayama, E., Taniyama, T., and Sakata, T. (1989). Exogenous expression of mouse interferon y cDNA in mouse neuroblastoma C1300 cells results in reduced tumorigenicity by augmented anti-tumor immunity. Proc. Natl. Acad. Sci. USA 86, 9456-9460. 237. Watkins, J. F., and Chen, L. (1969). Immunization of mice against Ehrlich ascites tumor using a HamstedEhrlich ascites tumor hybrid cell line. Nature 223, 1018-1022. 238. Weinberg, J. B., Matthews, T. J . , Cullen, B. R., and Malim, M. H. (1991). Productive human immunodeficiency virus type 1(HIV-1) infection of nonproliferating monocytes.]. Erp. Med. 174, 1477-1482. 239. Wu, G. Y., Wilson, J. M., Shalaby, F., Grossman, M., Shafritz, D. A., and Wu, C. H. (1991). Receptor-mediated gene delivery in vivo. Partial correction of genetic analbuminemia in Nagase rats. I . Biol. Chem. 266, 14338-14342. 240. Yanuck, M., Carbone, D. P., Pendleton, C. D., Tsukui, T., Winter, S. F., Minna, 3. D., and Berzofsky, J . A. (1993). A mutant p53 tumor suppressor protein is a target for peptide-induced CD8+ cytotoxic T-cells. Cancer Res. 53, 3257-3261. 241. Zoller, M., Strubel, A., Hammerling, G., Andrighitto, G., Raz, A., and Ben-Ze’ev, A. (1988). Interferon-y treatment of B16 melanoma cells: Opposing effects for nonadaptive and adaptive immune defence and its reflection by metastatic spread. Int. 1. Cancer 41,256-266.

INDEX

AAP, see Proteins, acute phase response N-Acetylcysteine effect on apoptosis, 250,262-263 inhibition of HIV lymphocyte death, 279 Acquired immunodeficiency syndrome associated lymphopenia, and lymphocyte PCD, 214 exacerbation, 16 murine retroviral models, 233-237 Addressin mucosal, MadCAM-1, 360 vascular, role of GlyCAM-1, 359 Adhesion cascade, 381-389 flowing leukocytes to platelets, 355 leukocyte-endothelial, 376-378 strong, role of integrins, 383-388 tight, pro-T cells to endothelium, 393 Age associated replacement of Vy6 T cells by Vy4,303-304 related extrathymic expansion of V62 T cells, 309 AIDS, see Acquired immunodeficiency syndrome 3-Aminobenzamide, effect on cytolysis, 252-253 Anergy y6 T cells, 313 and peripheral tolerance to cancer, 421 resulting from tolerization processes, 271-272 surviving T cells, 260 T cell, cyclosporin A effect, 262 Ankyrin repeats, interaction with NF-K re1 domain, 3-4

Antibodies to CD2, role in T cell death, 226 F23.1, induction of thymic cell depletion, 274 Antigen-presenting cells for antitumor immunity, 436-437 modification, 438 T cell surface molecules in communication with, 112-139 Antigens and antigen recognition, 318-324 based vaccination strategies, 435-437 CD2, role in T cell death, 226 CD4 and CD8, on IELs, 306-307 CD4-8-, majority of y6 T cells as, 102 CD4/CD8 coreceptor, role in negative selection, 171-172 CDw60, on CD4-8+ T cells, 108-109 mycobacterial, recognition, 321-322 recognition, 124-128 selfand foreign, 297-298 recognition, 319-320 tumor, 418-422 Apoptosis Bcl-2 effects, 137 as consequence of cell damaging agents, 237-240 control during intrathymic development, 168-174 hypothetical steps, 241 inducing signals, 89 induction via TCR/CD3 complex, 216-219 as mechanism of T cell negative selection, 166-168 modulation, functional consequences, 272-280 regulation, multiple pathways, 265-268

455

456

INDEX

and signal transduction pathways, 268-269 susceptibility of murine lymphocytes, 234 target cell, Ca”-independent, 226 T cells immunopharmacological manipulation in uiuo, 254-255 pharmacology, 211-296 role of p53, 138 Aspirin, inhibition of NF-Kactivation at high doses, 19-20 Ataxia telangiectasia, 67-69 Aurin tricarboxylic acid effect on thymic cellularity, 262 prevention of T cell PCD, 251 Autoantigens, Ku, and V(D)J recombination and DNA repair, 55-60 Autoimmune disease induction, prevention by apoptosisinhibitory drugs, 278 treatment, 273-275

