Advances in Immunology Volume 65

ADVANCES IN

Immunology VOLUME 65

This Page Intentionally Left Blank

ADVANCES IN

Immunology EDITED BY

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

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

VOLUME 65

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3 2 1

CONTENTS

ix

CONTRIBUTORS

NF-IL6 and NF-KB in Cytokine Gene Regulation

SHIZUOAKIRAAND TADAMITSU KISHIMOTO I. Introduction 11. NF-IL6 111. NF-KB N.Protein-Protein Interaction in Gene Regulation V. Cytokine Gene Regulation VI. Cytokine Induction in NF-ILG Family Knockout Mice VII. Cytokine Induction in NF-KB Knockout Mice VIII. Conclusion References

1 1 11 16 25 29 30 32 33

Transporter Associated with Antigen Processing

TIMELLIOTT I. Introduction 11. ABC Transporters 111. Gene Structure of TAP and Its Regulation IV. TAP Protein Structure V. TAP Polymo hism VI. Function of e TAP Complex VII. TAP and MHC Class I Assembly VIII. TAP in Disease IX. Concluding Remarks References

3:

V

47 56 58 61 71 75 87 92 96 96

vi

CONTENTS

NF-KB as a Frequent Target for Immunosuppressive and Anti-Inflammatory Molecules

PATRICK A. BAEUERLE AND VIJAY R. BAICHWAL I. Introduction 11. Glucocorticoids and Other Steroid Hormones 111. Cyclosporin A and FK506 IV. Rapamycin V. Salicylates VI. Antioxidants and Inhibitors of Enzymes Generating Reactive Oxygen Intermediates VII. Anti-TNF-a Antibodies and Gold Compounds in Treatment of Rheumatoid Arthritis VIII. ImmunosuppressiveActivity of CAMP IX, The Bacterial Metabolite Spergualin X. The Fungal Metabolite Gliotoxin XI. Viral Strategies to Control NF-KB XII. Conclusion References

111 118 120 121 121 122 123 124 125 126 127 128 132

Mouse Mammary Tumor Virus: Immunological Interplays between Virus and Host SANJrVA.

LUTHER AND HANS ACHA-ORBEA

I. Introduction 11. Mouse Mammary Tumor Virus 111. Structure of the SAg Protein

IV. Immune Res onse to MMTV V. T and B Ce Response to Endogenous Mtv VI. Com arison with Other SAgs MI, Conc usions References

t

P

139 140 157 167 196 208 211 212

IgA Deficiency

PETERD. BURROWS AND MAXD. COOPER I. 11. 111. IV. V. VI. VII. VIII.

Introduction Clinical Manifestations of IgA Deficiency IgA Structure, Production, and Function IgA Deficiency Viewed in the Context of the Genesis of IgA-Producing Cells Relationship of I D with Common Variable Immunodeficiency Genetic Suscepti dity for IgAD and CVID Pathogenesis of IgA Deficiency Conclusions References

!f

245 246 248 251 256 256 260 263 263

vii

CONTENTS

Role of Cellular Immunity in Protection against HIV Infection

SARAHROWLAND-JONES, RUSUNGTAN,AND ANDREWMCMICHAEL I. II. 111. IV. V. VI. VII. VIII.

Introduction Cellular Immunity in the Control of Other Viruses CTL Effector Mechanisms HLA and HJY Infection The Nature of HIV-Specific CTLs Measurement of HIV-Specific CTLs Role of HIV-Specific CTLs in the Natural History of HIV Infection Does HIV Escape from the CTL Response? IX. Therapeutic Implications of the Importance of HIV-Specific CTLs X. Conclusions References

277 278 280 284 286 287 290 311 317 322 323

High Endothelial Venules: Lymphocyte Traffic Control and Controlled Traffic GEORGKRAALAND REINAE. MEBIUS

347 348 350 351 352 352 358 359 360

I. Introduction 11. Structure of High Endothelid Venules 111. Role of HEVs and Lymphocyte Migration IV. In Vitro HEV Binding Assay V. Molecules Determining HEV-Lymphocyte Interactions VI. L Selectin VII. Integrins and Their Role in Lymphocyte-HEV Interactions VIII. CD44 and Lymphocyte Homing IX. Homing Rece tor Ligands on High Endothelid Cells X. Additional Mo ecules on High Endothelid Venules Involved in Lymphocyte Migration XI. Adhesion and Extravasation X I . Adhesion Cascade and Specificity of Lymphoc e Homin XIII. Regulation of the Unique Features of the Hig Endothe d Venule XIV. Concluding Remarks References

365 365 369 372 379 380

INDEX CONTENTS OF RECENTVOLUMES

397 407

f

Ti!

vii

CONTENTS

Role Cellular Immunity Left in Protection This ofPage Intentionally Blank against HIV Infection

SARAHROWLAND-JONES, RUSUNGTAN,AND ANDREWMCMICHAEL I. II. 111. IV. V. VI. VII. VIII.

Introduction Cellular Immunity in the Control of Other Viruses CTL Effector Mechanisms HLA and HJY Infection The Nature of HIV-Specific CTLs Measurement of HIV-Specific CTLs Role of HIV-Specific CTLs in the Natural History of HIV Infection Does HIV Escape from the CTL Response? IX. Therapeutic Implications of the Importance of HIV-Specific CTLs X. Conclusions References

277 278 280 284 286 287 290 311 317 322 323

High Endothelial Venules: Lymphocyte Traffic Control and Controlled Traffic GEORGKRAALAND REINAE. MEBIUS

347 348 350 351 352 352 358 359 360

I. Introduction 11. Structure of High Endothelid Venules 111. Role of HEVs and Lymphocyte Migration IV. In Vitro HEV Binding Assay V. Molecules Determining HEV-Lymphocyte Interactions VI. L Selectin VII. Integrins and Their Role in Lymphocyte-HEV Interactions VIII. CD44 and Lymphocyte Homing IX. Homing Rece tor Ligands on High Endothelid Cells X. Additional Mo ecules on High Endothelid Venules Involved in Lymphocyte Migration XI. Adhesion and Extravasation X I . Adhesion Cascade and Specificity of Lymphoc e Homin XIII. Regulation of the Unique Features of the Hig Endothe d Venule XIV. Concluding Remarks References

365 365 369 372 379 380

INDEX CONTENTS OF RECENTVOLUMES

397 407

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CONTRIBUTORS

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

Hans Acha-Orbea (139), Institute of Biochemistry and Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, 1066 Epalinges, Switzerland Shizuo Akira (l),Department of Biochemistry, Hyogo College of Medicine, Nishinomiya, Hyogo 663, Japan Patrick A. Baeuerle (lll),Tularik Inc., South San Francisco, California 94080 Vijay R. Baichwal (lll),Tularik Inc., South San Francisco, California 94080 Peter D. Burrows (245), Division of Developmental and Clinical Immunology, Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294 Max D. Cooper (245),Division of Developmental and Clinical Immunology, Departments of Medicine, Pediatrics, and Microbiology, University of Alabama at Birmingham and the Howard Hughes Medical Institute, Birmingham, Alabama 35294 Tim Elliott (47), Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom Tadamitsu Kishimoto (l),Osaka University Medical School, Department of Medicine 111, Osaka 565, Japan Georg Kraal (347), Department of Cell Biology and Immunology, Vrije Universiteit, Amsterdam, 1081 BT Amsterdam, The Netherlands Sanjiv A. Luther (1391, Institute of Biochemistry and Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, 1066 Epalinges, Switzerland Andrew McMichael (277), Molecular Immunology Group, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom Reina E. Mebius (347), Department of Cell Biology and Immunology, Vrije Universiteit, Amsterdam, 1081 BT Amsterdam, The Netherlands ix

X

CONTRIBUTORS

Sarah Rowland-Jones (277), Molecular Immunology Group, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom Rusung Tan (277), Molecular Immunology Group, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom

A D V A N C E S IN IMMUNOLOGY, VOL. 65

NF-IL6 and NF-KBin Cytokine Gene Regulation SHIZUO mu*AND TADAMMU KISHIMOTO~

*ckl#mmMt d Biochmishy, &ago Colbge d Medicine, Nishinom+, Hyqo 663,k p n ; and tOsaka Uninwriv hbdbl schod, &pntnwnt d Medicine I//, suik, Osaka 565, kpan

1. Introduction

The immune system is regulated through a complicated network madulated by a variety of cytokines and their cognate receptors. Transcriptional activation of inflammatoryresponse genes, such as the genes for cytokines, their receptors, cell adhesion molecules, and acute phase proteins, is regulated by a specific assembly of transcription factors on the enhancers and promoters of these genes. Accumulating evidence indicates that a relatively small number of transcription factors play a critical role in achieving the high level of orchestration required for the complex gene expression involved in the immune response. These include the NF-KB,NF-IL6, CREW ATF, Jun-Fos, STAT, and NF-AT families of transcription factors. On the other hand, dysfunctional regulation of these transcription factors may induce immunologically mediated diseases. In this review, we highli ht how protein-protein interactions between transcription factors may mo & I late the activation of the cytokine genes. Particular attention is directed to two important families of transcription factors, NF-IL6 and NF-KB.

2

It. NF-116

A. STRUCTURE AND FUNCTION OF NF-IL6 NF-IL6 was originally identified as a nuclear factor binding to a 14-bp palindromic sequence (ACATTGCACAATCT) within an IL-1 regponsive element in the human IL-6 gene (Isshiki et al., 1990).Cloning the cDNA encoding human NF-IL6 revealed that it has a high degree of homology with CEBP in the carboy-terminal basic and leucine zipper domains, responsible for DNA binding and dimerization, respectively (Akira d al., 1990).NF-IL6 recognizes the same nucleotide sequences as CEBP. Both proteins bind to a variety of the divergent nucleotide sequences with different affinities, and the consensus sequence is T(T/G)NNGNNAA(T/ G). NF-IL6 homologs in other species have been cloned under the names AGP/EBP (Chang et al., 1990), LAP (Descombes et al., 1990), IL-6DBP (Poli et al., 1990), rNFIL-6 (Metz and Ziff, 1991), C/EBP/3 (Cao et al., 1991), CRP2 (Williams et al., 1991), and NF-M (Katz et al., 1993). The 1

Copynght 0 1987 by Acadenuc Press dl nghts of reproduchon in any form reserved w65-277W37$2500

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SHIZUO AKIRA AND TADAMITSU KISHIMOTO

NF-IL6 gene is intronless, and it produces two proteins--LAP (liverenriched transcriptional activator protein, equivalent to NF-IL6) and LIP (liver inhibitory protein)-by the alternative use of two AUG initiation codons within the same open reading frame (Descombes and Schibler, 1991). LIP contains DNA binding and dimerization domains but is devoid of the N-terminal transcriptional activation domain and therefore behaves as an antagonist of LAP-induced transcription. The ratio of these two forms varies depending on the cell type and on the developmental stage and can be altered to activation dominance by IL-6 or other extracellular signals such as retinoic acid (Descombes and Schibler, 1991; Hsu et al., 1994).

B. ACTIVATIONOF NF-IL6 THROUGH PHOSPHORYLATION NF-IL6 activity is regulated by phosphorylation. Transient expression of a series of site-directed mutants of NF-IL6 and subsequent phosphopeptide mapping identified three phosphorylated residues: Ser-231 and Thr235, both located within the serine-rich domain (SRD) adjoining bZIP, and Ser-325 that is located within the leucine zipper. The amino acid sequence immediately surrounding Thr-235 (SSPPGTPSP)coincides with the consensus for MAP kinase recognition. In fact, a synthetic peptide containing Thr-235 was phosphorylated in vitro by purified MAP kinases. When vectors expressing NF-IL6 or oncogenic p2lras were cotransfected with an IL-6 promoter-luciferase gene reporter construct, simultaneous expression of both NF-IL6 and oncogenic p2lras resulted in a dramatic synergisticstimulation of the reporter gene. Two dimensional phosphopeptide mapping showed that oncogenic p2lras expression markedly augmented Thr-235 phosphorylation. Furthermore, the substitution of Ala for Thr-235 resulted in the loss of ras-dependent NF-IL6 activation. These results demonstrate that NF-IL6 is activated through phosphorylation of Thr-235 by a ras-dependent MAP kinase cascade (Nakajima et al., 1993). The molecular mechanisms of NF-IL6 transcriptional activation through phosphorylationon Thr-235 have been clarified (Kowen-Leutzet al., 1994). NF-M, the chicken homolog of NF-IL6, is a critical transcription factor required for the expression of chicken myelomonocytic growth factor (cMGF),which is distantly related to G-CSF and IL-6 (Burk et al., 1993; Katz et al., 1993). In vmyb- or v-myc-transformed cells, NF-M is the target of activated kinase oncogenes, which induce cMGF gene expression, resulting in autocrine growth stimulation. Analyses of functional domains of NF-M demonstrated that the amino-terminal domains contribute to the transactivating function of the protein, whereas the internal portion (amino acids 116-229) between bZip and the amino-terminal activating domains inhibits the transcriptional activation. A consensus sequence for MAP kinase located within the inhibitory domain is conserved between avian

3

NF-IL6 AND NF-KB IN CYTOKINE GENE REGULATION

and mammalian NF-IL6. NF-M was activated by deleting the entire region that harbors the MAP kinase site or by a point mutation at the MAP kinase site that mimics the negative charge of a phosphate residue. These results suggest that the inhibitory regions within NF-IL6 mask the transactivation domain and prevent its interaction with the basic transcription machinery. NF-IL6 is also phosphorylated within the leucine zipper in response to increased intracellular calcium concentrations via the activation of a calcium-calmodulin-dependent kinase (Wegner et al., 1992). In addition, the CAMP-mediatedphosphorylation of NF-IL6 is associated with nuclear translocation and transcriptional activation (Metz and Ziff, 1991). NF-IL6 is activated through phosphorylation of the N-terminal domain by PKC (Trautwein et al., 1993). Thus, NF-IL6 is, in fact, activated via multiple signaling pathways (Fig, 1). C. C/EBP FAMILY MEMBERS Following NF-IL6, several other C/EBP family members have been molecularly cloned. Currently, there are five known members of the C/EBP family (Fig. 2). These include C/EBP (Landschulz et al., 1988), NF-IL6, I G B P (also referred to as GPE-1-BP and CIEBPy) (Roman et al., 1990; Nishizawa et al., 1991) , NF-IL6P (CKBPS) (Kinoshita et al., 1992; Kageyama et al., 1991; Cao et al., 1991; Williams et al., 1991), and CHOP-10 (gadd153) (Ron and Habener, 1992).The genes encoding these proteins map to different loci of different chromosomes;C/EBP maps to human chromosome 19q13.1 (mouse chromosome 7) (Birkenmeier et al., 1989), NF-IL6 to human chromosome 20q13.1 (mouse chromosome 2) (Hendricks-Tayloret al., 1992), NF-IMP to human chromosome 8 q l l (mouse chromosome 16) (Cleutjens et al., 1993), and CHOP-10 to human chromosome 12q13.1-q13.2 (Park et al., 1992).

I I

PBC

I

NH2

NF NF-IL6

R-X-X-S/T

P-X-S/T-P

SRELST

SGSSGSLSTSSSSSPPGTPS

COOH

lP1 1319

159 170 218 237

262

306

345

FIG.1. Phosphorylation of NF-ILG. P, proline rich; S, serine rich; L, leucine.

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SHIZUO AKIRA AND TADAMITSU KISHIMOTO

m

COOH

CmBP

P

G

181190

282272

LLLLL

NHa

NF-IL6

318

358

COOH

P

P

13 19

8

169170 217231282

308

345

COOH

NHa

COOH

NH?.

Ig/EBP 62

NHZ

98

150

cnnw

LLL

I

FIG.2. CEBP family members. DNA, DNA-bindingdomain; P, proline rich; G, glycine rich; S, serine rich; L, leucine; E, glutamic acid; D, aspartic acid; T, threonine.

C/EBP is expressed in adipose, liver, and placental tissues that play vital roles in energy metabolism (McKnight et al., 1989). C/EBP mRNA increases markedly during the differentiation of 3T3-Ll preadipocytes to adipocytes (Birkenmeier et al., 1989). CEBP can transactivate the promoters of adipocyte-specific genes, a fatty acid binding protein 422 (aP2), stearoyl-CoA desaturase 1 (SCDl), and insulin-responsive glucose transporter-4 (GLUT4)(Christyet al., 1989).CEBP is an important regulatory factor in adipocyte differentiation. Premature activation of a constitutively expressed, but inactive, CEBP-estrogen receptor fusion protein causes premature expression of aP2 mRNA relative to the normal differentiation program (Umek et al., 1991). Other studies have also presented similar results (Lin and Lane, 1992; Freytag and Geddes, 1992). The ectopic expression of CLEBP by retroviral vectors induces adipogenesis directly in otherwise nonadipogenic NIH-3T3 cells (Freytag et al., 1994). In contrast, antisense CEBP RNA suppresses several adipose-specific mRNAs such as aP2, SCD1, and GLUT4, as well as triglyceride accumulation during the differentiation of 3T3-Ll preadipocytes (Samuelsson et al., 1991; Lin and Lane, 1992). CEBP may also be involved in controlling the expression of genes of which the products are critical to liver function (Park et al., 1993; Tae et

NF-IM AND NF-KB IN CYTOKINE GENE REGULATION

5

al., 1995; Nerlov and Ziff, 1994; Friedman et al., 1989). Actually, mice deficient in C/EBP become profoundly hypoglycemic and die several hours after birth due to deficient glycogen stores in the liver (Wang et al., 1995). The C/EBP protein is virtually absent in degenerating liver and hepatoma but is expressed at a high level in terminally differentiated, mature liver hepatocytes, suggesting that C/EBP expression is inversely correlated with cell proliferation (Birkenmeier et al., 1989; Friedman et al., 1989).In fact, transfection of a gene encoding a C/EBP-estrogen receptor fusion protein into 3T3-Ll adipoblasts caused transient growth arrest in vitro when cells were exposed to estrogen (Umek et al., 1991). Likewise, growth was transiently arrested when a C/EBP expression vector was cotransfected with a P-galactosidase reporter gene into several cell lines (Hendricks-Taylor and Darlington, 1995). An inducible C/EBP gene transfected into human hepatoma cell lines resulted in the reversible arrest of cell proliferation (Watkins et al., 1996). Furthermore, C/EBP appears to play the opposite role to that of c-myc in cell proliferation. C/EBP overproduction represses the growth of c-myc-transformed adipoblasts, which are normally unable to terminally differentiate (Freytag and Geddes, 1992). C/EBP inhibits proliferation of human fibrosarcoma cells by inducing p21/WAF-l/SDI-l protein (Timchenko et al., 1996). NF-ILGP is normally expressed at a low level but is markedly and rapidly induced in many tissues by LPS or by several inflammatory cytokines, including IL-1, TNF, and IL-6, as is NF-IL6 (Kinoshita et al., 1992). In Hep3B cells the activityof NF-IL6 is enhanced by IL-6 via posttranslational modulation, whereas NF-ILGP is transcriptionally induced by IL-6 (Ramji et al., 1993). NF-IL6P may function as a transcriptional regulator in the genes such as prostaglandin endoperoxide synthase 2 and complement C3 (Inoue et al., 1995; Juan et al., 1993). Ig/EBP was originally cloned as a nuclear factor that binds to functionally important C/EBP binding sites in immunoglobulin gene enhancers and promoters (Roman et al., 1990). Ig/EBP was also cloned as a transcription factor binding to the regulatory elements in the G-CSF, IL-4, and a-feto protein genes, respectively (Davydov et al., 1995a; Nishizawa et al., 1991; Thomassin et al., 1992). I@BP alone has no transcriptional activity, Ig/ EBP is ubiquitously expressed in normal adult tissues but is most abundant in the early stages of B lymphocytes (Cooper et al., 1994). CHOP was cloned as a protein interacting with the bZip domain of NFIL6 (Ron and Habener, 1992). Although CHOP can form heterodimers with other members of the C/EBP family, the heterodimer cannot bind to several known C/EBP binding sites because there are several amino acid substitutions in the highly conserved basic regions in all bZIP proteins. Therefore, CHOP has been considered as a negative modulator of the

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SHIZUO AKIRA AND TADAMITSU KlSHlMOTO

activity of other C/EBP family members. However, recent evidence demonstrated that the CHOP-C/EBP heterodimers can bind a select and relatively specific subset of genes that contain special CEBP sites (Ubeba et al., 1996). CHOP is identical to gadd153, one of a set of growth arrest and DNA damage-inducible genes isolated from Chinese hamster ovary (CHO) cells (Fornace et al., 1989). Microinjection of a CHOP expression plasmid or protein into proliferating NIH-3T3 cells induces growth arrest at the Gl/S boundary (Baroneet al., 1994). In human myxoid liposarcoma, which is a tumor of the adipose tissue, the CHOP gene is fused to TLS (Crozat et al., 1993). TLS-CHOP stably introduced in NIH-3T3 cells causes transformation to a tumor phenotype. There are differences in the temporal expression of CEBP family members during development or differentiation. Inflammation is accompanied by the acute phase response, which is characterized by significantalterations in the serum levels of severalplasma proteins, known as acute phase proteins (APPs).APPs are mainly synthesized by the liver. The concentrations of several plasma proteins, such as C-reactive proteins, serum amyloid A, fibrinogen, and complement protein C3, increase dramatically, whereas there is a decrease in plasma albumin, transfemn, and transthyretin (Baumann et al., 1989;Akiraand Kishimoto, 1992).NF-IL6 and NF-IL6P bind the regulatory elements in the promoters of a variety of acute phase protein genes and activate the transcription of these genes (Natsuka et al., 1991; Isshiki et al., 1991;Poli et al., 1990;Chang et al., 1990).During the acute phase response, the expression of three CEBP isoforms dramatically changes; the steadystate mRNA levels of CEBP decrease significantly in the liver, lung, and fat tissues of LPS-treated mice, whereas the steady-state levels of NF-IL6 and NF-IL6P dramaticallyincrease in many tissues (Isshiki et al., 1991;Alam et al., 1992).Although NF-IL6 mRNA levels were dramatically increased by LPS, the induction of NF-ILGP mRNA was much more striking. Replacement of CEBP by NF-IL6 and NF-ILGP duringthe acute phase may account for the transcriptional activation of a variety of acute phase protein genes as well as the decrease in the transcriptional activity of others such as the albumin gene. The process of adipocyte differentiation is associated with the induction of C/EBP isoforms. The terminal differentiation of cultured 3T3-Ll fibroblasts to the adipogenicphenotype is potently stimulated by dexamethasone (DEX) and methylisobutylxanthine (MIX). When stimulated with adipogenic stimulants, C/EBP (CEBPa) is not expressed during the first 2-4 days of the 3T3-Ll differentiation program. Much evidence indicates that the expression of NF-IL6 (CIEBPP) and NF-ILGP (CEBPS) plays an important role in adipogenesis. DEX activates the gene encoding NFIL6P whereas MIX directly induces NF-IL6 gene expression. During

NF-ILG AND NF-KB IN CYTOKINE GENE REGULATION

7

the early phase of differentiation, high levels of NF-IL6P and NF-IL6 accumulate. These transcription factors diminish during the terminal phase of differentiation and are replaced by CEBP. The presence of a C E B P binding site within the CEBP promoter indicates that NF-IL6 and/or NFIL6P are responsible for CEBP activation (Vasseur-Cognet and Lane, 1993; Christy et al., 1989).Precocious expression of either NF-IL6 or NFILGP, and particularly NF-IL6, stimulated adipogenic conversion of 3T3L1 cells (Yeh et al., 1995).Likewise, conditions that prevented the expression of functional NF-IL6 effectively blocked terminal differentiation. Conditional ectopic expression of NF-IL6 in NIH-3T302 cells exposed to dexamethasone activated the expression of an adipocyte-specific nuclear hormone receptor, PPARy, that stimulates the conversion of these fibroblasts into committed preadipocytes (Wu et al., 1995). Neither ectopic expression of NF-IL6 nor addition of dexamethasone alone can induce PPARy expression, but when present together, they have a synergistic effect on the adipogenic program. These results suggest that the enhanced expression of NF-IL6 converts multipotential mesenchymal precursor cells into preadipocytes that respond to adipogenic inducers, including dexamethasone and PPAR activators, to differentiate into adipocytes. TNF causes a dramatic morphological dedifferentiation of adipocytes in culture (Torti et al., 1985). This process is preceded by a decrease in the DNA binding activity and protein levels of CEBP and a reciprocal increase in NF-IL6 levels, indicating that the TNF-induced changes in the transcription factors that bind CEBP sites are important in the pathogenesis of inflammation-induced atrophy of adipose tissue (Stephens and Pekala, 1991; Ron et al., 1992; Williams et a!., 1992). Like adipocyte maturation, myelomonocytic differentiation is paralleled by a distinct expression profile of CEBP proteins. CEBP, NF-IL6, and NF-IL6P are expressed in myelomonocytic cells of bone marrow but not in erythroid or lymphoid cells (Scott et al., 1992) CEBP is expressed at a high level in dividing myeloblasts and diminishes to low levels during their terminal differentiation into polymorphonuclearleukocytes and macrophages. Conversely, NF-IL6 is expressed at low levels in dividing myeloblasts and induced at high levels during terminal differentiation into macrophages (Natsuka et d.,1992; Scott et al., 1992). NF-M, the chicken homolog of NF-IL6, is specifically expressed in myelomonocytic and eosinophilic cells of the chicken hematopoietic system, When an estrogen-inducible form of NF-M was stably expressed in a multipotent progenitor cell line and exposed to P-estradiol, the forced NF-M expression caused the downregulation of progenitor-specific surface markers and the upregulation of differentiation markers restricted to cells of the eosinophil and myeloid lineages (Mtiller et al., 1995). In addition

8

SHIZUO A K I M A N D TADAMITSU KISHIMOTO

to the onset of differentiation, cell death was induced with typical apoptotic features. These results suggest that NF-M plays an important role in commitment along the eosinophil lineage and in the induction of apoptosis. The expression and activity of CiEBP family members also change at different developmental stages in B cells. The negative regulator Ig/EBP is predominant in B cells; activator NF-IL6 increases more in mature B cells and is induced by the LPS activation of splenic B cells (Cooper et al., 1994). Thus, the CiEBP sites do not function as activator sites in early B cells but do so when these cells terminally differentiate. BY NF-IL6 D. TARGET GENESREGULATED NF-IL6 is expressed at an undetectable or a minor level in all normal tissues, but it is significantly induced by stimulation with LPS, IL-1, TNF, or IL-6. NF-IL6 may be responsible for regulating a variety of genes involved in inflammatory and immunological responses including cytokine and acute phase protein genes (Akira and Kishimoto, 1992). NF-IL6 was first cloned as a protein binding to the IL-l-responsive element of the IL-6 gene from a hgtll cDNA expression library of LPSstimulated human peripheral monocytes by South-Western blotting. With similar approaches, NF-ILGcDNA has been isolated as a DNA-binding protein recognizing the regulatory regions of a number of genes. These include the genes for albumin (Descombes et al., 1990),al-acid glycoprotein (Chang et al., 1990), cfos (Metz and Ziff, 1991), IL-4 (Davydov et al., 1995b), Pglycoprotein (Combates et al., 1994), topoisomerase I (Heiland and Knippers, 1995),a-fetoprotein (Thomassin et al., 1992), aromatase cytochrome P450 (Todaetal., 1995),pregnancy-specificglycoprotein (Chenet al., 1995), and human immunodeficiency virus type 1 (HIV-1) (Tesmer et al., 1993). Besides the cytokine and acute phase protein genes, NF-IL6 may be involved in regulating the genes for collagen a l(1)(Houglum et al., 1994), insulinlike growth factor (Nolten et al., 1994; Rodenburg et al., 1995), acetyl-CoA carboxylase (Tae et al., 1994), alcohol dehydrogenase (van Ooij et al., 1992), inducible nitric oxide synthase (Lowenstein et al., 1993; Nunokawa et al., 1994), ceruloplasmin (Bingle et al., 1993), serum amyloid A3 (Huang and Liao, 1994), &casein (Doppler et al., 1995), prostaglandin endoperoxide synthase 2 (Sirois and Richards, 1993; Inoue et al., 1995),phosphoenolpyruvate carboxykinase (Park et al., 1993),aspartate aminotransferase (Garlatti et al., 1993),Fas (Wada et al., 1995),and placental lactogen (Stephanou and Handwerger, 1995). Activated macrophages secrete cytokines and proteinases. NF-IL6 is predominantly expressed in macrophages, but not in lymphoblasts. NFIL6 expression is dramatically induced during macrophage differentiation (Natsuka et al., 1992; Scott et al., 1992). NF-IL6 binding motifs are also

NF-ILG AND NF-NB IN CYTOKINE GENE REGULATION

9

found in the functional regulatory regions of genes specifically induced in activated macrophages, such as IL-6, IL-la, IL-8, TNF-a, G-CSF, nitric oxide synthase, and lysozyme (Nishizawa and Nagata, 1990; Natsuka et al., 1992; Mukaida et al., 1990, Pope et al., 1994; Lowenstein et al., 1993; Goethe and Phi van, 1994). Bretz et al. (1994) showed that the ectopic expression of NF-IL6 conferred the LPS-inducible expression of IL-6 and monocyte chemoattractant protein 1 (MCP-1) upon lymphoblasts, which normally do not display the LPS induction of these inflammatory cytokines. The expression of NF-IL6 antisense RNA blocked the LPS induction of IL-6 expression in a macrophage cell line. These results showed the critical role of NF-IL6 protein in the activation of endogenous IL-6 and MCP-1 genes by LPS. In contrast, the TNF-a-induced synthesis of G-CSF was abolished in human fibroblasts by both antisense oligodeoxyribonucleotides and ribozyme-mediated specificelimination of NF-IL6 transcripts,whereas TNF-a-inducible synthesisof GM-CSF and IL-6 was not abolished (Kiehntopf et al., 1995). Consistent with these results, NF-IL6 knockout (KO) mice presented abnormal G-CSF induction in macrophages and fibroblasts, but the induction of IL-6 was not impaired in NF-IL6” macrophages (Tanaka et al., 1995; Screpanti et al., 1995). IL-6 induction could be fully compensated for by other NF-IL6 family members, such as NF-IL6P (C/EBPG), C/EBP, and Ig/EBP, as well as other different transcription factors. These results suggest that NF-IL6 may be necessary, but not essential, for IL-6 induction.

E. NF-IL6 IN VIRALINFECTION C/EBP was originally purified as an enhancer core-binding protein for enhancers of simian virus 40, Moloney murine sarcoma virus, and polyomas virus (Johnson et aZ., 1987). However, because C/EBP expression is restricted to tissues, such as the liver, adipose tissue, myeloid cells, and placenta, and it is not distributed in the same manner as the expression of these viruses, it is likely that other members of the C E B P family of transcription factors are also involved in viral expression. C E B P binds to both the 5’ end of the long terminal repeat (LTR) enhancer (nucleotides -225 to -188 bp of the SR-D strain) and the gap enhancer (nucleotides 813-872 bp) of Rous sarcoma virus and other avian retroviruses (Sears and Sealy, 1992; Zachow and Conklin, 1992). Multiple C E B P element motifs are found in the avian leukosis virus LTR enhancer (Bowers and Ruddell, 1992). Mutation of these C/EBP motifs reduces LTR-driven transcription and viral titers in fibroblasts, indicating that these sites are important for LTR enhancer function (Ryden et al., 1993). The C E B P motifs are absent in endogenous avian retroviral LTRs, which correlates with the very low transcriptional activity of these viruses (Habelet al., 1993).

10

SHIZUO AKIRA AND TADAMITSU KISHIMOTO

There are three NF-ILG(C/EBP) binding sites in the HIV-1 LTR (Tesmer et al., 1993).Region I includes NREl (positions -178 to -158 bp of the LTR) and appears to represent the preferred NF-IL6 binding sequence. In fact, NF-IL6 was isolated as a factor that regulates HIV-1 transcription through interaction with the NREl region. Region I1 flanks and overlaps one of the NF-KB binding sites. Region 111, which is weakly protected, is located upstream from region I. Regions I and I1 include a consensus NF-IL6 binding site, but a consensus sequence is not apparent in region 111. Although NF-IL6 binds NRE1, which exerts repressive effects on LTR-mediated transcription, NF-IL6 can activate transcription from the HIV-1 LTR. In the promonocytic cell line U937, NF-IL6 activity is increased when the cells are activated and HIV-1 LTR transcription is augmented (Henderson et al., 1995). The NF-IL6 binding site in the NREl region should, in principle, bind other members of the CEBP family, and one of them might repress HIV-1 transcription. The C/EBP site in the feline immunodeficiency virus (FIV) LTR is necessary for its efficient replication and is also involved in the inhibition of FIV LTR-directed gene expression by pseudorabies ICP4. Mutation of the CEBP site reduced the basal promoter activity and prevented efficient FIV replication in a feline kidney cell line as well as the inhibition of FIV LTR-directed gene expression by ICP4 (Kawaguchi et al., 1995). C/EBP interacts with the core promoter in at least five major areas of the human hepatitis B virus (HBV) and three other sites in the HBV enhancer (Yuh and Ting, 1991).Transient cotransfection of C/EBP expression vectors and the core promoter in the context of either the native hepatitis B virus genome or the luciferase reporter gene demonstrated that C/EBP at low concentrations modestly activates expression from the core promoter but represses at high concentrations (Lopez-Cabreraet al., 1990) Although whether C/EBP-dependent activation and repression of the core promoter or the enhancer activity have any significance remains to be examined, it is conceivable that variable levels of CEBP in infected hepatocytes modulate HBV infection. C/EBP family proteins interact with human papillomaviruses (HPVs). The expression of the HPV16 early gene, including E6- and E7-transforming genes, is regulated by several cellular proteins binding to the noncoding region (NCR),such as glucocorticoid receptor protein, nuclear factr I (NFl),and AP-1, all of which are positive regulators. NF-IL6 specifically binds to the HPV16 NCR and represses the early gene expression of HPV16 through competition with other transcriptional activators, such as NF-1 and AP-1 (Kyo et al., 1993). Consistent with this finding, a decoy study using nuclease-resistantoligomers containing NF-IL6 binding sites demonstrated that the levels of both HPVll transcripts and HPV DNA replication

NF-ILG AND NF-KB IN CYTOKINE GENE REGULATION

11

increase in cultured foreskin keratinocytes containing replicating HPVll DNA after depleting the NF-IL6, indicating that NF-IL6 is a repressor of HPV11 in keratinocytes (Wang et al., 1996). The transforming gene product, ElA, of adenovirus transactivates all the early genes of the virus as well as a subset of cellular genes and represses SV40, immunoglobulin heavy chain, and insulin enhancer-linked promoters. NF-IL6 regulates E1A-responsive promoters in the absence of E1A. NF-ILG alone is sufficient to complement the E1A deletion mutant d312 in viral infection (Spergel et al., 1992).These results show that NFIL6 acts as a sequence-specificcellular nuclear factor that regulates E1Aresponsive genes in the absence of E1A. 111. NF-KB

A. STRUCTURE AND FUNCTION OF NF-KB

NF-KB was originally characterized as a K immunoglobulin enhancer DNA-binding protein. It is, in fact, involved in the regulation of many genes activated during inflammatory, immune, and acute phase responses. Binding sites for NF-KB were identified in the regulatory regions of some cytokine genes (including the TNF, lymphotoxin, IL-6, IL-8, and P-IFN genes), the IL-2 receptor, class I and class I1 histocompatibility antigen, several acute phase response genes, as well as several viral enhancers including HIV-1 (Lenardo and Baltimore, 1989). The active form of NFKB is most frequently composed of the two DNA-binding subunits, p50 and p65 (RelA). The cloning of genes encoding the p50 and the p65 subunits of NF-KBhas revealed a family of NF-KB/rel proteins with high homology to the protooncogene c-rel and the Drosophila maternal effect gene dorsal (Kieran et al., 1990; Ghosh et al., 1990; Nolan et al., 1991). Members of this family share a highly conserved region of about 300 amino acid residues called the Re1 homology (RH) domain, which is responsible for both DNA binding and dimerization of the proteins. The consensus for NF-KBbinding is 5’-GGGPuNNPyCC-3’.The slight asymmetry of the binding motif accounts for the distinct DNA-binding specificities of p50 and ReIA. The p50 subunit prefers binding to the first half-site containing the three GC pairs, whereas RelA shows a preference for the second halfsite, which is usually more degenerate (Urban and Baeuerle, 1991; Kunsch et al., 1992).The N-terminal half of the RH domain is involved in contacting DNA, whereas the C-teminal half of the domain is responsible for RH-RH domain interaction. RelA and p50 can be independently transported into the nucleus due to a conserved cluster of positively charged amino acid residues in the C-terminal end of their RH domains that serve as nuclear location signals (NLSs).Proteins containing mutations in the NLSs are no

12

SHIZUO AKIRA AND TADAMITSU KISHIMOTO

longer translocated within the nucleus. The p50 protein has very little extra sequences, apart from the RH domain, and lacks transcriptional activity. Re1 A harbors transcription-activating domains in its C-terminal portion.

B. RELFAMILY To date, the RH domain has been found in six proteins: c-ReZ (v-mZ), dorsal, p50 (p105),p52 (plOO), RelA (p65), and RelB (Fig. 3). c-Rel was identified through its oncogenic derivative, v-rel, found in the avian leuke-

FIG.3. NF-KBfamily members.TA, transcriptional activation; NLS, nuclear localization signal; GRH, glycine-rich hinge.

NF-ILG AND NF-KB IN CYTOKINE GENE REGULATION

13

mogenic reticuloendotheliosis retrovirus that induces fatal B cell lymphomas in infected chickens (Gilmore, 1991; Wilhelmsen et al., 1984). Dorsal protein is involed in the control of dorsal-ventral axis formation in the early embryo of Drosophila (Govind and Steward, 1991). Originally, p52 cDNA was cloned by its homology to p50/p105 (Schmid et al., 1991). p52 was also independently discovered as part of a fusion protein, called lyt10, in a chromosomal breakpoint associated with B cell leukemia (Neri et nl., 1991). Like p50, p52 is generated from a precursor called p100. The plOO precursor has a structure similar to that of p105 and it is also an immediate early activation gene of human peripheral blood T cells (Bours et al., 1992). RelB was isolated as a serum-induced gene from a mouse fibroblast cDNA library (Ryseck et al., 1992). It is most closely related to c-rel but contains a putative leucine zipper domain in a long amino-teminal segment as well as a carboxyl-terminal transcritional activation domain. Although the amino-terminal regions of the NF-KB/Rel family members are highly conserved and perform similar functions, the carboxy terminals of these proteins differ significantly. These family members can be divided into two classes. One includes the precursors p105 and plOO that contain ankynn-like repeats. The other does not contain ankynn-like repeats and harbors sequences important for gene activation. These include Re& cRel, and RelB. Apart from forming homodimers, most NF-KB/Rel family members can form heterodimers with each other in vitro. The dimerization domain of p50 is located in the carboxy-terminal part of the RH domain. The dimerization and DNA-binding regions of p50 are separated. A mutant p50 unable to bind to DNA but able to form homodimers or heterodimers reduces the DNA-binding activity of the NF-KB/Rel family in uitro. This mutant can also act as a transdominant negative regulator in vivo by almost completely abolishing the inducible transcriptional activity of the HIV-1 and MHC class I promoters (Logeat et al., 1991). Although grouped into the same family, each individual NF-KB/Rel family protein or heterodimer complex may differ in DNA-binding specificity and transcription activity for a particular KBsite (Liou and Baltimore, 1993). Among the 10 possible combinations of the five known NF-KB/Rel proteins, RelB can form a heterodimer with p50 or p52 but not with p65 or c-Rel. RelB binds to KB sites with only weak d n i t y as a homodimer, whereas a RelB-p50 heterodimer shows high binding activity. RelB attenuates p50 KBand lacks transactivating potential. The transactivation activity of c-Re1 is considerably weaker than that of ReIA. RelA, p50, and c-Re1 are the major components of NF-KB complexes, binding to most of the known &-acting KB sites. Each KB site preferentially binds a specific set of NF-KB complexes as detected by EMSA. It is likely that these complexes play a major role in regulating target genes with this preferential KB site.

14

SHIZUO AKIRA A N D TADAMITSU KISHIMOTO

C. IKB FAMILY The IKBfamily proteins bind to one or more of the NF-KB/Rel proteins and thereby inhibit the nuclear translocation of the NF-KB/Rel proteins by masking the nuclear localization signal and, consequently, DNA-binding activity. Members include k B a , IKBP, IKB?, the protooncogene bcl-3, and the Drosophila gene cactus (Fig.3).The IKBproteins are homologous to the carboxy terminus of p105 and p100. All these proteins contain the so-called ankynn-like (ANK) repeat motif. The ANK repeat is a 33-amino acid motif, first identified in the yeast cell cycle-control proteins, and it functions as a protein-protein interaction motif. The IKBproteins contain between five and seven ANK repeats that represent the minimal requirement for physical interaction with the DNA-binding subunits and for inhibitory activity. IKBproteins prevent the binding of NF-KB to DNA as well as the nuclear translocation of NF-KBproteins. These properties are based on direct protein-protein interaction involving the ANK repeats and the C-terminal half of the NRD domain. Free I K Bcan ~ enter the nucleus and dissociate preformed NF-KB-DNA complexes, resulting in inhibition of KB-dependent transciption. IKBCY is identical to MAD3, which was isolated by the differential screening of a human cDNA library (Haskill et al., 1991). Its avian homolog pp40 was identified and cloned by virtue of its association with c-rel in avian cells (Davis et a!., 1991).In most cells, IKBCY is the predominant form of IKB.The bcl-3 gene was cloned from a recurrent chromosomal translocation in human B cell chronic lymphocytic leukemias (McKeithan et al., 1987). All the rearrangements in this locus occur in the 5’ regulatory region of the gene and result in the increased expression of wild-type Bcl-3 (Ohno et al., 1990). This protein contains seven ANK repeats. Bcl-3 inhibits the DNA-binding activity of not only NF-KBbut also p50 in vitro. The function of Bcl-3 is controversial. Bcl3 acts as a gene activator by removing p50 homodimers from the KB sites, thus allowing the p50-RelA complexes to bind DNA (Franzoso et al., 1992).The Bcl-3-p50 complex activates transcription through specific KB sites (Bourset al., 1993).The Drosophila gene, cactus, is genetically defined as a negative regulator of dorsal. I K Bcan ~ arise from alternative splicing of p105, has specificity for p50, and appears to be limited to mouse B cells (Inoue et al., 1992; Liou et al., 1992). The protein p50 is not synthesized as a nuclear DNA-binding protein but rather in the form of an inactive, cytoplasmic precursor of p105. The N-terminal half contains p50; the C-terminal half constitutes an IKBprotein with seven ANK repeats. These two functional domains are linked by a flexible glycine-rich region that presumably allows intramolecular inhibition of the p50 portion by the IKBportion. Protein p105 is proteolyticdy

NF-ILG AND NF-KB IN CITOKINE GENE REGULATION

15

processed to yield p50 and IKB.Proteolyticdy released p50 is more likely inhibited by either unprocessed p105 or Reh-IKB. D. ACTIVATIONOF NF-KB NF-KB is mainly a complex of p50 and relA. NF-KB is present in the cytoplasm of most cells as an inactive form complexedwith IKB.Stimulation by a number of agents results in the dissociation of the IKB-NF-KB complex (Baeuerle and Henkel, 1994; Baldwin, 1996). Subsequently, the NF-KB heterodimer migrates to the nucleus, where it binds to its cognate DNA binding sites and activates transcription. Various agents activate this factor, including mitogens [phorbol 12-myristate lSacetate(PMA) and lectins], cytokines (IL-1 and TNF), viruses (HIV-1 and cytomegalovirus), parasites, double-stranded RNA, and agents that provoke oxidative stress. The genes that encode p105, p100, and I K B are ~ highly inducible in response to the stimuli that activate NF-KB activity in many cell types examined, whereas the c-re1 gene is predominantly expressed in lymphoid cells and can also be induced by the same stimuli. NF-KB activity is also regulated during B cell differentiation (Liou and Baltimore, 1993).In preB cells, NF-KBprotein is maintained in an inactive form, although it can be activated by stimulating cells with PMA or LPS. By contrast, mature B cells constitutively express NF-KB activity in the nucleus. Studies on this constitutive NF-KBactivity suggest that it is predominantly a p50-cRe1 complex (Liou et al., 1994; Miyamoto et al., 1994). Constitutive NFKB activity may be required to initiate IgK gene expression in B cells. In macrophages, the PMA- and TNF-a-induced complex is p5O-RelA. Also in mature macrophages, NF-KB becomes constitutively active. Activation of NF-KB simply requires the disruption of the interaction between IKBand DNA-binding subunits. Phosphorylation events control NF-KB activation. Studies have demonstrated that proteasomes are also responsible for activating NF-KB(DiDonato et al., 1995;Chen et al., 1995). Proteasomes are multisubunit protease complexes that selectively degrade intracellular proteins. Most of the proteins are tagged for destruction by ubiquitination, which involves the attachment of multiple chains of the 76-amino acid protein ubiquitin to the protein to be degraded (Hilt and Wolf, 1996). Degradation of I K B in ~ vivo requires the signal-induced phosphorylation of both serines 32 and 36 near its amino terminus. In fact, phosphorylation-defectivemutants of I K Bdo ~ not undergo inducible polyubiquitination and IKB degradation (Brown et al., 1995; Traenckner et al., 1995). Phosphorylation does not result in the dissociation of the inactive NF-KB-IKB~ complex but rather in the conversion of these proteins into efficient substrates for the protein ubiqutination machinery. Thus, the scenario is as follows: Upon cell stimulation, IKBis phosphory-

16

SHIZUO AKIRA AND TADAMITSU KISHIMOTO

lated at serines 32 and 36, rapidly modified to yield high-molecular-weight forms by the multiubiquitination of nearby lysine residues of IKB, and subsequently degraded. The p50 subunit is also derived from p105 by ubiquitin- and proteasome-dependentprocessing (Palombellaet al., 1994). The processed p50 form assembles with p65 to form NF-KB, which is retained in an inactive form in the cytoplasm by binding to IKBproteins. Active oxygen radicals may also play an important role in the activation of NF-KB activity (Bauerle and Henkel, 1994).One common intracellular reaction induced by many, if not all, NF-KB-activating stimuli is oxidative stress. NF-KB is posttranslationally activated by low concentrations of hydrogen peroxide. Activation of the NF-KB in response to all inducing agents tested so far is blocked by a variety of chemically distinct antioxidants. These observations suggest that NF-KB is an oxidative stressresponsive transcription factor and that reactive oxygen intermediates play a messenger function in its activation. TNF-a and IL-lP are potent inflammatory cyokines and good NF-KB inducers in many cell types. The generation of ceramide in response to TNF-a and IL-lP may be critical in initiating the events leading to NF-KBactivation, although there is some evidence against a role for ceramide in the activation of NF-KB. TNF-a or IL-lP induces the activation of an acid sphingomyelinase through the production of 1,2-diacylglycerolby phosphatidylcholine-phospholipaseC. Sphingomyelinase then leads to the release of ceramide and activates NFKB activity. Currently, the interaction between NF-KB activation through IKBphosphorylation and degradation, oxidative stress, and ceramide is not well understood. IV. Protein-Protein Interaction in Gene Regulation

The combinatorial effects of transcription factors are very important in gene regulation. The cis elements in the promoters and the factors with which they interact do not function independently. Cooperation between transcription factors and higher order complex formation on the promoter appears to be necessary.