B B7, interaction with CD28, 133-134 BALB invariant delta chain, 304, 310, 312, 315, 320 B cells clonal deletion, 257 naive, homing, 350 Bimolecular reactions, simultaneous, 390 Blast cells, localization to gut, 348-349 Blood, fetal, containing pre-T cell, 140 Bloom syndrome, and DNA ligase deficiency, 65-66 Bone marrow derived B cells, clonal deletion, 257 derived cells, and deletion of thymocytes, 170 C Cancer potential of antigens, 437 therapy, gene transfer as, 417-454 Carbohydrates epitopes, tumor-associated alterations, 420 as selectin ligands, 358

Catabolic cascades, inhibition at effector level, 247-253 CD3/TCR complex ligation, 248 mediated peripheral T cell deletion, 219-224 monoclonal antibodies to, 300 second messenger triggered by, 230-232 Cell adhesion molecules CD31 redistribution to cell border, 395 six-lg domain molecule, 375 endothelial, and lymphocytes, 352 GlyCAM-I, as selectin ligand, 359 intercellular, see ICAM-1; ICAM-2; ICAM-3 L1, 200-kDa glycoprotein, 375-376 production during inflammation, 10 VAP-1 and L-VAP-2,377-378 vascular, see VCAM-1 Cell cycle interventions, 248-250 regulation of V(D)J recombination and DSB repair, 70-74 regulatory kinase ~ 3 4 ‘ blockade, ~~, 267 Cell cycle checkpoints Go-GI, and induction of apoptosis, 249-250 mechanisms, 66-69,71-74 mutations in, 29 Cell death ataxia telangiectasia cells, 69 deficient regulation, and disease states, 212 intrathymic, 166-168 T cells, see T cell death Cell degradation, redundant effector pathways, 269-270 Chemokines, in integrin activation, 378-379 Chromosomes 1, CD1 locus, 319 selectins on, 353 2, radiation hybrid with xrs cells, 57 5, transfer into XR-I cells, 52 8, complementation of scid defects, 50-51 14, rearrangement in ataxia telangiectasia, 68

457

INDEX

19, integration of adeno-associated virus, 424-425 22, location of p70K",57 errors, 72-74 Cisplatin, and apoptosis, 237-240 Cleavage model, V(D)J recombination, 38-39 Cleavage reactions, in V(D)J recombination pathway, 30, 33-35 Clones, self-reactive, 213 Cloning, cytokine genes, 425 Coding ends with P nucleotides, 37-38 and RSS ends, passage through DSB intermediates, 33-36 TCHG, hairpin accumulation at, 48 Coding joints formation, microheterogeneity, 39-40 microhomology, 40-42 V-3 cells, 51-52 Coding junctions and RSS junctions, 42 and scid mutant features, 46-48 Coeliac disease, and yG TCR expression, 329 Complement C3, in complement activation pathways, 9-10 C5a, attracting niyeloid cells, 380 Cosignals, inactivation of death programs, 243-245 CTLA-4, T cell receptor for B7, 134 Cycloheximide, effect on apoptosis induction, 265-267 Cyclosporin, effect on thyniocyte death, 173 Cyclosporin A autoimmune side effects, 278-279 blockade of C D 4 + 8 +thymocytes, 267 effect on PCD induction, 261-262 and thymic T cell maturation, 314-315 Cytokines, see also specijic cytokines alarm, 394-395 induced by NF-KB, 10-12 produced by different vector systems, 429-430 regulation of apoptosis, 229-230 role in peripheral clonal deletion, 271-272 in signaling, 369 synthesis coordination by NF-K, 1-2

Cytotoxicity, antibody-dependent cellular, 223

D Deletion autoreactive T cells, 272-275 CD4'8+ thymocytes, 166-168, 170-171, 259 clonal lymphocyte, physiology, 270-272 nascent bone marrow B cells, 257 and peripheral tolerance to cancer, 42 1 peripheral, in uitro models, 215-219 peripheral T cells, CD3/TCRmediated, 219-224 reduced level in Glo or Clo coding ends, 40 RSS junctional, 54 y6 T cells, 313 Dendritic cells, as target for vaccination strategies, 435-436 Development, intrathymic control by C X TCR, ~ 151-165 pre-T cell receptor, 142-151 control of apoptosis during, 168-174 T cells, 139-174 Dexamethasone, induced T cell death, 262-265 Diversity combinatorial, in leukocyte-endothelial cell recognition, 389-390 N region, 301-303 DNA damage, and p53 accumulation, 258 DSBs, role in V(D)J recombination, 32-36 encoding MHC class I molecule, 433 fragmentation, 246-248, 251-253, 262-264,270-271 -Ku complex, 55-57, 61-62 naked and cytotoxic T cell responses, 436-437 linkage of cell-surface protein, 423 nuclear, fragmentation, and PCD, 212-213

458

INDEX

-protein intermediates, 38-39 recombinant, and carcinogenesis, 417 repair, see Double-strand break repair; Repair synthesis repair syndromes, 62-69 transcription inhibition, 240 DNA ligase, deficiency in Bloom syndrome, 65-66 DNA-PK, see Protein kinase, DNAdependent Double-strand break repair relevance of joining activity, 45 and V(D)Jrecombination, 29-85 cell cycle regulation, 70-74 and Ku autoantigen, 55-60 Drugs cytotoxic, and apoptosis, 237-240 therapeutic, and leukocyte homing,

395-396 DSB repair, see Double-strand break repair

Effector cells activated in immune response, 298 cytolytic, generation by CD4-8+ a/3 T cells, 108 trafficking to vascularized tumors, 432 variance with introduced gene, 427 End-joining and nonhomologous recombination,

43-45

and scid mutation, 50 in w s group mutants, 54 Endonuclease in apoptotic cell degradation, 269-270 blockade, in apoptosis inhibition, 251 Endothelial cells, adhesion to leukocytes, 376-378,381-389 Endothelium, leukocyte transmigration,

388-389

Enterotoxin A, staphylococcal action on TCR, 323 deletion of Vp3+ and V P l l +R lymphocytes, 222 Enterotoxin B, staphylococcal beneficial effect, 274-275

induction cytokines, 271-272 thymocyte apoptosis, 216 VP8' thymocyte depletion, 259-260,

262

pleiotropic effects in oioo, 219-224 Eosinophils, recruitment, 431 Epidermal cells, dendritic expressing Ly-5, 299 reactivity, 320 Thy-1', 301-303 Vy5V61, colonization of skin, 317 Epithelial cells thymic, induction of positive selection,

164-165

and thymocyte deletion, 170 Epithelium, intestinal, derived T cell lineages, 110-112 Estradiol, depletion of CD4+8+ thymocytes, 227-229 Extravasation activated T cells in skin, 392 lymphocytes, in high endothelial venules, 347

F Farnesyltransferase inhibitors, in cancer therapy, 417 Feedback loop, NF-KB induction by cytokines and vice versa, 7 Fibroblasts, induction of positive selection, 164-165

FK-506

antiapoptotic effects, 267 inhibition of negative selection,

261-262 Fragmentins or granzymes, cytotoxic T cells containing, 240 peptide hydrolysis by, 243 Free radicals, formation or action, inhibition, 250

G G-CSF, see Granulocyte colonystimulating factor

INDEX

G e n e rearrangements cis-acting elements for, 30-31 D N A sequencing, 33-36 endogenous, 37 lymphoid-restricted, 43 TCR, developmentally programmed, 308-309 TCRy, 142 V a TCR, 112 Vy, developmentally regulated program, 301 Vy6, 31 1 VYJY, 314 Genes, see ulso Major histocompatibility complex; Transgenes

bcl-2

antiapoptotic, 233 apoptosis-regulatory, manipulation, 253-258 characterization, 136-137 defective, and T cell resistance to PCD, 215 inhibition of apoptosis, 250 overexpression, 168, 268, 270 c-myc overexpression and cell death, 249 role in thymocyte apoptosis, 169 cytokine cloning and expression, 425-427 upregulated by NF-KB, 10-12 in DSB repair and V(D)J recombination, 45-55 immune-response, 155 natural and artificial mutations, 90-101 nw-77, encoding orphan steroid receptor, 229 p53 association with apoptosis, 138 dependent pathways in immunodeficiency syndromes, 66-67 role in PCD regulation, 258 pin-I,and PCD, 258 RAG1 and RAG2 active in immature lymphocytes, 311 and signal joint product formation, 48 transcription by CD44-25+ cells, 141

transfection into XR-1 GI2 cells, 53 transient transfection into Bloom syndrome cells, 65 into nonlymphoid cells, 51 and V(D)J recombination, 32-33 responsive, induced by NF-KB, 5-6 suicide, 428-429 TCR, 119-120 TCR-a and -p and -y and -6 generation of diversity, 115-1 16 genomic organization, i 14-1 15 rearrangement and expression, 116-119 transcription inhibition, 243 TCH-P, induction of development, 142-144 Vy4, expressed in lung, 304 G e n e silencer, TCRy, 120 G e n e transfer as cancer therapy, 417-454 retroviral-mediated, 437-438 techniques, 422-425 Glucocorticoid receptors and antigen-driven T cell deletion, 258-260 linomide effects, 264-265 and T cell fate determination, 137-138 Glucocorticoids effect on CD4+8+thymocytes, 166- 170 induced thymocyte apoptosis, 245-246 and T cell death, 227-228,269 Glycoproteins CD28 and negative selection, 172 and T cell costimulation, 133-134 CD44, and T cell development, 134 CD45 characterization, 131-132 role in negative selection, 172 GM-CSF, see Granulocyte-macrophage colony-stimulating Factor Granulocyte colony-stimulating factor, and GM-CSF, in immune response, 10-11 Granulocyte-macrophage colonystimulating factor and antitumor immunity, 429-432 effect on genetically modified cells, 427