A. NF-IL6 AND NF-KB Both NF-IL6 and NF-KB binding sites are frequently found in the promoter region of the genes involved in inflammatory and immune responses (Fig. 4). Studies with the deletion mutants of the IL-6 gene demonstrated that these two sites are essential for IL-6 expression. Also, in the IL-8 gene, the sequence between -94 and -71 bp, composed of two regulatory elements, NF-KB(-80 to -71 bp) and NF-IL.6 binding sites (-94 to -81 bp), is minimally essential and sufficient to allow IL-8 gene expression

NF-ILG AND NF-KB IN CYTOKINE GENE REGULATION

IL-6

IL-8

17

-158 -145 -75 -66 ACATGCACAATCT -------- G G G A T m C C NF-IL6 NF-KB

--

NF-KB -72 -95 TCAGTTGCAAATCGTGGA A m C C NF-ILG

-190

G-CSF

SAA2

AT

NF-KB -179 CAGAGATTCCK A A ~ C A C A A I I NF-IL6 NF-IL6

-184 -171 -91 -82 AGGTTACACAACTG---------GGGAClTCC NF-IL6 NF-KB

NF-IL6 -557 7 1 -536 CCACAGTTqGGATTTCCGAACC NF-KB

-178 G A l T G C m A CTGGAAATTCC

-200

ICAM-1

dNF-ILG NF-KB

FIG.4. NF-IL6 and NF-KBbinding sites in inducible genes. G-CSF, granulocytecolonystimulating factor; SAA2, serum amyloid A2; AT, angiotensinogen; ICAM-1, intercellular adhesion molecule 1.

to be induced by IL-1, TNF, or PMA (Mukaida et al., 1990). NF-IL6 and NF-KB binding sites also interact in several other genes such as serum amyloid A1 (Li and Liao, 1992; Betts et al., 1993), serum amyloid A3 (Shimizu and Yamamoto, 1994), angiotensinogen (Brasier et al., 1990), GCSF (Dunn et al., 1994), intercellular adhesion molecule 1 (Hou et al., 1994),MGSNGRO (Altmeyeret al., 1995),TNF-stimulated gene 14 (Shattuck et al., 1994), and cyclooxygenase-2 genes (Yamamoto et al., 1995). A hgtll expression library screening using radiolabeled NF-KB p50 as a probe led to the isolation of NF-IL6 cDNA clones in addition to several clones of the NFKB/rel family members, which provided a first indication of a direct protein-protein interaction between NF-IL6 and NF-KB (LeClair et al., 1992). Subsequent studies have provided evidence for functional and physical interaction between NF-IL6 and NF-KB (Stein et al., 1993; Matsusaka et d.,1993; Kunsch et al., 1994).

18

SHIZUO AKIRA AND TADAMITSU KISHIMOTO

Stein et al. (1993) demonstrated that the transcriptional effect of the interaction between the NF-KBand CEBP families depends on the nature of the interacting proteins and the context of the binding site. When C/ EBP sites are present, the action is synergistic between the CEBP and NF-KB family members. Of particular importance is that a single C/EBP binding site is sufficient to function as a target for the functional synergy. In contrast,when KBbinding sites are present, the NF-KB-CBBP interaction results in decreased transcriptional activity. Inconsistent with the results of Stein et al., Vietor et al. (1996) have reported that C/EBP causes the transcriptional activation of some genes containing a KB element but lacking CEBP binding sites.

B. INTERACTION BETWEEN NF-IL6 AND AP-1 TNF-stimulated gene 6 (TSG-6) encodes a protein expressed during inflammation. The NF-IL6 and AP-1 families functionally cooperate to activate the TSG-6 gene by TNF or IL-1 through a promoter region that contains NF-IL6 sites (-126to -119 and -92 to -83 bp) and an AP-1 element (-126 to -119 bp) (Klampferet al., 1994).Deletion analysis and substitution mutagenesis showed that both sites are necessary for activation by TNF or IL-1. The basic leucine zipper region of NF-IL6 mediates a direct association with the bZIP regions of Fos and Jun in vitro (Hsu et al., 1994). NF-IL6 homodimers can bind to both NF-IL6 and AP-1 sites, whereas Fos and Jun cannot bind to most NF-IL6 sites. Activation of a reporter gene linked to the NF-IL6 site by NF-IL6 is repressed by Fos and by Jun in transient transfection assays. Thus, the interaction of NF-IL6 and AP1family proteins may contribute to the differential regulation of promoters during inflammation. The regulation of NF-IL6-responsive promoters during inflammation undoubtedly will be modulated by the concentrations and the ratios of NF-IL6 isoforms in the nucleus and possibly by AP-1 proteins through protein-protein interactions. Likewise, AP-l-dependent transcription may be regulated by NF-IL6 as a result of the association of NF-IL6 with AP-1 family proteins. By means of South-Western blotting, a cDNA encoding the human C/ EBPy was cloned from a cDNA expression library from human Jurkat T cells as a nuclear factor that binds to the positive regulatory element-I (PRE-I) in the human IL-4 gene (Davydov et al., 1995a). C/EBPy does not activate PRE-l-mediated transcription, but it interacts with members of the AP-1 family. Fos does not bind to PRE-I by itself or in combination with Jun. However, Fos interacts with CBBPy to form an additional complex on the PRE-I site. Jun does not interact with C/EBPy to form an additional complex but significantly enhances CBBPy binding to PRE-

NF-IL6 AND NF-KB IN CYTOKINE GENE REGULATION

19

I. These data indicate that CIEBPy changes the DNA-binding specificity of other transcription factors and recruits them to unusual DNA sites. C. NF-IL6 AND CREB A site located between -2782 and -2729 bp of the human prointerleukin1P gene functions as a strong LPS-responsive enhancer. LPS induces the formation of an NF-IL6-CREB heterodimer at the enhancer (Tsukada et al., 1994). Cotransfection studies using NF-ILG and CREB expression vectors showed that NF-IL6 transactivates the enhancer in the presence of LPS, whereas CREB acts either positively or negatively upon NF-ILGdependent transcriptional activation, depending on its CAMP-regulated phosphorylation state; the presence of unphosphorylated CREB inhibits, whereas phosphoCREB activates the IL-1P gene. Taken together, these results demonstrate that the enhancer element is a specialized LPSresponsive sequence that can be modulated by CAMP as a result of the involvement of NF-IL6-CRE-binding protein heterodimers. D. INTERACTION BETWEEN NF-IL6 AND STAT FAMILY Site-directed mutagenesis studies of the promoter regions of hepatic acute phase genes, including haptoglobin, hemopexin, CRP, and a2macroglobulin, revealed the IL-6 response elements, type 1 and type 2 (Akiraand Kishimoto, 1992).Type 1IL-GREs, characterized by the consensus sequence T(T/G)NNGNAA(T/G),are identical to the C/EBP binding motif. This element binds members of the CEB P family, of which NFIL6 and NF-IL6P are implicated in the regulation of acute phase protein genes by IL-6. The type 2 element consists of the hexanucleotide motif, CTGGGA , The nuclear factor, called acute phase response factor (APRF), binds the hexanucleotide (Wegenka et al., 1993). Molecular cloning of a cDNA encoding APRF revealed that APRF is a member of the STAT family, so it was renamed STAT3 (Akira et al., 1994; B o n g et al.,1994). Many acute phase protein genes, such as a1 acid glycoprotein, C-reactive protein, third component of complement, fibrinogen, and haptoglobulin, harbor both NF-IL6 and STAT binding motifs in their promoter regions, indicating that these two families synergistically activate acute phase protein genes. It is established that some cytokines can direct Ig isotype switching. Germline Ig heavy chain transcripts initiate upstream of their corresponding switch regions and are absolutely required for isotype switching. IL4 induces transcription of the germline CE genes in activated B cells and cells in this population subsequently undergo switch recombination to IgE. An IL-4-responsive element (residing at -126/-79 relative to the transcription initiation site) was identified in the 5’ flanking region of germline CE

20

SHIZUO AKIRA A N D TADAMITSU KISHIMOTO

gene and this segment binds the following three transcription factors: STAT6, one or more members of the C/EBP family, and NF-KB-~~O. Mutations of any of the binding sites for these three factors abolish or reduce IL-4 inducibility of the epsilon promoter. Furthermore, two binding sites for STAT6 and C/EBP can transfer IL-4 inducibility to a minimal cfos promoter (Delphin and Stavnezer, 1995). Data from STAT6 knockout mice demonstrated that STAT6 is essential for isotype switching to IgE (Takeda et al., 1996). Milk protein gene expression is regulated by a complex interplay among hormonally and developmentally regulated transacting factors. Several casein gene promoter regions capable of confemng hormonal inducibility to reporter genes in mammary cells are composite response elements, containing putative binding sites for the same set of hormonally and developmentally regulated factors: namely, C/EBP, MGF/STAT5,and the glucocorticoid receptor (Raught et al., 1995). MGF/STAT5 is a member of the STAT family and is absolutely required for lactogenic hormone induction of &casein gene transcription. The rat P-casein gene has four binding sites for C/EBP isoforms in the hormone response region between -220 and -132 bp, to which NF-IL6 (CIEBPP) and NF-IL6P (C/EBPS) bind in mammary epithelial cells. In close proximity to the C/EBP binding sites are binding sites for MGF/STAT6 and the glucocorticoid receptor. Mutational and deletion analyses demonstrated the functional importance of the C/EBP binding sites for the hormone-regulated transcription of the P-casein gene. C/EBP isoforms may synergize with STAT6 in the pcasein gene. Ciliary neurotrophic factor (CNTF) and LIF regulate vasoactive intestinal peptide (VIP) gene expression through a cytokine response element (CyRE) that interacts with members of the STAT transcription factor family. The CyRE STAT site is insufficient to mediate full transcriptional activation by CNTF/LIF. Three C/EBP binding sites are known within the VIP CyRE. The CEBP-related sites in addition to a STAT site are necessary for CNTFLIF-dependent transcriptional activation by the VIP cytokine response element (Symes et al., 1995). Growth hormone (GH) regulates the tissue-specific expression of a broad range of genes involved in growth, metabolism, and differentiation. GH belongs to the cytokine superfamily. Signaling through the GH receptor is mediated by both Ras-dependent and Ras-independent JAK-STAT pathways; the hormone-induced association of two receptor cytoplasmic domains results in the activation of JAK2, which then activates downstream components of the ras-MAP kinase pathway and cytoplasmic STAT proteins, mainly STAT5 Besides the phosphorylation of STATs, JAK2 activation induces phosphorylation of the SH2-containingadaptor molecule Shc

NF-IM AND NF-KB IN CYTOKINE GENE REGULATION

21

and the association of Shc with the Grb2-Sos complex. The nucleotide exchange factor Sos then promotes the formation of p2lRas (GTP),initiating a cascade of phosphorylation events that culminate with the phosphorylation of specific transcription factors in the nucleus. Adding GH to 3T3F442 preadipocytes led to a rapid increase in the binding activity of C/ EBP. The increase in CEBP binding activity was the result of an increase in the synthesis of NF-ILG(C/EBPP) and NF-ILGP(CIEBP6). GH exerts its effects on C/EBP isoforms at two levels: the transcriptional activation of NF-IL6P and translational activation of NF-IL6 (Clarkson et al., 1995). GH-responsive promoter regions have been described for Spi2.1, somatostatin, insulin, c-fos, and the CYP2C13 isoform of cytochrome P450. STAT5 binds to the growth hormone response element (GHRE) (-149 to -115 bp) on the rat Spi2.1 promoter (Bergad et al., 1995).Two C E B P binding sites are known in its immediate 3’-flanking sequence (-114 to -41 bp). The activity of GHRE is potentiated by proximal 5’ downstream sequences that contain C/EBP binding sites (Paquereauet al., 1992).These results imply that the Spi2.1 transcriptional activation by GH is regulated by the synergistic action of C/EBP and STAT family members. The synergistic activation of ICAM-1 by TNF-a and IFN-7 is also mediated by p65/p50 and p65/c-Rel and the interferon-responsive factor Statla (p91) (Jahnke and Johnson, 1994). E. INTERACTION BETWEEN NF-IL6 AND PU.l The transcription factor PU.l, the product of the Spi-1 protooncogene, is a hematopoietic-specific member of the ets family that is expressed principally in monocytes/macrophages and B lymphocytes (Macleod et aZ., 1992). Screening of a B cell cDNA expression library using radiolabeled PU.l protein as a probe isolated a number of clones interacting with PU.l protein. Among them, one clone encoded NF-ILGP. PU.l and NF-IL6P functionally cooperate to synergistically activate transcription and physically interact (Nagulapalliet aZ., 1995). PU.l and CEBP proteins regulate expression of the GM-CSF receptor a gene (Hohaus et al., 1995). Other C/EBP family target genes in myeloid cells such as G-CSF and the receptor for M-CSF are also regulated by PU.l.

F. INTERACTION BETWEEN NF-IL6 AND THE GLUCOCORTICOID RECEPTOR Glucocorticoids function by binding to specific cytoplasmic receptors, allowing the complex to translocate into the nucleus. Glucocorticoid receptors (GRs) are members of the steroid hormone receptor superfamily, all of which contain a homologous DNA-binding domain and divergent Cterminal ligand-binding domains. Activated GRs bind to a specific DNA

22

SHIZUO AKIRA A N D TADAMITSU KISHIMOTO

element named the glucocorticoid response element (GRE) and induce gene activation. al-acid glycoprotein (AGP)is a major acute phase protein synthesized primarily by the liver, AGP synthesis is synergistically augmented by glucocorticoids and inflammatory cytokines. A GRE is located between positions -120 and -107 bp in the 5'-flanking region of the AGP gene. However, the maximal induction by glucocorticoid requires another sequence located immediately downstream of this GRE. NF-IL6 and C/ EBP bind to the downstream sequence (Alam et al., 1993). Transient cotransfection with glucocorticoid receptor and NF-ILG expression vectors showed that NF-IL6 and ligand-activated glucocorticoid act synergistically to transactivate the AGP gene and that the maximal transcriptional activation of the AGP gene requires expression of intact NF-IL6 and the glucocorticoid receptor (Nishio et al., 1993). Transcriptional synergism was still evident even when one of the two factors lacked either its DNA-binding or transcriptional activation functions. A direct protein-protein interaction between these two distinct transcription factors is also demonstrated. Glucocorticoid has a synergistic effect by inducing the genes for some C/EBP isoforms (Cao et al., 1991). BETWEEN NF-IL6 AND MYB G. INTERACTION The oncogenev-myb of avian myeloblastosis virus encodes a transcription factor that can specifically transform cells of the myelomonocytic lineage. v-myb is a structurally altered form of c-myb, which is highly expressed in immature cells of all hematopoietic lineages and plays an essential role in the proliferation of hemotopoietic progenitor cells. Both v-myb and cmyb specifically recognize the motif, PyAAC(G/T)G, and activate promoters containing this binding sequence. One of the known natural Myb target genes is the chicken mim-1 gene, which is directly activated by c-myb and its promoter contains several Myb consensus binding sites. However, Myb alone does not induce m i m l expression. Myb activates the mim-1 gene by cooperatingwith members of the C/EBP family of transcription factors, such as CIEBPa, CIEBPP, and CIEBPG (Burk et al., 1993). A composite Myb-CIEBP response element consisting of closely spaced Myb and C/ EBP binding sites is both necessary for synergistic activation of the mim1 promoter and sufficient to confer synergistic Myb and C/EBP responsiveness onto a heterologous promoter (Mink et al., 1996).The lysozyme and neutrophil elastase genes are also activated by Myb in combination with C/EBP family members (Burk et al., 1993; Ness et al., 1993; Oelgeschliiger et al., 1995). OF INFLAMMATORY CYTOKINES BY GLUCOCORTICOID H. REPRESSION AND ESTROGEN Glucocorticoid hormones (GHs) are potent immunosuppresive drugs and are clinically applied to suppress immune and inflammatory responses.

NF-IL6 AND NF-XB IN CYTOKINE GENE REGULATION

23

Glucocorticoids work as immunosuppressants in part by inhibiting cytokine gene transcription. The GRE consensus sequences have not been identified in the GH-mediated inhibitory elements of the cytokine promoters. Earlier studies suggested a mechanism of GH-mediated transcriptional repression involving the physical interaction of GR and AP-1 (Jonat et al., 1990; YangYen et al., 1990). This interaction, termed cross-coupling, results in GHmediated repression through AP-1-responsive elements. The zinc finger region of GR is a critical domain for the physical interaction with and repression of AP-1. GH and estrogen downregulate the expression of inflammatory cytokines such as IL-6 and IL-8 by the transcription factors NF-KBand NF-IL6 via a direct interaction between these factors and the GR or estrogen receptor (ER) (Ray and Prefontaine, 1994; Ray et al., 1994; Scheinman et al., 1995b; Stein and Yang, 1995). The physical and functional interaction depends on the DNA-binding domain of GR or ER and on the Re1 homology domain of NF-KBand the bZIP region of NF-IL6. A novel mechanism of glucocorticoid-mediated repression of NF-KB activities was identified (Auphan et al., 1995; Scheinman et al., 1995a). GHs induce transcription of the I K Bgene. ~ The increase in I K BmRNA ~ results in an increased rate of I K B protein ~ synthesis, which effectively inhibits NF-KB activation. Although stimulation by TNF or IL-1 causes the release of NF-KB from I K B ~in, the presence of GH, this released NF-KBquickly reassociates with synthesized I K B ~thus , markedly reducing the amount of NF-KB that translocates to the nucleus (Fig. 5). BETWEEN NF-IL6 AND OTHER I. INTERACTION TRANSCRIPTION FACTORS The rat CYP2D5 gene encodes a cytochrome P450, a superfamily of enzymes, many of which are involved in oxidative metabolism of foreign compounds. Transfection studies using a series of CYP2D5 upstream DNA CAT gene fusion constructs identified a DNA element between -55 and -156 bp that conferred transcriptional activity in HepG2 cells. DNase I footprinting revealed a region protected by both HepG2 and liver cell nuclear extracts between -83 and -112 bp. This region displayed some sequence similarity to the Spl consensus sequence and bound the Spl protein. CYP2D5 promoter activity was markedly increased by cotransfection with a vector expressing NF-IL6. NF-IL6 alone was unable to directly bind the -83 to -112 bp region of the promoter but produced a ternary complex when combined with HepG2 nuclear extracts or recombinant human Spl. A poor CiEBP binding site is present adjacent to the Spl site, the mutagenesis of which abolished formation of the ternary complex with the CYP2D5 regulatory region. These results showed that NF-IL6 and Spl can work in conjunction, possibly by protein-protein interaction, to activate the CYP2D5 gene (Lee et al., 1994).

24

SHIZUO AKIRA AND TADAMITSU KISHIMOTO

FIG.5. Mechanisms of glucocorticoid-mediated inhibition of cytokine production. The activation of NF-KB involves the targeted degradation of cytoplasmic inhibitor, IKB, and the translocation of NF-KBto the nucleus. Nuclear NF-KBinduces transcription of cytokine genes. In the presence of glucocortimids IKB synthesis is increased as a result of increased transcription. Nuclear translocation of NF-KB is inhibited by the reassociation of NF-KB with newly synthesized IKB.On the other hand, activated glucocorticoid receptors physically associate with NF-KB and NF-IL6 and repress the DNA binding of these transcription factors, which results in the transcriptional repression of cytokine genes.

The monocyte-specific expression of the macrophage colony-stimulating factor (M-CSF) receptor is regulated by the transcription factors PU.l, AML1, and C/EBP. AMLl is a member of the core binding factor (CBF)or polyomavirus enhancer binding protein 2 (PEBPB) family of transcription factors (Ogawa et al., 1993; Wang et al., 1993). The members of the CBF family consist of heterodimers between DNA-binding a subunits and a p subunit (CBFP) that does not bind DNA directly but that enhances the binding of the a subunit. All the CBFa proteins contain a Runt domain, which is similar to the protein product of the DrosophiZa pair-rule gene runt, that encodes an early acting segmentation protein that regulates the expression of other segmentation genes. AMLl was identified by studying one of the most frequent chromosomal translocations found in AML,

NF-IL6 AND NF-KB IN CYTOKINE GENE REGULATION

25

t(8;21)(q22;q22).In uitro binding analysis revealed that the Runt domain of AMLl physically interacts with C/EBP. Transfection studies showed that C/EBP and AMLl in concert with the AMLl heterodimer partner, CBFP, synergistically activate the M-CSF receptor by more than 60-fold. These results demonstrated the physical and functional interactions between AMLl and the CEBP transcription factor family (Zhanget d., 1996). IL-6 gene expression is constitutively upregulated in many neoplastic cell lines. In HeLa cells, wild-type human or murine p53 preferentially repressed the IL-6 promoter, whereas the p53 mutant Val-135 and Phe132 upregulated IL-6 promoter activity in these cells. An intact NF-IL6 binding site in the IL-6 promoter was a requirement for upregulation of the IL-6 promoter by these p53 mutants, suggesting that NF-IL6 is a target for p53 modulation (Margulies and Sehgal, 1993). In functional experiments,wtp53 blocked transcriptional activation of the IL-6 promoter by NF-IL6. In contrast, the p53 mutant species Val-135 and Phe-132 enhanced NF-IL6-mediated gene activation. Wild-type RB interacts through its SV40 T antigen-binding domains with NF-IL6 both in ultro and in cultured cells (Chen et al., 1996). This interaction occurs in monocyte/macrophage precursors precisely when the cells differentiate and continues in terminally specialized cells. Furthermore, RB directly activates NF-IL6 by enhancing its binding to cognate DNA sequences and by increasing the transcription of a gene containing NF-IL6-binding elements in its promoter sequence. In addition to negatively regulating transcription factors such as E2F to prevent quiescent cells from passing a restriction point in G1, RB may positively regulate NF-IL6, a factor important for differentiation. V. Cytokine Gene Regulation

A. IL-1 GENEREGULATION Transient transfection demonstrated that two regulatory regions control the induction of the IL-1P gene: the upstream induction sequence (UIS) located between positions -3134 and -2729 bp and the promoter-proximal regulatory elements between positions -131 and +12 (Shirakawa d al., 1993; Auron and Webb, 1994) (Fig. 6). The UIS is extremely responsive to activation by LPS alone or superresponsive to LPS+dibutyryl CAMP. This region contains two independent enhancer regions, -2782 to -2729 and -2896 to -2846 bp, that appear to act cooperatively. The latter contains a c-AMP response element, whereas the former has a pseudosymmetric cyclic AMP response element binding site that binds heterodimer of NFIL6 and CREB-likeproteins (Tsukadaetal., 1994).The proximal regulatory elements are necessary for the tissue-specific expression and bind NF-IL6

26

SHIZUO AKIRA A N D TADAMITSU KISHIMOTO

-3

729

-

I

v

-7

-5;

NF-IL6

\

-3134

-2729

-297

-286

\

"7'

PU.l NF-116

/

-1 81

+12

P R E FIG.6. Cis-regulatory elements and transcription factors involved in IL-1/3 expression. UIS, upstream induction sequence; CRE, cyclic AMP response element; PRE, proximd regulatory element.

and PU.l. NF-KBalso plays a role in regulating IL-lP expression. Promoter deletion analyses identified two p65-responsive regions between -2800 and -2720 bp and between -512 and -133 bp; classical NF-KB binds to a consensus site at -296/-286 bp (Cogswell et al., 1994). Within an 80-bp region between -2800 and -2720 bp, there is a CRE-like 2761/-2753 bp element in addition to the KB -2751/-2741 bp element. Mutation of the CRE-like but not the KBelement caused a specific loss of p65 transactivation. It is likely that the bZIP family members and RelA form a complex that binds to sequences such as CREB/RelA and NF-ILG/RelA. Direct binding of NF-KB to DNA is not required, but NF-KB regulates I L l p gene induction via direct association with NF-IL6 in the absence of a cognate DNA binding site. NF-IL6 binding sites near the transcriptional initiation site are also necessary for the LPS induction of IL-lp (Zhang and Rom, 1993; Godambe et al., 1994). B. IL-6 GENEREGULATION There are five known functional cis-regulatory elements, namely, CAMP response element (CRE), AP-1, NF-IL6 (Isshiki et al., 1990), Spl (Kang et al., 1QQ6),and NF-KB sites in the promoter region of the IL-6 gene (Fig. 7). An additional NF-IL6 site (positions -72 to -63 bp) was identified in the IL-6 promoter (Zhang et al., 1994) (Fig. 7). These motifs are highly conserved among mammals. Zhang et al. (1994) examined the importance of each element in IL-6 expression in monocytes. Lipoarabinomannan (LAM) from the mycobacterial cell wall and LPS potently induce IL-6 gene expression in peripheral blood monocytes. By deletion analysis and

NF-ILG AND NF-xB IN CYTOKINE GENE REGULATION - 1 63

-287

AP-1 -277 CRE

NF-ILG

-1 4 5

CAlTGCACAATCT

27

-75 N F - a - 6 6 GGGAlllTCC

~ A T AAATGT C

-87 S'NF-JL6 - 7 6

FIG.7. Functional &-regulatory elements in the IL-6 promoter. CRE, cyclic AMP response element; SRE, serum response element.

chloramphenicol acetyltransferase assays in the human myelomonocytic leukemia cell line THP-1, both LAM- and LPS-inducible IL-6 promoter activities were localized to a DNA fragment at positions -158 to -49 bp, where two NF-IL6 and one NF-KB motifs are located. Site-directed mutagenesis of one or more of these motifs within the IL-6 promoter demonstrated that they both have positive regulatory activity. Deletion of all three elements abolished the inducibility of IL-6 promoter activity by both LAM and LPS, showing that the NF-IL6 and NF-KBsites mediate IL-6 induction in response to both LAM and LPS. Similarly, Dendorfer et al. (1994) identified the regulatory elements involved in IL-6 gene activation by prostaglandin E l , its second messenger CAMP, and by LPS in the mouse monocyhc cell line PUS-1.8. Mutations within four regulatory elements (AP-1, CRE, NF-IL6, and NF-KB)significantly reduced, but did not completely abrogate, the inducibility by prostaglandin E l or its second messenger CAMP. However, LPS-induced promoter activity was almost completely abolished by mutations in the NF-KB site, suggesting that a single regulatory element is crucial for LPS inducibility, whereas prostaglandins and cAMP act through multiple, partially redundant regulatory elements. These results indicate that the activity of at least four transcription factors is simultaneously required to maximally induce IL-6 gene transcription upon stimulation with cAMP or LPS, but the contribution of each regulatory element to the transcriptional activation of IL-6 gene appears to vary depending on the stimulus. Hypoxia induces IL-6 expression in endothelial cells (ECs).Transfection of ECs with a deletion chimeric IL-6 promoter-chloramphenicol acetyltransferase construct identified a hypoxia response element at -225/ -111 bp of the IL-6 gene. Electron mobility shift assays using -225/-111 bp as the labeled probe demonstrated enhanced binding activity in nuclear extracts of hypoxic ECs, and the hypoxia-induced band displayed a super-

28

SHIZUO AKIRA AND TADAMITSU KISHIMOTO

shift with antibody to NF-IL6. These results suggest that the hypoxiainduced activation of IL-6 transcription results from the activation of NFIL6 (Yan et al., 1995). The deregulated production of IL-6 is implicated in the pathogenesis of some HIV1-associated diseases. The molecular mechanisms underlying the abnormal IL-6 secretion of HIV-infected cells include transactivation of the IL-6 gene by HIVl. TAT transactivates the human IL-6 promoter and it requires a minimal region located between -172 and -52 bp, which contains both NF-IL6 and NF-KBbinding sites (Scalaet al., 1994).DNAprotein binding experiments showed that tat-transfected cells express a consistent increase in KB and NF-IL6 binding activity, suggesting that TAT protein plays a role in the pathogenesis of some HIV-associated diseases by modulating the expression of host cellular genes. C. IL-8 GENEREGULATION Transcription of the IL-8 gene requires the activation of a combination of either NF-KBand AP-1 or NF-KB and NF-IL6, depending on the cell type (Mukaida et al., 1994).The functional interaction of the NF-IL6 and NF-KB families in IL-8 gene regulation was examined (Matsusaka et al., 1993; Stein et al., 1993; Kunsch et al., 1994). Transient cotransfection assays demonstrated that RelA and members of the NF-IL6 family can functionally cooperate in transcriptional activation of the IL-8 gene. Electrophoretic mobility shift analysis indicated that NF-IL6, as well as other related members of this family, binds specifically to the NF-IL6 site in the IL-8 promoter, whereas RelA, but not p50, binds specifically to the NF-KB site. Mutational analyses of RelA demonstrated that the C-terminal transactivation domain and the DNA-binding domain are required for synergistic activation with NF-IL6. The IL-8 gene is induced by protein X of the hepatitis B virus (HBVX). Both NF-KB and CEBP binding sites are essential and sufficient for the induction of the IL-8 gene by HBV-X (Mahe et al., 1991). D. G-CSF GENEREGULATION The G-CSF promoter harbors a region (-200 to -165 bp) that is required for activation of the G-CSF gene by TNF-a and IL-1P (Shannon et al., 1992). This region contains the decanucleotide CK-1 element similar to the NF-KBbinding site and two repeated sequences that resemble NFIL6 binding sites. The equivalent regions of the mouse G-CSF promoter are required for LPS induction in mouse macrophages (Nishizawa and Nagata, 1990).The cytokine response region that contains the CK-1 decanucleotide element and the two NF-IL6 concensus elements independently confers TNF-dL-1P inducibility on a heterologous promoter. Similar to

NF-ILG AND NF-KB IN CYTOKINE GENE REGULATION

29

IL-8, RelA but not p50 binds to the CK-1 element. Electrophoretic mobility shift studies showed that RelA and NF-IL6 cooperatively bind to the cytokine response region and form a RelA/NF-ILG ternary complex (Dunn et al., 1994). The transactivator protein, Tax, from the human T leukemia virus type 1, transactivates the human G-CSF promoter. Mutations in either the CK1 element or the adjacent NF-IL6 element of the TNF response region of the G-CSF promoter reduced Tax activation, suggesting that the ReW NF-IL6 complex is also required for Tax function (Himes et al., 1993).

E. IL-4 Activity of the IL-4 promoter is localized to several cis-acting elements located within the first 300 bp from the transcriptional site. Five repeated elements, PO to P4, that share the common consensus ATITCCNNT are located between -40 and -250 bp, and each interacts with the T cell-specific factor NF(P). NF(P) contains a member of the calcineurin-dependentNFAT family of transcription factors and may also include less characterized factors (Rao, 1994). The IL-4 promoter Y box -114 CTGATTGG-107 significantly enhances overall promoter activity. Two sites, -85GTGTAATA-78 and -245GTGTAATT-238, which share sequence identity to the OAP region within the IL-2 promoter, are adjacent to the NF(P) binding sites P1 and P4 and the two are also responsible for transcriptional activation. NF-ATp and AP-1-related factors cooperatively activate the transcription of reporter genes driven by composite OAP/P sequence repeats (Rooney et al., 1995). It is considered that AP-1 family members interact with the OAP site to stabilize NF-AT interaction with the P sequences. NF-IL6 is also a transcription factor that binds to the OAP site in Jurkat cells and some T cell lines (Davydovetal., 1995b).Overexpression of NF-IL6 enhances transcription of the human IL-4 promoter, suggesting that NF-IL6 is involved in transcriptional activation of the human IL-4 promoter in T cells, although interaction of NF-IL6 with other transcription factors such as the NF-AT family remains unknown. VI. Cytokine Induction in NF-IL6 Family Knockout Mice

A. NF-IL6KO

NF-IL6 KO mice are viable but highly susceptible to facultative intracellular organisms, such as Listeria monocytogenes and Salmonella typhimudurn, due to impairment of bacteria killing by activated macrophages (Tanaka et al., 1995). The induction of various cytokines was examined in NF-IL6 KO mice. RNA was prepared from several sources (resident peritoneal macrophages and proteose peptone-elicited peritoneal macro-

30

SHIZUO AKIRA AND TADAMITSU KISHIMOTO

phages with or without stimulation by LPS in uitro).Reverse-transcribed cDNA was amplified by primers for 9 cytokines (TNF-a, IL-lP, IL-6, IL10, IL-12, MIPla, G-CSF, GM-CSF, and M-CSF). The induction levels of all cytokines in the NF-IL6 KO mice were comparable to those in wildtype mice, with the exception of G-CSF. Induction of G-CSF mRNA was also impaired in NF-IL6-deficient embryonic fibroblasts and bone marrowderived fibroblasts, but not in endothelial cells, demonstrating that NFIL6 is involved in G-CSF gene expression in a tissue-specific manner. This observation is consistent with the finding that both antisense oligonucleotides and ribozyme-mediated specific elimination of NF-IL6 transcripts abolished the TNF-a-induced synthesis of G-CSF in human fibroblasts. The cytokine induction of NF-1LG-I-embryonic fibroblasts in response to LPS was also examined. Besides G-CSF, several cytokines, such as IL-1P, IL-6, TNF, IL-12, and GM-CSF, were significantly decreased in NF-IL6”fibroblasts compared with the wild-type, indicating that NF-IL6 is actually involved in the LPS-mediated induction of IL-1P, IL-6, TNF, IL-12, and GM-CSF in embryonic fibroblasts (T. Tanaka and S. Akira, 1996). B. C E B P KO MICE Analysis of the homozygous CEBP-deleted mice demonstrates that C/ EBP is critical to the production and maintenance of life-sustaining metabolic fuel levels in the neonate (Wang et al., 1995). At birth, C/EBP/-mice were indistinguishable from their littermates. The gross morphology of the major organs was normal, as were birth weights. However, the C/ EBPL neonates became lethargic several hours after birth and died from hygoglycemiawithin8 hrpostpartum. In these mutant mice, the amounts of glycogen synthase mRNA were 50-70% of normal and the transcriptional induction of the genes for the gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, was delayed. Albumin mRNA was reduced by 50% in the mutant mice. The hepatocytes and adipocytes of the mutant mice failed to accumulate lipid, and expression of the gene for uncoupling protein, the defining marker of brown adipose tissue, was reduced. However, the mRNAs for fatty acid synthase, GLU4, and 42YaP2 were unaltered in the CEBP-deficient mice. Niether NFIL6 (CIEBPP) nor NF-IL6P (CEBPS) mRNA amounts were elevated to compensate for the lack of CEBP. The effects of C E B P deficiency on cytokine gene expression have not yet been characterized. VII. Cytokine Indudon in NF-KBKnockout Mice

A. ~ 5 KO 0 MICE Mice lackingthe p50 subunit of NF-KBshowno developmental abnormalities but exhibit multifocal defects in immune responses involving B

NF-IUi AND NF-KB IN CYTOKINE GENE REGULATION

31

lymphocytes and anonspecific response to infection. B cells do not proliferate in response to bacterial LPS and antibody production is impaired (Shaet al., 1995; Snapper et al.,1996). Total serum Ig is approximately 4-fold lower than in the knockout mice and IgE was reduced approximately40-fold, suggesting an important role of p50 in heavy-chain class switching. In fact, p50 is involved in inducing the germline E transcript, which is responsible for switching to IgE. Mice lacking p50 cannot remove L. monocytogenes and are more susceptible to infection with S. pneumoniae but are more resistant to infection with murine encephalomyocarditis (EMC) virus. The resistance to infection with EMC virus suggested that the absence of p50 was actually augmenting antiviral responses. Indeed, virally infected fibroblasts lacking p50 had augmented induction of IFN-/3 transcription in comparison to control fibroblasts. The expression of many genes, such as immunoglobulin K light chain, class I MHC, and TNF-a, was not affected by the absence of p50. In contrast, the expression of genes such as IL-6 was reduced, whereas the transcription of genes such as IFN-P was augmented in the absence of p50. The p501 macrophages stimulatedwith LPS, TNF-a, and IL-la release were normal, but IL-6 release was decreased several-fold relative to control macrophages. Thus, it is likely that p50 negatively and positively regulates expression of the IFN-P and IL-6 genes, respectively.

B. R E L A / P ~KO ~ MICE Disruption of the RelA locus leads to embryonic lethality at 15 or 16 days of gestation, concomitant with massive degeneration of the liver by programmed cell death or apoptosis (Beg et al., 1995). IKB and GM-CSF are TNF-a responsive and contain NF-KB sites. The normal induction of the p50/RelA heterodimer by TNF is lost in the RelA knockout mice. Embryonic fibroblasts from RelA-deficient mice are defective in the TNFmediated induction of mRNA for I K Band ~ GM-CSF, although basal levels of these transcripts are unaltered, indicating that RelA controls inducible, but not basal, transcription in NF-KB-regulated pathways. C. C-REL KO MICE Mice with an inactivated c-rel gene develop normally, and cells from all hemopoietic lineages appear normal. However, humoral immunity is impaired and mature B and T cells are unresponsive to most mitogenic stimuli (Kontgen et al., 1995). Proliferation of B cells induced by LPS, CD40, or anti-IgM is defective. IL-2 levels in Rel” T cells stimulated with concanavalin A, anti-CD3, or anti-CD3/anti-CD28 were low or undetectable, whereas much higher levels of IL-2 are present in the culture supernatant of Rel-’- T cells incubated with PMA and ionomycin, indicating the involvement of c-re1 in a specific intracellular signal pathway required for

32

SHIZUO AKIRA A N D TADAMITSU KISHIMOTO

IL-2 transcription. Anti-CD3- and anti-CD28-treated Rel-/-T cells make low or no detectable levels of IL-3, IL-5, GM-CSF, TNF-a, and IFN-y. The ability of exogenous IL-2 to restore T but not B cell proliferation indicates that the T cell-proliferative defect arises from a lack of IL-2 production and that Re1 regulates the expression of different genes in B and T cells. The genes regulated by Re1 that are critical for B cell proliferation are yet to be identified. Exogenous IL-2 also restores IL-5, TNF-a, and IFN-y, but not IL-3 and GM-CSF expression to approximately normal levels. In contrast, lipopolysaccharide-stimulated Re1-l-macrophages produce higher than normal levels of GM-CSF. These findings show that Re1 functions as an activator or repressor of gene expression and is required for the production of IL-3 and GM-CSF by T lymphocytes (Gerondakis et al., 1996). D. RELB KO MICE The expression of RelB is mainly restricted to lymphoid tissues such as the thymus, spleen, and lymph node in the adult mouse. In the thymus, RelB transcripts are confined to the medulla and high levels are expressed in the nucleus of interdigitating dendritic cells. Mice homozygous for the disrupted relB locus have phenotypic abnormalities including multifocal, mixed inflammatory cell infiltration in several organs, myeloid hyperplasia, splenomegalydue to extramedullaryhematopoiesis, and a reduced population of thymic dendritic cells (Burkly et al., 1995; Weih et al., 1995). The induced expression of “classical”NF-KBmel-regulatedgenes, such as IFNy, TNF-a, and IL-2, in the liver and lung of RelB-deficient mice suggests that RelB complexes do not play a major role in the regulation of these genes. E. I K BKO ~ MICE I K BKO ~ mice have been generated. These mice are apparently normal at birth but then die approximately 7 days later due to wasting. The I K B ~ KO mice have a small spleen and thymus, and granulopoiesis is enhanced. Cytoplasmic retention and the rapid nuclear translocation of NF-KB can occur in the absence of I K B ~which , actually results in a sustained NFKB response. The expression of some genes, such as G-CSF and VCAM1, is upregulated. An I K B deficiency ~ in mice results in a severe and widespread dermatitis (Beg et al., 1995; Klement et al., 1996). VIII. Conclusion

The regulated transcription of genes expressed in the immune system depends on the combinatorial activation of several transcription factors

NF-ILfi AND NF-KB IN CYTOKINE GENE REGULATION

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including the NF-IL6, NF-KB, Fos-Jun, CREB/ATF, NF-AT, and STAT families. Even in activation of the same gene, the combinatorial assembly of the transcription factors on the promoter seems to vary depending on the tissue or in response to individual extracellular signals. Indeed, gene knockout experiments have demonstrated the involvement of various transcription factors in the tissue- and signal-specific activation of certain genes. It is important to identify the target genes regulated by transcription factors involved in the immune response in a tissue- and signal-specific manner. This will increase understanding of the immune response at the molecular level and allow further development of new therapeutic intervention strategies against immunologically regulated diseases by blocking the activity of transcription factors instead of using steroid hormones. ACKNOWLEDGMENTS Studies on NF-IL6 in the laboratory of the authors presented in this article were supported in part by grants from the Ministry of Education of Japan. We thank T. Tanaka for the figures and a critical reading.

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Wang, H., Liu, K., Yuan, F., Berdichevsky, L., Taichman, L. B., and Auborn, K. (1996). CIEBPP is a negative regulator of human papillomavirus type 11in keratinocytes.]. Virol. 70,4839-4844. Wang, N.-D. Finegold, M. J.. Bradley, A., Ou, C. N., Abdelsayed, S. V., Wilde, M. D., Taylor, L. R., Wilson, D. R., and Darlington, G. J. (1995). Impaired energy homeostasis in C/EBPa! knockout mice. Science 269,1108-1112. Wang, S., Wang, Q., Crute, B. E., Melnikova, I. N., Keller, S. R., and Speck, N. A. (1993). Cloning and characterization of subunits of the T-cell receptor and murine leukemia virus enhancer core-binding factor. Mol. Cell. Biol. 13, 324-3339, Watkins, P. J., Condreay, J. P., Huber, B. E., Jacobs, S. J., and Adams, D. J. (1996). Impaired proliferation and tumorigenicity induced by CCAAT/enhancer-binding protein. Cancer Res. 56, 1063-1067. Wegenka, U. M., Buschmann, J., Luttticken, C., Heinrich, P. C., and Horn, F. (1993). Acute-phase response factor, a nuclear factor binding to acute-phase response elements, is rapidly activated by interleukin-6 at the posttranslational level. MoZ. Cell. Biol. 13, 276-288. Wegner, M., Cao, Z., and Rosenfeld, M. (1992). Calcium-regulated phosphorylation within the leucine zipper of CIEBPP. Science 256,370-373. Weih, F., Carrasco, D., Durham, S. K., Barton, D. S., Rizzo, C. A., Ryseck, R.-P., Lira, S. A., and Bravo, R. (1995). Multiorgan inflammation and hematopoietic abnormalities in mice with a targeted disruption of RelB, a member of the NF-KB/Rel family. Cell 80, 331-340. Wilhelmsen, K. C., Eggleton, K., and Temin, H. M. (1984). Nucleic acid sequences of the oncogene v-re1 in reticuloendotheliosis virus strain T and its cellular homolog, the protooncogene c-rel. J. Vlrol. 52, 172-182. Williams, P. M., Chang, D. J., Danesch, U., Ringold, G. M., and Heller, R. A. (1992). CCAAT/enhancer binding protein expression is rapidly extinguished in TA1 adipocyte cells treated with tumor necrosis factor. Mol. Endocrinol. 92, 1135-1141. Williams, S. C., Cantwell, C. A., and Johnson, P. F. (1991). A family of C/EBP-related proteins capable of forming covalently linked leucine zipper dimers in vitro. Genes Deu. 5, 1553-1567. Wu, Z., Xie, Y., Bucher, N. L., and Farmer, S. R. (1995). Conditional ectopic expression of C/EBPP in NIH-3T3 cells induces PPARy and stimulates adipogenesis. Genes Deu. 9,2350-2363. Yamamoto, K., Arakawa, T., Ueda, N., and Yamamoto, S. (1995). Transcriptional roles of nuclear factor K B and nuclear factor-interleukin-6 in the tumor necrosis factor adependent induction of cyclooxygenase-2in MC3T3-El cells.]. Biol. Chem. 270,3131531320. Yan, S. F., Tritto, I., Pinsky, D., Liao, H., Huang, J., Fuller, G., Brett, J., May, L., and Stern, D. (1995). Induction of interleukin 6 (IL-6) by hypoxia in vascular cells. Central role of the binding site for nuclear factor-IL-6. ]. Bfol. Chem. 270, 11463-11471. Yang-Yen, H.-F., Chambard, J.-C., Sun, Y.-L., Smeal, T., Schmidt, T. J., Drouin, J., and Karin, M. (1990). Transcriptional interference between c-Jun and the glucocoriticoid receptor: Mutual inhibition of DNA binding due to direct protein-protein interaction. Cell 62, 1205-1215. Yeh, W.-C., Cao, Z., Classon, M., and McKnight, S. L. (1995). Cascade regulation of terminal adipocyte differentiation by three members of the C/EBP familyof leucine zipper proteins. Genes Deu. 9, 168-181. Yuh, C. H., and Ting, L. P. (1991). C/EBP-like proteins binding to the functional box-a and box-p of the second enhancer of hepatitis B virus. Mol. Cell. Biol. 11,5044-5052.

46

SHIZUO AKIRA AND TADAMITSU KISHIMOTO

Zachow, K. R., and Conklin, K. F. (1992). CArG, CCAAT, and CCAAT-like protein binding sites in avian retrovirus long terminal repeat enhancers. J. Virol. 66, 1959-1970. Zhang, D.-E., Hetherington, C. J., Meyers, S., Rhoades, K. L., Larson, C. J., Chen, H.-M., Hiebert, S. W., and Tenen, D. G . (1996). CCAAT enhancer-binding protein(C/EBP) and AML1(CBFa2) synergisticallyactivate the macrophage colony-stimulatingfactor receptor promoter. Mol. Cell. Biol. 16, 1231-1240. Zhang, Y., and Rom, W. N. (1993). Regulation of the interleukin-l/3 (IL-1p) gene by mycobacterial components and lipopolysaccharideis mediated by two nuclear factor-IL6 motifs. Mol. Cell. Biol. 13, 3831-3837. Zhang, Y., Broser, M., and Rom, W. N. (1994). Acctivation of the interleukin 6 gene by Mycobacterium tuberculosis or lipopolysaccharide is mediated by nuclear factors NFIL6 and NF-KB. Proc. Natl. Acad. Sci. USA 91,2225-2229. Zhong, Z., Wen, Z., and Darnell, J. E. (1994). Stat3: A STAT family member activated by tyrosine phosphorylationin response to epidermal growth factor and interleukin-6. Science 264,95-98.

This article was accepted for publication on 27 September 1996.

ADVANCES IN IMMUNOLOGY. VOL. 65

Transporter Associated with Antigen Processing TIM EUlOll Nuffidd Dqmriment of Chkd Medicine, UnivcKlify d O M , John Radclfi Hospital, O M OX3 W, U n k d Kingdom

1. Introduction

The transporter associated with antigen processing (TAP) literature is large, but not yet too large to include in a single review. Although I have made every effort to make a comprehensive survey, I appreciate that I have missed some contributions, and I apologize to my colleagues who I may have inadvertently omitted and to those who feel misinterpreted because this is a highly personal account of the literature. I have included many of my own speculations that may become rapidly and embarrassingly out of date. I hope that some of them, although not being informative in the most literal sense, might help to stimulate discussion around this fascinating immunologically related molecule. A. THENEED FOR A TRANSPORTER Throughout the late 1970s, after the discovery that the recognition of viral antigens by cytotoxic T lymphocytes (CTLs) was MHC class I restricted (1-4), most immunologists believed antiviral CTLs recognized viral glycoproteins expressed at the surface of infected cells. This belief persisted even after it was shown that nonglycosylated proteins, which do not form part of the viral coat, were also target antigens for CTLs. These included influenza matrix protein (MP) and nucleoprotein (NP) (5-8). Several investigatorstried to reconcile this new data with the current model for CTL recognition by attempting to demonstrate the presence of small amounts of these internal proteins at the surface of infected cells (9-13). Neither MP nor NP contain a classical signal sequence that would target them to the secretory pathway, and exactly how these antigens might have appeared at the cell surface was the focus of lively debate for several years. Townsend and colleagues, having established that a cloned nucleoprotein gene could be recognized by anti-influenza A-specific CTLs, when expressed in uninfected target cells (14),went on to try and identify signal sequences within nucleoprotein that might direct it to the cell surface (15). They found that relatively short, nonoverlapping polypeptide fragments of NP could sensitize transfected target cells to lysis, indicating that no special signal sequence was needed for their recognition at the cell surface. Also, the two ends of the molecule (containing two different CTL epitopes) 47

Copynght 0 1997 by Academic Press All nghts of repmduction in any form reserved.

0065-27776/97$25 00

48

TIM ELLIOTT

could be transported to the cell surface independently of one another. Furthermore, a genetically engineered influenza A hemagglutinin (HA) molecule that lacked the ER translocation signal sequence and that was rapidly degraded in the cytoplasm, was as good as native HA in sensitizing target cells for lysis by HA-specific CTLs (16). A year later Townsend showed that synthetic peptides added to the exterior of cells were as effective at sensitizing MHC class I-matched target cells as virus infection (17).Together, these observations led him to the prescient conclusion that MHC class I-restricted antigens were synthesized in the cytoplasm and underwent partial hydrolysis there, and that the resulting peptide fragments were transported out of the cytosol by a novel transport mechanism that did not require a special signal sequence (15). It was these fragments, then, that were recognized by CTLs in association with class I MHC molecules. Subsequent experiments have indeed established that the primary site for antigen degradation is the cytosol, and that peptides first come into contact with MHC class I molecules in the lumen of the endoplasmic reticulum (reviewed in Ref. 18). Viral epitopes are therefore transported to the cell surface bound to MHC class I molecules. The presence of proteolytic activity in the cytosol and expression of MHC class I heavy chains and beta 2-microglobulin (D2m) in the ER is, however, insufficient for the presentation of endogenous antigens to CTLs. This was first indicated by the observation that in certain recombinant strains of laboratory rat (for example, the rl recombinant in which the rat MHC class I region from the a haplotype is expressed on the c haplotype background), the MHC class I allele RT1.Aa was unable to present some (al1o)antigensand was transported to the cell surface very slowly compared to its expression on the native RTla haplotype background (19-21). The genetic factors responsible for these phenomena were called class I modifiers (cim).Careful genetic analysis mapped the cim phenomenon to a region between RT1.H and RT1.Ba (22), and established that it existed in two allelic forms, cima(which allowed the presentation of doantigens by RT1.Aa and facilitated rapid intracellular trafficking) and cimb (which prevented the presentation of these epitopes and led to prolonged retention of RT1.Aa in the ER). Cfmawas dominant over cimb. Other studies that showed that the expression of class I molecules was necessary but not sufficient for antigen presentation came from the study of mutant cell lines. One of these was RMA-S, a derivative of the murine thymoma 28.7, which had undergone chemical mutagenesis followed by repeated selection for loss of MHC class I expression using anti-class I antibodies plus complement (23, 24). Townsend et al. showed that the class I molecules expressed by RMA-S were unable to present endogenous antigens to CTLs followingviral infection even though their intrinsic ability

TRANSPORTER ASSOCIATED WITH ANTIGEN PROCESSING

49

to present preprocessed peptide fragments when added to the cell culture medium was unimpaired. Furthermore, he showed that the class I heavy chain was unable to associate stably with p2m in RMA-S unless peptides were added to the cell culture (25). He reasoned that the two phenomena may be the result of a single genetic lesion that results in the inability of RMA-S to supply newly synthesized MHC class I molecules with peptides generated in the cytosol. This, he suggested, was most likely to be a defect in the putative transport mechanism for peptides out of the cytosol which he had proposed earlier. A second mutant cell line called LBL721.174, again selected for loss of MHC class I expression (26), was found to have an identical phenotype to RMA-S with respect to its ability to assemble stable heavy chain (HC)/P2m heterodimers and present viral antigens to CTLs (27). LBL721.174 had been shown previously, by complementation analysis, to have a large deletion in the class I1 region of the MHC that is contained within the equivalent region in the rat MHC to which the cim phenomenon maps (22, 28). Thus, a connection was made between the phenomenology observed in the rat cim phenomenon and that observed in the mutant antigen presenting cells RMA-S and LBL721.174. Also, the key to this connection lay somewhere in the MHC class I1 region and most likely involved a gene or genes that were responsible for delivering peptides to the lumen of the ER and/or loading newly synthesized class I molecules with them. B. A SUITABLE CANDIDATE Is DISCOVERED It was against this background that four groups independently described a candidate gene for a factor that would transport peptides across the ER membrane from the cytosol into the ER where they could assemble with newly synthesized class I molecules. Deverson et al. (29), searching for the gene(s) responsible for the cim phenomenon, identified two abundant cDNAs that they called mtpl and mtp2 (MHC linked transporter protein) by screening a cDNA library using an overlapping set of mouse cosmids covering the cim region as probes. Monaco et al. (30, 31), in their quest to identify two polymorphic proteins in the mouse that they had mapped to the region of the MHC responsible for the cim phenomenon, found 4 new genes. Two of these turned out to be the genes they were huntingLMP2 and LMP7 (32) (see elsewhere in this volume for their immunological relevance), whereas the other 2 homologous genes they called HAM1 and HAM2 (histocompatibility antigen modifier). The other two groups independently identified one of the human counterparts to these new genes. Trowsdale et al. (33,34), who were at the time mapping the human MHC, identified 5 new genes within the region deleted in LBL721.174 by probing cosmid and YAC clones covering that region onto cDNA librar-

50

TIM ELLIOlT

ies and identifylng clones with clusters of restriction sites containing the motif C(G),. These regions, also called CpG islands, are often found at the 5’ end of genes. Two of these seemed appropriate candidates for a peptide transporter and were called RING4 and RING11 (really interesting new gene). Spies et al. (35, 36)used chromosome walking across the region deleted in LBL721.174 to identify genes that might be responsible for its class I-loss phenotype. Having generated a series of overlapping cosmid clones that spanned the region between DRA and the steroid 21hydroxylase B locus, these were used to probe a cDNA library for new genes. Using this technique, 11 new genes were identified and mapped to their genomic locations. Four of them lay outside the deletion in LBL721.174. By looking for the presence of the remaining 7 genes in a series of mutant B cell lines related to LBL721.174, but carrying different deletions, it was possible to correlate the “LBL721.174 phenotype” (i-e., low class I expression) with the absence of 3 of the genes. One of these (Y3) was present but not transcribed in another mutant cell line (.61)with low class I expression, clearly indicating its importance for class I expression. It was duly called psfl (peptide supply factor). The location of the genes in the human, mouse, and rat major histocompatibility complex is shown in Fig. 1.Only the human genes have been mapped at this resolution,

I

- 300 kb

RTl

FIG.1. Colinearity of the human (HLA), mouse (H-2), and rat (RT1) MHC class I1 regions. Homologous loci are aligned. Terminology is as for the HLA loci unless otherwise indicated, and the scale is intended as a rough guide for the human map only; otherwise, the maps are not shown to scale.

I

TRANSPORTER ASSOCIATED WITH ANTIGEN PROCESSING

51

with the location of the rodent genes based on the colinearity of the human, mouse, and rat genomes in this region (37). On sequencing their newly discovered genes, it turned out that RING4 and PSFl (Y3) were the same gene, with the rat counterpart being mtpl and the murine HAM1. Similarly, the highly homologous genes RING11 and PSF2 were shown to be identical, with mtp2 and HAM2 being the corresponding rodent homologs. When the obvious similarity between these genes and a large family of ATP-binding cassette proteins (ABC proteins) (38) was established, each group recognized how appropriate these genes were for the putative role as peptide transporters. The ABC protein family includes members as diverse as P-glycoprotein (Pgp)-the product of the mammalian multidrug resistance (MDR) gene-and the hemolysin B transporter of Escherichia coli (HylB, see Table I ) . Most exciting was the fact that all the ABC proteins described to date were involved in the transport of small molecules across membranes, it fact that almost certainly contributed to the choice of names for these latest family members. The term transporter associated with antigen processing, or TAP, was coined in an attempt to clarify the growing literature during the early 1990s and was officiallystandardized in 1991 by a WHO nomenclature committee for factors in the HLA system (39).Thus, RING4, PSFl(YS), mtpl, and HAM1 were renamed TAP1, whereas RING11, PSF2, mtp2, and HAM2 were renamed TAP2. When alignments between TAPl and TAP2 and other members of the family are compared (Fig. 2), it is clear that most conservation is seen in a stretch over 200 amino acids around the two characteristic motifs present in the nucleotide-binding domains of all family members [Walker A and B motifs]. In 1982, Walker and colleagues identified two short amino acid sequence motifs that are present in most if not all nucleotide-binding proteins. The A motif has the general sequence G-X-X-G-X-G-K-S-T, in which the K residue is important for nucleotide binding. The B motif has the general sequence (Hydrophobic),-D, in which the D binds to MgATP) (40, 41). Little homology is shared over the N-terminal regions. It was subsequently found that the mutant cell line RMA-S carried a TAP2 gene with a C to T point mutation at nucleotide position 97 that introduced a premature stop codon after amino acid number 32 (42). Class I expression and antigen presenting activity could be restored to RMA-S by transfecting it with a functional mouse TAP2 gene (43, 44). Similarly, cell surface expression of class I in LBL721.174 could be restored by transfecting it with both TAPl and TAP2 (45). Several other cell lines with defective TAP genes have since been described, some of which are listed in Table 11. Thus, it did indeed appear that the putative peptide transporter-first suggested by Townsend to be necessary for class I-restricted

TABLE I SOMEMEMBERS OF THE ABC TRANSWRTER FAMILY ~~_______________

Subfamily PgP

CFl-R PMP TAP

Hly B

Members

Function

Domain organization“

MDR1, MDR3 (mouse and man), Export of hydrophobic drugs T-N-T-N MDR2 (mouse), Pgpl, Pgp2, Pgp3 (hamster), mdr49, mdr65 (D. melunogaster) CFTR (man, mouse, Xenupus, Chloride channel T-N-R-T-N cow, dogfish) PMP 70 (man) Import of polypeptides into T-N (+T-N) peroxisomes T-N T-N Export of peptides from TAP1, TAP2 (man, mouse, rat, gorilla) cytosol to ER lumen CyaB (B. pertussis) T-N + T-N Cyclolysin export LktB (P.haemolytica, A.

+

~

Notes

A 13th exon encodes an additional “R” domainb No evidence for homodimerization

0Ctimmycetowmitan.s)

HlyB (E. coli, P. OuZgatis) MsbA

ValA (F. rwvicidiu)

OPP

MsbA (E. coZi) OppABCDF (S. typhimuriurn) AmiABCDEF (S. pneumoniae) SpoOK (B. subtilis)

Whitehrod scarlet

(D. mehnogaster)

Leukotoxin export Hemolysin export

+ T-N N +T+N +T

T-N

Oligopeptide import

Eye-pigment precursor import

T-N

+ (T-N)

OppB-F encode the transporter, whereas OppA is a periplasmic substrate-binding protein essential for transport function No evidence for homodimerization

It is assumed that a functional transporter comprises two NBDs (N)and two TMSs (S),although mixed complexes comprising more than two of each have not yet heen formally ruled out for most transporters. Attention is drawn to those cases in which multimelization has not been noted. * The R domain is a cytoplasmic, hydrophilic, phosphorylated domain that is thought to regulate the channel activity of CFTR.Interestingly, Pgp has a vestigd “domain” between its N and C halves that is also phosphorylated and may be related to the channel function that has been reported for Pgp (134,135).

C - m d r l Mouse C - m d r l Human N - m d r l Mouse N - m d r l Human TAP2-B R a t TAP2 Mouse TAP2 Human T A P l Rat T A P l Mouse TAPl Human HlyB E c o l i

c-mdri nouse C - n d r l Human

N-mdrl Mouse N-ndrl H u m a n TAP2-B Rat TAP2 Mouse TAP2 Human T A P l Rat T A P l House TAPl Human HlyB E c o l i

C - m d r l Mouse C-mdrl Human ~ - m d r lnouse N-mdrl H u m a n TAP2-B R a t TAP2 Mouse TAP2 H u m a n TAPl R a t T A P l Mouse T A P l Human HlyB E c o l i

c-mdrl nouse C-mdrl Human ~ - m d r i nouse N-mdrl Human TAP2-B R a t TAP2 Mouse TAP2 H u m a n TAPl Rat T A P l Mouse TAPl Human HlyB E c o l i

.............................................................................................................. ..................................... ....................................... ..................................... .......................................

...................................................................................... ................................... ...................................

MRLSHPRPWASLLLVDLALLGLLQSSLGTLLPPGLPGLWLEGTLQLG~WG M R L S Y L R P W V S L ~ W L L L G L L Q G S L G N L L P G L W P L

................................... M R L P D L R P ~ S L L L V D A A L L ~ ~ P L G T L L ~ L P G L ~ E G T L ~ G L W G U ~ R G L L G ~ ~ L L L P ~ ~ T P L ................MAAHAWPTARLLLLLVDWLLLRWLPGIFSLLVPEVPLLR~AVGLSRWAI~L~GVL ........ AGARGWLAALQPLVAALGLLPGL

................MAAHAWPTAALLLUYDWLLLRWLPGIFSLLVPEVPLL~~PLLR~AVGLSRWAILG ........... H

R

S

S

R

C

P

A

P

R

G

C

.................................

AGARGWLAALQPLVAALGLLPGL R C L P G A S L A W L G T V L L L L A D W V L L R T A L L HDSCHKIDYGLYALEILAQYHNVSVNPEEIKHRFDTDGTGLGLTSWLLAAKSLELKIDRWfIFLPRLVW

111 lt2 220 . . L C r N E R C K S K D E I D N L D M S S K D S G S S L I R R R S T R K S l C O P I N G G M P ~ S V I F S K G P ENAADESKSEIDALMSSNDS~SLI~~VRGSQAQD~LST~DESIPWS~I~T~PY~~CAIINGG~P~AIIFSKIIGWTPJDD

... ........................... ..........................

H n E e D L K G R A D I W F S ~ K K S ~ ~ A V S V L T E l P N H D L E G O R N G G A K R K N P F K S ~ ~ ~ S ~ R Y ~ D K L ~LG P L !TM ~L VIF G I EHM T D I F A " F F S L R A L V G S ~ T ~ V ~ ~ ~ V ~ ~ ~ IPHYSCRITIDILGGDPD ~ P ~ ~ ~ F P F S L R A L V G G T A S T S W R V ~ A S W G W L L A O Y O A V A L S W A V W Q E . N R T L N I ( R L L K L S R P D L P F L I ~ F ~ W A ETLIPRYSGRVIDILGGDFD T V S ~ V A ~ P A R V A S R P W S W L L V G Y G A A G L S W S L W A V L S P P ~ Q E ~ Q ~ K L S ~ D L ETLIPHYSGRVIDILGGDFD P L L V ~ F ~ ~ A S ~ S A W W L L R E G ~ G L ~ ~ ~ S ~ P ~ ~ G G ~ A P S G ~ ~ GWAIPFFTGRITDWILQDKT ~ F L D S ~ G ~ L ~ A S F R K L S A W S T L R M j D S A G L L Y W N S R P D A F A I S W l U L P A A EMAIPFFTGPJTDWILQDKT A L F R E L I S W G A P O S A D S T R L L H W G S H P T A P W S Y A A A L P A l S S WAIPFFTGRLTDWILQDGS REDGRHPILTKISKEVNR~TFDLEQRNPRVLEQSEFEALYIKYRRIFIETLWSVP~LFALITPLFFQVVMDKVLVHRG

$'

L

~

I

I

L

V

S

305 221 2t3 3t4 PETQRQNS N L F S U F L I L G I I S F I T F F L ~ F T F G ~ G E I L ~ ~ K S ~ Q W S ~ D D P ~ ~ ~ Q ~ G A T G S ~ V PETI[RPNS N L F S L L F L A L G I I S F I T F F L o G F T F G ~ G E I L ~ ~ R ~ Q W ~ D D P ~ A L ~ ~ ~ G A I G S ~ V VSK NSTNHSEADI(RAHFAKLe~A~I~GVLIVAYIQVS~C~QI~IRQ~F~I~QEI~D..VHDVGTWTRLTDWSKINeGIGDKIGM

...................

...

...................

LEDIJlSNITNRSDINDTGFEEDHTRYAYYYSGIG~V~YIQVS~C~GRQI~I~QFF~I~QEI~D..VHDVGEWTRLTDDVSKINIGDKIGM

....................... ....................... ............. A P S P A m M . ............ VPsPTRNl ............. ALTI'ITRNL ....................... PDAFASAI PDAFASAI PWASAI

PSTL.

.............

FFMCLF

FFMCLP

7

FFMCLFS WLMCILTI WLMSILTI TLUSILTI NVITYALSV

1

SLSAGCRGGSFLFAESRIWIREQLPSSL~QDLAFFQE..TK ELNSQLSSDTSLMSQWLSLNANI SFSAGCRGGSFLFTMSRIWIFCSQLFSSLLXQDLGFFQE..TK E L N S R L S S L 7 T S L M S R W L P P I SLSAGCRGGCFTlTMSRINIREQLFSSLLRQDLGFFQE..TK E L N S R L S S ~ L M S N W L P L N A N V T V L E F A G D G I Y N T T H G H H H S R V H G E V F ~ ~ Q ~ G F F L K . . N PSITSRVTEGTSNVCESISDKLNL T A L E F A S I X i I Y N I T M G H R ~ ~ ~ Q ~ F F L K . . SITSRVTEDTANVCESISDTLSL NP AVLEFJGCGI~SHI&EVFGAVLXQETEFFQQ..N IMSRVI'EDTSTLSDSLSENLSL Y W F E I I L S G L R T Y T P A H S T ~ I ~ ~ P ~ L ~ P I S Y F~E~S ~V VRG. . ELDQIRNFLTGQALT .

306 415 4t5 IFPNIANLGTGIIISLIYGWQLTLLL~VPITAIAGWEMlMLSWALKDKKELEGSGKIAT~~~SLTREQKFE~AQSLQIPY~GITFF~ ITPNIANLGTGITISFIYGWQLTLLLLAIVPIIATAGVVEHKHLSWALKD~ELEWLGKIA~I~~SLTQEQKFE~AQSLQVP~SL~IFGITFSFT FFQANATPFGGFIIGFTRGh'KLTLVILAISPVLGLSAGIWAL R K A G A V A E E V L A A I R T V I A ~ ~ L ~ ~ E ~ I ~ T ~ I ~ G ~ P F P S H A r r ~ G F N G F T R ~ L T L V I ~ S ~ G L S ~ ~ ~ I LA K A G A V A E E V L A A I R T Y I A P G G P K K E L E I ( Y N l h n E E A K R URSLVKWGLYYFMIWSPRTPLSLLDLPLTIAAEKVYNPRH DAVAKAGQVVRE&VGGLQFGAEEQEVElY KEALERCRQLWWRRDLEKSLY LVIQ

LLRSLVKWGLYFFML4WSPQLTFLSLLDLPLTIAAEKVYNPRH URSLVKWGLYGFMLSISPQLTLLSL~~IAAE~ FLWYLGRGLCLLAFMIWGSFYLTVVTLLSLPLLFLLP~iWY

LLWYLGRALCLLVFMFWGSPYLTLWLINLPLLFLLPKKL FLWnVRGLC~I~GSVSLTMTrLITLPLLFLLPKKVGKW

SVLDLLFSLIFFAVMWYYSPKLTLVILFSLPCYAAWSVFISPIL

DAVRKAOQWREAVGGLQTVRSFGAEEQNSKYRULLERCRQL~L~D~LVIR DAVARAGQWReAVGGLQTVRSFGAEEHEVCRYKEALWXQLYWRRDLERALYLLVR E S L A K S M V A L E A L S A S F ~ G ~ Q K F R Q K L E E M S W ( S M V A L E A L S A M P T Y R S F ~ E G E A O K F R Q K ~ ~ ~ ~ A ~ SLAKSSQVAIEALSAHPTVRSFANEEGEAPKFREKU2EIKTLNQ~VAYA~S~ RNADNPSFLVESVTAI~IKAHAVSPQIlTNlWDKQLG~~GF~TIG~I

FIG.2. An amino acid alignment between the known TAP proteins and some other members of the ABC transporter superfamily using a gap penalty of 10. Exon boundaries for human TAPl and TAP2 are marked and extrapolated to other TAPS.The Walker A and B motifs are marked.

~

C

I

516

c-mdr1 MOUSe C-mdrl Human N-mdrl House N-mdrl Human TAPZ-B Rat TAP2 Mouse TAP2 Human TAPl Rat TAPl House TAPl Human HlyB Ecoli

6t7

718

Q A H M Y F S Y R A C F R P G A Y L ~ W ~ F ~ L ~ S A I Y f G QAHMYFSYAGCFRFWLYLVAHKLMSFEDVLLVFSAVYfGANKISAAHIIMIIEKTPLIDSYSTEGLMPNTLEGNVTFG~~PTRPDIPVLQG ~LIYASYALAFWYGTSLVISKEYSIGQVL~FSVLIGAFSVGQASPNIEAFANARGAAYEVFKIIDNKPSIDSFSKSGHKPDNIQG"lHFSYPSRKWQI~G ~LIYASYRW\FWYGTTLVLSGEYSIGQVL~FSVLIG~SVGQASPSI~~GRAYEIFKIID~PSIDSYSffiG~PDNIKG~EF~FSYPSFX~I~G GMQVLILNVGVQQILAGEVTRGGLLSFLLYQEEVGHHV YYHYGDHtSNVGAAeKVFSnDRRPNLP..NPGTLAPPRLEGRVEFQDVSFSYPSRPEK MQVLILNCGVQQILAGEVTRGGLLSFLLYQEEVGQ YYHYGDMLSNVGAAEKVFSYLDRKPNLP..QPGILAPPWLEGRVEFQDVSFSYPRRPEKP HLGVQMLMLSCGUXK%GELMGSLLSfMlYQESVGSW LWIYGDM[rSNVGAAEKVFS~RQPNLP..SPGTLAPITMGVVKFQDVSFAYPM(PDR SGHLLRVGILYLGGQLVVRGAVSSGNLVSFVLYQLQFTRR LSIYPSMQKSVGASEKIFEYLDRTPCSP..LSGSLAPLNHKGLVKFQDVSFAYP"VQ SGMLLRVGILYLGGQLVIRGTVSSGNLVSF'JLYQLQFTQAV LSLYPSHQKAVGSSEKIFEYLDRTPCSP..LSGSLAPSNMKGLVEFQDVSFAYPNQPRVQ S G H L L K V G I L Y I G G Q L ~ S W L V S S ~ ~ Y Q M Q ~ Q LSIYPRVQKAVGSSEKIFEnDRTPRCP..PSGLLTPLHLEGLVQFQDVSFAYPNRPDVL A QLIQKTVMIINLWLGAHLVISGDLSIGQLIAFNHLAGQIVAWIRLAQIWQDFWVGIS~RUiDVLNSPTES YHGKLTLPEINGDITFRNIRRYKPDSPV.ILDN

F

$

...

3

632 524 Walker-A at9 C-rndrl Mouse L S L E V K K G Q T L A L V 0 8 S O ~ Q L L E R P I D P ~ G S V F L E G K E I K Q ~ Q ~ ~ Q L G I V S Q E P I L F ~ S I ~ I A Y G D N S R W S Y E E I ~ I H Q F I D S L P D K Y C-mdrl Human L S L E V K K G Q T L A L V ~ S o C Q ~ Q L L E ~ Y D P L A G ~ L L E G K E I ~ Q W L ~ L G I V S Q E P I L F ~ S I A ~ I A Y G D N S R W S Q E E I ~ ~ I ~ F I E S L P ~ Y N-mdrl House L N L K V K S G Q T V A L V ( B T 8 o C Q ~ Q ~ R L Y D P L D L R E I I G W S Q E P V L F A 1 T I ~ I R Y G R E D . . V T M D E I E K A Y K E A N A Y D F I M L P H Q F N-mdrl H u m a n W L K V Q S G Q T V A L V ~ 0 C O ~ Q L M Q R L Y D ~ E ~ ~ D I R T I ~ L R E I I G W S Q E P V L F A 1 T I A E N I R Y G R E N . . V T M D E I E K A V K E A N A Y D P I ~ L P ~ F TAPI-B Rat LTFTLHPGKWALVQPUQSQKSWWLLLQNLYQFTGGQLLLEGEPLVQYDHHYLHR VGQEPVLFSGSVKDNIAYG.L.RDCEDAQVHARAQAACADDPIGEM"NG1 VGQEPVLFSGSVKDNIAYG.L.RCCEDAQVMAAAQAACADDFIGEWl"G1 TAP2 Mouse LTFTLHPGTVPALVQPUQSQKSTVAALLQNLYQPTGGQLLLEGEPLTEYDHHYLHR TAP2 Human LTPFLRPGEVTALVQOTVAALLQNLYQPTGGQVLLDERPISQYEHCYLHS SVGQEPVLFSGSVR"IAYG.L.QSCEDDKVMAAAQAAHADDP1QEMEHGI LTPFLYPGKVTALVOPgOBaKSTVAALLQNLYQPTGGKVLLEGEPLVQYDHHYL AAVGQEPLLZGRSFREXIAYG.LTRTFTMEEITAVAMESGPISGFPWY TAPl Rat TAPl House L T P T r H P G n ~ A A L L Q N L Y Q P T G G Q L L L ~ ~ V Q Y D H HAAVGQEPLLFGRSFRENIAYG.WRTFTMEEITAVAVESGAHDPISGF~Y Y ~ TAPl Human LTPTLRPGEVTALVQPUQEQKSWAALLQNLYQPTGGQLLLEGKPLPQYEHRYLHR AAVGQEPQVFGRSLQ~IAYG.LWRPTHEEIT~~SGAHSPISGLPQGY HlyB Ecoll I N L S I K Q G W I G I V O I 1 8 B 8 T L T K L I Q R P Y I P ~ ~ L I E G H D ~ P ~ ~ Q V ~ Q D ~ ~ S I I ~ I S ~ . . N P G n S V E X V I Y A A R ~ G ~ P I S E L R E G Y

.f

633

C-mdrl House C-mdrl Human

742 9t10 Wa1knr-B lot11 NTnVGDKGTQLSGGQKQ~AI~~QPM~TSAL~ES~Q~D..KAREGRTCIVIAHRLSTIQNADLI~QNGKVKEHGTMWLLAQKGIYFS~SV STKVGDKGTQLSGGQKQRIAIARALVRQPH~TSAL~ES~Q~D..KAREGR~IVIAHRtSTIQNADLI~QNGR~GTHWLLAQKGIYFS~SV

N-mdrl Mouse D T L V G E R C A Q L S G G O K Q R I A I ~ ~ ~ I ~ T S A D . . K A R E G R ~ I V I A H R L S T I A G F E G G V I V E ~ ~ D E ~ G I Y F ~ ~ N-mdrl Human D T L V G E R G A Q L S G G Q K Q R I A I ~ ~ P K ~ T S A L ~ S ~ W Q V A L D . . K R R K G R T T I V I A H R L S T I A G F D ~ I ~ G ~ D E ~ K G I Y F ~ ~ TAP2-B Rat E K G S Q L A V G Q K Q R W A P R V L I W ~ T S A L D A....ECEQ ?WRSQEDRTHLVIAHRLHTVQN~VLVLK~QL~~L~EQDWAHLV~~ TAP2 Mouse E K G G Q L A V G Q K Q R L A I ~ ~ P R ~ I ~ T S A L DQCEQ A Q N W R S Q G D R T M L V I A H R L H T V Q ~ V L ~ ~ R L ~ ~ D W ~ V ~ ~ E TAP2 Human EKGSQLAAGQKQRLAIAPRVLI~TSALDV QCEQ QWNSRGDRTVLVIAHRLQTVQ\IQRAHalLVLQEGKLQKLAQL................ TAPl R a t ETGNQLSGGQRQAVALARRLIFXPUILaDDATSALDAGNQ QRLGYESPEWASRTVLLITQLSLRERAHHILFLKEGSVCEQGTHLQL24XRGGCYRSMVEA TAPl Mouse ETGNQLSGGQRQAVALARALIRKPLLLILDDATSALDAGNQ QRLLYESPRRASRTVLLITQQLSLAEQAHHILFLREGSVGEQG TAPl Human EAGSQLSGGQRQAVALARALIRKPCVLILDDATSALDANSQ EQLLYESPERYSRSVLLITQHLSLVEQADHILFLEGGAIRZGGTHQQLMEKKGCYWAMVQA H l y E Ecoli N T I V G E ~ A G L S G G Q R Q R I A I A R A L V " P K ~ I ~ ~ T S ~ D ~ S ~ I ~ . . K I C K G R T V I I I A H R L S ? D R I I ~ K G K I V E Q G K H K E L L S E P E S L Y S Y L Y Q

i

.... ....

743

C-mdrl Mouse C-mdrl Human N-mdrl Mouse N-mdrl Human TAP2-B Rat TAP2 Mouse TAP2 Human TAPl Rat TAPl Mouse TAPl Human HlyB Ecoli

QAGAKRS. . QAGTKRQ. . WAGNEIE QTAGNEVEL A. ....... A. .......

.

.........

LAAPSD.. . LAAPAD.. PADAPE.. . LQSD.. ...

.

FIG.2. Continued

TABLE I1 A SELECTIOIV OF CELLLINESWITH DEFECTIVE P E P ~ I DTRANSPORTERS E TAP function Name LBL721.174 T2 .134 BM36.1 HI436 ST-EM0 RMA-S CMT.64

TAPl

TAP2

-

-

Large deletion in MHC I1 region

-

+

Small deletion in MHC I1 region 2-bp deletion in NBD leading to nonfunctional ORF Single base substitution in NBD leading to R659Q Single base substitution in TMD leading to premature stop at 253 Single base deletion leading to premature stop Low constitutive levels of expression-presumably from mutation in 5’UT region. Expression is IFN-y restorable Low constitutive levels of expression-presumably from mutation in 5’UT region. Expression is IFN-y restorable Nonfunctional TAP in a xenogeneic antigen presentation system. Restored by transfection with rat TAPl and TAP2 Low constitutive levels of expression-presumably from mutation in 5‘UT region. Expression is IFN-y restorable Low constitutive levels of expression. Expression is IFN-y restorable

+

-

-

+

+ +

-

-

-

-

-

-

-

-

-

-

-

BC2 BHK

4A1,6D1 EE2H3, EE8D2

Defect

Species

References

Human B cell 26, 28 BxT cell hybridoma 176 Human B cell 26, 28 Human B cell 55 Human small cell lung carcinoma 103 Human B cell Murine thymoma

104 24,42

Murine lung carcinoma

177, 178

Murine fibrosarcoma

178

Syrian hamster kidney epithelium

179

Murine fibrosarcoma Murine embryonic stem cell lines

180

181

56

TIM E L L I O T

antigen presentation-had been discovered. At this stage, however, the only indication that the genes encoded a protein that transported peptides was their obvious sequence similarity to other ABC transporters. At the time, none of these had been shown to transport peptides that were long enough to constitute T cell epitopes, and it was a further 3 years before the first convincing evidence emerged that the TAP proteins were indeed peptide transporters-by which time a wealth of information was available to show that the TAP proteins were essential for loading MHC class I molecules with antigenic peptides that could be presented to T cells. II. ABC Transporters

Several excellent reviews have been written on the structure and function of the ABC transporters (38). Here, it is only necessary to go into brief detail. The family of ATP-binding cassette transporters has almost 60 members found in both prokaryotes and eukaryotes. They are characterized by a conserved nucleotide-binding domain (NBD) containing two related and highly conserved ATP-binding motifs [the Walker A and B motifs (40, 41)]. Their second structural feature is an integral membrane component thought to consist of around 6 membrane spanning segments, here called the transmembrane domain (TMD). The functional unit comprises two NBDs and two TMDs (Fig. 3). In many bacterial members of the superfamTransmembrane Domain (TMD)

\

Nucleotide Binding

!!i!i!i

Monomer e.g., CITR PgP

Domain (NBD) Dimer e.g., TAP D. rnelanogasier wbs

Tetramer e.g., Opp

FIG.3. Different ABC transporters comprise four domains that are synthesized as a single polypeptide (monomer), as two polypeptides (dimer),or as four polypeptides (tetramer).See text and Table I for examples.

TRANSPORTER ASSOCIATED WITH ANTIGEN PROCESSING

57

ily each of the components are encoded on a single polypeptide-the functional unit being a tetramer (for example, oligopeptide permease of E . coli);other members of the family, including TAP and the Drosophilu mehogaster white, brown, and scarlet gene products, probably function as a dimer of two polypeptides each containing an N-terminal hydrophobic domain and a C-terminal ATP-binding domain. Pgp and the cystic fibrosis transmembrane conductance regulator encode all four units on one polypeptide and so function as monomers. Interestingly, when the N- and Cterminal halves of these monomeric transporters are expressed as separate polypeptides, they dimerize and form a functional complex (46). The mammalian ABC transporters can be grouped into four main subfamilies: the P-glycoproteins, the cystic fibrosis transmembrane conductance regulators, the peroxisomal membrane proteins (PMPs), and the TAP proteins (Table I). Based on a comparison of the ATP-binding domains of 57 members of the family, Hughes (47) has studied the evolution of these three subfamilies and found that Pgp and TAP are more related to one another than either is to CFTR [this is interesting because it suggests that the gene duplication events that are implied in the evolution of the Pgp and CFTR polypeptides (which encode all four components on one polypeptide) occurred independently] (see Fig. 4).The PMPs show no homology to other members of the family outside the ATP-binding domain itself, and, even here, homology is restricted to the Walker motifs and their immediate surrounding regions (48).Both PgP and TAP are in fact more closely related to the HlyB and MsbA ABC transporters in bacteria, which probably function as dimers, than they are to CFTR. On closer

CFTR-N CfTR-C

Fic. 4. The evolutionary relationship between three of the four major subgroups of the mammalian ABC transporters: Cystic fibrosis transmembrane conductance regulator (CFTR), TAP, and P-glycoprotein(PgP), with the prokaryotic hemolysin exporter (HylB). X representsan ancestral TAP gene that duplicated,givingrise to the two modernTAP genes.

58

TIM E L L I O T

inspection, it turns out that they share greatest similarity with HlyB and MsbA subfamily members from purple bacteria rather than those from gram-positive bacteria. Since it has been suggested that the mitochondria of eukaryotes originated as endosymbiotic purple bacteria (49) it may well be the case that TAP (and Pgp) was originally of mitochondrial origin, and was subsequently transferred to the nuclear genome. Based on the sequence homology and gene organization of the region spanning LMP2 and TAPl and that spanning LMP7 and TAP2 (Fig. l),it has been suggested that duplication of a primordial LMP/TAP unit gave rise to the current gene cluster. Beck et al. (50) have gone further, suggesting that the primordial unit (marked X in Fig. 4) was more related to LMP7RAPB because this contains fewer A h repeats within its introns, and that these are of a more conserved or ancient type. [Alu sequences are very short transposable elements (originally derived from an endogenous 7SL RNA gene) that, at about 500,000 copies, constitute about 5 % of the human genome. This number is thought to have accumulated over the past 60 million years or so and during that time has mutated away from the ancestral sequence]. They have also drawn attention to a series of inverted repeats flanking the ABC domain (exons 8-10) of TAP1, but not TAP2, that may have been involved in this duplication. 111. Gene Shuchmre of TAP and Its Regulation

The genomic structures of the human TAPl and TAP2 genes have been determined (50)and consist of 11exons each (Fig. 5; Table 111). Eight of the 11 exons are the same length, and the remaining 3 exons differ in length by 100 (exon l),3 (exon 9), and 78 (exon 11)nucleotides. The exon boundaries for the homologous exons of TAPl and TAP2 are of the same type. Figure 5 also shows that there is little conservation of intron size between homologous regions of the TAPl and TAP2 genes. Although the genomic structure of the rodent TAPs has not been determined, an alignment of the nucleotide sequences corresponding to the amino acid sequence alignment shown in Fig. 2 suggests that this arrangement of exons might be preserved. Homology is highest between rat and mouse TAPl (79% at the DNA level and 77% at the protein level). Human and rat TAPl are 76% homologous at the nucleotide level (70%protein), and human and mouse TAPl are 64% homologous at the nucleotide level (61% protein). Phylogenetic analysis (47)indicates that the degree of relatedness between the human TAPl and TAP2 genes is similar to that between each gene and its equivalent rodent homolog. Consequently, the human TAPs are 64% homologous at the protein level. As Table I11 shows, this homology is concentrated in the regions encoded by exons 8 and 10. These exons encode sections of the ATP-binding domains that contain the characteristic

TRANSPORTER ASSOCIATED WITH ANTIGEN PROCESSING

59 1282

A

1201

i

TAPl IS

TAP2 IS

1526

1763

2j29

FIG.5. An alignment of the 11 exons of human TAPl and TAPL, showing the length of each joining intron. The ISREs are also shown upstream of exon 1.

Walker A and B motifs. Homology between the remaining equivalent exons is between 49 and 65%. Assuming conservation of the exon structure between species, other exons are more similar between species than they are between TAPS of the same species. For example, the short exons 3 and 6 of human TAPl would be 72 and 85%homologous to the corresponding exons in the rat, but only 65 and 49% homologous to the corresponding exons in human TAP2. This observation may have important consequences when it comes to determining the relationship between TAP structure and function (see below). The 5' flanking sequences of genes contain information that determines the initiation, tempo, and timing of transcription. The TATA box, which normally lies 19-27 bases upstream from the transcriptional start site of most eukaryotic genes, is missing from the TAP genes, although they contain two putative GC-rich promoter elements (a so-called NF-KBbox 131 bases upstream of exon 1 and an SP/GC box 117 bases upstream) (51). Site-directed mutation of the SP/GC box of TAPl leads to a threefold reduction in its basal transcription, whereas mutation of the NF-KB box has no effect (52). In addition, a sequence corresponding to an interferony ( IFN-y) stimulated response element (ISRE)resides 198bases upstream of TAPl and 573 bases upstream of TAP2 (51).These regions are clearly

TABLE I11 COMPARISON OF THE NUCLEOTIDEAND PROTEIN SEQUENCES OF HUMAN TAPl AND TAP$ EXONBY EXON RNA“

Size

Protein %

Exon

TAPl

TAP2

TAPl

TAP2

TAPl

TAP2

Homologyb

1 2 3 4 5 6 7 8 9 10 11 Total

598 115 131 206 198 129 189 174 163 137 204 2244

493 115 131 206 198 129 189 174 160 137 126 2058

1-598 599-713 714-844 845- 1050 1051- 1248 1249-1377 1378-1566 1567-1740 1741-1903 1904-2040 2041-2244

1-493 494-608 609-739 740-945 946-1143 114-1272 1273-1461 1462- 1635 1636-1795 1796- 1932 1933-2058

1-2w 201-238 239-282 283-350 351-416 417-459 460-522 523-580 581-635 636-680 681-748

1-165 166-203 204-247 248-315 316-381 382-424 425-487 488-545 546-599 600-644 645-686

57 55 65 58 62 49 63 95 60

The exon boundaries are from Ref. 51.

* Percentage similarity with a gap penalty of 3 using the “best fit” option in the “ G C G software package. Numbers in bold indicate residues derived from triplets that span the exon splice site. Exons 1, 3, and 9 are type 1 and exon 2 is of type 2.

80

58

TRANSPORTER ASSOCIATED WITH ANTIGEN PROCESSING

61

functional because both TAPl and TAP2 transcription is upregulated by IFN-P, IFN-.)I and TNF-a, (but not others such as TGF-P, IL-1P, and IL-4) (53,211). IFN-.)Ihas a more profound effect that TNF, but the two act synergistically. The ISRE works in conjunction with the NF-KB box to regulate TAP expression because mutation of the former by site-directed mutagenesis, although having no effect on the basal level of TAPl transcription abrogates its TNF-a, inducibility (52). In the presence of stimulatory cytokines, TAPl transcription increases to a maximum level over 6 hr with a half-time of around 2 hr. This is considerably more rapid than the rate of increase seen during IFN-y-stimulated upregulation of MHC class I synthesis, which has a half-time of around 12 hr and reaches maximum levels in 48 hr-by which time TAPl transcription has returned to basal levels (211).Thus, it may be important that upregulation of TAP expression precedes that of MHC class I expression in order to ensure that, by the time that class I expression reaches a maximum, the supply of peptides to the ER is not limiting. This would ensure that, during an active immune response, the fraction of unloaded MHC class I molecules is kept to a minimum, thereby avoiding their accumulation in the ER where they could compete for ER chaperones and proteases essential for the survival of the infected host cell. It is interesting that the ISRE-like sequence upstream of TAP2 is functional because it is only weakly homologous to the consensus ISRE sequence and lies beyond the region in which experimentally defined ISREs have usually been found (up to 500 bases upstream).

N. TAP Protein Structure A. TAP FUNCTIONS AS A HETERODIMER By homology with other members of the ABC family of transporters, a functional TAP protein would be expected to be either a homodimer of TAPl or TAP2 or a heterodimer. Indeed, immunoprecipitations with antisera against TAPl coprecipitate the TAP2 protein (54, 55). The mutant cell line .134, in which the TAPl gene is deleted, has a phenotype identical to LBL721.174, which has both TAPl and TAP2 genes deleted (28). This suggests that TAP2 cannot function as a homodimer. Normal expression of class I molecules in ,134 can be reversed by transfection of a TAPl gene (56). Similarly, the mutant cell BM36.1, which has a normal TAPl but defective TAP2, has an LBL721.174-like phenotype (55), suggesting that the human TAPl protein cannot form a functional homodimer either. These results strongly suggest that a functional TAP complex is therefore a heterodimer. In support of this is the observation that, in in vitro transport assays (57-59) no activity has yet been recorded for systems in which only

62

TIM ELLIO’lT

a single chain is expressed. However, it is worth noting that RMA-S, which has a normal TAPl but defective TAP2 gene (see above), can present an endogenously synthesized vesicular stomatitus virus (VSV)-derivedepitope to CTL followinginfection even though most other CTL epitopes cannot be presented (60,61). Also, a limited subset of apparently peptide-dependent dospecific CTLs can recognize RMA-S (62,63). These results suggest that either the TAPl/mutant TAP2 heterodimer has some residual peptidetransporting activity or that TAPl homodimers display a low level of transporter activity. In support of the latter possibility, Gabathuler et al. (64)found that introduction of the rat TAPl gene alone into the mutant murine cell line CMT.64, which lacks detectable TAPl and TAP2 mRNA (see Table 11),was sufficient to restore their ability to present a VSV-derived epitope following infection. It may be, therefore, that TAPl can dimerize to form an inefficient transporter, perhaps with a much restricted substrate specificity.