460

INDEX

expressing tumor cells, 438 and G-CSF, in immune response, 10-11 Granulocytes, nonrecirculating, 350-35 1 Granzynies and apoptosis, 226 or fragmentins, cytotoxic T cells containing, 240 Grooves, MHC molecules mutations, 161 peptide antigens bound to, 330 Gut, associated tissues, homing to, 391-392 H Hairpin resolution model, for P nucleotide formation, 36-38 Hemopoietic cells, induction of positive selection, 164-165 Hepatocyte growth factor, effects on lymphocytes, 379-380 Herpes simplex, thymidine kinase gene, 434 High endothelial venules lymph node, 353,359,390-391 lymphocyte binding to, role of CD44, 376-377 lymphocyte extravasation in, 347 HIV-1, see Human immunodeficiency virus, type 1 Homing to gut-associated tissues, 391-392 leukocytes and therapeutic drugs, 395-396 integrins in, 362 molecular basis, 389-390 naive B cells, 350 peripheral lymph node, 390-391 skin, 392 Homing receptors, tissue-specific, 316-318 HTLV-1, see Human T cell leukemia virus, type 1 Human herpes virus, type 6, and HIV-1, exacerbation of AIDS disease progression, 16 Human immunodeficiency syndromes, and DNA repair syndromes, 62-69

Human immunodeficiency virus enhanced apoptotic decay, 233-237 inhibition of lymphocyte death, 279 lymphocytic tendency to undergo PCD, 214 and mitosis, 424 type 1, and NF-K, 14-17 Human T cell leukemia virus, type 1, tax protein, 17-18 Hyaluronic acid, CD44 binding to, 376-377

I ICAM-1 interaction with LFA-1 in negative selection, 172 Mac-1 receptor, 370 recruitment of leukocytes to inflammation site, 395 rolling cells containing, 381-382 ICAM-2, and ICAM-1, affinity for LFA1,373-374 ICAM-3, five-Ig domain molecule, 373-374 IELs, see Lymphocytes, intraepithelial Immunity antitumor, antigen-presenting cells for, 436-437 epithelial and mucosal, 297-298 to infections and diseases, role of IELs, 326-329 innate and gene products regulated by NFK, 20-21 role of NF-KB and re1 proteins, 1-27 systemic enhancement, 428-429 and MHC class I1 molecules, 433-434 tumor-specific, 418-419 Immunodeficiency, prevention by apoptosis-inhibitory drugs, 278-280 Immunodepression, systemic, and T cell PCD, 214 Immunoglobulins gene rearrangements, 31, 37-41 aberrant, 63

46 1

INDEX

IgA, antibody concentration on mucosal surfaces, 298 superfamily molecules, 372-376 Immunostimulation, by apoptosisinhibitory drugs, 278 Immunotherapy, adoptive, 437-438 Infection immunity, role of IELs, 326-329 viral and bacterial, induction of NF-KB, 6 and lymphopenia, 233-237 switch from latency to productivity, role of NF-K, 16-17 Inflammation leukocyte recruitment during, 394-395 and T helper cell activation, 106 Inflammatory response, and NF-KB, 9- 14 Influenza virus, and memory yS T cells, 328-329 Inhibitory proteins, IKB description, 2-4 phosphorylation by TNFa, 8 rapid degradation and release o f NFKB, 4-5 Integrins activation, chemotactic molecules in, 378-381 a 4 p l and a 6 p l , 3 7 1 a 4 P 7 and aEp7,371-372

aEpi

defined by monoclonal antibody HML-1,317-318 expression on intraepithelial lymphocytes, 392 pl and p2, 370-371 characterization, 362-370 faniily of adhesion receptors and ligands, 366-367 LFA-1,369-370 subunits, biochemical characterization, 364-365 Interferon-a, effect on L-selectin cell surface density, 353-354 Interferon-y, secreting IEL, 329 Interleukin-1 in induction of AAPs by liver, 10-12 induction of NF-KB, 7-9 NF-KB-induced, 10-12

Interleukin-2 dependent cells, rescue, 244 inhibition of T cell death, 269 production by activated T cells, 18-19 stimulation of natural killer cells, 426-429 Interleukin-4, preferential rescue of Th2 cells, 244 Interleukin-6, inflammatory cytokine, interaction withNF-K, 12-14 Interleukin-7, and TCRp regulation, 142 Interleukin-8, inflammatory cytokine, transcription, 12 Interleukin-2 receptors a, p, and y chains, 134-135 production, 19 Intestine IELs, majority as CD8+,307 infections and diseases, immunity, 329 In uitro models CD3iTCR-mediated peripheral T cell deletion, 219-224 peripheral deletion, 215-219 Ionizing radiation and scid defect, 49-51 sensitivity of ataxia telangiectasia cells, 68-69 Irradiation, and apoptosis, 237-240