B. TAP ASSEMBLYAND INTRACELLULAR LOCALIZATION Very little is known about how the TAPl and TAP2 proteins are synthesized or how the heterodimer is assembled. Neither TAPl nor TAP2 appear to have a cleavable signal sequence. It may be that, as with the N-terminal transmembrane segment (TMS) of the yeast ABC transporter ste6 (65), the first TMS of TAPl and TAP2 can interact with the ER translocation machinery and double as a functional “stop transfer” sequence. This is also seen with many type 2 membrane proteins, which are synthesized in such a way as to be orientated with their N termini in the cytoplasm and their C termini in the extracellular or lumenal compartment. Other, more complex mechanisms of insertion could take place, however, in which each TMS interacts independently with the signal-recognition particle and acts as a stop transfer signal. This would result in stepwise assembly in the ER membrane (66). Russ et al. (67) have studied the kinetics of intracellular assembly of the TAPlflAP2 heterodimer using recombinant vaccinia-encoded human TAPS. In a pulse-chase analysis they have shown that the heterodimer assembles extremely rapidly (within 5 min of synthesis). Because this rate of assembly is similar to those of other oligomeric membrane proteins with single membrane spanning domains, it would appear that the complex transmembrane topology of the N-terminal domains does not appear to limit assembly. This would support the rapid appearance of new functional transporters in the ER membrane following transcriptional upregulation by cytokines. No information is available regarding the regions of the TAP proteins that are involved in dimer formation, or indeed which structural features favor heterodimerization over homodimerization, although it is generally

TRANSPORTER ASSOCIATED WITH ANTIGEN PROCESSING

63

assumed that these will lie in the hydrophobic N-terminal domains, where the major differences between the two chains lie. However, when the Pgp polypeptide was expressed as two homologous halves (similar to TAPl and TAPB), they assembled to form a functional transporter via associations between the NBDs as well as through the two TMDs (68). Relevant to this point are some observations regarding TAP heterodimerization made in the cell line BM36.1 (see above). This cell has a RMA-S-like phenotype as a result of a double base-pair deletion in the Walker B motif of TAP2. Like RMA-S, the defect could be reversed by transfection with a normal TAP2 gene. The double base-pair deletion results in a frameshift and consequent replacement of the C-terminal 52 amino acids of TAP2 with a novel extension 103amino acids long. This mutant TAP2 forms a complex with the endogenous, normal TAPl which is nonfunctional. When normal TAP2 is transfected into BM36.1, TAP function is restored. Interestingly, in transfectants, the mutant TAP2 protein no longer coprecipitates with TAPl suggesting that assembly with wild-type TAP2 is preferred over the mutant. Although this could simply be a consequence of overexpression of the transgene, another possibility is that assembly is more efficient with wild-type TAP2 than with the mutant (55).This would imply that regions in the ATP-binding domain were important in stabilizing the heterodimer. Whether ER resident chaperone proteins are involved in the assembly of the TAP heterodimer is not known. However, studies of Pgp assembly have suggested that Hsc70 could participate in the folding of the NBDs and calnexin in the folding of the N-terminal hydrophobic domain (68). Other groups have also implicated calnexin in the folding and quality control of other ABC transporters (69). C. SUBCELLULAR LOCALIZATION Antibodies raised to the TAP complex give perinuclear staining patterns of cytospin preparations that are consistent with their localization to the endoplasmic reticulum (67). In addition, confocal microscopy has shown that TAP colocalizes with the ER resident chaperone molecule BiP (67). In an attempt to determine the precise location of TAP, Kleijmeer et al. (70) performed high-resolution immunogold-electron microscopy on sections of parent LBL721 cells compared to the TAP-negative line LBL721.174. In addition to localizing specifically to the ER, a significant fraction of the gold-conjugated anti-TAP1 antibody used in the experiment localized to the Golgi membranes. Because TAP colocalized with p53, which is known to reside in the cis-Golgi network or intermediate compartment, the authors concluded that TAP expression is restricted to the early secretory pathway, which includes both ER and cis-Golgi. This distribution of TAPl was the same regardless of whether TAP2 was coexpressed.

64

TIM ELLIOTT

5~7--W6/327 400 500

F

300 0

-

c

200

C

a

g

g

100

100

0

0 0

0 -

0 0

0

0 -

N

0

0 -

0 0

N

pM DIGITONIN

b

0 0

N

pM DIGITONIN

p M DIGITONIN

500

AK1 400

u

g =

300

300

304

0

200

2w C

E

a

a

g

100 l 0

0

-

0

0 0

0

:

L 0 -

N

pM DIGITONIN

100

0

0

0 N

0 -

0 0 N

pM DIGITONIN

pM DIGITONIN

400 300

300

g

100 0

0

0

-

N

0 N

pM DIGITONIN

pM DIGITONIN

0

0 -

0 0

0

0

pM

0

-

0

0 N

DIGITONIN

FIG. 6. Localizing the C terminus of TAP1 to the cytoplasm using flow cytometry. Increasing concentrations of digitonin treatment allow increasing fluorescent staining of the TAP complex with an antiserum raised to the C terminus of TAP1. This is concurrent with decreasing staining of the extracellular domains of class I (monoclonal antibody W6/32) and ICAM-1, which are present both in the ER and at the ceU surface. Note that staining with WW32 is low in the presence of 200 @4 digitonin despite a high steady-state pool of

TRANSPORTER ASSOCIATED WITH ANTIGEN PROCESSING

65

The structural features of TAP that are responsible for its retention in the early secretory pathway are unknown. Neither of the TAP proteins contain a known ER retention signal (71-73), and TAP is the only one of the mammalian ABC transporters to be retained in the early secretory pathway. It is possible that a more general structural feature, such as the length of individual membrane spanning segments or oligomerization, could be responsible for its retention in the early secretory pathway, as has been shown for proteins that are retained later in the trans-golgi network (74). D. TOPOLOGY There appears to be no consensus among members of the ABC transporters for the direction of transport relative to the location of the ATP-binding domains. Thus, some members transport their substrates away from the nucleotide-binding domains (e.g., Pgp), whereas others transport toward them (e.g.,oligopeptide permease of bacteria). There is no a priori reason, therefore, to assume that the TAP heterodimer resides with its ATPbinding domains on the cytoplasmic side of the ER membrane. Immunogold staining of ultrathin cryosections using an antiserum raised to the Cterminal 25 amino acids of human TAPl (70) suggested that TAPl is orientated with this C-terminal epitope in the cytosol. In an attempt to confirm this, we have used flow cytometric analysis of digitoninpermeablized cells using the same antiserum. Concentrations of digitonin can be used that completely solubilize the plasma membrane but leave the ER membrane intact (as evidenced by the fact that immature, endoglycosidase-H-sensitive M HC class I molecules are not solubilized but mature endoglycosidase-H-resistantones are). Figure 6 shows that at this concentration, staining with the anti-TAP1 antiserum increases, whereas staining with antibodies recognizing the extracellular domains of either MHC class I or ICAM-1 (another cell surface glycoprotein expressed by B cells) decreases as the plasma membrane is solubilized, despite the fact that substantial amounts of both proteins are present in the ER of these permeabilized cells. These results are also consistent with the exposure of the C terminus-and hence the NBD-to the cytoplasm. Topological

ER-residentclass I in all cells tested (data not shown).This indicates that digitonintreatment is selective for the plasmalemma and does not permeabilize the ER membrane. (a) BM28.7, the parent cell line of the mutant BM36.1, has normal TAPl and TAP2 and expresses high levels of surface class I (55). (b) BM36.1 has normal TAPl but a defective TAP2 and, as a result, expresses low cell surface class I. Greater than 90% of the class I molecules synthesizedby BM36.1 are ER resident (55). (c)LBL72.174 lacks the genes encoding TAPl and TAP2. Class I expression is similar to that of BM36.1.

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predictions of the TAP heterodimer must therefore be consistent with this orientation. Predicting the topology of any transmembrane protein generally begins with the prediction of potential transmembrane spanning segments (TMSs) from hydrophobicity plots (75, 76). These tend to be around 20 amino acids in length and are bounded by either positively charged residues or tryptophan or tyrosine (77). Several rules have been established to help the subsequent orientation of these putative TMSs, notably that positively charged amino acids tend to be more prevalent in the cytoplasmic than in extracytoplasmic segments (75), and that, at least for the N-terminal TMSs, the net charge at each end of the hydrophobic stretch can influence its orientation with the more net-positive end residing in the cytoplasm (76, 77). Alongside such predictive algorithms, there are many experimental techniques available to investigate the topology of transmembrane proteins. These include making fusion proteins with truncated segments of the transmembrane protein (78), protease accessibility (79), analysis of posttranslational modifications that occur in specific cellular compartments such as N glycosylation (ER; Ref. 80) and phosphorylation (cytoplasm; Ref. 81),antibody mapping (82-84), and chemical modification (85).Using a combination of these theoretical and experimental techniques, the topology of the Pgp MDR1, TAP’Sclosest relative, has now been fairly well established (85,86), and comprises six TMSs and a cytoplasmic NBD. Several attempts have been made to predict the topology of the TAPl protein. The first of these predicted eight potential TMSs (29, 35) and a similarity was noted between the hydrophobicity plots of the N-terminal hydrophobic domains of rat TAPl and the other ABC transporters. It was clear from these comparisons that the TAP proteins diverged most from their closest phylogenetic neighbors at their N termini. This observation has been noted in other models of TAP based on the putative MDRl topology, resulting in six transmembrane segments and a long, hydrophobic cytoplasmic N-terminal segment (87). Momburg et al. (88)have used the Kyte-Doolittle algorithm (89)to predict the topology of TAP2 amving at a protein that spans the membrane 10 times. Similarly, when the primary sequence of TAPl was analyzed by an algorithm for predicting transmembrane protein topology, which is based on a database of naturally occumng transmembrane proteins with known structure (90, 91), 10 potential membrane spanning regions of between 18 and 28 amino acids were predicted. These are very similar to the regions predicted by the Kyte-Doolittle algorithm. When the same algorithms were applied to the human MDRl N-terminal half (residues 1-700), they

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correctly predicted the six TMSs proposed for Pgp that have subsequently been confirmed by experimental analysis. As shown in Fig. 7 and Table IV, the first six predicted TMSs of TAPl are encoded by exons 1 and 2 with TMSa-d and half of TMSe residing in exon 1 and the remainder of TMSe and TMSfresiding in exon 2. Both algorithms place a putative TMS (from the region starting at residues 187-190 and ending at residues 206-208) across the boundary between exons 1 and 2 (residue 201). Two pairs of putative TMSs appear to be encoded within exons 4 (TMSg and -h) and 6 (TMSI and j ) . It has been suggested that exons could code for individual structural units of proteins (reviewed in Ref. 92). Certainly this appears to be the case for proteins such as hemoglobin and lysozyme, in which separate exons code for substrate-orientating residues, the catalytic center, and structural subregions of the protein. Similarly, MHC class I molecules are encoded by (at least) seven exons that can be treated as distinct structural units-even to the extent of being stable when expressed singly or in pairs (93-95; T. Elliott and E. Rigney, unpublished observations). Go (96) has shown that there is good correspondence between compact structural units (which she has called “modules”) and the exons that encode them. Thus, it may be a valuable exercise to consider predicted structural subregions of the TAP proteins, such as putative TMSs, alongside their exon structure. The fact that there does appear to be fairly good correspondence between predicted structural features and individual exons (Table IV; and Fig. 7 ) indicates that the prediction may be valid. This agreement could also help in predicting functional subregions of the molecule. Thus, a TAPl protein with 10 TMSs and a cytoplasmic C terminus would also have a cytoplasmic N terminus. When the “positive inside” rule is applied to this topology (considering only the N-terminal hydrophobic domain), the putative cytoplasmic side would have 26 positive charges and the lumenal side would have 8. The NBD would contribute a further 19 positive charges to the cytoplasmic side of the protein. Thus, this orientation is in accord with the positive inside rule. The charge difference rule also predicts this orientation for the first putative TMD (charge difference at N terminus minus charge difference at C terminus = +2) and correctly predicts the orientation of the following 4 TMSs. The cytoplasmic to lumenal orientation of the first putative TMS has been confirmed experimentally in a bacterial system in which a fusion protein comprising the N-terminal 39 residues of TAPl followed by a mature form of P-lactamase that, when expressed alone in E. coli, is sequestered inside the cell (equivalent to the cytoplasm of an eukaryotic cell), The fusion protein, however, was inserted into the membrane in such a way as to expose the P-lactamase moiety to the

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extracellular space (equivalentto the ER lumen), thus conferring resistance to ampicillin (97). The intervening sequences between putative TMSb and TMSc, TMSc and TMSd, TMSd and TMSe, and TMSe and TMSf are predicted to be fairly short (less than 20 amino acids) and hydrophilic, whereas that between TMSa and TMSb is predicted to be fairly short and hydrophobic. The sequence between the pairs of putative TMS encoded by exons 4 (g and h )and 6 (i andj) is predicted to be very short and hydrophobic. A large, hydrophilic intervening sequence links TMSfto the TMS pair encoded by exon 4, and this to the TMS pair encoded by exon 6. These probably comprise two further structural units because they are encoded by exon 3 and exon 5, respectively, and would reside in the cytoplasm according to this predicted topology. Taking this approach, the large cytoplasmic C-terminal NBD might be thought of as comprising two highly conserved modules (exons 8 and 10) that contain the Walker motifs, two homologous adapter modules (which are around 60% conserved between TAPl and TAPB), and a C-terminal module (exon 11)that varies in length between human TAPl and TAPS, which can be as short as 23 amino acids in some TAP2 alleles (see below). One feasible model for the TAPl structure is, therefore, the one shown in Fig. 8. Two lines of evidence support this model. First, when a comparison was made between the hydrophobicity plots of TAPl (1-748) and MDR1-N (1-700), whose topology has been established experimentally,a remarkable similarity was seen between the six TMSs of MDRl and putative TMSe-j of TAPl (Fig. 7). This similarity was enhanced when the short stretch of amino acids between MDR 89 and 111was treated as an insertion unique to MDRl (see Fig. 2 for sequence), Interestingly, this short stretch is highly glycosylated (the human protein contains three N-glycosylation sequons) and may constitute a structural feature that has evolved for the MDR function. Thus, the structure N terminal to this region (containing TMSa-d, encoded by the first exon) appears to be unique to TAP. Interestingly, when a prediction [using the method of Hofmann and Stoffel (91)] of the membrane spanning regions and their orientation was made using only the TAPl sequence corresponding to exon 1, the same four TMSs were predicted as when the complete sequence was subjected to the same

FIG. 7. Hydrophobicity plots for TAPl and TAP2 and the human MDRl encoded P-glycoprotein using the Hofman-Stoffel algorithm. Predicted TMS for TAF'1, as they appear in Fig. 8, are labeled a-j. Note that this algorithm only strongly predicts 6 or 7 potential TMSs for TAPB.

69

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3000,

TMpred output for TAPl/TAP2 r

I

I

I

I

1

i->o

..........

2000 1000

..........................

0

- 1000 -2000

-3000

-4000

-5000

-6000

0

200

1

I

I

400

600

800

1000

1200

1400

30

TMpred output for MDRl 3000 i->o o->l

....

2000

loo0 0

-loo0 -2m

-3000 -4000 -5000

-6000

-7000

0

200

400

600

800

lo00

1200

1400

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TABLE IV POSSIBLE STRUCTURAL “MODULES”ENCODED BY TAP1 EXONS Exon

1 2 3 4 5 6 7 8 9 10 11

-

Possible corresponding structural unit N-terminal TAP-specific module (4 transmembrane segments with short linker polypeptides) 2 transmembrane segments‘ Large hydrophilic module (cytosolic) 2 transmembrane segments Large hydrophilic module (cytosolic) 2 transmembrane segments Hydrophilic module (cytosolic) Module containing a Walker motif Hydrophilic module Module containing a Walker motif Hydrophilic module

‘The fifth TMS, predicted by several different algorithms, spans the boundary between exons 1 and

2 (see text).

I

I1

a

h

c

d

e

I

4 6 Lumen nn

f

g

u

h

i

j

Cytoplasm

3

FIG.8. A model for the structure of TAP1. This model comprises a cytoplasmic NBD made up of polypeptide encoded by exons 7-11 and an N-terminal TMD made up of six N-terminal TMS encoded by exons 1 and 2 (a-f) and two pairs of TMS encoded by exons 4 and ( g and h) and 6 (i and j), linked together on the cytoplasmic side of the ER membrane by hydrophilic stretches encoded by exons 3 and 5. Applying this structure to the TAP2 protein, asterisks show the location of amino acid residues that differ between TAE’2s of transporters with restrictive specificities and those that are more permissive. These are the following: positions 4, 262,266, and 374, which are charged; Q/N, L, and S/A, respectively, in the permissive transporters (rat TUB-A and human TAP2); and Y,R, F, acidic, and R, respectively, in the restrictive transporters (rat TAP2-B and mouse TAP2).

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predictive algorithm. This suggests that exon 1 may indeed constitute a TAP-specific structural module. The second line of evidence comes from the observation that TAPl does not appear to be glycosylated.TAPl contains four N-glycosylation sequons at positions 227, 250, 279, and 302 (TAP2 does not contain any). One of these (227) is predicted to lie at the ER boundary of a TMD, and another (302) at a similar cytoplasmic boundary and so would be inaccessible for glycosylation. The other two lie in the hydrophilic module encoded by exon 3, which according to this model should reside in the cytoplasm. Consistent with this orientation are the observations that TAPl immunopurified from B cells, as a heterodimer with TAPS, is endoglycosidase-F and endoglycosidase-H resistant (S. Springer and A. Townsend, personal communication, 1993), and the SDS-PAGE mobility of TAPl expressed in Drosophilu cells is unaffected by the glycosylation inhibitor tunicamycin (67).A small amount of TAPl can be labeled with [l-3H]mannose when it is overexpressed from recombinant vaccinia virus, but this is not endoglycosidase-H sensitive and it did not bind to the lectin concanavalin A, and was therefore dismissed as an experimental artefact. Correspondence between the hydrophobicity plots of TAP2 and TAPl is fairly good over the region spanning putative TMSc-j and the NBD but not as good as the correspondence between the two halves of MDR. There is considerable similarity between TAP2 and MDRl l-700 and, to a greater extent, MDRl 701-1280. As noted earlier, structural elements thought to be unique to TAP might reside at the N termini of both TAPl and TAPB. It is interesting to note that, using the Hoffman-Stoffel algorithm, this region of TAP contains only three putative TMSs, regardless of whether the whole sequence or just the first exon is considered. This raises the possibility that TAPl and TAP2 could be inserted into the membrane with their N termini on opposite sides (another possibility is that the first TMSs of both TAPl and TAP2 are inserted with their N termini orientated toward the cytoplasm, but TAP2 adopts a “leave-one-out” topology in which only eight of the nine potential TMSs span the membrane. This has been observed experimentally for a number of integral membrane proteins expressed in bacteria (see Ref. 98). Thus, although a persuasive argument can be made for the putative structure for TAPl shown in Fig. 8, the final solution of its topology awaits further experimental evidence. V. TAP Polymorphism

The alleles that have been described to date for the human and rat TAP proteins comprise a series of dimorphisms at various positions along the length of the polypeptide (99-102). TAP alleles are therefore defined in

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terms of a particular combination of these dimorphisms (see Tables V and VI). In addition to polymorphisms at the protein level, several other silent polymorphisms have been described for both TAPl and TAPS, but these will not be discussed here. In human TAP1, two dimorphic sites, one in the TMD and one in the NBD, define 4 alleles. Of these, only 3 have been observed, with TAPlA being by far the most common in the Caucasian population, with a frequency of 81% (101). A rare variant of TAPlA has been described that has an R to Q substitution at position 648 (100). In addition, a naturally occurring null allele has been reported in which residue 659 of TAPlC was changed from arginine to glutamine, resulting in a nonfunctional transporter (103). Four main dimorphic sites in the

TABLE V ALLELESOF HUMAN TAPl AND TAP2 Amino acid position: human TAPl alleles Allele

333 (TMD)"

637 (NBD)b

659 (NBD)

Frequency

TAPlA TAPlB TAPlC Putative TAPlCR659Q

I V I V I

D G G D G

R

81% 15%

R R R

4%

N/A One report"

Q

Amino acid position: human TAP2 alleles Allele

687 253 (TMD) 379 (TMD) 565 (NBD) 665 (NBD) (NBD) Frequencyd

TAP2A TAP2B TAP2C TAP2D TAP2E Putative Putative Putative TAP2R253X

R R R

R R R R

R STOP

V V I

I V I V I -

A A A T T T T A

-

T A T T T A A A -

STOP

Q

STOP STOP STOP

Q Q Q -

62% 26% 7% 4%

1% N/A N/A N/A One family reportedc

Note. Numbers indicate the amino acid position at which a dimorphic substitution has been iden-

tified. a

Transmembrane domain.

* Nucleotide binding domain. See text for description. In Caucasians.

TABLE VI ALLELES OF RAT TAP2 h i n o add position

AUele

MHC haplo-

46

73

92

105

107

109

165

167

168

217

218

262

265

266

352

374

380

394

424

455

464

526

527

€43

TAP2-A H S L TAPZA' T A B - B Y R TAF2-B' V

N

V

D

G

V

W

M

F

A

E

Q

S

L

V F V

S

Q

V

Q

N

L

Q

L

K

V

R 1 Kh,h.n,u

4

25

L

T

L

G

V

A

G

T

L

T

M

R

P

F

E

R

C

R

K

V

K

adf.g.(q,s)

c,

k, m

Note. From data compiled by Joly et d.(102) from 56 laboratory strains of Rattus nomgicus. TAPBB is more closely related to mouse TAP2 than is TAP2-A (96.3%similarityl92.3% identity compared to 95.2%similarityl91.28 identity).

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TAP2 gene have been described that could give rise to 16 possible alleles (99-101), with a rare variant of one of these ([TAP2-C(lOO)]R651C). However, in two separate analyses, the dimorphisms at positions 665 and 687 only occurred in two of the four possible combinations (99,101). Thus, it is thought that there are 8 major alleles of human TAPS, of which 5 have been observed to date. Four of the observed alleles terminate at residue 687. The most frequent allele identified to date is TAPSA, which is present in 62% of the population. In addition, a null allele of TAP2 has been described in a Moroccan family that has a mutation in codon 253 resulting in premature termination and a nonfunctional TAP complex in two homozygotic siblings (104) [the most severely affected child suffered from chronic colonization of the lungs by bacteria from birth, whereas her brother did not become affected until he was 6 years old, when he presented with similar symptoms. Peripheral blood lymphocytes from both children, but not from their heterozygous parents, had low surface expression of MHC class I, contained low (but not zero) numbers of CDS’cUp T cells, and displayed poor NK and CTL activity]. Thus, allelic variation among human TAPSdoes not appear to generate a great deal of structural diversity (Table V). This is in contrast to the rat TAP2 protiens that, although being less polymorphic (rat TAP2 is diallelic,with each allele having two variants; see Table VI),display more structural diversity between the 2 major alleles. Thus, 25 dimorphic sites, distributed fairly evenly throughout the length of the protein, define two groups of alleles called TAP2-A and TAP2-B (102). Rat TAPl appears to be monomorphic among all inbred strains of laboratory rat investigated to date. Little sequence information is available for the mouse TAP proteins, However, Gaskins et al. (53) have identified five TAPl alleles and seven TAP2 alleles by restriction fragment-length polymorphism (RFLP) analysis of over 15 inbred strains of laboratory mice (Table VII). At this stage, the structural correlates of these RFLPs are not known and could theoretically all be silent with respect to protein sequence. In general, because the TAP genes are located close together in the MHC, all MHC haplotypes are associated with one TAP allele. However, the distance between TAP and MHC class I region allows for a low frequency (around 0.1% in rat) of recombination, resulting in “mixed haplotypes.” In laboratory-bred rats, such an event has resulted in recombinant strains such as rl, in which the RT1-A” MHC class I allele, which is normally inherited along with the TAPS-A allele in strain RTla rats, has recombined with the TAP2-B allele from the rat strain RTlc. The RT1Ad molecule in this recombinant rat strain assembles slowly and is unable to present some endogenous antigens-an observation that is central to the cim phenomenon (see above). The reason for this is that structural polymorphism displayed by the two rat TAP2 alleles results in their

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TABLE VII ALLELES OF MOUSETAPl AND TAP2“ Mouse TAPl alleles Allele

Mspl

Xba 1

MHC haplotype

TAPla TAPlb TAPlc TAPld

lb 2 3 3

1 2 2 2

a, b, d, f, k, p, q, r, u, v, z NOD M. spretus Unique to strain VLnJ

Mouse TAP2 alleles Allele

BarnHl

Stul

Xhol

TAP2a TAP2b TAP2c TAP2d

IC

1 1 2 2

1 2 2 2

3 1 2

2 3 2

TAP2e TAP2f TAP2g

1 1

2 1 1

3

MHC haplotype a, k, p

f b, j, q, u, z NOD and strain RIIIs/J

d V

Strain POSCwEi

a Compiled from 21 laboratoly strains of MILSmusculus by Gaskins d d.(53). ’’ Alleles are defined by restriction fragment-length polymorphisms of genomic

DNA. Numbers 1-3 refer to different restriction enzyme digest patterns. Thus, an Mspl digest could generate characteristic fragments of 3.8 (1). 2.3 (2). or 4.5 kb (3). An Xbal digest could yield fragments of 10 (1) or 8 kb (2). As for TAP1, the TAP2 deles are defined as restriction fragment-length polymorphisms for genomic DNA. Three diagnostic restriction enzymes were used to generate the following patterns of digest products. A BamHl digest could yield fragments of 4.3 (1). 3.4 (2), or 10 kb (3)..A Stul digest could give fragments of 9.5 and 8.8 (l),9.5 and 4.0 (2),or 9.5 kg (3).An Xhol digest could give fragments of 15, 9.5, and 5.5 kb (1); 17, 9.7, and 8.0 kb (2); or 22, 17, 12, and 9.7 kb (3).

transport of different sets of peptides (see below). Rat TAP-2A transports peptides that can bind to all rat class I molecules (including RT1-A”), whereas TAP-2B transports a pool of peptides that only bind very poorly to RTl-Aa. Because the RT1-Aaallele shows very strong linkage disequilibrium with the TAP-2A allele, these differences went unnoticed until recombinant rat strains were studied in which RT1-Aawas brought into cis association with a TAP-2B dele. The discovery of the cim phenomenon was therefore a key to determining the substrate specificity of the TAP complex, as is discussed below. VI. Function of the TAP Complex

Experiments with cell lines lacking a functional TAP complex have shown that both a functional TAPl and TAP2 protein are required for the antigen

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presenting function of MHC class I-bearing cells. Laboratory mice with experimentally deleted TAP genes also lack stable cell surface expression of MHC class I molecules. These mice have greatly reduced numbers of CD8+ T cells in both central and peripheral lymphoid organs, suggesting that a functional TAP complex is essential for the generation of MHC class I-restricted T cells during development (105). These observations were made before it was known that the TAP complex was indeed a peptide transporter. Such a demonstrationwas achieved only once in vitro transport assays were developed. These are discussed below. A. IN VITRO TRANSPORT ASSAYS Early attempts to define the biochemical function of TAP compared the uptake of peptides by microsomes prepared from normal and TAP-negative cell lines in the presence and absence of ATP. Using an in vitro translation system comprising HLA-B27 mRNA, microsomes from the cell lines T1 (TAP+) and T2 (TAP-), and a biotinylated HLA-B27-binding peptide, Levy et al. (106)demonstrated that the TAP-dependent assembly of HLAB27 with peptide required the hydrolysis of ATP. However, they went on to suggest that this was not a demonstration of TAP-mediated ATPdependent peptide transport because an independent measurement of peptide uptake by microsomes (accumulationof peptide by the ER chaperone BiP) showed no difference between T1 and T2 and was ATP independent. It is unfortunate that the authors chose to interpret their results in this way because, with the privilege of hindsight in the light of more convincing evidence that TAP is an ATP-dependent peptide transporter (see text that follows), the Levy results are rather illuminating. It is clear from this and other studies that microsome preparations are permeable to short peptides that will accumulate in the lumen down a concentration gradient (107-109). Live endoplasmic reticulum is not permeable in this way. However, what is clear from the Levy experiments is that peptides that are taken up in this nonspecific way are, by and large, denied access to newly synthesized class I molecules. Thus, very little peptide-driven assembly of HLA-B27 was observed in T2 microsomes or in T I microsomes in the absence of ATP, despite good nonspecific peptide uptake by both microsomes. Convincing peptide-driven assembly of class I molecules was seen only in microsomes that contained TAP and when ATP was present-a result that almost certainly shows that class I molecules efficientlyassemble only with peptides that are delivered to the ER by TAP. This result is worth bearing in mind when assessing the efficiency of class I loading with peptides that have been delivered to the ER via TAP-independent means (110, 111).One other intriguing point to emerge from this study is that, certainly in the absence of TAP, BiP is unable to transfer its bound peptides

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to awaiting empty class I molecules-a point that is relevant to speculations that BiP might act as an ER chaperone for peptides awaiting an appropriate MHC class I receptor. A very similar experimental setup to the one described by Levy et al. (106) was used by Shepherd et al. (112) in one of the first reports to suggest that TAP was in fact an ATP-dependent peptide transporter. In this assay, the accumulation of a labeled peptide (FAPGNYF'AL) by liver microsomes prepared from normal C57BV6 or TAP knockout BV6 mice was compared. These experiments clearly showed that both the rate and the extent of peptide uptake were greater when TAP was present, and that this uptake was ATP dependent. The accumulation of peptide in this experiment (and presumably in the Levy experiment) is dependent on there being an appropriate class I receptor for the incoming peptide. This fact was exploited by Androlewicz et al. (113) in another early report showing that TAP transported peptides in an ATP-dependent way. Here, T1 and T2 cells penneabilized by the bacterial toxin streptolysin-0 (SL0),which only affects membranes with high cholesterol content and hence avoiding the ER and Golgi membranes while effectively permeabilizing the plasmalemma, were used instead of microsome preparations. Like Levy et al., they demonstrated the ATP-dependent uptake of labeled peptide by class I present in T1 but not in T2. In this way, Yang and Braciale (114) have calculated that the mouse TAPlaRAP2e heterodimer observes Michaelis-Menten kinetics for transport of an index peptide, with a K , of around 661 nM and a V,, of around 3 X mollmidcell. Another way of retaining peptides in the ER was exploited by Neefjes et al. (57)in the third of the initial reports on TAP function. By incorporating an N-glycosylation sequon into a labeled peptide, this group was able to follow its appearance in the ER lumen of SL-0-permeabilized cells. On entering the lumen of the ER, such peptides can act as acceptors for a branched-chain carbohydrate. By isolating glycosylated, and therefore transported, peptide by lectin affinity chromatography, Neefjes et al. convincingly demonstrated TAP-dependent transport, which required the hydrolysis of ATP, and they were able to calculate that each cell had the capacity to transport 2 X lo4 peptides per minute across the ER membrane-more than enough to saturate the 100 or so class I molecules synthesized in the same time. An important practical point to emerge from these studies was that peptide transport assays using microsomes or permeabilized cells work only when there exists a mechanism for peptide retention in the lumen, be this class I binding or N glycosylation. It is important to note that experiments that measure the ability of a peptide to compete with an index peptide in these kinds of assay are very indirect ways of measuring transport

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because the unknown peptide will compete for the retention mechanism (class I binding/glycosylation)as well as for transport itself.

B. LENGTHSPECIFICITY The early demonstrationsof peptide transport utilized peptide substrates of 9-12 amino acids and established that both a free amino and carboxy terminus are required for their transport (115, 116). Peptides that are synthesized either entirely or partially from D amino acids are not transported (115,117).However, a retroinverso peptide of D amino acids, which preserves the spatial topochemistryof the side chains while exchanging NH for CO groups and CO for NH groups, has been shown to be a substrate for TAP (117). Moreover, the same group has shown that peptides that incorporate one or two isosteric peptide (double) bonds are actually transported more efficiently than normal L amino acid peptides (117). Several reports have shown that peptides of up to 23 residues can compete effectively with nonamer index peptides in indirect measurements oftransport byTAP (57,112,113,115,118).In these experiments,however, it is difficult to determine whether the long peptide, or a shorter version of it (produced as a result of limited proteolysis by contaminants in the transport reaction), is the actual competitor. This issue has been addressed by two groups who, by placing the labeled amino acid ([ '251-]iodotyrosine) at the N terminus of a model peptide substrate and an N-glycosylation sequon at its C terminus, were able to directly measure TAP-dependent transport of peptides of varying length. Both groups showed that peptides of 16 residues or more could be transported. In one study, the transport of a 13mer by a human TAP was as efficient as a nonamer (116) (this 13mer was produced as a result of limited proteolysis of a l6mer during the transport assay). In a second study, transport of a 12mer by the rat TAPlEAP2-B was around 80% as efficient as a nonamer and transport of a l6mer about 20% as efficient (119). This study also indicated that some TAP alleles may be more permissive than others with respect to the range of peptide length that they are able to transport because rat TAP1/ TAP2-A was less efficient at transporting these longer peptides than rat TAPlEAP2-B. This observation has recently been confirmed (120). This study also showed that transport of a 40mer can be demonstrated in a direct transport assay. Peptides of 7 residues or less are transported very inefficiently (120) or undetectably (116, 119), a result that is supported by several indirect measurements showing that peptides of 7 residues or less are ineffective in competition assays (e.g., Ref. 115). C. SEQUENCE SPECIFICITY The cim phenomenon indicated that some degree of functional polymorphism accompanied the structural polymorphism seen in the rat TAP2

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gene. The in vitro transport assays described previously provided a way of investigating this functional polymorphism and enabled scientists to determine whether different TAP alleles transported different sets of peptides. A huge amount of data is now available on the effect of peptide sequence on its ability to be transported by the human TAPlRAPB, the mouse TAPldI'APBc, the rat TAPlRAPB-A (cim"), and rat TAP1/ TAPB-B (cimb)heterodimers. This information is summarized in Table VIII. Momburg et al. (121) have compared the substrate specificity of each of these heterodimers using a direct transport assay. They found that the rat TAPlRAPB-A transporter is more permissive than the TAPlRAP2-B transporter in that the former will transport peptides with any residue (except proline) at their C termini but the latter will transport only peptides ending in hydrophobic or aromatic residues. (Among the hydrophobic residues tested at the C terminus, I, L, and V were much more efficiently transported than A, M, and C.) An indirect study of the C-terminal residue preferences of these two transporters (actually TAPlRAPB-A' and TAP1/ TAPB-B') confirmed the observation that the TAPB-A (cim') allele was the more permissive of the two (118). However, this study indicated that peptides with acidic and some polar C-terminal residues could compete with the index peptide. This difference could reflect a difference in the substrate specificity between the two related TAPB-B alleles or simply the fact that semi-intact cells were used in one system and microsomes in the other. Alternatively, it could reflect the fact that peptides with acidic C termini can bind to both TAPB-A and TAPB-B efficiently, but only TAPB-B can transport them. Further comparisons between the ability of peptides to compete and their ability to be transported in the same system are required to address this possibility. Differences in the specificity between rat cim" and cimh alleles are restricted to these differences at the C termini of peptide substrates. Momburg et al. (121) have also shown that, like the rat TAPlRAP2-A allele, the human TAPlEAP2 heterodimer is permissive and will transport peptides with any residue except proline at their C termini. The specificity of the murine TAPldI'APBc allele, however, resembles the rat TAP11 TAPB-B more closely in that peptides with hydrophobic or aromatic C termini are preferred. Peptides with basic, acidic, polar, or glycine C termini are transported by murine TAPldI'APBc much less efficiently than by either the human TAPlRAP2 or the rat TAPlRAP2-A transporter. This restricted specificity of the murine TAPla/TAPBc complex has been confirmed by an indirect assay (114). The effect on transport efficacy of different amino acid side chains at other positions in the peptide substrate has also been investigated (122). The main finding is that proline is not tolerated at the first three positions

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TABLE VIII SEQUENCE SPECIFICITV OF THE TAP TRANSPORTERS ~

Rat TAPlmAP2-A Position in peptide Amino acid side chain

2

1

Basic Acidic Hydrophobic Aromatic Polar G P

3

4

5 0

6

7

8 0

-

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

-

-

0

0

0

0

0

0

-

7

8

9

-

-

9

0

0

0

0 oh Oh

-

0

Rat TAPlmAP2-B ~

~

Position in peptide Amino acid side chain

1

Basic Acidic Hydrophobic Aromatic Polar G P

0

2

3

4

5

-

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

-

0

0

0

0

0

0

-

0

0

0

0

-

7

8

-

0

0

-

Q

-

-

0

6 -

-

0

0

-

0

0

-

Mouse TAPlafI'AP2c Position in peptide Amino acid side chain

1

2

3

4

Basic Acidic Hydrophobic Aromatic Polar G P

0

0

0

0

0

0

-

0

0

0

0

0

0

0

0

-

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0 0

-

-

0

0

0

0

-

-

_

5 0

6 -

-

0

@

0

0

-

Human TAPmAP2 Position in peptide Amino acid side chain

1

2

3

4

Basic Acidic Hydrophobic

5

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

6

7 0

-

8 0

-

0

9 0 0

0

(continues)

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TABLE VIII (Continued) Human TAPlmAP2 Position in peptide Amino acid side chain

1

2

3

4

5

6

7

Aromatic Polar G P

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

-

0

0

0

0

-

-

_

-

8 -

9 0

a Compiled from direct transport assays and indirect assays in which test peptides were used to inhibit the transport of an index peptide. Each experiment used model Smer peptides and variants thereof in which each position was substituted with other amino acids. The effect on peptide transport was then assessed. Residues that were tolerated are designated 0, those that were preferred are designated 0,and those that were not tolerated are designated -. In competition assays using the Rat TAPbTAP-A' transporter, peptides ending in either M or F did not compete with an index peptide. In competition experiments using the Rat TAPbTAPP-B' transporter, acidic residues at PS mmpeted efficiently but aromatic residues did not. Peptides ending in H (basic) and T (polar) side chains were poorly transported in direct transport assays. In indirect assays, H and K (basic) and T and Q (polar) were poor competitors.

of a potential substrate for any of the transporters. This is a significant finding because many human class I alleles, including more than 50% of the B locus alleles that have been investigated to date, require proline at position 2 of a nonamer to bind. This fact, taken together with the observations that (i) TAP can transport peptides longer than 9 amino acids and (ii) peptide trimming has been observed in the ER (111, 123), strongly suggests that the processing of longer precursors in the ER, giving rise to peptides of a length and sequence that is optimal for binding to class I, may be an obligate process-at least for some class I alleles. Neefjes et al. (122)have also indicated that an acidic residue at positions 6 or 7 can enhance the transport, by all TAPs, of a model nonamer. It is not yet clear to what extent other subtle differences between the substrate specificityof different TAPs indicated by these experiments are functionally significant. For example, the apparent preference for G at position 1 for the cim",but not cimb,allele or the inability of cimb,but not &ma,to tolerate G or basic residues at position 2. These differences are not mirrored in human and mouse TAPs. A study of the ability of the rat TAPlEAP2-A heterodimer to transport peptides with large side chain extensions has shown that a polymer of up to 9 lysine residues attached via an isopeptide bond to the peptide backbone of a model Smer hindered neither binding to nor translocation by the transporter. Extensions of between 10 and 20 lysines were not transported, but surprisingly they were still able to bind to TAP (117).

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No functional polymorphism has yet been demonstrated among either mouse or human TAP alleles. In fact, Obst et al. (124) have shown that the specificity of all human transporters that they tested (TAPlmAPZA, TAP 1B/TAP2B,TAPlC/TAPBC, TAPlA/TAP2B, TAPlA/TAPSC, TAP1A/ TAP2E, TAPlBRAPBA, and TAPlBmAPBD) was identical. Similarly, no difference in substrate specificity was seen among three murine transporters (TAPla/TAP2c, TAPla/TAP2e, and TAPlnAP2a). Thus, there appears to be two functional subgroups of TAP transporters: a permissive group, which includes the human transporters and rat transporters containing the TAP2-A allele, and a group with a more restricted specificity that includes the murine transporters and rat transporters containing the TAPS-B allele. By comparing the chemical nature of the amino acids at all the variant sites between the two rat TAP2 alleles with the equivalent amino acids in the mouse and human genes, it might be possible to infer which of these are responsible for the differences in function that are observed between the two groups. At first sight, this would appear fruitless because the rat TAP2 alleles are much more closely related to one another than they are to either mouse or human TAPS. However, by concentrating on the 25 amino acids at positions that define the two rat TAP2 alleles, a correlation between structure and function begins to emerge (see Table IX).Eighteen of these are identical or similar in the human and mouse sequences. The remaining 7, however, divide the four transporters into two groups that correlate with their function. Thus, within the N-terminal transmembrane domain, the permissive transporters (rat TAPS-A and human TAP2) have a charged residue at position 4, Q or N at 262, L at 266, S or A at 374, and Q at 424, whereas the restrictive transporters have Y at 4, R at 262, F at 266, an acidic residue at 374 and R at 424. In addition, rat TAP2B has two unique substitutions at positions 455 (polar in other TAP2s but K in TAP2-B) and 526 (Q in other TAPS but K in TAPS-B). It is interesting that two of these substitutions are polar in the permissive group to basic in the restrictive group (positions 262 and 424), which, if they were to reside in the peptide binding site of TAP, could inhibit the transport of peptides with basic amino acids at their C terminus (see text that follows). This difference may be exaggerated for TAP2B, which also has polar to basic substitutions at positions within the NBD (positions 455 and 526). When these seven residues are located on the structural model for TAPl (assuming a correspondence between TAPl and TAP2 in this region), they all reside in the cytoplasm and hence would be in a suitable position, topologically at least, to form part of a peptide binding site (see Fig. 8).

TABLE IX A COMPARISON OF AMINO ACIDRESIDUES AT POSITIONS THAT DEFINE THE Two ALLELESOF RAT TAP2 WITH EQUNALENT POSITIONS IN HUMANAND MOUSE TAP2 Amino acid position

Allele

4

25

RatTAF'2-A H S H u m a n T A P 2 RatTAP2-B Y R M o u s e T A F ' B

46

73

92

L N V D G L T A L T L Y G L T V

105

107

109

165

167

168

217

218

262"

265

266"

352

D G G G

G G V G

V A A V

W G G G

M T T T

F L L L

A T T T

E M M M

Q

S P P P

L L F F

V V V V

N R R

374" 380 394 S A E D

Q R R R

V C C C

424'

455

Q Q R R

N S K

Q

464 526 L L V L

Q Q

K Q

527 L V V L

a It is interesting to note that, i n particular, positions 262,266,374, and 42A define the two functional groups comprising (i) rat TAP2-A and human TAPP, which have a permissive specificity (see Table 8); and (ii) rat TAP2-B and mouse TAP2, which are restrictive (see Table VIII).

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This idea is supported by two recent studies. In the first, a point mutation at position 374 in human TAP2 from A to D resulted in an alteration in the pattern of peptides that could be transported by heterodimers containing the mutant TAP2 from a permissive to a more restricted specificity (125). The second study by Momburg et al. (126), using transporters incorporating chimeric TAPS-A/TAP2-B proteins, has shown that changing amino acids at positions 374 and 380 from the “permissive” to the “restrictive” sequence results in a transporter with a substrate specificity more like that of the restrictive phenotype. Similarly, placing the permissive sequence in the context of the TAP2-B partially relieves the block against the transport of peptides with small hydrophobic, polar or basic C-terminal side chains. They also found that the pair of residues at positions 217 and 218 may have some role in controlling the transport of peptides with basic C termini. In all the TAP2s, except for rat TAP2-B, position 217 is T and 218 is M, whereas A217 and E218 are unique to TAP2-B (see Table IX). Evolution of the TAP2-A allele may have occurred under pressure from the appearance of class I molecules that could accommodate a basic residue at the C terminus. Like the modem mouse, the ancestral rodent may have originally expressed only class I molecules with an F pocket, which could accommodate peptides with hydrophobic C termini (the peptide-binding groove of MHC class I molecules is lined with four highly polymorphic and two less polymorphic “pockets,” which together govern the binding specificity of each allele. The F pocket defines one end of the peptidebinding groove into which fits the C-terminus and C-terminal side chain of the peptide ligand. The specificity of the F pocket is largely controlled by residue 116). A single mutation at position 116 in the peptide-binding groove of this class I molecule from tyrosine to aspartic acid may have been sufficient to alter the specificity of an ancient class I molecule to prefer peptides with a basic C terminus (see Ref. 127 for discussion). Over time, the RT1-Aa allele has segregated with the TAP2-B allele as might be expected if efficient antigen presentation function were to be maintained by haplotypes containing the new MHC class I allele (128; E. Joly, personal communication, 1996).

D. PEPTIDEAND NUCLEOTIDEBINDING Unlike peptide transport by TAP, peptide binding to TAP is ATP independent. Thus, in the absence of ATP, peptide binding and transport can be uncoupled. In this way, three independent studies using three different TAP/peptide systems have measured the equilibrium binding constant of TAP for peptide at between 3 and 6 X lO-’M (58, 120,129). van Endert et al. (58), using peptide libraries of different lengths, have shown that only peptides of between 8 and 16 amino acids will bind. Uebel et al.

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(129) have also used restricted peptide libraries to dissect the relative importance of each residue of the model peptide RRYNASTEL for binding to TAP. They found that R1, R2,and Y3 all had a stabilizing influence on binding, whereas N4, S6, T7, and E8 were destabilizing. A5 and L6 were neutral. It is perhaps significant that in transport assays, human TAP disfavors peptides with a penultimate negatively charged residue such as E8, which was destabilizing in this study. These experiments suggest that the specificity of TAP for the transport of peptides of a certain length and sequence might be governed by an initial peptide binding event-although more work needs to be done to determine the extent of this correlation. Peptide-binding is sensitive to temperature, pH, and the presence of ATP or nonhydrolyzable analogs (58).Thus, 55% of bound peptide can be released from the TAP complex by raising the temperature to 50°C for 5 min. Binding of model peptides to TAP has a pH optimum of 7.8, and extremes of pH (less than 3 or greater than 11.5) can effect the release of 80% of bound peptide. In general, TAPl and TAP2 appear to be required for formation of the peptide binding site. However, some peptide binding to TAPl has been shown to occur in the absence of TAP2 (87).This may have been nonspecific binding, as suggested by the authors, or may reflect the fact that TAPl may be able to form homodimers with a low capacity for transporting peptides (see above). Peptides into which a photoactivatable side chain has been incorporated have been used to probe the sites of contact between TAP and its substrates (87, 130). All peptides tested to date cross-link to both chains of the heterodimer, but the extent to which each chain is labeled by the photoactivatable group varies depending on the peptide sequence (but apparently not where in the sequence the photoactivatable group resides). Thus, in one study (130) peptide QVPLRPMTYB (where B is para-azidophenylalanine-derivatized lysine) labeled TAPl to a far greater extent than TAP2, whereas ABAYAAEEF labeled TAP2 more than TAP1. This difference was probably not due to the relative position of the B residue, as indicated by a second study (87)that showed that the peptides TBDNKTRAY and TYDNKTRAB (where B is L-(trifluoromethyldiaziarinyl phenylalanine) both labeled TAP2 more than TAP1, whereas the peptides RYBANATRSA and TNKTRIDGQBY both preferentially labeled TAP1. There is therefore no evidence to date to suggest that the two chains of the TAP heterodimer interact with distinct sites on the peptide substrate. Nijenhuis et al. (87), using this photocross-linking technique, have mapped the part of the peptide binding site that is contributed by the TAPl chain to a region between residues 376 and 487. In the model structure for TAPl described previously (and indeed for the one postulated by the authors), this stretch of TAP1, which spans the region encoded by

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exons 6 and 7 , comprises two transmembrane segments and a hydrophilic module residing in the cytoplasm. It is particularly interesting that two of the residues that have been suggested could influence transport specificity governed by TAP2 alleles reside in a similar position in TAP2 (positions 374 and 424; see preceding text). Also, in studies with Pgp, drug-labeling studies have localized the substrate binding site of MDRl to the cytoplasmic ends of TMS6 and TMSl2 and the cytoplasmic loop between T M S l l and TMSl2 (131, 132). According to the model in Fig. 8, these regions might be equivalent to the cytoplasmic end of the final TMS in both TAPl and TAP2 that is consistent with both the photocross-linking study of TAPl (87) and the observation that residues in this region of TAP2 might control TAP specificity (see preceding text). The binding of nucleotides to both native TAP heterodimers and isolated nucleotide-binding domains, expressed as recombinant proteins, has been investigated. Russ et al. (67) have shown that TAPl and TAP2 can be photolabeled to the same extent with 8-azido ATP, and that this is inhibitable by both ATP and EDTA. This suggests that each ATP-binding domain contains an ATP and a Mg2' binding site. ATP bound as well as CTP and UTP, with GTP and ADP binding very poorly and AMP not at all. Thus, it is highly likely that in vivo (where the concentrations of ATP and GTP are similar) ATP is the preferred substrate. Similar results have been seen for the isolated TAPl NBD (133),although the relative affinitiesof different nucleotides were slightly different (ATP > GTP > ADP > CTP > AMP). In this study, the isolated TAPl NBD did not display any ATPase activity, but the authors attributed this to misfolding of the recombinant protein rather than to any true functional difference. However, a study of the nucleotide-bindingproperties of each TAP chain expressed in the absence of the other is required to address this more fully.

E. A MODELFOR TRANSPORT-THE CYTOPLASMIC SIDE The evidence presented previously strongly suggests that peptide binding to TAP precedes its translocation across the ER membrane. TAP could act as a true transporter in which peptides are sequentially bound and translocated across the ER membrane or it could act as a peptide channel that is activated by peptide binding, allowing the diffusion of peptides into the ER lumen down a concentration gradient. Other members of the ABC transporter family have been shown to have both functions, for example, Pgp transports small organic compounds specifically but also acts as a voltage-gated Ca", Nat, and Kf channel (134-136). Although most data are more consistent with a transporter function for TAP, one or two observations have been made that are inconsistent with this model. For example, van Endert et al. (58)have shown that, although in general the

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efficiency of peptide translocation by TAP correlates well with its relative affinity for different peptides, one example was found in which a low molar excess of a putative “competitor” peptide actually increased the transport of an index peptide. One explanation, which the authors favor, is that the increased concentration of peptide on the cytoplasmic side of the ER membrane inhibited the diffusion of transported index peptides back out of the ER down a concentration gradient [a phenomenon that has been described in detail elsewhere (112, 115)] resulting in more efficient accumulation of the index peptide in the lumen. An alternative explanation is that the competitor peptide is more efficient at opening a channel than the index peptide. The observation that ATP binding releases bound peptide from TAP, even in the absence of hydrolysis, strongly suggests that ATP binding to the NBD induces a conformational change in the TAP complex-changing it from a high- to a low-affinity receptor for peptide. Similar observations have been made with other members of the ABC transporter family (137). Presumably, as suggested by van Endert d al. (58),the translocation event follows on rapidly from ATP binding and is accompanied by ATP hydrolysis. ATP hydrolysis may be required to drive a conformational change in TAP that both facilitates peptide transport and regenerates the high-affinity peptide binding site. This leaves the TAP complex, at the end of a transport cycle, with a high affinity for peptide and a low affinity for ATP (see Fig. 9). This model predicts at least three conformational changes in the TAP heterodimer per transport cycle. One interesting observation that follows from this model is that upon ATP binding to the peptide-loaded TAP heterodimer (resulting in a reduced affinity for peptide) two competing processes are set in motion. The first is dissociation of peptide from the complex and the second is translocation of the peptide through the complex (which is dependent on the hydrolysis of ATP). It is entirely possible that some peptides, while binding to the high-affinity TAP complex efficiently, dissociate from the low-affinity complex at a rate that exceeds their rate of transport. This would be observed experimentally as a peptide that worked efficiently as a competitor in indirect transport assays but that was poorly transported in direct assays. The transport of peptides with acidic C termini by the rat TAPl/TAPB-B allele might be such an example, as is the extreme example of the branched peptides studied by Gromme et al. (117) (see preceding text). MI. TAP and MHC Class I Assembly

TAP could supply peptides to newly synthesized MHC class I molecules simply by raising the concentration of free peptide in the lumen of the

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1 High Ka peptide binding rite

High Ka ATP binding site

\

f

peptide

b

\ Low Ka ATP

(a)

binding site

J

Low Ka peptide binding site

P

3

FIG.9. A model for the relationship between ATF’ binding and hydrolysis and peptide binding and transport by TAP. The “resting” TAP heterodimer (a) has a high-affinity peptide binding site and a low-&nity ATP binding site. Peptide binding to this complex (1) exposes a high-affinity ATP binding site. Upon ATP binding (2),the high-&nity peptide binding site is lost with the resulting dissociation of peptide that is then transported across the ER membrane (3) upon the hydrolysis of ATP, which also regenerates the high-affinity peptide binding site (4).

ER. Evidence suggests, however, that the TAP heterodimer may play a much more active role in the assembly of class I molecules with peptides. Indeed, reinterpretation of experiments performed by Levy et al. (106) (see above) suggests that bulk flow of peptides into the lumen of the ER may not be an efficient way of delivering them to MHC class I molecules, and that peptides need to be delivered to newly synthesized class I molecules by TAP for efficient assembly to occur. By choosing appropriate detergents for solubilizing cells, it is possible to coimmunoprecipitate MHC class I with antibodies raised to the TAP complex (138, 139). These experiments, therefore, demonstrate that a fraction of both mouse and human class I molecules is physically associated with the TAP complex in the ER of living cells. The association appears to be with the TAP1 chain because coprecipitation of MHC class I was seen in RMA-S (138), which expresses a truncated TAPS, and has even

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been observed in cells that do not express the TAP2 chain (140). Pulsechase analysis revealed that the rate of egress of class I molecules from the early secretory pathway corresponded with their rate of release from TAP (138). This suggested a model in which empty, newly synthesized class I molecules bound to TAP and were thus retained in the ER until an appropriate peptide was delivered to the peptide binding groove. This idea was supported by experiments that showed that dissociation of class I from TAP was enhanced by the binding of peptides delivered to the ER in artificial systems using permeabilized cells and added peptides (138, 139).Also consistent with this idea is the observation that, when the supply of peptides to the ER is limited (for example, by poisoning the proteolytic enzymes used to generate peptides in the cytosol) the TAP:class I interaction is prolonged, and complexes accumulate in the ER (141). Class I molecules associate soon after their assembly with p2m (142). For example, the murine allele H2-Db can be found in association with TAP as early as 5 min after its synthesis, reaching a maximum after around 10 min (138). Because the release of class I molecules from TAP closely mirrors their release from the ER, these results indicate that for most of their time in the early secretory compartment, class I molecules are associated with TAP. It has been known for some time that other chaperone molecules are involved in the assembly of MHC class I molecules (Ref. 143 and reviewed elsewhere in this volume). In particular, calnexin associates with free HC and is almost certainly involved in the early stages of class I assembly (143). In mice, calnexin can also be found in association with HC:P2m heterodimers (144,145).It appeared likely that class I molecules assembled on calnexin were transferred to TAP for peptide loading. However, Suh et al. (141) have shown that calnexin forms ternary complexes with TAP and class I. They estimate that up to 80% of H2-Db,which is associated with TAP, is simultaneously associated with calnexin and that the ER resident pool of H2-Dbcomprises around 50%complexed to both calnexin and TAP, 10%with TAP alone, and 40%with calnexin alone. Interestingly, whereas peptide binding by class I releases them from TAP, they are not released from calnexin. This suggests that release of loaded class I molecules from TAP might actually precede their release from calnexin. Thus, calnexin may accompany class I molecules further along the assembly line than was originally thought and may be the factor governing their release into the trans-Golgi network. It is unlikely, however, that release from calnexin is the rate determining step in class I maturation because the rate of class I release from TAP is very similar to its rate of release from calnexin (and its rate of release from the ER).

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In humans, where calnexin can only be seen in association with free HC [and not HC:/32m complexes (146, 147)], this later stage of class I chaperoning appears to have been adopted by the homologous ER chaperone calreticulin. Sadasivan et al. (148) have shown that calreticulin forms ternary complexes with class I and TAP, and that approximately half of the ER resident pool of HC:/32m heterodimers exists in this form, with the rest forming binary complexes with calreticulin. When TAP is absent, the entire pool is associated with calreticulin. It is likely that, whereas in the mouse, class I molecules are assembled on calnexin, which conducts them to TAP and accompanies them briefly after their release, in humans, the association of /32m with HC signals their release from one Ca2+-binding chaperone (calnexin)to another (calreticulin). This idea is shown in Fig. 10.

FIG.10. A model for the role of TAP in the assembly of newly synthesized human MHC class I molecules. Class I heavy chains in a nonnative conformation first associate with calnexin (CX), probably via a lectinlike interaction. Upon p2m binding, the HC adopts a native conformation and dissociates from CX. This “empty” receptor might now enter the secretory pathway and appear at the cell surface, unless it is retrieved by a second calciumbinding chaperone, calreticulin (CRT). The CRT:Class I complex then associates with the TAP:tapasin complex in the ER whereupon class I is loaded with peptides supplied by TAP. Once loaded, the CRT:class I complex dissociates from TAP and rapidly separates, releasing free CRT and loaded class I molecules into the secretory pathway. When TAP is absent or when class I is in excess, it is likely that CRT acts as a sorting point for empty class I complexes-chaperoning them to an appropriate site for degradation somewhere in the early secretory pathway.

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The same group has also shown that a further molecule, which they have called tapasin, is essential for the interaction between class I and TAP. This molecule was first seen as a 48-kDa glycoprotein in immunoprecipitates of TAP from digitonin lysates (149). Tapasin is missing in the recently described cell line .220 (150, 151) in which some class I alleles are unable to associate with TAP and, as a result, remain in the ER as unstable “empty” molecules. Sadasivan et al. (148) have shown that complexes containing TAP, tapasin, HLA class I, and calreticulin can be isolated from digitonin lysates using antibodies to either calreticulin or TAP. A Triton X-100 labile TAP:tapasin complex forms in the absence of class I, and a tapasin:class 1:calreticulin complex (which is Triton X-100 stable) is observed in the absence of TAP. These results imply that tapasin bridges TAP and a class 1:calreticulin complex. Furthermore, because the interaction between tapasin and calreticulin appears to be class I dependent, it is likely that there is a direct interaction between tapasin and class I. These results are consistent with the hypothetical scheme for class I assembly outlined in Fig. 10. Whether tapasin simply acts as a tether for class I or whether it fulfills a more active role in peptide loading of class I molecules is not known. Tapasin could, for example, itself bind to peptides delivered to the ER by TAP and pass them on to class I molecules. This is reminiscent of the role played by the oligopeptide-binding protein of bacteria (OppA), which binds to peptides of two to five amino acid residues in the periplasm and delivers them to the Opp transmembrane transporter complex (OppB-E) (152). It is not yet known whether a similar molecule is involved in the interaction between TAP and class I in the mouse. Although it is clear from these experiments that tapasin is needed for class I to interact with TAP optimally, it is still unclear whether MHC class I molecules contact TAP directly or only via a tapasin bridge. Indeed, in the cell line .220, which lacks tapasin, different class I alleles behave very differently. The expression of some are normal, whereas others (e.g., HLA-A1 and HLA-B8, which do not associate with TAP in .220) do not assemble efficiently with endogenous peptides (151). Suh et al. (141) have shown that the extracellular domains of class I are sufficient for their peptide-regulated interaction with TAP and that the region of the a3 domain around residue E222 is important in maintaining the interaction. Similarly, Carreno et al. (153) have implicated both the region of the a3 domain around 227 and p2m in the association between TAP and class I. It is not clear whether the inability of these mutant molecules to associate with TAP impairs their ability to present endogenous antigens. Recently, residues in the a 2 domain have been shown to be important in controlling the interaction between TAP and class I. First, Neisig et al. (154)

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have shown that allele-specificdifferences in the peptide-bindinggrooveand in particular residue 116in the F pocket-are important for determining the amount of class I that can be found associated with TAP in cell lysates. Although it seems unlikely that this residue could participate directly in the TAP:class I interaction, it may influencethe efficiencyof loading, and hence the steady-statelevels, of the TAP:class I present inside the cell. This is supported by the observation that those alleles that associate poorly with TAP in these experiments are loaded with peptides and transported to the cell surfacemost rapidly. Second,Lewis et al. (155)and Peace-Breweretal. (156) have shown that a T to K mutation at position 134 of HLA-A2.1 disrupts its interaction with TAP. This residue lies within a highly conserved, solventexposed region of the a 2 domain. What is special about this mutation is that it inhibits the ability of HLAA2.1 to present endogenous (TAP-dependent) peptide antigens implying that this region of the molecule has some role in maintaining a physiologicallyrelevant interaction with TAP. Not only do mutant T134K molecules fail to interact with TAP but also they are rapidly transported to the cell surface as empty receptors. One might predict that if this behavior were due to the lack of a direct interaction between class I HC and TAP, in cell lines that lack TAP class I molecules should also traffic rapidly as empty molecules. We have shown that this is not the case (155). In LBL721.174, the majority of unloaded wild-type A2.1 molecules are degraded in the early secretorycompartment and the few that do escape degradation are transportedto the cell surface slowly.This led us to the conclusion that the T to K mutation disrupts an interaction with an accessory molecule that is responsible both for the retention on the ER of class I molecules awaitingloadingand for the sortingof unloaded molecules to the degradative pathway when these are in excess (Fig. 10).One strong candidate for this accessory molecule is, of course, tapasin-although it should be pointed out that in the tapasin-negative cell line ,220, wild-type HLA-A2.1 is expressed normally (150);other candidates are the ER resident chaperones calnexin and calreticulin. Indeed T134K does not associate with calreticulin as does wild-type HLA-A2.1(J. Lewis andT. Elliott, unpublisheddata, 1996).Thus, according to the scheme shown in Fig. 10, newly assembled empty receptors might enter the secretory pathway once they have dissociated from calnexin unless they are rescued by calreticulin, which engages them in the peptide loading process. Failure to bind to calreticulin could lead to the early release of unloaded class I molecules into the secretory pathway as indeed T134K molecules appear to be (155). VIII. TAP in Disease

A. TAP POLYMORPHISM AND DISEASE ASSOCIATION Early observations that allelic differences in the TAP genes could lead to differences in antigen presentation in recombinant strains of rats led to

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the speculation that an association may exist between different TAP alleles and autoimmune diseases that involved MHC class I-restricted T cells. Despite the lack of demonstrable functional polymorphism among the human TAPS (124), several groups have sought an association between TAP alleles and a number of diseases with autoimmune etiology. These are summarized in Table X. As might have been expected, no significant association has been recorded for many of these diseases. In cases in which such an association has been suggested, it is difficult to determine whether this is with the TAP allele itself or with other genes in the MHC (e.g., HLA class 11). For example, although a striking absence of TAPBB was found in insulin-dependent diabetes mellitus (IDDM) patients in one study (157), closer analysis revealed that the phenomenon was caused by linkage disequilibrium between the TAP and the HLA-DR locus, which has been known for a long time to be associated with IDDM. Another TABLE X STUDIES OF TAP ASSOCIATION WITH AUTOIMMUNEDISEASES Disease Insulin-dependent diabetes mellitus (IDDM)

TAP association TAP2B

Ankylosing spondylitis TAPlB Bechets disease Reiters syndrome

TAPlC TAPlC, TAPPA

Rheumatoid arthritis Atopic dermatitis Multiple sclerosis Coeliac disease Juvenile rheumatoid arthritis

None None None None None

Primary billiary cirrhosis Systemic lupus erythrromatosis Kidney graft rejection Psoriasis

None None None None

Notes

References

Possibly protective. Possible TAPcontrolled low MHC class I expression on B cells from NOD mice Increased from 1.9 to 17% in a subgroup of patients with extraspinal disease Decreased from 1 to 0% Increased from 1.9 to 13%; increased from 55 to 77%. This is independent of B27 association

157, 163, 182188

Possible increased TAF'lB, TAPBC, and TAP2D is likely to be due to linkage disequilibrium with DR8

167 189 166

190 191, 192 193, 194 195 196

197 Chinese

198 199 200

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study (158) found a dominant protective effect of the TAP2B allele in IDDM, which the authors argue is independent of the protective effect of the DRB1"02DQB1"0602 haplotype that has been described previously. That MHC class I-restricted T cells are important in diabetogenesis comes from observations made in the nonobese diabetic (NOD) mouse model in which CD8' lymphocytes are required (but not sufficient) to transfer the disease from an affected adult to a syngeneic neonate (159).The NOD strain of mouse expresses a novel TAPl allele (TAPlb) and the fairly common TAP2d allele (160). Some investigators have claimed that NOD lymphocytes express low levels of class I, which can be rescued by transfection with other TAPl or TAP2 alleles (161,162), implying that the TAP2d/ TAPlb transporter is inefficient at loading H2N0Dclass I with peptides. If this were the case, it has been suggested that poor presentation of isletspecific antigens may impair the development of self-tolerance, which could lead to autoimmune disease. This result is controversial, and other groups have found no difference in the expression and IFN-y inducibility of TAP or the expression of class I on NOD lymphocytes (163, 164). The major spondyloarthritic diseases, ankylosing spondylitis (AS),Reiters syndrome (RS), and reactive arthritis, are strongly associated with HLAB27 (165). Although the role of HLA-B27 in this association is unknown, it has been suggested that it could present self-epitopes to autoimmune CTLs, and that the repertoire of peptides available for presentationgoverned by polymorphic TAPS-could be one factor in determining why some B27 individuals develop the disease while most do not. In support of this controversial idea is the observation that a small sample of RS patients studied by Barron et al. (166) displayed an elevated frequency-of both the rare TAPlC allele and the common TAP2A allele (see Table X). In a similar study of 85 AS patients (167), however, a similar increase in the frequency of the rare TAP alleles TAPlC and TAPlE was ascribed to linkage disequilibrium between these alleles and HLA-B27. In summary, no contribution from the TAP genes has been found for the development of most autoimmune diseases studied so far, and in those cases in which it has the results seem to be conflicting. B. TAP DYSFUNCTION IN TUMORS TAP downregulation or dysfunction has been observed in a number of different tumors, as shown in Table XI. In every case, this leads to low or no class I expression at the tumor cell surface, as might be expected. It has been argued that there is a selective advantage to tumor cells that have lost class I expression because they could escape detection by tumorspecific CTLs (168, 169). This is an appealing concept that becomes ever more likely as evidence continues to accumulate that CTLs may indeed

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TABLE XI TAP DYSFUNC:TION LEADING TO Loss OF MHC CLASSI EXPRESSION IN A VARIETY OF TUMORS Tumor Non-small cell lung carcinoma Small cell lung carcinoma Primary and secondary breast cancer Cervical carcinoma Colorectal carcinoma Adenoma Prostate cancer Murine fibrosarcoma Human villous trophoblad

(I

TAP dysfunction TAPl protein loss" Low but inducible TAPl and TAP2 mRNA TAPl protein loss" TAPl protein loss',c TAPl protein loss" TAPl protein loss"

Low TAPl and TAP2 mRNA Low TAPl and TAP2 mRNA

References 201, 202b 203 204 205, 206 207, 208 207 209 180 210, 212, 213

Immunohistochemistry with anti-TAP1 antisera. mRNA not investigated; TAP2 not investigated.

* Claw I loss in these tumors led to increased susceptibility to NK cells (214, 215).

' In these studies, loss of TAP expression correlated with metastatic tumors (cf. primary tumors). Although obviously not neoplastic, human villous trophoblasts express low classic class I in the first trimester of pregnancy, apparently as a result of low TAP expression. They are included in this list for the sake of completeness.

be involved in immune surveillance-and the elimination of newly arising neoplasms. However, Salcedo d al. (170) have shown that loss of class I in non-small cell lung carcinoma leads to their increased susceptibility to lysis by NK cells-another cell type thought to be involved in the ablation of tumors in viva C. VIRALINHIBITORS OF TAP Although a role for CTLs in tumor immunity has yet to be firmly established, their importance in combating viral infections is now fairly well established. It is interesting to note that at least one virus has evolved a mechanism for blocking its own presentation to CTLs by inhibiting the function of TAP. Herpes simplex virus expresses a 9-kDa immediate early protein, ICP47, which binds to TAP and inhibits peptide translocation (171, 172). Further analysis revealed that the ICP47 binding site required both TAPl and TAPS, and that it competes with peptides for binding to the complex but not for transport (173). In fact, the affinity of ICP47 for human TAP has been measured at KD = 5 X (173, 174) compared to K,'s of up to 4 X lo-' for peptides (58).Its affinity for murine TAP is 100-fold lower, consistent with the species specificity of the ICP47 effect (175).

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IX. Concluding Remarks

Discovery of the TAPS provided the key with which to understand the link between antigen processing and antigen presentation. Having established its primary function as a peptide transporter, we need to answer a multitude of questions in order to fully understand how this molecule fits into the whole process of T cell immunity. What is its structure? How is peptide binding and peptide transport coordinated? What is its role in the assembly and quality control of newly synthesized class I molecules? How did the TAP genes evolve? These are all questions that will fuel the activity of immunologists, biochemists, and biologists alike for many years to come.

ACKNOWLEDGMENTS I thank P. Cresswell, U. Gileadi, E. Joly, T. Hansen, J. Neefjes, S . Springer, and D. Williams for sharing their unpublished data and especially Uzi Gileadi for informative discussions and performing the sequence alignment shown in Fig. 2. Thanks also go to A. Glithero, J. Lewis, A. Lucassen, E. Rigney, and P. Wood for helpful discussions and comments. T. E. is a Wellcome Trust Senior Fellow in Basic Biomedical Science.

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198. Savage, D., Ng, A. S., Howe, C. H., Ngai, S. J., Darke, F. C., and Hui, K. (1995). HLA and TAP associations in Chinese systemic lupus erythematosus patients. Tisme Antigens 46, 213-216. 199. Chewier, D., Giral, M., Braud,V., Bourbigot, B., Muller, J., Bignon, Y. J., and Soulillou, J. (1995).Effects of MHC-encoded TAP1 and TAP2 gene polymorphism and matching on kidney graft rejection. Tmnsplantation 60, 292-296. 200. Fakler, J. W., Schmitt, E. M., Vejbaesya, S., Boehncke, W. H., Steny, W., and Eiermann, T. H. (1994). Analysis of TAP2 and HLA-DP gene polymorphism in psoriasis. Hum. Immunol. 40,299-302. 201. Restifo, N. P., Esquivel, F., Kawakami, Y., Yewdell, J. W., Mule, J. J., Rosenberg, S. A., and Bennink, J. R. (1993). Identification of human cancers deficient in antigen processing. J. Exp. Med. 177, 265-272. 202. Korkolopoulou, P., Kaklamanis, L., Pezzella, F., Harris, A., and Gatter, K. (199F). Loss of antigen-presenting molecules (MHC class I and TAP-1) in lung cancer. Br. J. Cancer 73(2), 148-153. 203. Fisk, B., Ioannides, C., Agganval, G. S., Wharton, J., O’Brian, T. C., Restifo, A. N., and Clisson, B. (1994). Enhanced expression of HLA-A,B,C and inducibility of TAP1, TAP-2, and HLA-A,B,C by interferon-gamma in a mukidrug-resistant small cell lung cancer line. Lymphokine Cytokine Res. 13(2), 125-131. 204. Kaklamanis, L., Leek, R., Koukourakis, M., Gatter, K., and Harris, A. (1995). Loss of transporter in antigen processing 1 transport protein and major histocompatibility complex class I molecules in metastatic versus primary breast cancer. Cancer Re.s. 55(22), 5191-5194. 205. Cromme, F., Airey, V. J., Heemels, M., Ploegh, T. H., Keating, L. P., Stem, J. P., Meijer, L. C., and Walboomers, J. (1994). Loss of transporter protein, encoded by the TAP-1 gene, is highly correlated with loss of HLA expression in cervical carcinomas. /. Exp. Med. 179(1),335-340. 206. Cromme, F., Van Bommel, V. P., Walboomers, J. J., Gallee, M., Stem, W. P., Kenemans, L. P., Helmerhorst, T., Stukart, M. M., and Meijer, C. (1994). Differences in MHC and TAP-1 expression in cervical cancer lymph node metastases as compared with the primary tumours. Br. J. Cancer 69(6), 1176-1181. 207. Kalamanis, L., Townsend, A,, Doussis Anagnostopoulou, I., Mortensen, A. N., Harris, A., and Gatter, K. (1994).Loss of major histocompatibilitycomplex-encoded transporter associated with antigen presentation (TAP) in colorectal cancer. Am. J. Pathid. 145(3),505-509. 208. Keating, P., Cromme, J. F., Duggan Keen, V. M., Snijders, P., Walhoomers, F. J., Hunter, M. R., Dyer, D. P., and Stem, P. (1995). Frequency of down-regulation of individual HLA-A and -B alleles in cervical carcinomas in relation to TAP-1 expression. Br. J. Cancer 72(2), 405-411. 209. Sanda, M., Restifo, G. N., Walsh, P. J., Kawakami, C. Y., Nelson, W., Pardoll, G. D., and Simons, J. (1995). Molecular characterization of defective antigen processing in human prostate cancer. 1.Natl. Cancer lnst. 87(4), 280-285. 210. Roby, K. F., Fei, K., Yang, Y., and Hunt, J. (1994).Expression of HLA class II-associated peptide transporter and proteasome genes in human placentas and trophoblast cell lines. lmmirnology 83(3), 444-448. 211. Epperson, D. E., Arnold, D., Spies, T., Cresswell, P., Pober, J.. and Johnson, D. R. (1992).Cytokines increase transporter in antigen processing-1 expression more rapidly than HLA class I expression in epithelial cells. J. ImmunoZ. 149, 3297-3301. 212. Roby, K. F., Gershon, D., and Hunt, J. S. (1996). Expression of the transporter for antigen processing-1 (Tap-1) gene in subpopulations of human trophoblast cells. PZacenta 17, 27-32.

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213. Clover, L. M., Sargent, I. L., Townsend, A., Tampe, R., and Redman, C. W. G. (1995). Expression of TAP1 by human trophoblast. Eur. J. Immunol. 25, 543-548. 214. Franksson, L., George, E., Powis, S . , Butcher, G., Howard, J., and Karre, K. (1993). Tumorigenicity conferred to lymphoma mutant by major histocompatibilitycomplexencoded transporter gene. ]. Exp. Med. 177(1), 201-205. 215. Salcedo, M., Momburg, F., Haemmerling, C.,and Ljunggren, H. (1994). Resistance to natural killer cell lysis conferred by TAPlA genes in human antigen-processing mutant cells. 1. Zmmunol. 152(4), 1702-1708. This article was accepted for publication on 1 October 1996.

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ADVANCES IN IMMUNOLOGY. VOL. 65

NF-HB as a Frequent Target for Immunosuppressive and Anti-Inflammatory Molecules PATRICK A. BAEUERLE AND W A Y R. BAICHWAL Tuhrik Incorpombd, Soulfi'San Fmncko, California 94080

1. Introduction

Any immune or inflammatory reaction requires the de novo synthesis of special proteins that are usually not required for a day-to-day life of cells. These include cell adhesion molecules allowing transmigration of leukocytes and lymphocytes into inflammed tissue, chemokines attracting macrophages, and inflammatory cytokines that serve to amplify and spread the primary pathogenic signal. The predominant mechanism by which these proteins are newly synthesized involves an inducible transcriptional initiation of their respective genes. This is governed by preexisting transcription factors that, when activated, bind to regulatory regions of such genes and initiate a program of gene expression. Primary signals for this event can, for instance, be components of bacterial cell walls, such as lipopolysaccharide (LPS). The inflammatory cytokines TNF and IL-1 are then newly synthesized and trigger the same genetic program as LPS in cells that have never encountered the primary pathogenic signal. Eventually, this can lead to an avalanche of cytokines causing severe systemic effects, including toxic shock syndromes. Apart from these acute reactions, aberrant expression of inflammatory cytokines also plays a major causative role in chronic inflammatory autoimmune diseases such as rheumatoid arthritis (reviewed in Feldmann et al., 1996) and multiple sclerosis (reviewed in Steinman, 1996). The transcription factor NF-KBhas been recognized as a major regulator of pathogen- and inflammatory cytokine-inducible gene regulation (reviewed in Baeuerle and Henkel, 1994). This is evident, on one hand, when the endogenous and exogenous conditions that can trigger the activation of NF-KBare considered (Tables I and 11).On the other hand, it becomes apparent if the functions of genes are considered that were reported to be inducibly regulated by NF-KB binding motifs in promoters and enhancers (Table 111).It is important to point out that not every stimulus will activate every target gene in a given cell type. On the contrary, there are pronounced cell type-specific differences with respect to inducing conditions that relate to the expression of receptors and other genetic factors. At the level of DNA, NF-KB synergizes with many additional transcription factors that 111

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TABLE I EXOGENOUS NF-KB-INDUCING CONDITIONS' Class Bacteria and products

Viruses and products

Eukaryotic parasite Xenobiotics

Condition

Mycobaderium tuberculosis Listerla monocytogenes Shigella flexneri Helicobacter pylori Lipopolysaccharide (lipid A) Exotoxin B Toxic shock syndrome toxin 1 Muramyl peptides G(Anh)M Tetra Staphylococcus enterotoxin A (superantigen) Human immunodeficiency virus type 1 (HIV-1) Human T cell leukemia virus type 1 (HTLV-1) Hepatitis B virus (HBV) Herpes simplex virus type 1 Human herpes virus-6 Herpes virus Saimiri Newcastle disease virus Cytomegalovirus (CMV) Sendai virus Sindbis virus Epstein-Barr virus (EBV) Adenovirus Double-stranded RNA intermediates Tax (from HTLV-1) iel (CMV) Tat (HIV-1) gpl60 (HIV-1) X (HBV) MHBs' (HBV) HVS13 (Herpes Saimiri) EBNA2 (EBV) Latent membrane protein (EBV) Hemagglutinin (influenza) EW19K (adenovirus) E1A (adenovirus 5 )

Theileria pamu Antigens (T and B cells) Cycloheximide Anisomycin Phorbol esters Concanavalin A Phytohemagglutinin Calcium ionophores Okadaic acid Calyculin A

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TABLE I --Continued Class

Environmental hazards

Condition Tunicainycin Brefeldin A Monensin Cyclopimonic acid Thapsigargin Tax01 Nocodazol Calchicine Podophyllotoxin Vinblastine 1-P-u-arahinofuranosyl cytosine Kainic acid N-methyl-maspartate hthralin Pyrogallol Aphidicolin Tetrachlorocarhon Chromiiim (VI) Cobalt Nickel Pervanadate Crocidolite asbestos fibers UV-A, UV-B, UV-C light Ionizing radiation Photosensitization Ozone Silica particles

Based on studies reported in the literature. References are available from the authors n p n request.

may determine cell type-specific expression of genes and the input from other signal transduction pathways in their regulation. NF-KBusually plays the role of a powerful genetic switch that-although necessary-is rarely sufficient for inducible expression of a particular gene. Through the synergistic interaction of NF-KB with other factors in gene regulation a great degree of specificity is achieved without limiting the unique potential of NF-KB to coordinately induce a broad antipathogenic genomic response. A hallmark of NF-KBis that it is kept in an inactive form in the cytoplasm waiting to be activated by a multitude of inainly adverse stimuli (Baeuerle and Baltimore, 1988a). The latency is achieved by binding of an inhibitory protein, called IKB,to a potentially DNA-binding dimer of NF-KBsubunits (Baeuerle and Baltimore, 1988b; reviewed in Beg and Baldwin, 1993).

TABLE I1 ENDOGENOUS NF-KB-INDUCING CONDITIONS' Class

Condition

Inflammatory cytokines

Tumor necrosis factor-a Lymphotoxin (TNF-0) Interleukin-1 Interleukin- 17 Leukemia inhibitory factor Interleukin-2 Platelet-derived growth factor Insulin Nerve growth factor Macrophage colony-stimulating factor IgM receptor ligand (antigen) CD3 ligand (antigen) CD2 ligand CD28 ligand (B7-1) CD4 ligand (gp120) CD30 ligand CD40 ligand CD35 ligand (complement) CD 1lb/CD18 ligand (complement) IyGA/E ligand Fit-1 ligand Fcy2a receptor ligand (IgG2a) Thrombin Angiotensin I1 Platelet activating factor f-Met-Leu-Phe Leukotriene B4 12(R)-Hydroxyeicosatrienoic acid HzOz Oxidized low-density li oprotein Advanced glycated en products Amyloid protein fragment (PA4) Adherence (monocytes) P-selectin tethering of monocytes Fibronectin adherence (SMCs) Reoxygenation Hypoxia Liver regeneration Serum Shear stress Cold shock Hyperosmotic shock Hyperoxia Hemorrhage Anti-FadApo-1 L-Glutamate Depolarization Positive T cell selection

Growth factors

Immunoreceptor ligands

Mediators

Modified proteins Cell adhesion Stress reactions

Others

B

O Based on studies reported in the literature. References are available from the authors upon request.

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TABLE I11 TARGET GENESFOR NF-KB' Class Viruses

Immunoreceptors

Cell adhesion molecules

Cytokines and growth factors

Chemokines

Target gene Human immunodeficiency virus 1 (HIV-1) Simian immunodeficiency virus (SIV) Cytomegalovirus (CMV) Adenovirus E3 region JC virus Simian virus 40 (SV40) Immunoglobulin K light chain Interleukin-2 receptor a-chain T cell receptor /32 Major histocompatibility complex class I (H-2Kb) &Microglobulin Invariant chain Ii Platelet-activating factor receptor Tissue factor CDllb CD48 CD69 Endothelid leucocyte adhesion molecule 1 (ELAM-1) Vascular cell adhesion molecule 1 (VCAM-1) Intercellular cell adhesion molecule 1 (ICAM-1) Mucosal vascular addressin cell adhesion molecule 1 (MadCAM-1) @Interferon Tumor necrosis factor-a Lymphtoxin (TNF-/3) Interleukin-10 Interleukin-2 Interleukin-3 Interleukin-6 Interleukin-8 Interleukin-12 Granulocyte/macrophage colony-stimulating factor (GM-CSF) Granulocyte colony-stimulating factor (G-CSF) Macrophage colony-stimulating factor (M-CSF) GROa, CROP, GROylmelanoma growth stimulating activity (MGSA) Proenkephalin Monocyte chemoattractant protein 1 (MCP-1) Macrophage inflammatory protein la (MIP-la) Macrophage inflammatory protein 2 (MIP-2) IP-10 KC (continues)

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TABLE 111-Continued Class

Target gene

Angiotensinogen Serum amyloid A precursor Factor B C-reactive protein C4b binding protein Urokinase-type plasminogen activator Lipopolysaccharide binding protein (LPS-BP) Transcription factors and subunits c-re1 NF-KB precursor p105 NF-KB precursor pl00 IKB-LX P53 c-myc Interferon regulatory factor 1 (IRF-1) Interferon regulatory factor 2 (IRF-2) Oxidative stress-related enzymes NO synthase and proteins Cyclooxygenase-2 12-Lipoxygenase Phospholipase A2 Ferritin H chain NAD(P)H: quinone oxidoreductase Others Vimentin Laminin B2 chain K3 keratin Lysozyme Proteasome subunit LMP2 Peptide transporter TAP1 A20

Acute phase proteins

" Genes that have been reported to be regulated by NF-KRsites in their prninoter region are listed. References 'ire available froiii the authors iipon request.

Both NF-KB subunits and IKB proteins belong to larger protein families that constitute a system that currently consists of 10 known members (reviewed in Verma et nl., 1995). Among the five NF-KB subunits, p6,5/ RelA plays an outstanding role as a potent transcriptional activator that is most frequently found in inducible complexes of various immune and nonimmune cell types. c-Re1 and RelB are likewise transcriptional activators that seem to have more prominent roles in immune cells. p50 and p52 have little or no transactivating capacity but form heterodimers of high DNA-binding affinity with the other three subunits. All NF-KB subunits share a 300-amino acid homology region (the Re1 domain) that is

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sufficient for DNA binding and dimerization. The transactivating domains reside in the unique C-terminal portions of RelA, c-Rel, and RelB. NF-KBdimers can potentially associate with five different IKBproteins, two of which represent precursor molecules for p50 and p52, called p105 and p100. The major players in inducible NF-KB activation are IKB-CY, -/3, and -E, of which IKB-CY is the best studied inhibitor. A structural characteristic of all IKBproteins is a cluster of five to seven ankynn repeat motifs that, as an entity, are required for binding to the Re1 homology domain of NF-KB subunits. In response to various signals, for instance, TNF, IL-1, and phorbol ester, IKB-CY undergoes phosphorylation on serines 32 and 36 within its regulatory N terminus. This does not release IKB-CY from NF-KB but turns the inhibitor into a substrate for ubiquitin-conjugating enzymes. Once ubiquitin is conjugated to lysines 21 and 22, IKB-CY is rapidly degraded by the proteasome (reviewed in Baldwin, 1996). The liberated NF-KB is then transported into the nucleus and will initiate transcription by binding to regulatory KB motifs in target genes. Although these downstream events are understood in some detail, it is still not known how TNF and IL-1 receptors trigger IKBphosphorylation. Neither has the IKB-CY kinase been molecularly identified nor are the proteins and messengers known that regulate IKBkinase(s) (or counteracting IKB phosphatases). More progress has been made recently in understanding TNF and IL-1 signaling at the plasma membrane. In the case of TNF receptors, a number of adaptor molecules were identified, called TRADD and TRAFs (Hsu et al., 1995; Rothe et al., 1994; Hsu et al., 1996a), that become recruited to the receptor upon ligand binding and are necessary for NF-KB activation. Early signaling events in both TNF and IL-1 action appear to involve protein kinases, called RIP (Hsu et al., 1996b) and IRAK (Cao et al., 1996), that become associated with the respective receptors but do not directly phosphorylate IKB-a. A role of NF-KB as an important genetic switch that rapidly turns on proinflammatory gene expression in response to mostly pathogenic conditions was proposed early on (Baeuerle and Baltimore, 1991). However, this assumption was mainly based on studies describing conditions that activate NF-KB in cell culture experiments and genes harboring cisacting KB motifs in their enhancers. Only recently has more compelling evidence for the role of NF-KB in autoimmune and inflammatory diseases been obtained. One kind of experiment explored the state of NF-KB activation in a number of human diseases and animal disease models. Aberrant NF-KB activationwas observed in smooth muscle cells and macrophages of atherosclerotic lesions (Brand et al., 1996), in synovial tissue of rheumatoid arthritis patients (Marok et al., 1996), in peripheral microglia of mice suffering from experimental autoimmune encephalomyelitis(Kalt-

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Schmidt et al., 1994), and in brain neurons adjacent to plaques of Alzheimer’s disease patients (Kaltschmidt et al., 1997). These studies were aided by immunocytochemical methods using antibodies specific for the nuclear form of the p65/RelA subunit that has been released from IKB(Kaltschmidt et al., 1995). Direct functional evidence for a role of NF-KB in immune regulation has come from recent genetic studies using transgenic and gene knockout mice (reviewed in Baeuerle and Baltimore, 1996). Various forms and degrees of immunodeficiencies are observed when distinct members of the NF-KB family are inactivated by gene disruption. The full importance of the system may become apparent only after single gene knockout mice are crossed to generate double and multiple knockouts because individual NF-KB subunits are in part functionally redundant. However, already at this stage, the various mouse models strongly support a key role of the NFKBIIKBsystem in an impressive number of immune regulatory processes. The effect of adenovirus-mediated expression of IKB-cxin endothelial cells supports the notion that the NF-KB transcription factor is a key regulator of proinflammatory genes (Wrighton et al., 1996). By this approach, activation of all known dimer combinations of NF-KB was prevented in a specific fashion. IKB overexpression resulted in a collective inhibition of LPS-induced NF-KB-regulated gene expression, including that of the VCAM-1, IL-1, IL-6, IL-8, and tissue factor genes. As a result, leukocyte adhesion to endothelid cells was fully suppressed. This provides a “proof of principle” that NF-KBactivation is an attractive target for antiinflammatory drugs. In the following sections, we will summarize studies showing that numerous molecules possessing immunosuppressive and antiinflammatory activity are now being recognized to target mechanisms controlling the activity of transcription factor NF-KB,which may well explain, at least partially, their pharmacological behavior. It. Glucocorticoidsand Other Steroid Hormones

Glucocorticoidsare among the most frequently used immunosuppressive and anti-inflammatory drugs and, despite severe side effects, are still the “gold standard” for the development of novel superior anti-inflammatory agents. Glucocorticoids associate in the cytoplasm with their heat shock protein-complexed receptor (reviewed in Tsai and O’Malley, 1994; Beato et al., 1995). This releases the associated heat shock proteins and allows dimerization and nuclear transport of the ligand-bound glucocorticoid receptor (GR). The DNA-binding form of the glucocorticoid-GR complex is expected to exert its anti-inflammatory action as a transcription factor at the level of gene expression. This can either occur through positive GR

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elements (GREs) or negative GREs, leading to either gene induction or repression. Despite the extensive clinical use of glucocorticoids and a precise understanding of their molecular biology, very little is known about the basis for their anti-inflammatory action. A number of proinflammatory genes that are inducibly upregulated by NF-KB were recently found to be subject to negative regulation by dexamethasone (Dex), an agonistic glucocorticoid, without an apparent dependence on repressing GREs. In the case of the IL-6 (Ray and Prefontaine, 1994), IL-8 (Mukaida et al., 1994), and ICAM-1 genes (Caldenhoven et al., 1995), the cis-acting elements mediating repression by Dex were identified in each case as the regulatory NF-KB motifs of the genes. However, in no case was direct binding of ligand-activated GR to the KB sites evident. A significant, though not complete, attenuation of KB-dependent transactivation was observed that required relatively high Dex concentrations between 0.1 and 1pM. NF-KB-binding activity from nuclei of Dextreated cells was also reduced, suggesting the hormone interfered with the activation of NF-KB. More detailed analyses of the NF-KB repression by Dex revealed two fundamentally distinct molecular mechanisms. One is independent of de nooo protein synthesis and involves a direct interaction of ligand-bound GR with the NF-KB subunits p65/RelA and p50 (Caldenhoven et al., 1995; Scheinman et al., 1995a). The DNA-binding domain with the zinc fingers and the ligand-binding domain of the GR are required. This interaction between NF-KB and GR does not necessarily abrogate the DNA binding of NF-KB.Preliminary data suggest that GR interacts with the transactivating C-terminal domain of p65/RelA, suggesting that GR may simply mask the strong activation domain of NF-KB, eventually substituting it with its own weaker one. This would explain the partial inhibition. A second mechanism is dependent on & now protein synthesis and involves the a possibly by yet undiscovtranscriptional upregulation of the 1 ~ B - gene, ered positive GREs in the gene’s regulatory regions (Auphan et al., 1995; Scheinman et al., 1995b). This also requires high concentrations of Dex in the range of 0.1-1 pM. The Dex-upregulated IKB-a is thought to recapture NF-KB that was liberated previously by signal-induced degradation of IKB-a. This mechanism may play a role in attenuating NF-KB activity under chronic conditions but, because of its requirement on protein synthesis, can hardly prevent NF-KB activation when given at the same time as the inductive stimulus. It is likely that both mechanisms equally contribute to the inhibition of NF-KB-dependent gene expression. In endothelial cells, no induction of IKB-a synthesis was observed (Brostjan et al., 1996). The strong inhibitory effect of Dex on TNF-a-induced gene

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expression in this cell type appeared to rely entirely on a physical interaction between GR and NF-KB. GR is not the only steroid hormone receptor that can physically interact and thereby interfere with NF-KB activity. Progesterone receptor and estrogen receptor likewise repress KB-dependent gene transcription (Caldenhoven et al., 1995; Stein and Yang, 1995). 111. Cyclosporin A and FK506

Cyclosporin A and FK506 exert their strong immunosuppressive effect by tethering the calcium-dependent phosphatase calcineurin to cyclosporin A- or FK506-binding proteins, respectively, thereby inactivating the enzyme’s function in Ca2+-dependentT cell signal transduction and gene activation (Schreiber and Crabtree, 1992). Prime targets for the calcineurin pathway are transcription factors belonging to the nuclear factor of activated T cells (NFAT) family, which have been shown to be important regulators of IL-2 gene expression (Durand et al., 1988; Jain et al., 1995; Rooney et al., 1995). However, NF-KB also was shown to be regulated in T cells by calcineurin and to coregulate IL-2 gene expression with NFATs (Schmidt et al., 1990; Frantz et al., 1994; Los et al., 1995).T cell activation and IL-2 induction require not only a Ca2+signal, which can be provided by calcium ionophores, lectins, or transfection of a constitutively active form of calcineurin, but also a second protein kinase C-dependent signal. Unlike NFAT and only in T cells, NF-KB shows a strong dependence on both signaling pathways for its full activation. Calcium ionophores (or active calcineurin) or phorbol esters alone are only suboptimal inducers, whereas a combination of both act synergistically. Hence, cyclosporin A or FK506 will only partially inhibit NF-KB that has been activated by a bimodal T cell-activating stimulus. NF-KB activation is completely inhibited by the two drugs when the factor is activated by the calcium-dependent pathway, but it is not affected when it is activated only by the PKC-dependent pathway. This partial inhibition of NF-KB activation by cyclosporin and FK506 in the nanomolar range may be of physiological relevance for IL2 gene expression because overexpression of 1 ~ B - can a also substantially block the activation of the IL-2 promoter in response to phorbol ester/ lectin treatment of T cells (Frantz et al., 1994). In contrast to the previously described situation, a recent study described the activation of NF-KB and IL-6 gene expression by FK506 (Muraoka et al., 1996). This is, however, only observed at pM concentrations of FK506 and may be the cause of adverse side effects of the drug in the kidney.