J Joining model, V(D)J recombination, 42-43

K Keratinocytes, expression of dendritic epidermal cell ligand, 320

1 Leishmaniasis, and y6 T cell accumulation, 326-328 Leprosy, and y6 T cell accumulation, 326-328 Leukemia cells, depletion, 272-275 Leukocytes adhesion to endothelial cells, 376-378. 381-389

462

INDEX

migration, 345-351 recruitment during inflammation,

394-395

and transendothelial migration,

388-389

Leukotriene B4, attracting myeloid cells, 38 1 LFA-I activation by chemokine, 381 affinity of ICAM-1 and ICAM-2,

373-374 integrins, 369-370 interaction with ICAM-1 in negative selection, 172 Li-Fraumeni syndrome, and mutant p53 allele, 66-67

Ligands positive selection, 160-164 TCR, inducing deletion of thymocytes, 170- 171 Linomide autoimmune side effects, 278-279 reduction of DNA fragmentation,

263-265 Listeria monocytogenes, and interferony-secreting IEL, 329 Long terminal repeats, HIV-1, 14-17 Lung diseases, immunity, 328-329 y6 T cell receptors of murine IEL, 303-304 occurrence of lymphocytes, 299 resident y6 T cell expansion, 321 Lymph node, peripheral, homing, 390-391 Lymphocytes B, see B cells helper T, see T helper cells hepatocyte growth factor effects, 379-380 HIV, and PCD, 214 intraepithelial CD4 and CD8 on, 306-307 effector potential, 324-326 and immune system, 297-343 origin, 308-311 role in immunity intestine, 329 lung, 328-329 skin, 326-328

T cell lineages, 110-112 as T cells, 298-300 as T cells bearing y6 TCR, 301 memory and effector, 347-350 naive, 389 resident pulmonary, CD4-8- aP T cells, 319 T, see T cells tumor-infiltrating, 437-438 turnover and clonal deletion, physiology, 270-272 Lymphoid precursors, maturation, role of

CD44,377 Lymphoma B cell, and GM-CSF, expression, 435 development, and bcl-2 gene overexpression, 270 T cell, depletion, 272-275 Lymphopenia chronic AIDS-associated, and lymphocyte PCD, 214 and viral infection, 233-237 Lymphoproliferation, treatment,

273-275

M Macrophages cosignals from, 243-245 present at vaccinating site, 431 Major histocompatibility complex class I µglobulin, 380 restoration by single peptides, 162 class I and class I1 and ap T cells, 107-112 deficient mice, 156 and yS T cell reactivities, 323-324 interaction with CD4 and CD8 coreceptors, 129- 131 ligation of aPTCR and coreceptor,

159

peptide binding for T cell recognition, 88 as potential tumor antigens, 420-421 role in y6 T cell function, 103-104 class 11, restricted antigen presentation, 319 determinants, polymorphic, 304

463

INDEX

and positive selection, 164 presentation, 330-331 Maturation, phenotypic, sequence, 141 Memory phenotype, peripheral T cells expressing, 216 Metastasis, and leukocyte homing, 396 Methylation, status in cis, role in gene rearrangements, 30-31 MHC, see Major histocompatibility complex Pz-Microglobulin, chemotactic for pro-T cells, 380 Migration, see QhTransmigration leukocytes, 345-351 Mitosis, and HIV, 424 Models, see also specific models leukocyte-endothelial cell recognition, 381-389 Mouse athymic nude, 309-311 CD2-deficient, 172 IELs, invariant y8 T cell receptors, 301-304 lprllpr, accumulation of CD4-8- ap T cells, 112 MHC class I-deficient, 313 Mtv-7+ and Mtv-7-, 222 p53-deficient, 169 scid and DSB repair and V(D)J recombination, 45-51 genetic reconstitution experiments, 142-143 lymphocytes, 317 rearrangement-deficient, 155-156 thymocytes, 37-38 self-reactive T cells, 274-277 transgenic bclZIscid, 48-49 H-2b and H-2d, 313-316 with knockout mutations, 30-33, 41,89 RAG-’-, 143-145 TAP-l-’-, 162-163 TCR-a, -p, -y, and -6 loci, 118-119 Mucosa, and immunity, 297-298 Mutants, V(D)J recombination/DSB repair and cell cycle arrest, 71-72 leading to chromosome errors, 72-74