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Iv. Rapamycin

Rapamycin, a macrolide extracted from Streptomyces hygroscopicus, can potently suppress T cell activation in vitro and prolong organ allograft survival and onset of autoimmune disease in vivo (reviewed in Sigal and Dumont, 1992). Rapamycin acts on IL-2 signaling and the costimulatory CD28-mediated signaling pathway that are both unresponsive to cyclosporin A and FK506. The drug can inhibit activation of the ribosomal p70 S6 kinase but it is unclear how this contributes to its immunosuppressive potential. Costimulation of T cells with phorbol ester and anti-CD28 leads to a long-term depletion of IKB-a and consecutive upregulation and nuclear translocation of c-Rel, which is not seen with the individual stimuli (Lai and Tan, 1994). The costimulatory effect is prevented if Jurkat T cells or primary T cells are treated with 10-25 n g h l rapamycin. Although the consequence of rapamycin treatment on KB-dependent gene expression and DNA binding of RemF-KB complexes was not investigated, the study identified another CD28-induced signaling reaction as a potential target for the potent immunosuppressive drug rapamycin. V. Salicylates

Salicylates, like aspirin, are widely used anti-inflammatory drugs. Treatment of chronic inflammatorydiseases needs much higher doses of salicylates than required for inhibition of prostaglandin H (PGH) synthase by covalent modification, suggesting that these drugs have additional prostaglandinindependent effects. This is supported by the finding that analogs, such as salicylate, which cannot alkylate PGH synthase, retain their anti-inflammatory potential (reviewedinWeissmann, 1991).This anti-inflammatorypotential at high doses may rely on the inhibition of NF-KB,as recently suggested by the work of Kopp and Ghosh (1994). Both salicylate and acetylsalicylate (aspirin)were found to inhibit NF-KBactivation and KB-dependent report gene induction with an IC50 value between 1and 2 mM in Jurkat T cells. Other nonsteroidal anti-inflammatory PGH synthase inhibitors, including acetaminophen and indomethacin, had no effect. Pierce et al. (1996) could demonstrate that 10-20 mM salicylate or aspirin prevents the TNF-ainduced expression on endothelial cells of E-selectin, VCAM-1, and ICAM-1, which are all encoded by NF-KB-regulated genes (see Table 111). The transmigration of neutrophils across TNF-a-treated endothelial monolayers was accordingly blocked by the salicylates. Endothelial cells treated with salicylate fail to inducibly degrade IKB-cxapparently because the drugs block phosphorylation of IKB.No effect was seen on TNF-a-induced phos-

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phorylation of transcription factor ATF-2, suggesting that salicylates inhibit a specific upstream event controlling IKB-akinase. The specificity of salicylates is a matter of dispute. Frantz and O’Neill (1995)observed that in Jurkat T cells sodium salicylate inhibits transcription factors AP-1 and CREB as well as intrinsic kinase activities with an IC50 around 5 mM, which is just twofold higher than the IC50 for NF-KB inhibition. Salicylate concentrations in the serum of 1or 2 mM are required for its anti-inflammatory effect and concentrations of 6.5 mM are toxic. The question arises whether salicylates can efficiently function as selective NF-KBinhibitors within such a narrow concentration range. In experiments using cultured endothelial cells, very minor effects of 10-20 mM salicylate were observed on cell survival, GAPDH transcript levels, and constitutive ICAM-1 expression. Hence, it is possible that lymphoid cells are more sensitive to the drug than endothelial cells. As will be the topic of the next section, salicylates are likely to exert their NF-KB-inhibitory effect as scavengers of reactive oxygen intermediates (Sagone and Husney, 1987). VI. Antioxidants and Inhibitors of Enzymes Generating Reactive Oxygen Intermediates

Inflammatory reactions are generally accompanied by the local production of reactive oxygen intermediates (ROIs), such as superoxide, hydrogen peroxide, hydroxyl radicals, and nitric oxide (Baggiolini and Thelan, 1991; Omar et al., 1991). High amounts of ROIs are produced and released by stimulated neutrophils and macrophages as chemical weapons to destroy microbes. As a side effect, bystanding cells are either killed or subject to various degrees of oxidative stress. B and T cells and nonlymphoid cells are also capable of producing small amounts of ROIs in response to inflammatory cytokines and numerous other conditions. These small amounts of ROIs are not sufficient to harm microbes or neighboring cells but may serve as second messengers of proinflammatory signaling cascades (Schreck and Baeuerle, 1991).As cytotoxic agents and inducers of proinflammatory gene expression, ROIs can considerably contribute to inflammatory reactions in various ways. This is why many compounds with ROI-scavenging or antioxidant properties, among them many natural products, exhibit potent anti-inflammatory activity (Sies, 1991). In some cases, the antioxidant activity of compounds comes from the inhibition of enzymes involved directly or indirectly in ROI production, including lipoxygenases, cyclooxygenases, phospholipases, and mitochondrial enzymes. In other cases, compounds act by directly reacting with (scavenging) and thereby neutralizing ROIs. a-Tocopherol (vitamin E) is a well-studied example of the latter class (reviewed in Liebler, 1993).

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Both ROI scavengers and inhibitors of ROI-generating enzymes have been shown to prevent activation of NF-KB in response to proinflammatory cytokines (reviewed in Schreck et al., 1992a; Meyer et al., 1993; Sen and Packer, 1996). Examples for the first class include the flavanoid apigenin (Gerritsen et al., 1995), curcumin (Singh and Aggarwal, 1995), vitamin E and derivatives (Suzuki and Packer, 1993),many thiols such as N-acetylL-cysteine (NAC; Staal et al., 1990), and dithiocarbamates (Schreck et al., 1992b). IC50 values can be in the mM range as seen with NAC or in the low pM range as seen with curcumin. An example of the second class is the anti-inflammatory drug tepoxalin, which specifically targets the peroxidase activity as an intrinsic part of cyclo- and lipoxygenases (Kazmi et al., 1994; Munroe et al., 1995).Some of the antioxidant inhibitors of NF-KBactivation were tested in in vivo models of inflammation and found to coordinately prevent expression of ICAM-1, ELAM-1, and VCAM (Gemtsen et al., 1995; Ferran et al., 1995; Chen et al., 1995). How do antioxidants inhibit NF-KB activation? Pyrrolidine dithiocarbamate was shown to prevent the de novo phosphorylation of IKB-a in response to a variety of inducers (Traenckner et al., 1995), suggesting that a common redox-sensitive step links receptor events to the IKB-a kinase. It is by no means clear what components and mechanisms are involved. One possibility is that redox-sensitive cysteines in kinases or phosphatases or their regulators become modified upon increases in cellular ROI production. This could occur by reversible addition of glutathione. Studies have shown that H202can induce tyrosine as well as serinehhreonine phosphorylation events (Heffetz et al., 1990; O’Shea et al., 1992) and the activation of a number of transcription factors, including NF-KB, AP-1, and Elk-1, and respective target genes (reviewed in Schulze-Osthoff et al., 1995). A role of H202as messenger in TNF-a signaling is evident from the observation that stable overexpression of the Hz02-degradingenzyme catalase is inhibitory for NF-KBactivation, which can be reversed by a catalase inhibitor (Schmidt et al., 1995a). Mitochondria were proposed as a source for the ROI (Schulze-Osthoff et al., 1992). Likewise, a fair number of other NF-KB-inducing conditions were shown to lead to an increased cellular production of H20z,which can be prevented by antioxidants (see, for example, Schmidt et al., 1995b). We anticipate that the anti-inflammatory potential of salicylates is related to their antioxidative potential as well. VII. Anti-TNF-cuAntibodies and Gold Compounds in Treatment of Rheumatoid A h i t i s

Rheumatoid arthritis (RA)is characterized by the chronic upregulation of multiple proinflammatory cytokines, including TNF-a, IL-6, IL-8, IL-

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1, and GM-CSF (Feldmann et al., 1996). The beneficial effect of antiTNF-a antibody in multiple clinical trials puts TNF-a overproduction into center stage of the autoimmune disease. TNF-a is among the most potent inducers of transcription factor NF-KB. Because inducible expression of the TNF-a gene is controlled by a number of KB-binding motifs in its upstream enhancer (Jongeneel, 1994), TNF-a may be able to establish a positive autoregulatory loop leading to a chronically enhanced expression of the cytokine in an auto- or paracrine fashion. This in turn leads to local or systemic TNF-a-induced upregulation of a number of other proinflammatory cytokines, chemokines, and cell adhesion molecules that are all encoded by NF-KB-controlledgenes (see Table 111).Hence, a prime effect of the anti-TNF-a antibodies is probably prevention of the proinflammatory gene regulatory cascade induced by TNF-a via NF-KBactivation. In support for a critical role of NF-KB in the disease, a recent study obtained evidence for a constitutive activation of the otherwise inducible factor in synovial tissue from rheumatoid arthritis patients (Marok et al., 1996). Gold compounds have been widely used in the treatment of RA (Leibfarth and Persellin, 1981). The molecular basis for their broad inhibitory effects on autoimmune antibody production, endothelial cell proliferation, and nonspecific anti-inflammatory effects is not understood. In such compounds, the aurous gold cation Au( I ) is complexed to a sulfur-containing ligand, such as thioglucose or thiomalate. Peak serum gold concentrations of 30-40 p M were measured in RA patients treated with these compounds, Williams et al. (1992) have observed that the IL-l-induced activation of a NF-KB- (and AP-1-) controlled reporter gene in 3T3 fibroblasts is suppressed by aurothioglucose with an IC50 in the range of 30-40 pM. Yang et al. (1995) have observed that a variety of clinically used gold compounds abrogate the DNA binding of NF-KB under cell-free conditions in the range of 100 pM. Aurothioglucose was most effective. Thioglucose or thiomalic acid on their own had no effect, whereas a gold salt without a thiol compound, HAuC14,was effective. The gold cation with its affinity for thiol groups could simply inactivate NF-KB DNA binding by direct binding to critical conserved cysteine residues in the DNA-binding domains of NF-KB proteins. Likewise, the gold cation could work as an oxidizing agent that covalently modifies NF-KB’Scysteine residues. Although it is conceivable that inactivation of NF-KB(and AP-1) is the prime mechanism by which gold compounds aid in the treatment of RA, this will be difficult to establish conclusively due to the pleiotropic activity of this reactive chemical. VIII. ImmunosuppressiveActivity of CAMP

Activators of CAMP-dependent protein kinase A (PKA), such as prostaglandin E2 (PGE2) or the drug forskolin, are known to suppress the

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expression of IL-2 (and IFN-7) in Thl cells (Rappoport and Dodge, 1982; reviewed in Phipps et al., 1991). Chen and Rothenberg (1994) were the first to notice that cAMP inhibits NF-KB activity in ELA.El T cells. The immunosuppressive effect of PKA on IL-2 was investigated in more detail by Neumann et al. (1995). PKA was found to act at least in part at a transcriptional level on the IL-2 promotedenhancer. Forskolin, PGE2, or a lipophilic cAMP analog caused a two- to fourfold inhibition of an IL-2 promoter-controlled reporter gene, which was dependent on an intact KBbinding motif at position -205. In contrast, with a construct lacking this site, forskolin even induced a threefold enhancement of transcription. Mutation of the major AP-l/NFAT site in the IL-2 promoter did not abrogate PKA inhibition. Likewise, a reporter construct harboring multimerized IL-2 KB motifs but not one with multimerized NFAT/AP-1 motifs was inhibited. In DNA-binding assays, both clonal T cell lines and isolated peripheral T lymphocytes showed an impaired activation of NF-KB by forskolin in response to a PMA/ionomycin treatment. The mechanism by which PKA attenuates the participation of NF-KB in IL-2 gene regulation seems complex and may occur at distinct levels. Impaired degradation of IKB and nuclear appearance of active DNAbinding NF-kB in response to forskolin suggests that PKA is involved in a negative cross-talk upstream from the inducible NF-KB-IKB complex, perhaps involving the Ras-Raf-ERK pathway. Another effect of forskolin is the induction of c-Re1 and p105 de novo synthesis (Neumann et ul., 1995). At least in transient transfections, overexpression of c-Re1 and p105 is found to attenuate the transcriptional activity of Rel/p65. Lastly, all NFKB subunits bear a potential PKA phosphorylation site (Arg-Arg-Pro-Ser) within their conserved Re1 homology region. Direct phosphorylation of NF-KB subunits by PKA may play an additional modulatory role in the T cell immunosuppressive effects of CAMP-increasing hormones and mediators. NF-KB-dependent downregulation of IL-2 is certainly not the only mechanism by which cAMP and PKA exert immunosuppressive influences. It will be interesting to find out which other NF-KB-regulated genes are inhibited by a cAMP increase. TNF-a may be one other example (reviewed in Haraguchi et al., 1995). Upregulation of IL-10, an immunosuppressive cytokine, is probably an equally important mechanism. Haraguchi et al. (1995) proposed that the immunosuppressive activity of retroviruses is due to viral transmembrane envelope proteins that act by increasing the host cell’s cAMP levels. IX. The Bacterial Metabolite Spergualin

Deoxyspergualin (DSG)currently undergoes clinical trials for treatments of transplant rejection and autoimmune disease progression and prevention

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of human anti-mouse antibody response (reviewed in Tepper et al., 1995). In animal models, the drug shows prolonged xeno- and allograft survival, suppression of T and non-T immune responses, and suppression of various autoimmune diseases, such as experimental allergic encephalomyelitis. DSG is a synthetic derivative of spergualin, a fermentation product of Bacillus luterosporus. The molecular mechanism of its action is unknown. At the cellular level, however, DSG shows multiple effects, some of which hint at a transcriptional effect involving NF-KB, such as downregulation of the NF-KBtarget genes encoding IL-1, MHC class I, and IL-2 receptor. The inhibitory effect of DSG on antibody production and B cell differentiation prompted Tepper et al. (1995) to investigate whether K light chain expression and NF-KB activation in the pre-B cell line 70Z/3 were affected by long-term treatment with DSG. After DSG treatment at 5 pg/ml for 2 or 3 days, pre-B cells became deficient for both mRNA and cell surface expression of Klight chain in response to LPS. No effects on the constitutive surface expression of MHC class I and CD45, or on p heavy chain mRNA levels, were observed over this period. DSG was found to inhibit in a dosedependent fashion the LPS-induced nuclear appearance of active NF-KB, a central transcriptional regulator of the K light chain gene. The IC50 was between 10 and 100 ng/ml. Future studies need to establish the specificity of this inhibition and find out at what level and by what kinetics DSG blocks the activation of NF-KB. X. The Fungal Metabolite Gliotoxin

The high mortality associated with Aspergillus infections is not well understood (Bodey, 1988; Bodey et al., 1992). A possible etiologic agent in aspergillosis is a toxic fungal metabolite called gliotoxin. Gliotoxin is secreted by the fungi and was shown to have profound immunosuppressive effect by virtue of inhibiting B, T, and macrophage cell functions and by causing apoptosis of immune cells. There is a vast literature about the effects of the fungal metabolite in uivo, in cell culture, and in cell-free systems (Waring et al., 1988;Taylor, 1971; Sutton et al., 1994).The confusingly pleiotropic effects of gliotoxin emerging from these experiments may be related to the use of very different concentrations of the compound. As an epipolythiodioxopiperazine derivative, gliotoxin has the potential to covalently react at high concentrations with sulfhydryl residues in polypeptides. Gliotoxin at relatively low concentrations was recently found to selectively inhibit the activation of NF-KB in a number of cell lines (Pahl et al., 1996). IC50 values in the order of 50-100 nM were observed. At these concentrations, gliotoxin did not detectably affect the DNA-binding activity

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of a variety of other transcription factors, including CREB, Oct-1, NFAT, or Statl; nor did it inhibit the tyrosine kinase activities of Lck and Fyn in intact cells. Likewise, gliotoxin prevented the induction of a ICAM-1 promoter-controlled luciferase gene by TNF, IL-1, and PMA (all activators of NF-KB) but not by y-interferon (an activator of Stats). Gliotoxin did not act by directly modifymg and inactivating NF-KB in intact cells, This only occurred at high pM concentration in vitro, suggesting that the redoxsensitive cysteine residues in the DNA-binding domains of NF-KBproteins remained intact under cell culture conditions. Rather, gliotoxin was found to prevent the degradation but not the inducible phosphorylation of IKBa.This shows that the fungal immunosuppressant very selectively interfered with NF-KB activation at just one of its many signaling steps. Additional experiments are required to find out whether the compound inhibits ubiquitin conjugation to IKB or proteasome-mediated degradation of the inhibitor. Not only gliotoxin’s immunosuppressive effect but also its proapoptotic effect may be related to its NF-KB inhibitory potential. As mentioned previously, cells from RelA knockout mice are extremely sensitized for cytotoxic stimuli, A similar phenotype should occur when NF-KBactivation is blocked by a potent inhibitor, such as gliotoxin. XI. Viral Strategies to Control NF-KB

NF-KBas an immediate early transcriptional regulator plays an important role in the antiviral response. This is evident from the activation of NFKB by a large number of viruses belonging to different classes (see Tables I and 11). Double-stranded viral RNA intermediates, viral transactivator proteins, and virion proteins overloading the endoplasmic reticulum have been identified as signals activating NF-KB. The role of NF-KB in an antiviral response is also evident from the genes that virus-activated NFKBcan potentially induce in concert with other transcription factors. The role of NF-KB in the expression of @interferon gene is well studied (reviewed in Maniatis et al., 1992). Another kind of antiviral response, i.e., an increased processing and presentation of viral peptides to cytotoxic T cells, is also transcriptionally coordinated by NF-KB. The factor has been shown to be involved in transcriptional upregulation of the genes encoding the proteasome subunit LMP2, the peptide transporter TAP1 (Wright et al., 1995),and MHC class I molecules and their associated /32 microglobulin chain (Israel et al., 1989). NF-KB thus appears as a prime target for viral strategies abrogating the cells’ primary antiviral response. Hence, it does not come as a surprise that a large virus has acquired an IKB-like protein during evolution. This is the African swine fever virus (ASFV), a

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170,101-nuc1eotide-1ongDNA virus of its own class, that encodes a 28kDa ankynn repeat-containing protein, called A238L (Yanez et al., 1995; reviewed in Baeuerle and Baltimore, 1996). The viral IKB protein shows all characteristics of a bonafide IKBprotein when overexpressed in cells or studied in uitro. Most important, it was noted that ASFV-infected cells are deficient for NF-KBactivation in response to TNF-a. Another viral protein that can attenuate NF-KBactivation is the adenovirus Sencoded protein E1B 19K (Schmitz et al., 1996). When transiently overexpressed, E1B 19K prevents the induction of NF-KB DNA-binding and transcriptional activity in response to expression of viral E1A 13s protein or exposure of cells to phorbol ester or TNF-a. Like the overexpression of an IKBprotein, E1B 19K prevents the appearance of active nuclear NF-KB. A small hydrophobic region in E1B 19K with homology to the antiapoptotic Bcl-2 protein is required for the inhibitory activity (Limbourg et al., 1996).Interestingly, Bcl-2 was also found to attenuate NF-KB activity-however, by a different mechanism (Grimm et al., 1996).The mechanism by which E1B 19K attenuates NF-KB activation is unknown. At the same time that viruses have evolved strategies to attenuate NFKB activation, they have also tried to optimally adapt and even exploit the apparently inevitable activation of the factor upon infection. Not only do viruses encode proteins that can activate NF-KB (see Table I ) but also they carry genes exquisitely controlled by the host’s transcription factor (see Table 111). A well-studied example of a virus that is even dependent on NF-KBactivation for its expression is the genomically integrated HIV1 provirus (Nabel and Baltimore, 1987). Two adjacent high-affinity NFKB binding motifs in the HIV-1 enhancer ensure that the proviral genome is rapidly expressed upon T cell activation. There is one other reason why viruses may want to establish a finely tuned balance between NF-KB inhibition and activation. Mice in which the RelA subunit of NF-KB has been inactivatedby homologous recombination die in utero from apoptosis of liver cells (Beg et al., 1995). Moreover, fibroblasts from such mice are far more susceptible to the cytotoxic effects of TNF-a than are cells from wild-type animals, suggesting that RelA has a role in protecting cells from apoptotic death, presumably via expression of antiapoptotic genes. Hence, by activating NF-KB, viruses may prevent their host cells from dying prematurely when exposed to TNF-a and other proapoptotic stimuli. XII. Conclusion

Currently, there is no other transcription factor system known to be such a frequent target for various anti-inflammatory and immunosuppressive

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molecules as the NF-KB/Rel system. This may be for several reasons. A simple reason is that the factor assumes a key role in mediating gene regulatory responses to pathogens and that even a partial inhibiton by pleiotropically acting inhibitors may have a considerable impact on the course of an inflammatory reaction. A second reason is that the signaling pathway by which NF-KB is activated offers many potential points of interference. These include specific targets in the signaling pathway, such as cytokine receptors, their associated adaptors, IKBkinase, and the DNAbinding NF-KBdimer itself, but also less specific targets, such as ubiquitinconjugating enzymes, the proteasome, nuclear transport proteins, or components of the basal transcription machinery. Because inflammatory reactions require de novo synthesis of many proteins, almost any agent that inhibits transcription or protein synthesis may have an anti-inflammatory effect. Despite this consideration it must be emphasized that the majority of inhibitory mechanisms described previously seem rather selective (see Fig. 1). An intriguing observation is that there are examples of pathogenic viruses, bacteria, and fungi that invested some of their genomic information in controlling the activity of the host transcription factor NF-KB (and other regulatory proteins). During evolution, this occurred in viruses by encoding proteins that more or less directly inhibit NF-KBactivation and in bacteria and fungi by encoding multiple enzymes that are involved in biosynthetic pathways producing secondary metabolites with NF-KB-inhibitory activity. It should be pointed out that the prime targets for immunosuppressive natural products are usually not the transcription factors per se but rather upstream components of signaling cascades leading to transcription factor activation. The obvious advantage for this target selection is that the effect of such inhibitors will be more pleiotropic and, therefore, would affect more than one transcription factor system at a time. There is a considerable overlap in stimuli that activate NF-KBand AP-1 (Angel, 1995).Therefore, it may not be too surprising to find that AP-1 activation is also a target for a number of the molecules described previously. There are a number of drugs and endogenous proteins with immunosuppressive activity that await to be tested for their effect on NF-KB. These include IL-10, a-melanocyte-stimulating hormone, vitamin D, and thalidomide. Based on a detailed understanding of the molecular events governing NF-KB activation, novel anti-inflammatory drugs can be developed. One attempt tries to exploit the fact that the inducibly phosphorylated IKBhas to be degraded by the proteasome in order to release transcriptionally active NF-KB. A number of synthetic peptide aldehyde proteasome inhibitors (Palombella et al., 1994;Traenckner et al., 1994)and a proteasome subunit-

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FIG. 1. Scheme for activation of NF-KB. As described in the text, a primary pathogen induces production of proinflammatory cytokines TNF and IL-1. The cytokines bind to receptors on target cells, which results in assembly of a signaling complex that includes the adaptor proteins TRADD and TRAFB and the kinases RIP and IRAK, respectively, for the TNF and IL-1 receptor. Through a series of uncharacterized steps, an IKBkinase is activated

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binding fungal metabolite, lactacystin (Fenteany et al., 1995),were found to potently inhibit NF-KB activation by virtue of stabilizing IKB. When tested in endothelial cell activation models, proteasome inhibitors completely prevented neutrophil adhesion and transmigration as well as TNFa-induced expression of ELAM-1, VCAM-1, and ICAM-1 (Read et aE., 1995). Because proteasome activity is also crucial for other transcription regulatory processes and cell cycle progression (reviewed in Pahl and Baeuerle, 1996), it remains to be determined whether proteasome inhibitors will be useful for treatment of acute and chronic human immune disorders. How dangerous can it be to block NF-KB? NF-KB certainly fulfills very useful functions in mediating inflammatory reactions and an antiapoptotic response. Hence, the worst effects of NF-KBinhibition may be immunodeficiencies and increased sensitivity of cells to apoptosis, as is suggested by results from transgenic animal models. There is no evidence so far to suggest that NF-KB/Rel proteins play a role in embryonic development. However, it must be pointed out that data from multiple NF-KB subunit gene knockouts are still missing. These may have much more severe phenotypes because of the lack of compensatory mechanisms. Drugs that are not specific for certain NF-KB complexes but that inhibit the entire pathway may produce a phenotype as expected for a multiple subunit gene knockout. Increased apoptosis and immune deficiency syndromes may arise when the entire NF-KB system is blocked for longer periods of time, in particular in combination with other proapoptotic (cytostatic) drugs. This could be one reason why more physiological NF-KB control mechanisms, such as steroid hormones or CAMP, seek to attenuate rather than to erradicate NF-KB activation. A partial inhibition of NF-KB activity may still allow an antiapoptotic response but will significantly reduce the expression of cell adhesion molecules, chemokines, and cytokines. Proper dosing and a suitable pharmacodynamic behavior of novel drugs may allow us to mimic characteristics of more physiological NF-KB-inhibiting mechanisms.

ACKNOWLEDGMENTS We are grateful to Romunda Craft for drawing the figure and putting together the references. In view of the large number of publications in this field we have not attempted

that phosphorylates IKB-CIon serine 32 and 36 resulting in ubiquitination and subsequent degradation of IKB by the proteasome. The released p5O/p65 heterodimer then translocates to the nucleus to activate transcription of target genes. The potential site of action of immunosuppressive and anti-inflammatory molecules discussed in the review is indicated. Dexamethasone can interfere with NF-KBboth by dampening transcriptional activation by p5O/p65 and by inducing the expression of 1 ~ B - a .

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to cite every study and we apologize to those colleagues whose work has not been referred to directly.

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Kaltschmidt, B., Volk, B., Baeuerle, P. A., and Kaltschmidt, C. (1997). Transcription factor NF-KBis activated in primary neurons by amyloid PA4 and in neurons surrounding early plaques from Alzheimer disease patients. Pmc. Natl. Acad. Sci. USA, in press. Kaltschmidt, C., Kaltschmidt, B., Lannes-Vieira, J., Kreuzberg, G., Wekerle, H., Baeuerle, P. A., and Gehrmann, J. (1994). Activation of transcription factor NF-KB in microglia during experimental autoimmune encephalomyelitis.J. Neuroimmunol. 55, 99- 106. Kaltschmidt, C., Kaltschmidt, B., Henkel, T., Stockinger, H., and Baeuerle, P. A. (1995). Selective recognition of the activated form of transcription factor NF-KBby a monoclonal antibody. Biol. Chem. Hoppe-Seyler 376, 9-16. Kazmi, S. M. I., Plante, R. K., Visconti, V., Taylor, G. R., Zhou, L., and Lau, C. Y. (1994). Suppression of NF-KB activation and NF-KB-dependent gene expression by tepoxalin, a dual inhibitor of cyclooxygenase and 5-lipoxygenase.J. Cell. Biochem. 57, 299-310. Kopp, E., and Ghosh, S. (1994). Inhibition of NF-KB by sodium salicylate and aspirin. Science 265,956-958. Lai, J.-H., and Tan, T.-J. (1994). CD28 signaling causes a sustained down-regulation of IKB-a which can be prevented by the immunosuppressant rapamycin. J. B i d . Chem. 269,30077-30080. Leibfarth, J. H., and Persellin, R. H. (1981). Mechanisms of action of gold. Agents Actions 11,458-472. Liebler, D. C. (1993). The role of metabolism in the antioxidant function of vitamin E. Citr. Reu. Toxicol. 23, 147-169. Limbourg, F. P., Staedler, H., Chinnadurai, G., Baeuerle, P. A., and Schmitz, M. L. (1996). A hydrophobic region within the adenovirus E l B 19 kDa protein is necessary for the transient inhibition of NF-KB activated by different stimuli. J. Biol. Chem., 271,2039220398. Los, M., Schenk, H., Hexel, K., Baeuerle, P. A., Droge, W., and Schulze-Osthoff, K. (1995). IL-2 gene expression and NF-KB activation through CD28 requires reactive oxygen production by 5-lipoxygenase. EMBO 1. 14,3731-3740. Maniatis, T., Whittemore, L. A., Du, W., Fan, C. M., Keller, A., Palombella, V., and Thanos, D. (1992). Positive and negative control of human interferon-0 gene expression. In Transcriptional Regulation, Part 2 ( S . L. McKnight and K. Yamamoto, Eds.), pp. 11931220. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Marok, R., Winyard, P. G., Coumbe, A., Kus, M. L., Gaffney, K., Blades, S., Mapp, P. I., Moms, C. J., Blake, D. R., Kaltschmidt, C., and Baeuerle, P. A. (1996). Activation of transcription factor NF-KB in human inflamed synovial tissue. Arthritis Rheum. 39, 583-591. Mattila, P. S., Ullman, K. S., Fiering, S., Emmel, E. A., McCutcheon, M., Crabtree, G. R., and Henenberg, L. A. (1990). The actions of cyclosporin A and FK506 suggest a novel step in the activation of T lymphocytes. EMBO J. 9(13), 4425-4433. Meyer, M., Schreck, R., and Baeuerle, P. A. (1993). H202and antioxidants have opposite effects on activation of NF-KB and AP-1 in intact cells: AP-1 as secondary antioxidantresponsive factor. EMBO /. 12, 2005-2015. Meyer, M., Schreck, R., Muller, J. M., and Baeuerle, P. A. (1994). Redox control of gene expression by eukaryotic transcription factors NF-KB,AP-1 and SRFRCF23 (C. Pasquier et al., Eds.), pp. 217-235. Birkhauser Verlag, Basel, Switzerland. Mukaida, N., Morita, M., Ishikawa, Y., Rice, N., Okamoto, %I., Kasahara, T., and Matsushima, K. (1994). Novel mechanism of glucocorticoid-mediated gene repression. J. Biol. Chem. 269, 13289-13295. Munroe, D. G., Wang, E. Y., MacIntyre, J. P., Tam, S. S. C., Lee, D. H. S., Taylor, G. R., Zhou, L., Plante, R. K., Kazmi, S. M. I., Baeuerle, P. A., and Lau, C. Y. (1995). Novel

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intracellular signalling function of prostaglandin H synthase-1 in NF-KB activation.

1.lnfamm. 45,260-268.

Muraoka, K., Fujimoto, K., Sun, X., Yoshioka, K., Shimizu, K., Yagi, M., Bose, H., Jr., Miyazaki, I., and Yamamoto, K. (1996). Immunosuppressant FK506 induces interleukin6 production through the activation of transcription factor nuclear factor (NF)-KB. 1.Clin. Invest. 97, 2433-2439. Nabel, G., and Baltimore, D. (1987).An inducible transcription factor activates expression of human immunodeficiency virus in T cells. Nature 326, 711-713. Neumann, M., Grieshammer, T., Chuvpilo, S., Kneitz, B., Lohoff, M., Schimpl, A., Franza, B. R., Jr., and Serfling, E. (1995).ReWp65 is a molecular target for the immunosuppressive action of protein kinase A. EMBO 1. 14(9), 1991-2004. Omar, B., McCord, J., and Downey, J. (1991). Ischaemia-reperfusion. In Oxidative Stress: Oxidants and Antioxidants (H. Sies, Ed.), pp. 493-528. Academic Press, London. O’Shea,J. J., McVicar, D. W., Bailey, T.L., Bums, C., and Smyth, M. J. (1992). Activation of human peripheral blood T lymphocytes by pharmacological induction of protein-tyrosine phosphorylation. Proc. Natl. Acud. Sci. USA 89, 10306-10310. Pahl, H. L., and Baeuerle, P. A. (1996). Control of gene expression by proteolysis. Cum. %in. Cell Biol. 8, 340-347. Pahl, H. L., Kraub, B., Schulze-Osthoff, K., Traenckner, E. B.-M., Parks, T., Myers, C., Waring, P., Muehlbacher, A., Czemilofsky,P. A,, and Baeuerle, P. A. (1996).The immunosuppressive fund gliotoxin is a selective inhibitor of transcription factor NF-KB./. Exp. Med. 183, 1829-1840. Palombella, V. J.. Rando, 0. J.. Goldberg, A. L., and Maniatis, T. (1994). The ubiquitinproteasome pathway is required for processing the N F - K B ~ precursor protein and the activation of NF-KB. Cell 78, 773-785. Phipps, R. P., Stein, S. H., and Roper, R. L. (1991).A new view of prostaglandin E regulation of the immune response. lmmunol. Today 12, 349-352. Pierce, J. W., Read, M. A., Ding, H., Luscinskas, F. W., and Collins, T. (1996). Salicylates inhibit 1 ~ B - phosphorylation, a edothelial-leukocyte adhesion molecule expression, and neutrophil transmigration. I. lmmunol. 156, 3961-3969. Rappaport, R., and Dodge, G. R. (1982). Prostaglandin E inhibits the production of human interleukin 2. J. E x p Med. 155, 943-948. Ray, A,, and Prefontaine, K. E. (1994). Physical association and functional antagonism between the p65 subunit of transcription factor NF-KB and the glucocorticoid receptor. Proc. Natl. Acad. Sci. USA 91, 752-756. Read, M. A,, Neish, A. S., Luscinskas, F. W., Palombella, V. J., Maniatis, T., and Collins, T. (1995). The proteasome pathway is required for cytokine-induced endothelial-leukocyte adhesion molecule expression. lmmunity 2, 493-506. Roederer M., Staal, F. J.. Raju, P. A., Ela, S. W., Herzenberg, L. A. (1990). Cytokinestimulated human immunodeficiencyvirus replication is inhibited by N-acetyl-L-cysteine. Proc. Natl. Acad. Sd. USA 87, 4884-4888. Rooney, J. W., Sun, Y.-I., Glimcher, L. H., and Hoey, T. (1995). Novel NFAT sites that mediate activation of the interleukin-2 promoter in response to T-cell receptor stimulation. Mol. Cell. Biol. 15, 6299-6310. Rothe, M., Wong, S. C., Henzel, W. J., and Goeddel, D. V. (1994). A novel family of putative signal transducers associated with the cytoplasmic domain of the 75 kDa tumor necrosis factor receptor. Cell 78, 681-692. Sagone, A. L., and Husney, R. M. (1987).Oxidation of salicylates by stimulated granulocytes: Evidence that these drugs act as free radical scavengers in biological systems.]. Immunol. 138, 2177-2183.

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Scheinman, R. I., Cogswell, P. C., Lofquist, A. K., and Baldwin, A. S., Jr. (1995a). Role of transcriptional activation of I K Bin~ mediation of immunosupression by glucocorticoids. Science 270,283-286. Scheinman, R. I., Gualberto, A,, Jewell, C. M., Cidlowski, H. A., and Baldwin, A. S., Jr. (1995b). Characterization of mechanisms involved in transrepression of NF-KB by activated glucocorticoid receptors. Mol. Cell. Biol. 15, 943-953. Schmidt, A., Hennighausen, L., and Siebenlist, U. (1990). Inducible nuclear factor binding to the KB elements of the human immunodeficiency virus enhancer in T cells can be blocked by cyclosporin A in a signal-dependent manner. 1.Virol. 64,4037-4041. Schmidt, K. N., Amstad, P., Cerutti, P., and Baeuerle, P. A. (1995a). The roles of hydrogen peroxide and superoxide as messengers in the activation of transcription factor NF-KB. Chem. Bwl. 2, 13-22. Schmidt, K. N., Traenckner, E. B.-M., Meier, B., and Baeuerle, P. A. (1995b). Induction of oxidative stress by okadaic acid is required for activation of transcription factor NFKB.J. Biol. Chem. 270, 27136-27142. Schmitz, M. L., Indorf, A., Limbourg, F. P., Stadtler, H., Traencher, E. B.-M., and Baeuerle, P. A. (1996). The dual effect of adenovirus 5 E1A 13s protein on NF-KB activation is antagonized by E1B 19 K. Mol. Cell. Biol. 15. Schreck, R., and Baeuerle, P. A. (1991). A role for oxygen radicals as second messengers. Trends Cell. Biol. 1, 39-42. Schreck, R., Albermann, K., and Baeuerle, P. A. (1992a). NF-KB: An oxidative stressresponsive transcription factor of eukaryotic cells [review]. Free Rudic. Res. Commun. 17,221-237. Schreck, R., Meier, B., Mannel, D., Droge, W., and Baeuerle, P. A. (1992b). Dithiocarbamates as potent inhibitors of NF-KBactivation in intact cells.]. Elrp. Med. 175,1181-1194. Schreiber, S . L., and Crabtree, G . R. (1992). The mechanism of action of cyclosporin A and FK506. Zmmunol. Today 13, 136-142. Schulze-Osthoff, K., Bakker, A. C., Vanhaesebroeck, B., Beyaert, R., Jacobs, W. A., and Fiers, W. (1992). Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation.]. Biol. Chem. 267, 5317-5323. Schulze-Osthoff, K., Los, M., and Baeuerle, P. A. (1995). Redox signalling by transcription factors NF-KB and AP-1 in lymphocytes. Biochem. Pharmacol. 50, 735-741. Sen, C. K., and Packer, L. (1996). Antioxidant and redox regulation of gene transcription. FASEB J. 10,709-720. Sies, H. (1991). Oxidotive Stress. Oxidants and Antioxidants. p. 650. Academic Press, London. Sigal, N. H., and Dumont, F. J. (1992). Cyclosporin A, FK-506, and rapamycin: Pharmacologic probes of lymphocyte signal transduction. Annu. Reu. Zmmunol. 10,519-560. Singh, S., and Aggarwal, B. B. (1995). Activation of transcription factor NF-KBis suppressed by curcumin (diferulolylmethane).1.Biol. Chem. 270, 24995-25000. Staal, F. J. T., Roederer, M., Herzenberg, L. A., and Herzenberg, L. A. (1990).Intracellular thiols regulate activation of nuclear factor KB and transcription of human immunodeficiency virus. Proc. Natl. Acud. Sci. USA 87,9943-9947. Stein, B., and Yang, M. X. (1995). Repression of the interleukin-6 promoter by estrogen receptor is mediated by NF-KB and CIEBPP. Mol. Cell. BloZ. 15, 4971-4979. Steinman, L. (1996). Multiple sclerosis: A coordinated immunological attack against myehn in the central nervous system. Cell 85, 299-302. Sutton, P., Newcombe, N. R., Waxing, P., and Mullbacher, A. (1994). In vivo immunosuppressive activity of gliotoxin, a metabolite produced by human pathogenic fungi. Infect. Immun. 62, 1192-1198.

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Suznki, Y. J.. and Packer, L. (1993).Inhibition of NF-KBactivation by vitamin E derivatives. Biochetti. Biophys. Res. Cotiitrmti. 193, 277-283. .Taylor. A. ( 1971). The toxicology of sporidesmin and other epipolythiodoxopiperazines. Microhiol. Toxiiis 7, 337-376. Tepper, M. A,, Nadler, S. G., Esselstyn, J. M., and Sterhenz, K. G. (1995). Deoxyspergualin inhibits K light chain expression in 70W3 pre-B cells by blocking lipopolysaccharideindriced NF-KB activation.J. Imtntcnol. 155, 2427-2436. Traenckiier. E. B.-M.. Wilk, S., and Baeuerle, P. A. (1994).A proteasome inhibitor prevents activation of NF-KB and stabilizes a newly phosphoylated form of IKB-a that is still bound to NF-KB. EMBO /. 13,5433-5441. Traenckner, E. B.-M., Pahl, H. L., Schmidt, K. N., Wilk, S., and Baeuerle, P. A. (1995). Phosphoiylation of human IKB-cx on serines 32 and 36 controls 1 ~ B - aproteolysis and NF-KB activation in response to diverse stimuli. EMBO J. 14, 2876-2883. Tsai, M.-J., and O’Malley, B. W. (1994). Molecular mechanisms of action of steroidkhyroid receptor superfainily members. A w w . Reo. Biodieni. 63, 451-486. Verma, I. M., Stevenson, J. K., Schwarz, E. M., Van Anhverp, D., and Miyamoto, S. ( 1995). ReVNF-KBAKB family: Intimate tales of association and dissociation. Genes Deu. 9, 2723-2735. Waring, P., Eichner, R. D., and Bullbacher, A. (1988). The chemistry and biology of the immiiiiomodulating agent gliotoxin and related epipolythiodioxopiperazines.Med. Res. Reu. 8, 499-524. Weissmann. G. (1991). Aspirin. Sci. Am. 264, 84-90. Williams, D. H., Jeffery, L. J., and Murray, E. J. (1992).Aurothioglucose inhibits introduced NF-KB and AP-1 activity by acting as an IL-1 functional antagonist. Biochim. Biophys. Acta 1180, 9-14. Wright, K. L., White, L. C., Kelly, A,, Beck, S., Trowsdale, J., and Ting, J. P. (1995). Coordinate regulation of the human TAP1 and LMP2 genes from a shared bidirectional promoter. 1. Exp, Med. 181, 1459-1471. Wrighton, C. J., Warbinek, R. H., Moll, T., Eytner, R., Bach, F. H., and de Martin, R. (1996). Inhihitian of endothelid cell activation by adenovirus-mediated expression of IKBLY, an inhibitor of the transcription factor NF-KB.J. Exp. Med. 183, 1013-1022. Yanez, R. J.. Rodriguez. J. M., Nogal, M. L., Yuste, L., Enriquez, C., Rodriguez, J. F., and Vinuela, E. (1995). Analysis of the complete nncleotide sequence of African swine fever virns. Virology 208, 249-278. Yang. J.-P,, Merin, T., Nakano, T., Kato, T., Kitade, Y., and Okamato, T. (199s).Inhibition of the DNA-binding activityin NF-KBby gold compounds in vitro. FEBS Lett. 361,89-96. Zlrou, L., Ritchie, D., Wang, E. Y., Barbone, A. G., Argentieri, D., and Lau, C. Y. (1994). Tepoxdin, a novel iinmunosuppressive agent with a different mechanism of action from cyclosporin A. J. Ztttitiuno~.153, 5026-5037. This article was accepted for publication on 27 September 1996.

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ADVANCES IN IMMUNOLOGY. VOL. 65

Mouse Mammary Tumor Virus: Immunological Interplays between Virus and Host SANJN A. LUTHER AND HANS ACHA-ORBEA I n s h b d B i a c h i s t r y and Ludwig inshk, far Cancer Reseaxh, lauwnne Bmnch, University of laumnne, 1066 Epa/inges, S w h d a n d

1. Introduction

It took nearly 100 years from the first description of mammary tumors in wild mice (Crisp, 1854)to the discovery that an extrachromosomal factor was responsible for the high incidence of mammary carcinomas in susceptible mouse strains (Lathrop and Loeb, 1918; Staff of the Roscoe B. Jackson Memorial Laboratory, 1933; Koretweg, 1934). Soon thereafter it was realized that this factor was transmitted maternally via milk to the offspring (Bittner, 1936). However, more time was required to convince researchers that the agent responsible for tumor formation was a retrovirus (Bryan et al., 1942; Bittner, 1948; Passey et al., 1950; Dmochovski and Grey, 1957; Bernhard, 1958). One of the reasons for the skepticism was the finding that tumorfree mouse strains were found to be infected with such retroviruses in the mammary gland. The presence and role of endogenous viruses was not known yet. Despite the difficultyof obtaining large quantities ofvirus (mostly from mouse milk or in lower amounts from supernatants of mammary tumor cell cultures) and the long incubation times for biological readouts (forcancer development, often more than 1 year), mouse mammary tumor virus (MMTV) has been in the center of cancer research from 1940 to the late 1970s. Only the most important older articles will be cited in this review. The work of this period was already extensively reviewed (Nandi and McGrath, 1973; Bentvelzen and Hilgers, 1980). Interest into MMTV was reawakened when it was found that MMTV represented the solution to a more than 20-year-old immunological puzzle. In the early 1970s Festenstein (1973) described that mixed lymphocyte reactions between different strains of mice could lead to extremely strong reactions even when the two mouse strains involved were major histocompatibility complex (MHC) compatible. He called the antigens responsible for this effect minor lymphocyte stimulating (Mls) antigens. It took over 20 years of frustrating trials to generate antibodies and to characterize biochemically and immunologically the nature of these Mls antigens. Finally it was shown that this effect was encoded by an open reading frame (orf) with previously unclear functions encoded by endogenous MMTV proviruses (for nomenclature see below) (Acha-Orbea et al., 1991; Choi 139

Copyright 0 1997 hy Academic Press

AU rights of repduction in any form reserved. 0065-2776~97S25.W

140

SANJIV A. LUTHER A N D IlANS ACHA-ORUEA

et al., 1991; Woodland et al., 1991b). These proviruses can be found in all commonly used mouse strains and in the majority of wild mice. Nearly all of them encode a functional superantigen (SAg). SAgs were originally described as bacterial antigens capable of binding to different MHC class I1 but not MHC class I molecules and of interacting with a large proportion of T cells sharing a T cell receptor (TCR) V/3 element (Janeway et al., 1989; Kappler et al., 1989a; White et al., 1989). Because the numbers of Vp elements are limited (25 for mice and SO 100 for humans), a large proportion (130%) of T cells will be triggered by a given SAg. This compares with 1 in 104106T cells for a classical peptideMHC complex; hence the name SAg. To date, SAgs have been described in bacteria (Staphylococci, Streptococci, Mycobacteria, Yersinia, and Mycoplasma), retroviruses (MMTV), other viruses (rabies, CMV, and EBV), protozoa, and even in plants (for reviews see Janeway et nl., 1989; Marrack and Kappler, 1990; Acha-Orbea, 1993; Kotzin et al., 1993; Scherer et al., 1993;Acha-Orbea and MacDonald, 1995; Fleischer et al., 1995, or specific references in section VI). Despite significant structural differences between these proteins, they all share the above definition in binding MHC class I1 molecules and interacting polyclonally with T cells sharing a TCR Vp element. For MMTV it was clearly shown that the role of these SAgs is to allow a much more efficient infection (Held et al., 199313).Their role for bacteria is less clear. Most likely, the presence of a SAg allows a slight advantage in the maintenance or propagation of the infection although the results are not completely clear yet (Rott and Fleischer, 1994). What makes SAgs interesting for immunologists and virologists is their profound effect on the immune system and the ability to follow the reacting T cells in viva as well as in vitro with antibodies specific for TCR V/3 elements. The characterization of SAg-induced immune responses gives insights into thymic and peripheral tolerance induction, unresponsiveness (anergy), antigen presentation, immune response, T B collaboration, as well as virus host interaction. Some of the older key findings as well as the newer developments will be discussed. II. Mouse Mammary Tumor Virus

Like all the members of the retrovirus family, MMTV encodes gag, pol, and env molecules in its 8.5-kb RNA genome (see Fig. 1). To synthesize these proteins the retroviral RNA has to be reverse transcribed and integrated into the genome of the host cell. What makes MMTV special is a long orf in the 3’ long terminal repeat (LTR). It encodes a SAg and is required for completion of the retroviral life cycle.