Mutations in cell cycle checkpoints, 29 grooves of MHC molecules, 161 natural and artificial, 90-101 scid, 45-51 sxil, 54-55 and transgenes, 147-149 v-3,5142 XR-1.52-53 xrs, 53-54 M ycobacteria, reactivity, 32 1-322 Myeloid cells, chemotactic molecules attracting, 380-381

N Natural killer cells apoptosis, 227 stimulation by interleukin-2, 426-429 Necrosis, versus apoptosis, 240 Neolactosylceramides, sulfoglucuronylcontaining, 358 Neutralization, apoptosis-inducing stimuli, 242-243 Neutrophils, senescent, 270 Nijmegen breakage syndrome, chromosome 7 and 14 translocations, 69 N region, diversity, 301-303 Nucleotides conjugate, 322 in gene rearrangements, 32 mismatched, 44 nontemplated, 308 palindromic, 36-38 two, overlap internal to coding end, 42

0 Oligonucleotides antisense, 249-250 in ligation reactions, 33 Onienn’s syndrome, with V(D)J recombination defects, 63-64

P Packaging cells, development, 424 PCD, see Programmed cell death

464

INDEX

Peptides bacterial formyl, attracting myeloid cells, 381 fusogenic, 423 role in positive selection, 161-164 Pertussis toxin autoimmune side effects, 278 and G protein-mediated regulation, 260-261 suppression of cell death, 246-248 Pharmacology, T cell apoptosis, 211-296 Phosphorylation IKB by TNFa, 8 TAM, by Src family protein tyrosine kinases, 127-128, 130 transcription factors by DNA-PK, 60-62 Plasma membrane, P-selectin translocation, 356 Platelet-activating factor, attracting myeloid cells, 381 P nucleotides, see Nucleotides, palindromic Programmed cell death abnormal regulation, 214-215 CD4-8- cells, linomide effects, 263-265 cytotoxic T cells, TCR-driven, 243 different types, classification, 265-267 HIV-induced, 236-237 induction by Apo-1/Fas, 135-136 leukocyte, 280-281 and nuclear DNA fragmentation, 212-213 role of reactive oxygen species, 250 and T cell persistence, 273-275 and T cell selection, 89 thy mocytes , 166- 170 Proliferative response, T cell clone, 126-127 Protease inhibitors, prevention of DNA fragmentation, 252 Protein kinase DNA-dependent, association with Ku, 60-62 ~ 3 4 ' ~ 'cell , cycle regulatory, blockade, 267 Protein kinase C, role in thymocyte apoptosis, 245-246

Protein kinase Ia, interaction with CD31 TCR complex, 232 Proteins acute phase responsive, NF-KBdependent, 9-10 Bcl-2 downregulation, 151 effect on apoptosis, 137 highly expressed in pro-B cells, 256-258 CD3 complex with TCR, see CD3/TCR complex E , 8, and y subunits, 127-128 T cell lines expressing, 122-124 CD4 and CD8, coreceptors, 129-131 c-Jun and c-Fos, 138-139 c-Myc, and thymocyte cell death, 138 -DNA complexes, 56-57 fusion expressing B cell lymphoma and GM-CSF, 435 L-selectin-IgG, 353, 358-359 G, in chemokine activation of integrin, 379 gp33 cloning of encoding gene, 145 covalent association of TCRp protein, 150 heat-shock, mycobacterial, recognition, 321-322 inhibitory, see Inhibitory proteins KB-dependent, inappropriate expression, 17-18 membrane-spanning Apo- l/Fas induction of PCD, 135-136 and negative selection, 172-173 role in T cell death, 226 Nur-77, nuclear hormone receptor, 137 oncogenic, recognition by T cells, 420-421 p50 and p 5 2 , 3 p70K",located on human chromosome 22,57 ras, mutant, 417 rel, see Re1 proteins tax, activation of NF-K, 17-18 TCR, intracellular selection, 120-121 viral-produced, 16-17

465

INDEX

5 and r )

cells in thymic cortex, 154-155

invariant chains, 127-128 and structure and assembly of TCR complexes, 121-124 Protein-tyrosine kinase p561ck interaction with CD4 and CD8 coreceptors, 129-131 role in signaling through pre-TCR, 150 phosphorylation of TAM, 127-128, 130 Protein-tyrosine phosphatase, CD45R(O), 246 Proteoglycans, CD44 binding to p-chemokine, 383 role in leukocyte-endothelial adhesion, 376-377

i nterleu kin-2-dependent cells, 244

scid coding junction products, 48-49 signal, 241 thymocytes, from PCD, 165 Retinoic acid, 9 4 s isomer PCD inhibition, 245 targeted to steroid receptor family, 259 Retinol, see Vitamin A RNA double-stranded, induction of NF-KB, 6, 11-12 negative strand transcript, 15-16 translation inhibition, 240 Rolling, leukocytes, 382-383 RSS, see Recombination signal sequences RU-38486, effect on CD4 '8' thymocyte deletion, 259

R Reactive oxygen intermediates induction of NF-KB,6-7 scavenging by U P S , 9 Reactive oxygen species generation, inhibition by Bcl-2, 256 role in PCD, 250 Recombination nonhomoIogous, and end-joining, 43-45 site-specific, see V(D)J recombination Recombination signal sequences junctions, and coding junctions, 42, 46,

53-55 in V(D)J recombination, 31-36 Re1 proteins and ankyrin repeats, 3-4 in innate immunity in vertebrates, 1-27 subunits, and promotion of HIV-1 transcription, 15 Repair, double-strand break, see Doublestrand break repair Repair synthesis, across double-strand gap, 44 Reproductive tract, female, invariant y8 T cell receptors, 303 Rescue CD4'8- cells, 158

Sarcoidosis, and TCR junctional regions, 328-329 Scatter factor, see Hepatocyte growth factor Scid syndromes, with V(D)J recombination defects, 62-63 E-Selectin downregulation by degradation in lysosomes, 357-358 ligands CLA, 250-kDa, and SSEA-1, 361-362 CLA+ memory T cells, 392 L-Selectin as pro-T cell homing molecule, 393 T cells positive and negative for, 390-391 widespread distribution on leukocytes, 353-354 P-Selectin cooperative ligand binding site, 354-357 ligands, 360 120-kDa, 361 PSGL-1,360-361 synthesis, induction by alarm cytokines, 394

466

INDEX

Selectins, monospecific ligands, 362 Selection extrathymic, 315-316 intracellular, 120-121 negative inhibition by FK-506, 261-262 T cells, 166-168 thymocytes, 165-174, 171-174 transgenic T cells, 316 positive C D 4 t 8 t cells, 158-159 inducing cells, 164-165 ligands, 160-164 T cells, intrathymic, 139-174 thymic, 311-315 Septic shock, experimental treatment, 280-281 Sialomucins, CD34, and selectin ligand, 359-360 Signaling, cytokines in, 369 Signal transduction by aP TCR complex, 124-128 by NF-KB, 5 pre-TCR, 145-146 Signal transduction pathway artificial inducers, 269 artificial triggering, 230-232 Bcl-2 effects, 256 DNA damage, 29 and intervention of apoptosis, 245-247 leading to NF-KB activation by TNFa, 7-9 and p53,67 TCR-mediated, relation of Apo-lIFas, 136 Skin homing, 392 infections, immunity, 326-328 invariant y6 T cell receptors of murine IEL, 301-303 Sodium salicylate, inhibition of NF-K activation at high doses, 19-20 Spermine, depletion, induction of DNA fragmentation, 252 Spleen lymphocyte migration, 346-347 lymphocyte recirculation, 350 Stem cells self-renewal, 422

switching, developmentally programmed, 308-309 Sugars, as selectin ligands, chemical structure, 355 Superantigens induced deletion and anergy, differential regulation, 272 mycobacterial, 321-323 SEA, see Enterotoxin A SEB, see Enterotoxin B T cells stimulated by, 260-262 Superoxide anion, relation to P-selectin,

355 Surface receptors, alternative, death induction by, 224-227 Systemic lupus erythematosus, overexpression of Fas variant, 274

T TAM, see Tyrosine-containing sequence motifs T cell death induction, 224-240, 238 inhibition, 240-265 by interleukin-2, 269 TCR-mediated, 215-224 T cell receptors a , rearrangement, 150-152

aP

developmental control, 157 induction of CD8+ T cell apoptosis, 223 specificity, 174 aP heterodimer role in signal transduction, 125-127 specificity, 124-125 ap and y6 complexes, 113-128 diversified in human, 305-306 in mice, 304-305 Y6 expression by fetal CD44'CD25+ cells, 141 invariant, 301-304 lung, 303-304 skin, 301-303

INDEX

tongue and female reproductive tract, 303 6, hairpin accumulation at, 48 complex, 128-136 expression at different anatomical sites, 300-306 gene rearrangements, 31, 36-41, 63, 68-69 developmentally programmed, 308-309 ligands inducing deletion of thymocytes, 170-171 male-specific, CD4-8+ thymocytes expressing, 261 mediated T cell death, 215-224 methodological breakthroughs in discovery, 88-89 oligoclonal expansion, 327 precursors, structure and function, 144- 151 signaling in CD4+8+thymocytes, 173- 174 transgenic, 154-156, 159, 166-167 VP-specific antibodies, 275 Vy5 and Vy6 subsets, 103-104 T cells