SUPERANTIGENS OF M M T V

141

As w illbe discussed, infectious MMTV requires an intact immune system for completion of its life cycle (Squartini et al., 1970; Tsubura et al., 1988; Held et al., 1993a).It infects B cells first after binding to a still unknown receptor structure. The outer viral membrane fuses with the cell membrane and releases the viral core into the cytoplasm. Thereafter, the viral RNA is reverse transcribed and has to integrate its genome into the host DNA to achieve transcription and translation of the retroviral genes (Weiss et al., 1985).Only then, the SAg, which is not detectable in the virus particle, can be produced and expressed on the cell surface. Later, T cells are also infected and the virus finally reaches, by still unknown mechanisms, the mammary gland. When the mammary gland gets receptive for infection, the virus is transferred and the viral life cycle is completed. The virus derives its name from the high incidence of mammary tumors which are induced by some of the MMTVs. These tumors are induced by insertional activation of nearby protooncogenes.

A. VIROLOGY

I . Endogenous and Exogenous Mouse Mamma y Tumor Viruses MMTV exists either as an infectious virus (exogenous virus or MMTV) which is transmitted maternally via milk during the first 2 weeks of life or as an endogenous provirus (Mtv)which is transmitted following Mendelian transmission rules. Infectious MMTV is found in high amounts in the milk of infected mice (up to 10" virus particles/ml or 1 mg/ml). Uptake of virus-containing milk leads to infection of the offspring. As will be discussed in more detail, B cells are infected and the SAg-induced T B collaboration is the key to the establishment of an efficient lifelong infection and finally for infection of the mammary gland. Several such infectious MMTVs have been described. The name of infectious MMTVs is usually composed of MMTV followed in parentheses by the mouse strain in which it was first isolated [e.g., MMTV(C3H)I. The currently used MMTVs are listed and characterized in Table I. Surprisingly, these infectious retroviruses seem to mutate only on rare occasions despite the error-prone reverse transcriptase. LTR sequences encompassing over 10% of the retroviral genome obtained more than 10 years apart from MMTV(C3H) were identical (Moore et al., 1987; A. Shakhov and H. Acha-Orbea, unpublished data). These results indicate that MMTV and its host have reached an equilibrium and therefore no selection of escape mutants is observed. Alternatively, MMTV exists as a germline transmitted provirus (Varmus et al., 1972; Cohen and Varmus, 1979, 1980; Groner and Hynes, 1980; Kozak et al., 1987). These proviruses derive from rare integrations into

142

SANJIV A. LUTHER A N D HANS ACHA-ORBEA

A

R U5 PB mRNA

rn7GpppGm

1({

PP U3 R

:I:’(/AAAAAAAAAA

gag

5‘ LTR

gag

poi

p$\

env

3‘LTR orf

CDNA and integrated -

I / I

U3 R U5

U3 R U5 PB rnRNAs after integration

-- -

gagPr77

bo( p21 I

A

I

p27

-

H

p14

/

4

I fs25%

gagPr160 lplol p21 MA p21 %of viral 7-16 1-8 proteins

I

envPt-73 % of viral proteins

gp52 20-31

I

p27 CA 16-20

I

C

1

fs 10%

I

p14 NC 7-11

gp36 16-23

-

9 kb, gag, Pro, POI 3.6 kb env 1.7 kb orf

I

I p30 p131 PR 2-7

? RT 4%

I? I IN <1%

I

velop env gp36 and gp52

Reverse transcriptase FIT Matrix MA

SUPERANTIGENS OF MMTV

143

germ cells and are transmitted from then on as endogenous mouse genetic information according to Mendelian rules. These endogenous loci are termed Mtv followed by a number (e.g., Mtu7). So far, numbers go from 1 to 53 with several numbers lacking due to mistakes in the attributions (see Table 111). On the average 28 endogenous Mtu loci are found in all the common mouse strains (Kozak et al., 1987; Scherer et al., 1993). Depending on the origin of wild mice, between 0 and 20 endogenous proviruses are found. In European mice, between 0 and 2 copies are found and in isolated valleys of Nepal up to 20 have been found (Cazenave et al., 1990;Jouvin-Marche et al., 1993).Although there are some inbred Mtvfree wild mouse strains, the genetic background, especially their MHC, is impeding further immunological research (Pullen et al., 1990a). Recently Mtv-free or single Mtv congenic mouse lines with defined MHC haplotypes were derived (Braun et al., 1995; Scherer et al., 1995). Most described proviruses have lost the capability of producing infectious viral particles. The exceptions are Mtvl (Heston and Derringer, 1952; Boot and Muhlbock, 1956; van Nie and Verstraeten, 1975; Michalides et al., 1981),Mtv2 (Heston and Demnger, 1952; Boot and Muhlbock, 1956; van Nie and Verstraeten, 1975; Michalides et al., 1981), Mtv3 (Nusse et al., 1980; Michalides et al., 1985; Hainaut et al., 1990),Mtv4 (Imai et al., 1983b; Luther et al., 1994), Mtv48 (Niimi et al., 1995) and Mtv53 (H. Acha-Orbea and A. Shakhov, unpublished data). Some of the latter ones have a name for their proviral locus as well as the derived infectious MMTV particle such as Mtu2 and MMTV(GR), Mtv4 and MMTV(SHN), and Mtu53 and MMTV(SW).Mtu3 only produces defective viral particles which do not contain env molecules (see Table I). Most endogenous Mtvs express a functional SAg. Env sequences, on the contrary, are often nonfunctional in these proviruses (Mtv6, Mtv7, and M t v 8 ) (Gunzburget al., 1984;Cho et al., 1995;A. N. Shakhov and H. AchaOrbea, unpublished data). Three viruses have been sequenced entirely [MMTV(BR6)MMTV(CSH),and MMTV( JYG)]. For envelope molecules only six sequences have been reported so far which are 96% homologous

FIG.1. Virus structure of mouse mammary tumor virus. (A) Viral genome before (mRNA) and after reverse transcriptiodintegration (cDNA)of MMTV as well as the known transcripts derived from the promotors in the 5’LTR. (B) Schematic representation of the MMTV proteins.The three gag precursorproteins (Pr77,PrllO, and Pr10) are obtained by introduction of one or two frameshifts (fs) during translation. The envprecursoris shown underneath. Thereafterthe precursor proteins are cleavedinto the different fragments.The same abbreviations as those in C are used. Below the abbreviations of the names of the molecules, the percentage of total viral proteins is indicated. (C) Schematic representation of an MMTV particle (adapted from Coffin, 1996).

TABLE I OF THE INFECCIOUS MMTV STAINS PROPERTIES CD4+ T Cell stimulation after injection (PO-LN)

CD4+ T cell deletion after injection (in blood)

CD4+ T cell deletion after transmission (in blood)

Maximal MiXimal extent of Half-max extent of Half-max I-E deletion deletion deletion deletion depen(96) inweeh dence (8) inweeh

Extent of Endogenous stimuMtv VP lation

VS

7,8

+++

7,8,14

30

ca. 3

8.2

+++

8.2,14

ND

ND

MMTV (RIIId)

8

+++

7,s

50

ca. 10

MMTV (SW) Mtu.53

6

+++

6,7,8.1,9

95

ca. 3

95

5-6

-

MMTV

6

+++

6,7,8.1,9

ND

ND

95

5-6

-

4

+++

30

ca. 3

95

5-6

+

MMTV strain

MMTV (SHN MMTV (FM)

Mtd

75 ca.90

ND

Tumor incidence: BALB/c (months) High (6-9)

4

Luther et d.(1994)

12

-

<8(LN)

-

Yoshimoto et d. (1994), Matsuzawa etd. (1995)

ND

-

Xenarios et d.

Low (>12)

(PG) MMTV (SIM)

References

High (6-12)

(unpublished observation) Held et d.(1992), Papiernik et d. (1992),AchaOrbea et d.(1992) Nishio et d.(1994), Luther et d. (1994) Maillard et d.(1996)

+ rp cn

MVIMTV ( C3H )

14

+

MMTV ( G R ) Mtu2

14

MMTV (I1 TES1.I)

14,lS

60

ca. 5

7s

3-8

KD

14

ND

ND

NT)

NT)

14

+++

14

80

ca. 2

ND

ND

MMTV (RIIIj) MMTV (C4)

14

+++

ND

ND

ND

ND

ND

2

2

95

ca. 3

95

6

MMTV ( V j

2

+++ +++

2

ND

ND

<2 (spl)

MMTV (CS) Mtu48 MMTV (I1

2 2

+++

2 2

ND 70

ND ca. 3

ca. 90% (spU ND ND

+++

ND ND

High (6-12)

Marrack et a!. (1991). Ignatowitz et nl. (19921. Acha-Orbea et a!. (1992), Pucrllo et a/. (19931, Penninger et RI. (1994) High (6-1 2) Arha-Orbra e t nl. (1992).Ferrick & az. (1992) Ando et al. (1995), Wajjwalku et al. (1995) Wajjwalku et al (1995) Intermediate Shakov et a!. (1993) (10-12) High Hodes et al (1993) Niimi et al. (1995) Ando d al. (1995)

TES2) Note. BALB/c mice were used in most assays. T cell stirnillabin was meawred 4 (lays after infec!on (Peak Day 4-6). T cell deletion was assessed with a virus dosr that induced a clearly meuurtllle stiiriulatiun on Day 4. The I-E dependencc shows the necessi? o f MHC class I1 I-E for the stimril;dion and deletion uf SAg-reactive T celIs. The Following known M M T V strains have not yet been analyzed on their effea on T cells: MMTV(RF). -(DDD). -CDDO), -(MB), and M t o 1 . It is pssihlc that MMTV(CS) and MMTV(I1 TESB! are identical hccausr thcy arc dcrivcd from tht? samr inniise crossing ;md code for the same M h SAg sequence. The T cell deletion by MMTV(I1 TES14) and MMTV(I1 TES12) was measured by injecting inilk containing both vinises (Ando et a!., 1995).Data on tumor incidence come from Hageman et al. (1981) and our own unpublished data. ND, not determined; LN, lymph node; PO-LN. draining popliteal lymph node; spl, spleen;

+ + +, strong; +, weak.

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SANJIV A. LUTHER AND HANS ACHA-ORBEA

at the amino acid level (Fasel et al., 1982; Majors and Varmus, 1983b; Moore et al., 1987; Nishio et al., 1994; Nakamura et al., 1996). Many MMTV SAg sequences are known and sequence homologies in the order of 8099%are found (see Fig. 3 for COOH-terminal SAg sequences) (Donehower et al., 1981, 1983; Fasel et al., 1982;Kennedy et al., 1982; BrandtCarlson et al., 1993). Because most of the polymorphism is localized in the 30 COOH-terminal amino acids the homology is much higher in the NH2-terminal 290 amino acids (92100%).

2. Viral Structure MMTV particles have a diameter of about 105 nm. Based on their acentric and highly condensed nucleocapsid they were called B-type particles to distinguish them from the concentric C-type particles. Inside the infected cells, immature intracytoplasmic A-type particles are found (80 90 nm) which contain mostly uncleaved gag proteins but lack envelope molecules (Nusse et al., 1979; Vaidya et al., 1980; Racevskis and Sarkar, 1982; Menendez Arias et al., 1992). They are the precursors of the viral core (Tanaka et al., 1972; Smith and Lee, 1975; Sarkar and Whittington, 1977; Tanaka, 1977; Cardiff et al., 1978). These A-type particles bud through the plasma membrane, thereby acquiring an outer lipid layer that contains viral envelope (env) proteins, and mature into B-type particles. In the viral particle two polyadenylated and capped positive-strand 8.5kb-long mRNA molecules (Schlom et al., 1973) are found associated with host tRNALpas primer for reverse transcriptase (Peters and Glover, 1980). In addition, reverse transcriptase (Duesberg and Cardiff, 1968; Marcus et al., 1976; Dion et al., 1977), protease, and gag proteins (Dickson and Skehel, 1974; Teramoto et al., 1974; Sarkar and Dion, 1975; Sarkar et al., 1976; Yagi et al., 1978) are found in the mature MMTV particle. Viral proteins are abbreviated with Pr for precursor protein, gp for glycoprotein, p for protein, and pp for phosphoprotein. These abbreviations are always followed by the molecular weight in kDa. On the viral surface characteristic spikes are detected by electron microscopy which represent the envelope proteins gp36 and gp52. Env molecules define receptor specificity and host range. The different molecules and their functions are indicated in Fig. 1. All retroviral genomes encode the internal structural proteins (gag), polymerase (pol), and env proteins. The genes for these proteins are localized between two LTRs. Often these genes contain slightly overlapping DNA sequences (for MMTV: 51 nucleotides polenv, 53 nucleotides envSAg). Capsid, protease, and polymerase proteins are translated from the full-length mRNA which is initiated in the U5 region of the 5’ LTR (see Fig. 1A). These proteins are encoded in three different reading frames

SUPERANTIGENS OF MMTV

147

and frameshifts during translation are required for production of the downstream proteins. Both protease and polymerase are carboxyl-terminalextensions of some of the gag polyproteins. The change of reading frame occurs quite frequently by slipping at stem loop structures or *-knots and leads to a gradient with about 4-fold lower expression of protease and 40-fold lower expression of pol compared to gag (Jacks et al., 1987; Moore et al., 1987).Depending on absence of frameshift (Pr77, gag only), one frameshift (PrllO, gagpro), or two frameshifts (Pr160,gagpropol) different precursor proteins are produced (see Fig. 1B). The env proteins are translated from a 3.6-kb mRNA which is initiated at the same site as the full-length RNA but has spliced out the gag, protease, and pol regions. The envelope protein is first synthesized as a 73-kDa precursor glycoprotein (Pr73) and then cleaved after a RAKR sequence into the hydrophobic transmembrane gp36 and the more hydrophilic surface gp52 (Dickson and Skehel, 1974; Parks et d., 1974; Teramoto et d., 1974; Sarkar and Dion, 1975; Redmond et al., 1984;for review see Dickson and Peters, 1983).Several lymphoma (L1210, ML, and GRSL2) and mammary epithelial cell lines have been derived which express predominantly the uncleaved gp73 precursor molecule (Stuck et al., 1964; Zak-Nejmark et al., 1978; Nusse et al., 1979; van Blitterswijk et al., 1979; Vaidya et al., 1980; Racevskis and Sarkar, 1982). These cells form immature A-type (8090 nm) instead of mature B-type (105 nm) particles which lack env proteins (MenendezArias et al,, 1992). Cells secreting MMTV express the cleaved gp52 and gp36 molecules but no uncleaved envelope at the cell surface, Interestingly, B cells from BALB/c mice express only the unprocessed form of the env molecules from Mtv8 and Mtv9 (Lopez-Cepero et al., 1995).This could correlate with the observation that so far no MMTV production has been detected from infected B cells. The U3 region of the LTR of MMTV is much longer than that of most other retroviral LTRs and contains a complex set of transcriptional regulatory elements including steroid hormone responsive elements. The 3' LTR of MMTV contains an orf whose 5' end overlaps in a different reading frame with the 3' end of the env gene and which encodes the SAg (see Section 111). The SAg is most likely translated from a 1.7-kb spliced subgenomic mRNA initiated at the same site as the full-length RNA but lacking gag, pol, and most of the env sequences. A second promotor for generation of a SAg message has been recently described located further upstream in the 5' LTR (Gunzburg et al, 1993). In the env gene a third promotor structure was found (Miller et al., 1992; Zhang et al., 1996). Several other spliced mRNA molecules containing 3' LTR sequences with unknown functions can be detected by Northern blot analysis (2.4 kb) or with the polymerase chain reaction (PCR). The PCR

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products contain additional portions of the env gene (A. N. Shakhov, W. Held, and H. Acha-Orbea, unpublished observations). 3. Transcriptional Regulation A unique characteristic of the MMTV genome is its responsiveness to steroidhormones. High-level MMTVexpression in mammary glandis regulated transcriptionallyby corticosteroid receptor-mediated activation (for review see Nandi and McGrath, 1973;Ringold et al., 1983; Gunzburg and Salmons, 1992).The steroid hormone progesterone is most important for the increase in MMTV production during pregnancy. Four horinone-responsive elements are localized between nucleotides -,50 and -202 relative to the mRNA start site. They contain overlapping recognition sites for activated progestin and steroid receptors (Buetti and Diggelmann, 1983; Hynes et al., 1983; Majors and Varmus, 1983a). Binding of these receptors induces a great increase in transcription. All the older studies analyzed MMTV antigen expression in mammary tissues, in mammary tumor cell lines, or in lymphomas such as L1210.It was recently found that addition of glucocorticoidsor activation of B cells with LPS or cytokines such as IL-4 or IL-2 together with IL5 led to upregulation of endogenous Mtv mRNA by different mechanisms (King and Corley, 1990; King et al., 1990; Woodland et al., 1990; Lund and Corley, 1991).The mRNA levels were much higher in stimulated B cells than in T cell lines and comparable to those of mammary tumor cell lines. The half-life of these mRNAs was not altered. Increase in MMTV mRNA expression in B cells does not always lead to a stronger SAg presentation. SAg expression at the cell surface, however, is strongly increased by addition of IL-4, which s e e m not to affect overall MMTV transcription but rather upregulates MHC class I1 expression (Woodland et al., 1990). The level of SAg presentation did not correlate well with the presumed 1.7-kb SAg message levels in these studies. The interpretation of the results was that de no00 class I1 synthesis is required in B cells to form a complex with SAg, which can then be brought to the cell surface (Lund et al., 1993). 4. MMTV Znfection and Transmission

During lactation the infectious form of MMTV is secreted in large amounts into milk, thus transmitting the virus to the offspring (a less efficient form of transmission is discussed in Section II,A,5). The suckling pups have high amounts of viral particles in the intestinal lumen but very few within the epithelial cells (Hainaut et al., 1983; Bevilacqua et al., 1989). Retroviral proteins such as gp52, however, were found in all epithelial cells of the ileum and less in the duodenum and jejunum (Gonella and Neutra, 1984; Bevilacqua et al., 1989; Desaymard et al., 1993; Karapetian

SUPERANTIGENS OF MMTV

149

et al., 1994). In 10-day-old mice gp52 was distributed in a clear gradient within the gut villi with the majority in the tips and virtually nothing in the crypts. Most gp52 concentrated in small apical vesicles and in the large vacuoles of enterocytes. The gp52 staining did not colocalize with the staining for the secretory component of IgA (Karapetian et al., 1994). Because neonatal endothelium is known to take up lumenal macromolecules by fluid-phase endocytosis during the first 2 weeks of life, these data are in agreement with a lysosomal degradative pathway (Bevilacqua et aE., 1989). This finding could explain the large quantity of virus required to ensure the penetration of a small number of viral particles that finally infect. Dramatic changes occur in the gut epithelium with age. Two or three weeks after birth the neonatal-type epithelium is replaced by adulttype epithelium and the stomach acidifies. It is likely that these events are responsible for the lack of infection by MMTV via the mucosal route when mice are older than 2 weeks (Acha-Orbea, unpublished results). Viral DNA, a sign of successful infection, can be detected exclusively in the Peyer’s patches of the small intestine between Days 4 and 9 after birth. The infection then spreads to the adjacent mesenteric lymph nodes and all sites where lymphocytes are found (Karapetian et al., 1994).Peyer’s patches are lymphoid areas specialized in antigen sampling and are overlaid by the follicle-associated epithelium (FAE) consisting of M cells, enterocytes, and dendritic cells (Keren, 1992; Kelsall and Strober, 1996; Neutra et al., 1996). The FAE of newborns differs from their adult counterparts by their capacity to internalize proteins and to accumulate them in large vacuoles. M cells lack an organized brush border and its associated glycocalix, which facilitates uptake of proteins and microorganisms. They have been shown to be the portal of entry for reovirus, poliovirus, and human immunodeficiencyvirus (Wolf et al., 1981; Sicinski et al., 1990; Amerongen et al., 1991). Clearly, MMTV envelope protein was found in FAE and accumulated beneath the epithelial layer in contact with Peyer’s patch lymphocytes (Karapetian et al., 1994). Several mechanisms have been proposed for MMTV uptake, such as transport by M cells, entry through breaches in the epithelium, or transport via already infected maternal milk-derived lymphocytes. However, no clear evidence for any of these mechanisms has been reported so far. An alternative hypothesis was that MMTV, which is coated with anti-env IgG, can be taken up after binding to and transport by neonatal Fc receptors. A key role for this mechanism was recently disproved by the use of p2 microglobulin-deficient mice. These mice lack classic MHC class I molecules and the neonatal Fc receptor, which belongs to the same family of molecules and requires P2 microglobulin for its function (Abrahamson et al., 1979; Simister and Mostov, 1989). These mice acquire no maternal immunoglobulins and have no

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SANJIV A. LUTHER AND HANS ACHA-ORBEA

binding of IgG to the lumenal side of the gut epithelium, but are normally infectable with MMTV (Velin et al., 1996). Recent results have shown that MMTV primarily infects B cells in the Peyer's patches as well as in peripheral lymph nodes after virus injection (for MMTV life cycle, see Fig. 2) (Held et al., 1992, 199313; 0. Karapetian and H. Acha-Orbea, unpublished data). Immature A-type particles can be found in infected B cells but so far it has not been possible to show production of infectious MMTV particles by these cells. As described previously, lack of env processing leads to accumulation of immature MMTV particles. To date, it is not clear whether dendritic cells or macrophages are infected in the beginning. Only much later is viral spread to b

Virus production in milk

t t MMTV penetrates gut epithelium (day 1 to 14) t Babies drink infectious milk

Virus infects B lymphocytes exclusively in Peyer's patches

t Superantigen presentation by B cells (day 4 onward)

t t

T-B interaction Amplification of integrated virus by B lymphocyte division

t t Emigration of B cells with integratedvirus (day 14) t Anergization of T cells

Spread of virus between lymphocyte subsets without detectable viremia

t Infectionof mammary gland after puberty

t Development of mammary tumors after integrationclose to proto-oncogene

FIG.2. Life cycle of infectious MMTV.

SUPERANTICENS OF MMTV

151

other lymphocyte subsets detected. The picture changes after several weeks when MMTV reaches the mammary gland epithelium where enormous numbers of infectious particles are produced (Karapetian et al., 1994). Infectious particles have so far only been detected in mammary gland and its tumors and in other exocrine organs (see below). It is not clear what is required for MMTV replication and how MMTV can reach the mammary gland without production of infectious particles in lymphocytes. One possibility is direct cell to cell transfer; another is the presence of rare lymphocytes which can produce infectious particles. Direct cell to cell transfer of proteins or viral material has been reported on many occasions, especially between cells having intimate contact such as lymphocytes but also for mammary epithelial cells (Gay et al., 1970; Olsen et al., 1981; Abraham et al., 1988). For a few retroviruses the interactions between their env molecules and cellular receptors required for specific infection of target cells have been determined (Weiss and Tailor, 1995). Often these receptors are very complex, as in the case of H N in which CD4 and a chemokine receptor are required for infection (for review see Hill and Littman, 1996). Such a receptor structure has not yet been found for MMTV. A second key feature of most retroviruses is that the target cell has to be in cell cycle for integration of the retroviral cDNA to occur (Weiss et al., 1985; Roe et al., 1993). One exception is HIV, which expresses a karyophilic gag protein that allows integration into cells that are activated but not in cell cycle (Bukrinsky et al., 1993; Trono, 1995). It is very likely that the target cell either has to be in cell cycle at the time of infection or has to enter cell cycle concomitantly or shortly after infection. Little is known about the mechanism of MMTV infection because no reproducible and efficient in vitro infection assay has been available until recently (Vacheron et al., submitted). This task is complicated by the fact that even in vivo only a very small percentage of cells can be infected (Held et al., 1993b; 1994a).

5. M M T V Tissue Distribution Endogenous and exogenous MMTVs have very similar tissue distribution and tissue-specific expression. It is clear that exogenous MMTV can only be expressed in cells infected by the virus. From these cells either viral mRNA expression or production of infectious secreted virus or cytoplasmic virus could be measured. These studies were performed with several different strains of MMTV including MMTV(GR), -(SHN), -(KF), -(C3H), -(RIII), and the infectious particle derived from Mtvl. In order to determine the tissue distribution either expression of viral proteins [gp52 (env) or gp28 (gag)] by ELISA and histology or, alternatively, injection of cells or tissue extracts followed by evaluation of tumor incidence were per-

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SANJIV A. LUTHER A N D HANS ACHA-ORBEA

formed. High expression was consistently found in organs with exocrine function. Strongest expression was found in normal lactating mammary gland and mammary tumors but high levels were also observed in nonlactating mammary glands and in salivary glands. Much lower expression of these proteins was detected in seminal fluid and male reproductive organs including seminal vesicles, coagulating gland, epididymis, and prostate (Rongey et al., 1975; Hendrick et al., 1978; Kozma et al., 1980; Tsubura et al., 1981; Imai et al., 1983a). The product of the endogenous Mtv4 was found to be expressed in quite high amounts in sebaceous glands in skin (Koizumi et al., 1992). Most important, many reports document MMTV expression in lymphoid organs (Dux and Muhlbock, 1968; Nandi et al., 1972; Gillette et al., 1974; Noon et al., 1975; Verstraeten et al., 1975; Charyulu et al., 1979; Sarkar, 1986; Tsubura et al., 1988; Waanders et al., 1993b). By electron microscopy A- and B-type MMTV particles were found in milk, prostatic gland, parotic gland, submandibular gland, coagulating gland, and seminal vesicles. Despite extensive search for mature B-type particles these were never found in lymph node or spleen (Rongey et al., 1975;Taniguchi, 1987). Extracts of mammary gland, salivary gland, seminal vesicles, and seminal fluid were able to infect naive mice upon injection, whereas extracts from neither infected lymphocytes nor blood plasma could infect naive mice (Bentvelzen and Brinkhof, 1977; Morimoto et al., 1985). Transfer of infectious MMTV to the mammary gland was, however, observed after injection of normal mice with histocompatible splenocytes, B cells, CD4+ T cells, or CD8+ T cells from infected donors, suggesting that infection of lymphocytes does occur (see below) (Nandi et al., 1971; Waanders et al., 199313). Injection of allogeneic infected lymphocytes or lymphocyte extracts obtained from infected mice resulted in the absence of measurable infection. These observations go hand in hand with the observation that B cells have never been seen to produce infectious MMTV. Transmission of the virus to the neonate by milk is clearly the most efficient way of infection. Inefficient, but clearly detectable horizontal transmission of MMTV has been described by several authors (Moore and Holben, 1978; Morimoto et al., 1985).Male to male, male to female, and, to a lesser extent, female to female transmission was seen. Most likely this infection occurs through biting. Alternatively, infectious particles have been found in sperm. These breeding studies were complicated by the presence of endogenous Mtvl or Mtv2, which as proviruses can induce cancer at fairly late time points. The studies argue for an in utero transmission in a few of the mice (Andervont, 1963). 6. Mtv SAg Expression Mtu DNA is present in every cell of the body and expression is determined by the MMTV regulatory sequences and the site of integration.

SUPERANTIGENS OF MMTV

153

Three types of experiments were undertaken to show expression in different tissues: Immunoprecipitation studies, SAg-mediated T cell stimulation in vivo and in vitro, and RT-PCR. To detect SAg expression indirectly by stimulation of T cells, expression of MHC class I1 molecules or alternatively transfer of SAg from MHC class II-negative to MHC class II-positive cells is required. Shortly after the description of the Mls loci, it was shown that B cells were the best presenters of these SAgs (von Boehmer and Sprent, 1974). Env expression was found on a small subpopulation of B cells (<5%) (Lopez et al., 1985). Peritoneal as well as bone marrow macrophages were found to be incapable of presenting endogenous MMTV SAgs to T cells; tissue macrophages were never tested (Molina et al., 1989; Webb et al., 1989). Contradictory results were obtained when dendritic cells or T cells were investigated (Hamilos et al., 1989; Webb et al., 1989, 1990; Larsson-Sciard et al., 1990; Waanders et al., 1993a). Splenic dendritic cells did not stimulate MMTV SAg responses or responses of Mtv SAg-reactive T cell hybridomas (Webb et al., 1989, and references therein). These results contradicted earlier studies, which indicated good presentation capabilities of dendritic cells for these SAgs. What was not addressed by the authors was whether there is a synergistic effect of dendritic cells when tested together with B cells. Such a synergistic effect was shown by several authors (Hamilos et al., 1989; Inaba et al., 1991; Mazda et al., 1991). Similarly, the role of T cells in SAg production and presentation was contradictory. It became clear that CD8+ and, to a lesser extent, CD4+ cells can induce a SAg response in vitro when class II-positive presenting cells are present (Larsson-Sciard et al., 1990). There is evidence that the method by which T cells are stimulated determines whether they produce SAg mRNA and protein. In one study, PCR analysis revealed SAg expression in CD8+ T cells, activated B cells, and at low levels in thymic epithelium. However, dendritic cells, macrophages, and CD4' T cells were negative (Jarvis et al., 1994). Contradictory results were obtained by authors using fresh dendritic cells ex vivu or CD4' T cells stimulated in vitro by antigen but not by polyclonal activators (Arase-Fukushi et al., 1993; Arase et al., 1995; Ardavin et al., 1996). Transfer of MMTV SAg from cell to cell has been observed by many authors (see also Section V,B,3) but was hard to obtain reproducibly in vitro (DeKruyff et al., 1986; Pullen et al., 1988; Speiser et al., 1989). B. T ~ J M O INDUCTION R BY MMTV 1 . M a m m y Tumors MMTV is responsible for the majority of mammary carcinomas in mice. More than 100years ago mouse strains were described with high incidences of mammary carcinomas (Crisp, 1854). Later, inbred mouse strains were

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derived with high, intermediate or low mammary carcinoma incidence. In high-incidence strains up to 100%of mice had mammary carcinomas by the age of 1 year (Hageman et al., 1981). It was finally shown that the tumor-inducing agent was present in milk and could be transmitted by foster nursing (Bittner, 1936). Only later did it become clear that MMTV was responsible for this effect. Mammary tumors often appear in two wavesone around 6 months and a second one around 9 months. Forced breeding increased the tumor incidence dramatically. This is best illustrated in the review by Hageman et al. (1981)in which many breeding experiments with different MMTVs are summarized (see Table I). For example, 100% of force-bred BALB/c mice fostered by C3H mothers developed mammary tumors after 6 or 7 months, whereas the frequency was 84%at 11 months in virgin mice. These results clearly reflect the role of hormones in MMTV biology (see Section II,A,3). Contrary to other oncogenic retroviruses, MMTV does not carry an oncogene in its genome. One of the steps involved in oncogenesis is the integration of the viral genome close to host protooncogenes. Through the action of the 3’ LTR promotor and enhancer, genes that lie within about 20 kb of the LTR can be transcribed. Frequent integrations close to such protooncogenes were described, and so far at least six of these integration (int) genes (int-l to -6) have been characterized. Surprisingly, some MMTVs are much less efficient in inducing mammary tumors (discussed below). Functions of some of the int genes have been determined (Nusse and Varmus, 1982; Etkind and Sarkar, 1983; Peters et al., 1983, 1989; Gallahan et al., 1987; Morris et al., 1991; Robbins et al., 1992; Durgam and Tekmal, 1994; Sarkar et al., 1994; Durgam et al., 1995; Marchetti et al., 1995; Tekmal and Durgam, 1995). Recently, the nomenclature for some of them was changed to Wnt (a hybrid between wingless and int) (for review see Nusse and Varmus, 1992). Several indications exist that show that integration close to a protooncogene alone is not sufficient for tumor induction: (i) mice transgenic for int genes have an increased risk of tumor formation but only few mammary gland epithelial cells transform; (ii) tumors are usually oligoclonal and their growth is often, at least in the beginning, hormone dependent; and (iii) more often than expected on a random basis, integrations in Wnt-l and int-2 are found in the same tumor (Peters et al., 1986). Different MMTVs and strains of mice show preferential integration close to different protooncogenes. In C3H mice most tumors have an MMTV integration close to Wnt-1 with a high frequency of an additional integration in int-2. Other MMTVs and other mouse strains tend to have lower frequencies of integrations in these sites (Etkind, 1989). For many mammary tumors the genes activated by MMTV insertion remain to be identified.

SUPERANTIGENS OF MMTV

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Wnt-l codes for a secretory glycoprotein which acts on neighboring cells; int-2 and hst-l are members of the fibroblast growth factor family, and int-3 is a presumptive transmembrane receptor (Nusse and Varmus, 1992). The link between overexpression of these proteins and cancer formation is unknown. Not all MMTVs which efficiently infect a particular mouse strain lead to high incidences of mammary carcinomas. For example, MMTV(C3H) induces mammary carcinomas within 1 year in nearly 100% of BALB/c mice (reviewed in Hageman et al., 1981), whereas MMTV(SW) promotes cancer development in only very rare occasions (Held et al., 1992). This low level of tumor incidence was found despite the high levels of MMTV in the milk of the infected mothers. There is only an indirect link between the SAg and tumor formation. The role of the SAg is to establish a productive B lymphocyte infection which is critical for the infection of the mammary gland (as will be explained in more detail in Section IV). There is only one report which claims an important role for the SAg as an oncogene (Mukhopadhyay et al., 1995). It seems unlikely that the MMTVs found today represent a major selective disadvantage for the mouse strains carrying them. Tumor induction appears in general after 6 months of age, an age when mice already have reproduced and rarely survive much longer in the wild. The antitumoral immune response will be covered in Section N,B,6.

2. Other Tumors In rare cases MMTV-induced tumors other than mammary carcinomas have been described. Most were found in lymphocytes. Originally, it was surprising that the lymphocytes, which are the first targets of infection, are much less susceptible to tumor induction. When it was shown that all these lymphomas had a deletion spanning part of the 3’ LTR it became clear that lymphocytes are most likely protected from tumorigenesis by MMTV. The regions deleted in all these tumors contained the 3’ end of SAg as well as many gene regulation elements (Ball et al., 1985, 1988; Gunzburg and Salmons, 1992). Although the molecular reasons for this protective effect are poorly understood, tumorigenesis in the lymphoid compartment before viral transmission would represent an end point in the viral life cycle. A particular case was reported with endogenous Mtv29. This Mtv is found in SJL mice, which develop so-called reticular cell lymphomas after 1 year of age. It was shown that these tumors are in fact follicular B cell lymphomas due to overexpression of the Mtv29 SAg (Zhang et al., 1996). The tumors grew due to secretion of cytokines by the continuously activated SAg-reactive T cells. Two groups made very similar observations; one

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describing TCR Vp17 (Katz et al.,1988) and the other TCR Vp16 expressing T cells (Tsiagbe d al., 1993a,b) as the responsible cytokine secretors. In this system overexpression of the SAg in old mice leads to tumor formation and overcomes neonatal tolerance, a situation which could also be regarded as a case of autoimmunity. An indirect effect of Mtu SAgs on tumor formation was observed with polyoma-induced tumors, Mouse strains expressing Mtv7 are protected from these tumors on the H-2k background due to clonal deletion of T cells expressing TCR Vp6. Vp6' CD8' T cells are crucial for elimination of polyoma-induced tumors; in their absence polyomas appear (Lukacher et al., 1995). 3. Role of MHC and Mouse Strain in Tumor Susceptibility

In older literature, many reports on the relative insensibility of certain mouse strains to mammary tumor induction by MMTV are found. One of the best investigated resistant mouse strains is represented by C57BU6 (Bentvelzen, 1968). One of the reasons for their resistance to MMTV(C3H)-induced mammary tumors was the poor SAg presentation in the absence of I-E molecules (Pucillo et al., 1993; Held et al., 1994b). Other factors were mapped to MMTV-induced serum interferon secretion, which was higher in C57BU6 mice than in BALB/c mice (DeMaeyer et al., 1974). Using MMTV(C3H) infection of recombinant inbred strains derived from C57BLJ6 (low susceptibility) and BALB/c mice (high susceptibility) resulted in intermediate susceptibilities. These results clearly indicated that several genes are involved in tumor susceptibility (Bentvelzen et al.,1972; Heston and Parks, 1977; Dux et al.,1978). In crosses between two resistant strains high susceptibility to MMTV(C3H)-induced tumors was found, illustrating the highly complex genetics of tumor susceptibility (Nandi, 1974). The MHC locus plays a major role in susceptibility to MMTV-induced tumors (for the importance of MHC class I1 in SAg presentation see Section 111,B). Its influence on susceptibility was investigated using H-2 congenic B10 mice (Muhlbock and Dux, 1971, 1974). Susceptibility to MMTV(C3H) tumors correlates well with I-E expression. There are, however, exceptions. The most striking is represented by B1O.M Sn, which expresses the H-2f MHC haplotype. These mice cannot express I-Ea or -P due to mutations. Nevertheless, they are highly susceptible. An explanation for this paradox is the very slow deletion of SAg-reactive T cells in this MHC haplotype observed in MMTV(GR) transgenic mice expressing H-2f but not in mice expressing H-2b (Acha-Orbea et al., 1993).

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157

111. Shucture of the SAg Protein

One of the distinctive features of SAgs is the requirement for MHC class I1 expressing SAg presenting cells and the recognition of this complex by T cells bearing SAg-reactive TCR V/3 elements. Clear evidence for bacterial as well as rabies virus SAgs binding with high affinities to MHC class I1 molecules exists (Fraser, 1989; Herrmann et al., 1989; Mollick et al., 1989; Lafon et al., 1992). Careful mutational analysis and cocrystals between bacterial SAgs and human DR molecules have been published (Kappler et al., 1992; Jardetzky et al., 1994; Fraser and Hudson, 1995; Hudson et al., 1995). OF THE MMTV SAg PROTEIN A. STRUCTURE

Originally the orf was described to function as negative or positive regulator of gene expression (van Klaveren and Bentvelzen, 1988; Salmons et al., 1990). It became clear that it is this orf that encodes the SAg protein but it is currently not known whether it encodes other functions. SAg proteins are never detected in virions. It has been estimated that less than one SAg molecule is present per virion based on the detection limits of the methods used (Held et al., 1994a; Acha-Orbea, unpublished results). SAgs are polymorphic (see Figs. 3 and 4) and will therefore be named with MMTV or Mtv followed by SAg. The SAg of MMTV(SW) will be called MMTV(SW) SAg and the SAg of Mtv7 will be called Mtv7 SAg for the rest of this review. The first descriptions of the SAg protein were by in vitro translation of viral RNA (Dickson and Peters, 1981; Dickson et al., 1981). In parallel the open reading frame was found by DNA sequencing (Donehower et al., 1981, 1983; Fasel et al., 1982; Kennedy et al., 1982). Later, these experiments were repeated using microsomal membranes. These latter studies clearly showed that SAg molecules are type I1 proteins (Choi et al., 1992; Knight et al., 1992; Korman et al., 1992). Type I1 proteins are proteins which are inserted into the membrane with its COOH terminus on the outside of the cell. Except for the in vitro translation experiments it was very difficult to demonstrate SAg protein biochemically. They are expressed at extremely low levels in SAg expressing cells. First descriptions of rather high levels of SAg expression came from phorbole ester-stimulated L1210 or EL-4 cells. Several MMTV-infected lymphomas have been shown to express high levels of this glycoprotein of 3437 kDa but sequence analysis revealed that in these tumors part of the SAg sequence has been deleted (Ball et al., 1985, 1988; Racevskis, 1986) (see also Section II,B,2). It has not been determined whether this molecule is found on the cell

M t v MMTV MMTV ( SHN) Mtv29 (RCSI MMTVIFMI MMTVIRIIId)f Mtv7 Mtv53 ,MMTV(SW)c MMTV I JYG) Mtv43 Mtv5O Mtv8 Mtv8 Mtv9 Mtv9 Mtvll Mtv30 KMTVISIM)

*z

Mtvl7 Mtvl7 Mtv23 Mtv-SHNa Mtv-M12

0

MMTV(C3Hl MMTV(GR)e MMTV(I1 TES141

Mtvld MtvCjd Mt~l3~ Mtv3 Mt~44~ Mtv-MA1 MMTVIC41 MMTVlC3H-Kl MMTV(V1 Mtv48.Mtv51a MMTV I11 TES2) Mtv-DDO

(DBA/2, C3HI (DBA/Z, BXDI (DBA/Z, BXDJ (C58/JI (NZWI

(MI1 (BdLB/cJ fC3Hl

(BdLB/cI (CSJ

SUPERANTIGENS OF MMTV

159

FIG 3. Alignment of polymorphic COOH-terminal MMTV SAg sequences. The last 70 C-terminal amino acids of all published Mtu-SAg sequences are aligned. A dash represents an identical amino acid as shown in the sequence of MMTV(SHN)SAg. According to the sequence homology in the polymorphic C terminus the Mtu SAg can be grouped into seven families. The amino acid sequences were derived from the following references: Mtul (Crouse and Pauley, 1989; Pullen et al., 1992; Korman et al., 1992), Mtu3 (Pullen et al., 1992), Mtu6 (Pullen et al., 1992; Korman et al., 1992), Mtu7 (Beutner et al., 1992; Held et al., 1992).Mtu8 (Kennedy et al., 1982; Donehower et al., 1983; Kuo et al., 1988), Mtu9 (Kuo et al., 1988; King et al., 1990),M t u l l (Crouse and Pauley, 1989), M t u l 3 (Pullen et al., 1992; Korman et al., 1992), Mtul7 (Kuo et al., 1988; Korman et al., 1992), Mtu23 (Ignatowiczet d.,1994), M u 2 9 (RCS) (Tsiagbe et al., 1993b; Zhang et al., 1996).Mtu30 (Scherer et al., 1995),Mtu43 (Rudy et al., 1992),Mt044 (Rosenwasser et d.,1993),Mtu48 (Niimi et al., 1995),Mtu50 (Niimi et al., 1994a,b),Mtu51 (Nakamura et al., 1996), Mtu53 (A. N. Shakov and H. Acha-Orbea, unpublished data), MtuMl2 (Ignatowicz et al., 1994), Mtu-MA1 (Jouvin-Marcheet al., 1992),Mtu-DDO (Jouvin-Marche,1993), Mtu-SHNa and Mtu-SHNb (Koizumi et al., 1994), MMTV(V) (Kang et al., 1993), MMTV(BR6x) (Moore et al., 1987), MMTV(C3H)(Majors and Varmus, 1983; Choi et al., 1991), MMTV(C3H-K) (Wellinger et al., 1986; Acha-Orbea et al., 1991),MMTV(C4) (Shakov et al., 1993), MMTV(FM) (Yoshimoto et al., 1994), MMTV(GR) (Fasel et al., 1992), MMTV(JYG) (Nishio et a/., 1994; Luther et al., 1994), MMTV(MB) and MMTV(RII1d) (I. Xenarios and H. AchaOrbea, unpublished data), MMTV(SHN) (Luther et al., 1994), MMTV(S1M) (Maillard et al., 1996), MMTV(SW) (Held et al., 1992), MMTV(I1 TESB) (Ando et al., 1995), and MMTV( I1 TES14) (Wajjwalku et al., 1995). Some corrected sequences were taken from Brandt-Carlson et al. (1993). The Vfl specificities were shown the following references: Mtul (Abe et al., 1988; Fry and Matis, 1988; Frankel et al., 1991; Pullen et al., 1992),Mtu3 (Abe et ol., 1988; Fry and Matis, 1988; Fairchild et al., 1991; Pullen d al., 1988, 1992; McDuffie et al., 1992; Scherer et al., 1995), Mtu6 (Abe et al., 1988; Fry and Matis, 1988; Woodland et al., 1991a; Frankel et al., 1991; Pullen et al., 1988, 1992; Jouvin-Marche et al., 1992; Braun et al., 1995), Mtu7 (MacDonald et al., 1988; Kappler et al., 1988; Happ et al., 1989; Okada et al., 1990; Braun et al., 1995; Scherer et al., 1995), Mtu8 (Vacchio et al., 1990; Dyson et al., 1991; Abe et al., 1991; Ryan et al., 1991; Woodland et al., 1992b; Tomonari et al., 1994; Braun et al., 1995; Scherer et al., 1995), Mtu9 (BU et al., 1989; Woodland et al., 1990; Vacchio et al., 1990; Dyson et nl., 1991; Woodland et al., 1991a,b; Abe et al., 1991; Ryan et al., 1991; Tomonari et al., 1994; Braun et al., 1995; Scherer et al., 1995). Mtull (Dyson et al., 1991; Abe et al., 1991; Ryan et al., 1991; Foo-Philips d al., 1992; Tomonari et al., 1994),M t u l 3 (Pullen et al., 1988, 1992; Abe et al., 1988; F I ~ and Matis, 1988; Frankel et al., 1991; Cazenave et al., 1990), Mtul4 (Scherer et al., 1995),Mtul7 (Scherer et al., 1995).Mtu23 (Ignatowiczet al., 1994),Mtu29 (RCS) (Tsiagbe et al., 1993a; Katz et nl., 1988),Mto30 (Scherer et al., 1995), Mtu43 (Rudy et al., 1992), Mtu44 (Fairchild et al., 1992),Mtu48 (Niimi et al., 1995),Mtu50 (Wajjwalku et d., 1993), M t d l (Niimi et al., 1995),Mtu53 (A. Shakov and H. Acha-Orbea, unpublished data), MtuM12 (Ignatowicz et al., 1994). Mtu-MAI (Jouvin-Marche et al., 1992), Mtu-DDO (Jouvin-Marche,1993), MMTV(V) (Hodes et d.,1993),MMTV(C3H) (Marrack et al., 1991; Choi et al., 1991), MMTV(C3H-K) (Acha-Orbeaet al., 1991), MMTV(C4) (Shakov et al., 1993), MMTV(FM) (Yoshimoto et al., 1994; Matsuzawa et al., 1995), MMTV(GR) and Mtu2 (Acha-Orbeaet al., 1991), Mtu2 (Ferrick et d.,1992), MMTV( JYG) (Nishio et al., 1994; Luther et ol., 1994), MMTV(MB) and MMTV(RII1d) (I. Xenarios and H. Acha-Orbea, unpublished data), MMTV(SHN) (Luther et d.,1994), MMTV(S1M)

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SANJIV A. LUTHER A N D HANS ACHA-ORBEA

surface. Due to lack of the COOH-terminal amino acids of the protein, these molecules cannot function as SAgs anymore. Either full-length SAg molecules or, more likely, gene regulation elements in this region of the LTR are inhibiting lymphocyte transformation. Four groups have reported monoclonal antibodies or antisera against COOH-terminal peptides of Mtv7 (Acha-Orbea et al., 1992; Haga et al., 1992;Winslow et al., 1992; Mohan et al., 1993).These reagents react with the native SAg because they clearly block SAg function. Despite the fact that all four groups were able to use these antibodies in biochemical experiments, such as immune precipitation and flow cytometry with overexpressing cells, only one group was able to detect cell surface expression on lipopolysaccharide-activated (but not on resting) SAg-presenting B cells by flow cytometry (Winslow et al., 1992). In this study B cells expressing MHC class I1 molecules were unable to present MMTV SAgs (H-2q) and T cells were negative. Immunoprecipitation from SAg overexpressing insect cells or transfectants revealed molecules of about 45 kDa, indicating glycosylation (BrandtCarlson and Butel, 1991; Acha-Orbea et al., 1992; Winslow et al., 1992, 1994). These molecules were not found on the cell surface and were modified with high-mannose oligosaccharides typical for endoplasmatic reticulum resident proteins (Winslow et al., 1994). These molecules had a short half-life and were of low abundance even in the transfected cells. Analysis of SAg expression using recombinant vaccinia viruses revealed

FIG.3. Continued (Maillard et al., 1996), MMTV(SW) (Held et al., 1992; Papiernik et al., 1992), MMTV(I1 TES2) (Ando et al., 1995), and MMTV(I1 TES14) (Wajjwalku et al., 1995; Ando et al., 1995). The references for the genetic linkage between Mls phenotypes and Mtv loci are listed in Table I of Acha-Orbea et al. (1993). “Mtu48 and Mtu51 differ by two nucleotide replacements in the N terminus of the SAg causing amino acid changes (Niimi et d.,1995; Nakamura et al., 1996). MMTV(CS) (Niimi et al., 1995) and MMTV( I1 TES2) (Andoet d., 1995)are both identical to Mtu48 in the C-terminal amino acid sequence of the SAg. bMtu44 can be put either in the second or sixth listed group because it is reactive with Vps specific to the two groups (Rosenwasser et al., 1993). Alternatively, Mtu44 could be linked to one of the three still unidentified Mtus in NZW mice. “MMTV(SW)is identical in the C-terminal amino acid sequence with the Mtu53 found in outbred Swiss mice (A. N. Shakov and H. Acha-Orbea, unpublished data). dA4tul, Mtu6, and Mtul3 do not differ in their C-terminal amino acid sequence of the SAg. Mtul and Mtu6 share the same amino acid sequence for the whole SAg but differ by eight nucleotides on the DNA level. Mtul3 has two amino acid changes in the N-terminal sequence compared to Mtul and Mtu6 (Pullen et al., 1992). “MMTV(GR)is also called Mtu-2 or DDD-Mtu-2 (Matsuzawa et al., 1990).fRIIImice from different sources harbor most likely different infectious MMTV strains. Therefore, we termed the MMTV from Dutch RIII strains MMTV (RIIId) and the one from Japanese RIII mice MMTV (RIIIj).