4

CD4+8-, heterogeneity, function, and specificity, 105-108 CD4 -8+, heterogeneity, function, and specificity, 108-109 CD4-8-, expression of VP8 TCR chains, 109-110 extrathymically derived, 111-112 negative and positive selection, 31 1 reactivities, 318-319 a p and yG differences, 300 lineage divergence, 14 I activation, 18-19 autoreactive, deletion, 272-275 Y6

extrathymically derived, 110-111 function, 104-105 heterogeneity, 102-103 reactivities, 319-324 self-tolerance, 313 specificity, 103-104

467

costimulation, molecules involved in, 132-134 development, 90-101 epithelial, clonotype dominance, 298 fate determination, 136-139 IELs as, 298-300 intrathymic development and selection, 139-174 lineages, 87-88 extrathymically derived, 110-1 12 thymus-derived, 102-110 negative selection, 166-168 paracortical area, 431 precursors, migration to thymus, 347 pro-, homing to thymus, 392-394 recognition, antigen processing for, 420 resident pulmonary CD4 and CD8 antigens, 306 y6 lineage, 303-304 surface molecules, in communication with antigen-presenting cells, 112- 139 V@+, SEB effects, 275 TCR, see T cell receptors Temperature, and apoptosis, 237-240 Terminal deoxynucleotidyl transferase, 117 Testosterone, depletion of CD4+8+ thymocytes, 227-229 Tethering, leukocytes, 382-383 T helper cells Tho, Thl, and Th2, characterization, 106-107 Th2, PCD induction, inhibition, 243 Therapy cancer, gene transfer as, 417-454 by inhibition of NF-K, 19-20 for lymphoid tumors, 282 Thymic cortex, related cells, and rescue, 154- 155 Thymidine kinase, expressing cells, killing, 434 T h ymocytes accumulation of TCRG rearrangements, 33 CD4+8+ commitment to CD4 or CD8 lineages, 156-160

468

INDEX

deletion, blockade by cyclosporin A, 267 depletion by testosterone and estradiol, 227-229 generation and turnover, 151-153 reduction in 6 mice, 146-149 TCR signaling in, 173-174 CD4-8+, mature, expressing malespecific TCR, 261 CD4+8- and CD4-8+, generation from CD4'8' pre-cells, 153-165 negative selection, 165-174 scid with broken DNA coding ends, 37-38 hairpin accumulation at TCRG coding ends, 48 Vj38', SEB-induced depletion, 259-260 Thymus colonizing cells, and TCRp expressing cells, 139-142 derived T cell lineages, 102-110 pro-T cell homing to, 392-394 T cell development and programming in, 88 TNFa, see Tumor necrosis factor a Tongue, invariant y6 T cell receptors of murine IEL, 303 Transcription, KB-dependent, 3 Transcription factors assembly, role in gene rearrangements, 30-31 Dif, in re1 family, characterization, 14 NF-IL6, 12-14 NF-K as inflammatory mediator, 14 inhibition, 19-20 NF-KB activation, 4-6 inappropriate, 14-18 description, 2-4 in innate immunity in vertebrates, 1-27 physiologic inducers, 6-9 phosphorylation, activation by DNAPK, 60-62 Transfomjng growth factor 8, production by endothelial cells, 358

Transgenes, and mutations, effect on T cell development, 147-149 Transglutaminase, and inhibition of apoptosis, 253 Transmigration, leukocyte-endothelial, 388-389 Triggering, leukocytes, 383 Tumor cells genetic modification, 425-435 transfected, effect on preexisting tumor, 432-433 Tumor necrosis factor a,induction of NF-KB, 7-9 Tumor necrosis factor receptors, p55, death domain, 135 Tumors, see also Cancer lymphoid, therapy, 282 nonimmunogenic, 419 Tyrosine-containing sequence motifs, 127-128, 130

V Vaccination antigen-based strategies, 435-437 cancer, strategies, 427, 433 efficacy of different clones, 430-431 and tumor-specific antigens, 418-420 VCAM-I ligand for a4Pl integrin, 374-375 recruitment of leukocytes to inflammation site, 395 V(D)J junctions encoding CDR3 region, 125 and generation of TCR gene diversity, 116-117 V(D)J recombination cleavage model, 38-39 and DNA repair, and Ku autoantigen, 55-60 and DSB repair, 29-85 cell cycle regulation, 70-74 initiation, 30-32 joining model, 42-43 relevance of joining activity, 45 and TCR gene expression, 116-117

469

INDEX

Vectors, viral, and gene transfer techniques, 422-425 Vitamin A binding to sex steroids, 229 proautoimmune potential, 278 Vitamin D3, binding to sex steroids, 229

Vitamin E, inhibition of HIV lymphocyte death, 279

W Weibel-Palade bodies, storage of Pselectin, 356-357