ATGaa1 ATGaa38

ATGaa 122

-

SUPERANTIGENS OF MMTV

161

NH;

----)

-

ATG aa 143

Transmembrane region aa 45-67 RARR 68-71

-

Potential N-linked glycosylation sites aa 79-81,89-91,93-95, 131-133, 146-148

RKRR aa 168-171 c- RGKR aa 193-196

ATG aa 163

Polymorphic region aa 290-320

COOH

FIG.4. Predicted structure of the SAg protein. Indicated are the three potential furine cleavage sites (RARR and RKRR) as well as the five potential start codons.

a short half-life of about 1.5 hr for the 45-kDa protein in COS cells (Krummenacherand Diggelmann, 1993).Most of the product remained in the high-mannose form and was most likely degraded in the endoplasmatic reticulum (Krummenacherand Diggelmann, 1993; Winslow et al., 1994). One group was able to analyze the cell surface form after iodination using two antibodies directed against the COOH or NH2 terminus of the molecule, respectively. Both antibodies precipitated the COOH-terminal iodinated 18.5-kDa polypeptide from the cell surface. The interpretation of the results was that the molecule was cleaved but still remained associated on the cell surface (Winslow et al., 1992, 1994). Unfortunately, to date it has been impossible to use extracellular recombinant SAg molecules which show SAg activity; therefore, the nature of the molecules with SAg activity still remains to be determined. There are five in-frame potential start codons (ATG) found in the part of the gene encoding the NH2-terminal180 amino acids of most described sequences. It is not clear which proteins are produced by infected lympho-

162

S A N J N A. LUTHER AND HANS ACHA-ORBEA

cytes or mammary gland cells, What became clear was that the most potent SAgs initiate from the first ATG and once the potential transmembrane region at positions 4567 is removed in either transgenic mice or after transfection of engineered molecules, no SAg function is detectable (Choi et al., 1992; Lambert et al., 1993).The full-length protein has the strongest activity, molecules starting at amino acid 38 have reduced activity, and shorter constructs fail to induce SAg stimulation after transfection. These results, however, do not exclude that these shorter forms could act as SAgs or have other unknown functions. Potentially, removal of the hydrophobic sequences could alter trafficking in the cell. The SAg proteins initiating at the first start codon are between 313 and 325 amino acids long. The predicted molecular weight of nonglycosylated proteins is around 38 kDa. It is a glycoprotein containing four to six Nlinked potential glycosylation sites in the NH2-terminal half of the molecule (positions 79, 80,90,94, 132, and 147).The consensus glycosylation motif for eukaryotic proteins is NXSI", where X is any amino acid. It seems unlikely that asparagine molecules which are very close together will be glycosylated. Therefore, most likely, a maximum of four glycosylations are found per SAg molecule after expression in insect cells or after biochemical analysis (Brandt-Carlson and Butel, 1991;Acha-Orbea et al., 1992;Winslow et al., 1992, 1994). The COOH-terminal 2537 amino acids are highly polymorphic. The known sequences can be grouped into seven families based on this polymorphism (see Fig. 3). This polymorphism correlates well with the TCR Vfi specificities of the SAgs (Acha-Orbeaet al., 1991; Choiet al., 1991).Several studies have implicated this part of the molecule in interactions with the TCR Vp elements or the TCR (Acha-Orbea et al., 1992; Winslow et al., 1992; Mohan et al., 1993; Yazdanbakhsh et al., 1993). The polymorphic COOH-terminal amino acids of SAg proteins are shown in Fig. 3. Low levels of homology were found between the polymorphic COOH termini of different families but there is an amino acid identity of over 93%in the rest of the molecule. Based on these sequences many predictions on the characteristics of the SAg protein were made, most of which were confirmed later. A schematic structure of the SAg protein is shown in Fig. 4. Most SAgs show six or seven cysteine molecules but intramolecular disulfide bridges seem not to be required for SAg function. Three are located in the previously described transmembrane region, one or two in the NH2-terminal intracellular portion (positions 14 and 35), and one or two in the extracellular COOH-terminal fragment (positions 115 and 264). Several stretches of basic amino acid motifs are found in SAg molecules. These sites were thought to be sites for processing by endoproteases recognizing di- or tetrabasic amino acids (Barr, 1991; Steiner et al., 1992).

SUPERANTIGENS OF MMTV

163

The presence of tetra- and dibasic motifs suggested potential cleavage by subtilisin-like proteases, also known as proprotein convertases. Furins are typical members of such mammalian proteases (also known as PACE). They recognize the motif RXWRR and are responsible for the processing of precursor proteins in the secretory pathway. Two or three such potential sites are found in all known MMTV SAgs. The first just after the transmembrane domain positions 6872, the second at positions 169172, and the third at positions 192195. Cleavage at position 71 by furins has been observed in vitro and this sequence is conserved in most MMTV SAg sequences (Park et al., 1995). Only three of the so far sequenced MMTV SAgs have mutations resulting in a change from RARR to RACR (MMTV(SW)), to RAHR (MMTV(RCS), and to SARR (MMTV(C3H-K). The first two still represent a more degenerate furine motif (RXXR), whereas the latter does not allow cleavage by furins, which require the presence of arginine at position -3 relative to the cleavage site. The SAg of MMTV(C3H-K) is not functional possibly because cleavage at this site is required to render the SAg functional (Acha-Orbea et al., 1991; Shakhov et al., 1993). It would render the SAg soluble and allow transfer of SAg from cell to cell as documented in vitro and in vivo. The second site also can be cleaved in vitro by furins and it was suggested that cleavage at this site is required for the production of the 18.5-kDa fragment. Mutating this site prevents generation of this fragment. Four functional SAgs, however, have not retained this site; they all have histidine at the potential cleavage site [(Mtv44, MMTV(IITES14), MMTV(FM), and MMTV(IITESS)]. The third site is only conserved in 16 of 28 sequences. Furthermore, in vitro this site is not cleaved by furins. Cell lines deficient in furins were incapable of SAg presentation; however, after supertransfection with the relevant furin gene, presentation can occur. This result is a good indication of the requirement for cleavage of the SAg but does not yet formally prove the necessity of cleavage of the SAg by furins. It could also be interpreted as being important for maturation of MHC class IT molecules (Mix and Winslow, 1996). The positive charge (CCK) in the presumed transmembrane region seems not to be important for SAg function. Often positive charges in transmembrane regions indicate interactions with negative charges on other proteins (Yazdanbakhsh et al., 1993). There are nearly identical SAg sequences which show slightly different TCR Vp specificities. Most likely this is due to different affhities for TCR Vp and/or different expression levels of the endogenous SAgs. This latter possibility is supported by the observation that the highly expressed Mtu6 and Mtv9 SAgs delete, in addition to Vp3, a T cell population expressing TCR Vp5 which is not or only poorly recognized by similar SAgs which

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are expressed at lower levels (Mtvl and Mtv8). When chimeric SAg molecules were tested for activity, the V/3 specificity correlated with the sequence at the COOH terminus (Yazdanbakhsh et al., 1993). In summary, it is not yet clear whether and where the SAgs are cleaved and which form of the SAg represents the functional SAg. It is also possible that other sites are cleaved by proteases such as granzymes. Several groups have introduced mutations in these sequences and the results are so far quite contradictory.

B. INTERACTIONS WITH MHC CLASS I1 There are several indirect arguments suggesting an interaction between MMTV SAgs and MHC class I1 molecules. First, stimulation of T cells by Mtu SAgs can be completely blocked with monoclonal antibodies to MHC class I1 antigens (Wall et al., 1983). Second, there is a clear hierarchy in Mtu SAg presentation by MHC congenic mouse strains. I-E molecules are the best presenters for all the described Mtu SAgs. In mouse strains lacking the I-E isotype a hierarchy between the gene products of the different IA alleles is observed for Mtv7 SAg: I-Ak > I-Ab > I-Ad % I-As % I-A" (DeKruyff et a!., 1986; Kappler et al., 1988; MacDonald et al., 1989a). None of the described MMTV SAgs is able to be presented by I-As molecules. MMTV SAgs can be divided in two major groups. One group requires expression of I-E molecules for presentation to occur, whereas the other can be presented by both I-A and I-E molecules (see Tables I and 111). Most likely, different SAg expression levels and/or lower binding affinity for I-A than for I-E are responsible for these observations. As for bacterial SAgs, MHC class I1 molecules from different species can present MMTV SAgs to human, rat, or mouse T cells. Most of these results have been obtained with endogenous Mtu SAgs. Each endogenous Mtu is present in a different chromosomal environment (see Table 111),and therefore can be expressed at different levels. However, very similar results have recently been obtained for presentation of SAgs from infectious MMTV by I-E, I-Ab, and I-As (Pucillo et al., 1993; Held et al., 1994b). Only one report showing direct interaction of MMTV SAgs with MHC class I1 has appeared in the literature (Mottershead et al., 1995). This is due to the difficulties encountered in producing recombinant MMTV SAg molecules. Bacterial recombinant SAg fragments containing the COOHterminal half of the SAg molecules have been used to measure binding to HLA DR1 and DR4. Results show binding to the /3 chain of the class I1 molecule. MHC class I molecules were used as negative controls. No biological function of these recombinant SAgs was detectable. These results are in conflict with experiments using SAg peptides to block SAg presenta-

SUPERANTIGENS OF MMTV

165

tion that indicate that the region lacking in this construction is responsible for binding (Torres et al., 1993). Several lines of evidence indicate that human MHC class I1 molecules can present MMTV SAgs. Transgenic mice expressing either HLA-DQ or HLA-DR delete the expected T cell populations (Zhou et al., 1991, 1992; Altmann et al., 1993).Analysis of L cells cotransfected with human MHC class I1 genes and M t v 7 or M t v 9 SAgs indicated good SAg presentation by DR1 and DR4, whereas several other DR alleles as well as DP and DQ isotypes were not able to present efficiently (Thibodeau et al., 1994). Structural polymorphisms between these MHC class I1 molecules might help to localize the important SAg interaction residues. Mutated human DR genes which did not interact with the two bacterial SAgs TSST-1 and SEB were used to show that bacterial and MMTV SAgs bind to different locations on the MHC class I1 molecules (Subramanyam et al., 1993; Thibodeau et al., 1994). WITH TCR Vp C. INTERACTIONS The first indication of the parts of the TCR implicated in MMTV SAg interaction came by analysis of TCR allotypes found in wild mice which had gained or lost the capability to interact with a particular MMTV SAg. The observed sequence polymorphisms could then be used to map the key amino acids in TCR-SAg interaction. M t v 7 SAg normally interacts with TCR Vp6, 7, 8.1, and 9, but not with TCR Vp8.Z. or TCR Vfi8.2b. It was observed that the highly homologous Vp8.2 sequences found in wild mice were able to interact with this SAg. Analysis of these TCR sequences of Vp8.2 T cells isolated from wild mice revealed a few amino acid changes compared to nonreactive Vp8.2 sequences (Pullen et al., 1990b; Braun et al., 1995). In addition, site-directed mutagenesis experiments confirmed important residues at positions 22, 70, and 71 on TCR Vp8.2 (Pullen et al., 1991). Similar studies with different Vp17 alleles derived from wild mice implicated residues 48 and 74 in recognition of Mtv SAg (Cazenave et al., 1990). The five amino acids described in the two studies are close together in the predicted TCR /3 chain structure. Position 48 is in CDR2, whereas the others (22,70,71, and 74) are located in CDR4 and are brought together to form a flat surface on the lateral side of the TCR, far away from the supposed peptide interaction site. Recent results show that TCR binding sites for bacterial and MMTV SAgs are different (Liao et al., 1996) (Fig. 5 ) . Similar analyses were made with rat TCR molecules. Different members of the TCR Vp8 family were analyzed for M t v 7 reactivity. Analysis of genomic rat TCR V/38 sequences which were supposed to interact or not to interact with M t v 7 SAg, implicated residues 9, 14, 62 (Lew 8.2 reacts

166

SANJIV A. LUTHER AND HANS ACHA-ORBEA

M MTV(SI M )

FIG. 5. Dendrogram of the murine TCR V/3 elements. This dendrogram shows the relationship between all variable regions of the mouse TCR /3 chain (based on peptide sequence alignments by Arden et al., 1995).The longer the line that joins two sequences, the greater the divergence; all the other parameters are arbitrary. The circles show the VP specificities of SEB and of some MMTV SAgs.

and F344 does not), 15, and 83 (Lew 8.5 reacts and F344 does not) (Gold et al., 1994). An independent study of rat TCR sequences responsive to Mtv7 SAg came to different conclusions (Herrmann et al., 1994). Comparison of cDNA sequences of T cell hybridomas from responder and nonresponder rat strains localized differences in TCR Vp8.2 (but not Vp 8.5) to amino acids 14, 50, 51, and 7073. Except for residue 26, which is found in the CDRl and involved in TCRMHC class I1 interactions, many of these residues implicated in SAg interaction are clustered in a loop of the predicted TCR structure corresponding to the hypervariable region 4 (HV4) of immunoglobulin heavy chain molecules. It is predicted that this part of the TCR Vp domain is localized on the lateral side of the molecule, relatively far away from the conventional peptidelMHC binding region as described previously.

167

SbPEhNTIGEhS OF MMTV

Human TCRSreact ~ lwith l A4tb SAgs when rksented in an appro i-iate MHC class 11 contekt (Labrtxqhe et al., 1993 ; Thibodeau et al., 994). Among the V/3 families of htlman T cells reacting with Mtt17,SAg conserved sequence6 were found in the predicted FlV4 loop. The human TCR Vfll2, -13, -14, -l&and -93 elements responding in this case wei-e the most homologous to the ~ d c t tnotme i ~ TCR sequences (Labrecque et al., lQ93b). Seveval reports have appeared in the literature which show that TCR V a is also involved in SAg recognition. Several Va elements were found rarely in MMTV SAg-reactive T cells. Among the Mtv7-reactive T cells a preference for V d l + T cells was found, whereas among the Mtv7nonreactive T cells V&B+ 7' cells were found at increased percentages (Smith t% d., "3;V d ~ h i oet al., 1992; Blackman et al., 1993; Waanders et d., IW3b).A mle for J elements has also been reported (Candeias et at., 1BOl; Pullen &td., 1996). Recent festlkts indicate that recombinant Mtv7 SAg proteins bind with high afhity to TCh Vfl rholedes (Mottershead et al., 1995). Over&, these studies indicate that the SAg binds to the lateral side of the TCR and MHC m d e d e s . The SAgTCR MHC interaction might result in an altered signding quality. Signalirig thmu h the TCR complex is different in response to Mt07 SAg compared to c assical antigens when T cell hybridomas were used. Proliferation was observed for SAgs as well as for classical antigens. MMTV SAg stimulation resulted in reduced calcium mobilization and reduced inositol phosphate changes. Activated B cells as antigen presenting cells, however, resulted in activation of the phosphatidyl inositol turnover. Signaling through the TCR complex was different in response to Mt07 SAg compared to classical antigens when T cell hybridomas wwe used. Proliferation was observed for SAgs as well as for classical antigens, MMTV SAg stimulation resulted in reduced calcium mobilization and reduced inositol phosphate changes. Activated B cells as antigen presenting cells, howewr, wsulted in activation of the phosphatidyl inositol turnover (O'hotirke et at., IS90: Caugker et al., 1991; Liu et d., 1991; Webb et ul., 1094; Weber et at., 1995). The role of bound peptides in this interaction is not known yet (Woodland et al., 1997).

1

1p

pl

IV. Immune Response to MMTV The immune response to MMTV can be divided into three major phases. Within hours after v i r u s uptake a T cell- and SAg-independent B cell activation occurs (see Section W,A). After 1or 2 days the infected B cells present the SAg on their cell shrface, which leads to a SAg-dependent T-B collaboration.This second phase leads to proliferation and differentiation of

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both T and B cells very similar to classical T-B interactions and peaks between days 4 and 6 (see Section IV,B). The third phase is characterized by a chronicimmune response which is observed lifelong after the infection. It is not yet clear whether this persistent infection is dependent on SAgs (see Sections IV,B,4 and IV,B,5). A. SAg- AND T CELL-INDEPENDENT IMMUNE RESPONSE TO MMTV Retroviruses (with the exception of HIV) are well known to require cycling cells in order to accomplish integration of reverse transcribed viral DNA (Varmus and Swanstrom, 1984; Coffin, 1996; Bukrinsky et al., 1993; Roe et al., 1993).Therefore, for MMTV two possible scenarios to achieve integration of viral DNA into the host B cell can be imagined: Either MMTV infects proliferating or activated B cells or it directly activates B cells by the infection process, e.g., by cross-linking of B cell surface molecules or by production of growth factors. Several observations argue for a mechanism of direct B cell activation with no involvement of the SAg protein. First, MMTV(SW)injection into lupus-prone Yaa mice did not lead to an increased efficiency of infection although these mice had large numbers of B cells in cell cycle (S. Izui and H. Acha-Orbea, unpublished observations).Second, addition of high doses of MMTV(C3H) to nylon-wool-purified B cells in vitro led to B cell proliferation by day 3 after coincubation (Lopez et al., 1985). Similarly, 4-6 days after injection of MMTV(SW) into BALB.D2 mice, which have deleted the MMTV(SW)-SAg-reactiveT cells, or into BALB/c nulnu mice increased B cell numbers, but not VP6’ CD4+ T cell numbers, were observed (Held et al., 1993a).Third, injection of normal or athymic mice with MMTV(SW)resulted in a rapid and transient activation of the majority of B cells independently of T cells. The infection was preferentially detected in the activated B cell population (C. Ardavin et al., submitted). Fourth, experiments using the reverse transcriptase inhibitor 3’-azido-3’-deoxythymidine (AZT)have shown that there are insufficient amounts of SAg protein present in the mature virion to induce a local immune response (Held et al., 1994a).Collectively, these data suggest that MMTV particles have the inherent capacity to activate resting B cells directly. The clearest evidence for T-independent B cell activation comes from the observation of a biphasic B cell activation during the first 3 days after infection of adult mice with MMTV (C. Ardavin et al., submitted). Within 18 hr up to 80% of all B cells present in the popliteal lymph node undergo polyclonal activation, as judged by upregulation of early activation markers such as CD69 and CD86 (B7-2).This upregulation occurs exclusively on B cells and is transient, as downregulation occurs between 21 and 29 hr after infection. Only a few hours later (36-92 hr), B cells and SAg-reactive

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T cells reexpress these activation markers. These two phases of B cell activation show clearly different characteristics. The first phase is SAg and T cell independent and does not involve any measurable cellular proliferation. In contrast, the second phase of B cell activation is absent in BIO.G (H-2’3)and BALB/c ndnu mice, which cannot present or respond to the SAg, respectively. Thus, this phase of B cell activation involves the presentation of the SAg molecule on the surface of infected B cells followed by the interaction of these B cells with the SAg-reactive T helper cells. The T cell dependence of this second phase is also reflected in the activation and cell division of both B cells and SAg-reactive T cells (see Section IV,B). It has been proposed that the first wave of polyclonal B cell activation is due to cross-linking of surface molecules present on most or all B cells but not T cells because such an activation is not seen after vaccinia virus infection. The virus integration preferentially detected among activated B cells (CD69’) suggests that this activation event possibly allows virus penetration and integration. Because only a fraction of the activated B cells contains viral DNA, the involvement of a second factor, such as a clonally distributed receptor, has been proposed. This interpretation will be discussed under Section IV,C. The loss of CD69+ B cells after 2129 hr could be explained either by the downregulation of the activation marker or by cell death or emigration of partially activated B cells. Histological analysis performed on draining lymph nodes 24 hr after infection shows that apart from an increased cell number, the distribution of cells as well as the number of cycling or dying cells (after a 2-hr pulse with BrdU) resembles a nondraining lymph node (Luther et al., 1997). Therefore, downregulation of the activation marker seems more plausible, but more conclusive studies are needed to settle this point. Similar observations were made with other viruses and T-independent antigens. Most antigens can induce B cell differentiation if help is provided by specific T helper cells. T helper cells recognize the antigen usually in the form of peptides complexed to MHC class I1 molecules. However, a heterogenous group of antigens has the inherent ability to activate B cells in nude mice, thereby bypassing the usual requirements for conventional antigen presentation and direct T cell help (Kindred, 1979).Antigens which can induce such a T-independent (TI) B cell activation have been classified in TI-1 and TI-2 antigens according to their activity in neonatal mice and in X-linked immunodeficient (xid)CBMN mice (Mond et al., 1979, 1995; Scher, 1982). B cells from CBA/N mice lack Bruton’s tyrosine kinase (Btk) and can be activated by TI-1 antigens but not by TI-2 antigens. Similarly, neonatal B cells respond to TI-1 antigens. TI-1 antigens comprise direct B cell activators such as mitogens. TI-2 antigens have characteristics embodied by polysaccharides, such as high molecular weight, repeating anti-

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genic epitopes (Feldman and Nossal, 1972; Dintzis et al., 1989), and inability to induce MHC class II-restricted T help (Harding et al., 1991). Although no cell contact with T cells is needed, TI-2-induced B cell differentiation is thought to be dependent on factors derived from antigen presenting cells, T cells, or NK cells. The mechanism underlying this B cell activation is still enigmatic (Coutinho and Moller, 1975; Mond et al., 1995). In contrast, TI-1 antigens activate B cells directly possibly by implicating surface immunoglobulins. One group, termed B cell SAgs, binds to particular families of Ig-VH domains independently of the light chain and D or J regions (Pascual and Capra, 1991; Zouali, 1995). The best studied B cell SAg is staphylococcal protein A, which has been shown to bind to the FR3 region of VH3-expressingB cells (a region homologous to the solventexposed CDR4 domain of the TCR Vp gene product involved in binding of T cell SAgs) (Sasso et al., 1988). SPA selectively activates VH3-expressing B cells and induces their differentiation into Ig-secreting cells (VasquezKristiansen et al., 1996). Other B cell SAgs are SED (Dominati-Saad et aE., 1996) or HIV a 1 2 0 (Berberian et al., 1993; David et al., 1995),which have been reported to rescue or delete VH subpopulations, respectively. Thus, B cell SAg can either activate or delete B cells expressing the same immunoglobulin VH region, possibly by cross-linking Ig molecules to various degrees. Viruses, bacteria, and parasites have been shown to be composed of T cell-dependent and -independent antigens (Feldman and Basten, 1971; Burns et al., 1975; Anders et al., 1984; Milich and McLachlan, 1985;Charan and Zinkernagel, 1986; Reinitz and Mansfield, 1990). The rigid array of repetitive epitopes on some enveloped viruses may allow extensive crosslinking of surface immunoglobulins (Burns et al., 1975). In support of this, haptens spaced approx. 10 nm apart most efficientlyactivate B cells (Dintzis et al., 1976,1989). The envelope proteins of VSV particles were calculated to be spaced 5-10 nm (Bachmann et al., 199313).Despite extensive search, none of the known second signals (CD40, complement receptor, tumor necrosis factor a,and NK cells) were required for B cell activation by VSV particles (Fehr et al., 1996). Rather, it seems that the pattern of antigen is important and sufficient. Although whole VSV particles behaved as TI-1 antigens, deaggregated VSV glycoproteins behaved like TI-2 antigens (Bachmann et al., 1995). The signal given to B cells by whole virus particles was potent enough to break B cell tolerance in mice transgenic for VSV glycoprotein, whereas deaggregated glycoprotein was not. It was postulated that B cell tolerance might be defined by antigen patterns rather than self-nonself recognition (Zinkernagel et al., 1990; Bachmann et al., 1993b, 1995; Zinkernagel, 1996).

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It is generally believed that direct B cell activation by viruses leads to a rapid and short-lived secretion of IgM antibodies (peaking on Days 3-5), lacks isotype switch, and allows no memory formation (Ahmed and Gray, 1996; Zinkernagel, 1996). Although IgG responses have been reported in T cell-deficient mice in the case of polyoma virus (Szomolanyi-Tsudaand Welsh, 1996) or high-dose injections of VSV (Freer et al., 1995), nothing is currently known about the signaling within these B cells or on their infection status. As for VSV, env molecules of MMTV are spaced 7.4 nm apart as determined by electron microscopic studies (Sarkar and Moore, 1974). The results of T cell-independent B cell activation early after MMTV encounter are in agreement with such a mechanism. In addition, MMTV infection leads to a very rapid generation of virus envelope-specific antibodies which is strongly increased upon help by SAg-reactive T cells (Luther et al., submitted). Interestingly, injection of high doses of MMTV(SW) into BALB.D2 or B1O.G mice generated a detectable antiviral antibody response, The results suggest that MMTV particles might activate virus envelope-specific B cells in a T cell-independent manner. This could point toward a critical role for B cell surface molecules in infection (see Section IV,C on virus receptor). Clearly, although T-independent B cell activation might be essential for early events in virus infection, the successful completion of the MMTV life cycle is strongly dependent on SAg-reactive T cells (Squartini et a!., 1970; Tsubura et al., 1988; Golovkina et al., 1992; Held et a!., 199313).

B. SAg-DEPENDENT IMMUNE RESPONSE TO MMTV 1. Neonatal T and B Cell Responses to Erogenous MMTV Milk-borne MMTV particles infect offspring during the first 2 weeks of life and require a SAg response to establish an efficient lifelong infection (Golovkina et al., 1992; Held et al., 1993b). On Day 4 after birth, pups nursed on MMTV(SW) milk showed detectable infection among Peyer’s patch B lymphocytes as detected by amplification of reverse-transcribed MMTV cDNA (Karapetian et al., 1994; 0. Karapetian, unpublished observations). One day later an increase in SAg-reactive VP6+ T cells became measurable and peaked on Days 6-9 after birth. This SAg reaction was observed in the Peyer’s patches, was never seen in other lymphoid organs, and was followed by a gradual decrease in the SAg-responsive T cell population approaching a near complete deletion in all lymphoid tissues of 2 to 4-month-old mice (see Table I ) (Held et al., 1992; Papiernik et al., 1992; Desaymard et al., 1993; Tucek et al., 1993; Karapetian et al., 1994). Viral intracellular DNA was detectable in the mesenteric lymph node

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from day 14 onward followed by lifelong infection in all lymphoid organs (Karapetian et al., 1994). Most likely this spread is initiated by emigration of infected cells from the Peyer's patches and later by infection of other lymphocytes (Waanders et al., 1993b). Peripheral deletion of the SAgreactive T cells started after 2-4 weeks and was complete for most viruses after 15 weeks (see Table I ) (Marrack et al., 1991; Held et al., 1992; Papiernik et al., 1992; Hodes et al., 1993; Shakhov et al., 1993; Luther et al., 1994; Maillard et al., 1996). Similar experiments using MMTV(C3H) resulted in deletion without prior detectable increase among the responsive T cells in the Peyer's patches (Karapetian et al., 1994). This observation is in agreement with studies on peripheral injection of MMTV into naive adult mice: MMTV(SW) induces a local increase followed by systemic deletion of the SAg-responsive T cells, whereas MMTV(C3H) induces systemic deletion of T cells with only a marginal increase of the SAg-reactive T cells. The fact that both MMTV strains are transmitted successfully from one generation to the other suggests that an increase in number (detectable by flow cytometry) of SAg-reactive T cells is not essential for virus survival. About 100 times higher dose of MMTV(SW), MMTV(CSH), or MMTV(C4) was required to induce a detectable increase in SAgreactive T cells than to cause clonal deletion (Held et al., 1994a; H. Acha-Orbea, unpublished data). Similar observations were made in CD4deficient mice. In these mice no deletion of SAg-reactive CD8' T cells was observed with MMTV(C3H) but transmission was clearly detectable (Penninger et al., 1994). The expression of the SAg of MMTV(SW)on Peyer's patch lymphocytes goes in parallel with a strong increase in size: On days 8 or 9 after birth the size of the patches is three times that of control mice. The usually scattered B cells within the neonatal Peyer's patch reorganize into B cell follicles upon infection. By day 10 after birth large amounts of IgG and IgA are secreted locally (M. Astori, 0. Karapetian, and H. Acha-Orbea, unpublished data). In addition, the infection seems to spread via the mesenteric lymph nodes to reach all other lymphatic organs after several weeks. At this time point viral DNA is clearly detectable in the thymus and mammary gland (Karapetian et al., 1994). The spread of MMTV infection has been investigated by transfers of purified cell populations from adult mice infected neonatally with MMTV(SW).Adoptive transfers into naive mice have shown that B, CD4', and CD8' lymphocyte subpopulations are infected and can induce clonal deletion. Isolation of lymphocyte subpopulations from these mice and transfer of B, CD4', and CD8' T cells show that each infected subset can infect the other two subsets. However, transfer of sera from infected mice never induced T cell deletion (Waanders et al., 1993b). Together, these

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observations indicate that MMTV does not induce a detectable viremia but probably spreads due to the proliferative and migratory properties of infected lymphocytes. Viral spread between lymphocyte subsets could occur by cell-cell contact (see Section II,A,4). All infectious MMTV strains reported so far lead to the progressive systemic deletion of SAg-reactive T cells, which is usually complete after several months (see Table I). This deletion is maintained lifelong, reflecting the persistence of the virus infection and of the SAg presentation. The expression of SAg of infectious MMTV is sufficient for VP-specific stimulation and deletion to occur (Acha-Orbea et al., 1991; Choi et al., 1991). However, it has been suggested that the simultaneous expression of MMTV-env and SAg induces a more efficient T cell stimulation but has no effect on deletion (Golovkina et al., 1995). What is more difficult to explain is that endogenous env molecules do not fulfill their proposed function (Mtul, -8, -11, and -14). Transfection of constructs containing only the SAg but not the env sequence into BALB/c derived A20 cells resulted in perfect presentation (Mtu8 and -9) and several monoclonal anti-env monoclonal antibodies and polyclonal anti-env sera do not inhibit SAg reactions in vitro (S. Vacheron et al., submitted). Conclusive experiments are required to settle h s point. MMTV infection leads to the deletion of T cells of both subsets (CD4' and CD8+) with the former being more affected. This process was observed for double-positive cells in the thymus and for mature CD4+ T cells in the periphery (Marrack et al., 1991; Held et al., 1992; Ignatowicz et al., 1992; Papiernik et al., 1992). This means that after initial infection of the gut the virus reaches the thymus. Peripheral deletion of CD4+, but not CD8+ cells, occurs equally in the absence or presence of a thymus with MMTV(C3H) (Ignatowiczet al., 1992).The reduction of CD8+ T cells in the periphery after infection seems to be due to thymic deletion followed by the slow dilution of the peripheral CD8+ T cell pool with new T cells. This probably reflects the lower activation threshold of thymocytes compared to peripheral T cells. Other MMTVs expressing stronger SAgs have not yet been tested for deletion of CD8' T cells in thymectomized mice. The extent and kinetics of neonatal deletion of CD4+ T cells depends on factors such as H-2 haplotype, mouse strain, and MMTV strain. No major differences between male and female mice were observed, although females have a slightly faster deletion kinetics (Marrack et al., 1991). However, there is a clear dependence on the MHC haplotype of the mouse strain. In I-E-positive mouse strains an efficient deletion of SAg-reactive T cells is observed with most MMTV strains. This is in contrast to I-Enegative mice in which several MMTV strains induced no or only a partial

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deletion (see Tqbble Z and Section STI,H) (Acha-Orbeast d., 1891: Chai d al., 1891; Pucillo et al., 109$ MdUard p t a!., 1BF)tN. MMTV strains, such as MMTV(SW), kWl3'(C4), and M~?'V(SlM),have been shown to delete virtually all SAg-reactive T ceUs ip the T-&positive BALBh strain within 2 or 3 mantb (see Table T). Others, such 84 MMTV(C3H) and MMTV(SHN), delete oqly 75% of all reactive T oefs in the same mouse strain. Currently, it is not clear whether the extent of deletion reflects a lower titer of infectious virus in the milk or a lower affinity for TCR or MHC. Coinfection with an I-E-dependent [MMTV(IITES 2)] and an I-E-independent [MMTV(IITES 14)] MMTV was seen in I-&negative mice (Ando et al., 1995).This could be due to either infection of B cells with both viruses qr bystander activation of B oells. An altprqgtjve mechanism of T ceU talerance is unresponsiveness*Such T cells do not proliferate to TCP crawlinking ~r rechdlengp with antigen and do not produce IL-2 (reviewedin Schwarb, I88ri).The responsiveness of the remain@ T cells that exprw EiAg-reaotive TCRs was investigated after neoqqtd infectiqp with the Vfi14-specifip MMTV(C3H) (Ignatwdcz et aZ., 199%).Tbe CD4' T cells resistant to depletion by the SAg respond poorly on a per cell basis to in vitrp stirnulatho with antibsdles against VP14 in terms of prolifer#inn (seg Seetian V,P,3). This phenomenon was more pronounced iq 4-weeE-old miw& &an in 4-week-old anes, pmbably reflecting anergy prior ta deletiinn of fj4prea~tiveT cells (in 4-week-old animals) and preferential expansion of T cells with poor SAg reactivity with age. Together, SAg-reactive T cells can have four different fates in response to the neonatal encounter with fie SAg of infeotiaus MMTV:sHmula#on, unresponsiveness, deletion, and nanreactiveness, The Arst three possibilities imply recognition of the SAg. It i s likely that the T cells expanding after SAg encounter become unresponsive and/or are deleted dmilar to injection of bacterial SAgs sych as QEB or a ells expressing endogenous Mtvs (Lilliehoiiket al., 1975; fiammeqsee et pl., 1848; Webb st al,, lQQ0; MacDonald et al., 1991; Renno et al., 1895). The stage of maturity of the neonatd jmmupe syatem was addressed in many different articles, the main conclusions being that tolsrance induction is similar in neonatal and adult mice, but fiat neonatal m i ~ haw e a reduced ability to mount an immune response (Fnrrithuber st al., 1888: Ridge et al., 1996; Sarzotti et al., 1996). The maturity af the pedpheral neonatal immune response was addressed recently with MMTV SAgs. In both studies footpad injection of MMTV(SW) or congenic B cells expressing an endogenous version of this virus, Mtv7, were used (LeBon et aZ., 1995; Astori et al., submitted).

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Injection of MMTV(SW) and detection of the SAg response measures either infection of the neonatal B cells or the ability of T and B cells to collaborate. The two studies came to different conclusions. In contrast to Peyer's patches, prior to 10 days after birth practically no SAg response is inducible with this virus. Thereafter, an increase of the SAg response is observed until day 30 after birth, when adult-type SAg responses are measured. Surprisingly, B cells start secreting antibodies in quantities similar to those of adult mice due to SAg-mediated help only after day 23 after birth (Astori et al., submitted), whereas before day 23 no antibody secretion is detected. The picture is different when adult Mtv7-expressing B cells are injected. By 6 days of age (the earliest time point measured) a SAg response is detected and neonatal T cells are able to help adult B cells to secrete antibodies.Therefore, neonatal B and T cells seem to be unable to cooporate in SAg-mediated antibody secretion during the first 22 days of life, although neonatal T cells are already mature in this respect. In contrast, Mtu7 congenic lymph node cells isolated from 12- to 18day-old mice induced an adult-type SAg T cell response but could not be induced to secrete antibodies in neonatal or adult mice (Astori et al., submitted). Different results were obtained by Le Bon et al. (1995),who showed that the induction of the SAg response is strongly reduced when neonatal or adult Mtv7 congenic splenocytes are injected. The differences between the two studies are most likely due to the source of injected cells (spleen by Le Bon et al. vs. lymph node by Astori et al.). The frequency of B cells in neonatal spleens is very low and Mtv-7 SAg expression increases strongly during the first 2 weeks of life (Niimi et al., 1994).

2. Adult T Cell Responses to Exogenous MMTV As shown in Fig. 3, the known MMTV strains fall into five families with different TCR Vp specificities. Intensity of SAg-mediated stimulation and speed and extent of deletion are characteristic for each MMTV strain. In the following a general description of the events after MMTV injection is given. The injection of milk-purified MMTV into the footpad of adult mice leads to a strong SAg response in the draining popliteal lymph node. The lymph node increases in cellularity, first visible on day 1 (2- to g-fold), reaching a peak (up to 40-fold) on days 4-6, and decreasing thereafter. During the first few days, infection is detected almost exclusivelyin B cells. With MMTV(SW),T cells expressing Vp6 increase from 10 to 20-40% of all CD4+ T cells in the draining lymph node within 4-6 days. This Vpspecific increase is not observed in the blood nor in nondraining lymphoid tissues of the same animal. At the peak of the response a decrease of SAgreactive T cells is observed in blood and in nondraining lymphoid tissues.

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This decrease most likely represents trapping of SAg-reactive cells in the draining lymph node rather than clonal deletion (Le Bon et al., 1996). This local T cell stimulationis followed by the progressive, systemicdeletion of most SAg-reactive T cells. Curiously, the draining lymph node is the only site where some SAg-reactive T cells are spared from deletion over several months. The lifelong deletion of the T cells reactive with MMTV(SW) in the other lymphoid organs reflects the persistence of the virus infection and SAg presentation in adult mice (S. Luther and H. AchaOrbea, unpublished data). The 10 to &@foldincrease in numbers of B cells and V/36-specific T cells within the popliteal lymph node 6 days after MMTV(SW) injection is due to several effects. During the first 2 days unspecific trapping occurs, characterized by the polyclonal accumulation of T and B cells and the almost complete lack of proliferative responses (Le Bon et al., 1996; Luther et al., 1997). The early accumulation of T cells, but not B cells, can be blocked by administration of anti-LFA-lp antibodies before MMTV injection (C. Ardavin and H. Acha-Orbea, unpublished). Between days 3 and 7, B cells and some SAg-reactive T cells enter cell cycle. In contrast, the cellular increase after injection of SEB (which is systemic even after subcutaneous injection) was shown to be due to SAg-reactiveT cells entering cell cycle (Renno et al., 1995, 1996). The phenomenon observed with MMTV is in agreement with previous studies showing the attraction and retention of lymphocytes during the first days after immunization followed by local lymphocyte activation, resulting in the release of effector-competent lymphocytesfrom the lymphoid organs after 5-7 days (Sprent and Webb, 1987). The activation of T cells after injection of MMTV(SW) is restricted to the SAg-reactive CD4+ T cells present locally and is manifested from day 3 onward by blastogenesis (up to 90% of all V/36+CD4'), incorporation of BrdU (up to 9% of all V/36+CD4+in a 2-hr BrdU pulse; up to 15%after 4 day BrdU exposure) (Le Bon et al., 1996); Luther et al., 1997), and modulation of activation markers (CD69+,CD62L-, and CD44') (Champagne et al., 1996; Le Bon et al., 1996). No upregulation of the IL-2 receptor a-chain (CD25) is observed (LeBon et al., 1996). It is likely that the SAg-reactive T cell subset is responsible for the peak of cytokine secretion (such as IL-2, IL-4, and IFN-7) observed 5 days after injection of MMTV(V) (Palmer et al., 1996). All proliferative T cell responses in the first few days are exclusively seen in the paracortex of the lymph node (Luther et al., 1997) (see Fig. 6). This local immune response can be efficiently blocked with monoclonal antibodies directed against the Cterminal Mtu7 SAg peptide (Acha-Orbeaet al., 1992; Held et al., 1993b).

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Paracortex

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FIG.6. Model proposed for the events of SAg-drivenT-B interaction allowing productive MMTV infection. The three boxes represent the different compartments of the popliteal lymph node draining the site of injection with infectious MMTV. We propose the following model: MMTV infects preferentially the B cells which express SAg in associationwith MHC class I1 products. T helper cells (CD4+)expressing the SAg-reactive TCR-Vfl determinant are activated, expand, and provide help to the infected B cells within the T cell-rich paracortex. Later in the response, these T cells undergo activation-induced cell death in a still undefined site. The activated B cells migrate into the medullary cords, where they expand and thereby amplify the virus infection. Most of these B cells then differentiate into IgG expressingblasts and later into plasma cells, some of which secrete virus-neutralizing antibodies. At a time when the medullary cord reaction has subsided, B cells start to proliferate and differentiate in germinal centers (GC) within the B cell follicle (cortex), where SAg-reactiveT cells are predominantly found. This chronic reaction could potentially give rise to long-lived infected B and T memory cells as well as virus-specific plasma cells (reproduced with permission from Luther and Acha-Orbea, 1996).

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The MHC haplotype strongly influences the extent of T cell stimulation and deletion. Similar to deletion after neonatal infection, MMTV infection results in the strongest T cell expansion and deletion in the presence of MHC I-E molecules (Held et al., 199413) (for more details see Section 111,B). With MMTV(SW), approximately 10-fold more virus needs to be injected into I-E-negative responder strains to obtain a T cell stimulation and B cell infection levels comparable to I-E-positive strains. The mechanism of extrathymic T cell deletion by the SAg of MMTV is still unresolved. Young Fas-deficient mice (MRL lprllpr) show a normal expansion and deletion of the T cells upon injection of MMTV(SW). MRL lprllpr mice older than 10 weeks have a reduced capacity to delete the SAg-reactiveT cells, most likely due to the starting autoimmune phenotype (Papiernik et al., 1995). Therefore, it seems that T cell deletion to MMTV SAg is Fas independent. 3. T Cell-Dependent B Cell Bflerentiation in Response to Exogenous MMTV B cells are the primary target of MMTV infection (Held et al., 1992, 1993b) and are activated in two waves. After the previously described T cell-independent B cell activation by MMTV, the second wave of B cell activation, detectable after 36-48 hr, is strictly dependent on the presence of the SAg-reactive T cells. Nude mice reconstituted with SAg-nonreactive V/314+T cells do not show a B cell response to MMTV(SW) unless they are reconstituted with the SAg-reactive V/3GtCD4' T cells (Held et al., 1993a). Similarly, mice expressing H-2q, which is unable to present MMTV SAgs, do not show this second activation phase (Ardavin et al., submitted). In vivo, B cells are stimulated by CD4' T cells, possibly by a combined action of cell contact and cytokines (Held et al., 1993a,b). Such a scenario could explain the interdependence and synchronicity of the T and B cell responses. It is further supported by several lines of evidence: (i) Mice lacking B cells could not be infected productively by MMTV and do not lead to measurable T cell deletion (Beutner et al., 1994), suggesting that B cells are essential for the establishment and persistence of MMTV infection; (ii)T cell proliferation is observed slightly before B cell proliferation (Held et al., 1993a), indicating prior activation of T cells by B cells; (iii) the level of detectable viral DNA within the draining lymph node increases more than 1000-fold between days 1 and 6 upon MMTV(SW) infection (Held et al., 1994a). This amplification of the virus infection was seen independently of reinfection, suggesting T cell- and SAg-dependent B cell division (Held et al., 1994a); B cells present MMTV SAg efficiently to CD4' T cells in in vitro infection experiments, and this interaction can be blocked with anti-MHC class 11, anti-TCR, anti-CD4, and anti-SAg

SUPERANTIGENS OF MMTV

179

antibodies in uitro (Vacheron et al., submitted); and (iv) B cells are the best presenters of endogenous Mtu SAgs (see Section V,A) and are polyclonally stimulated during the SAg-drivenT-B interaction (Augustin and Coutinho, 1980; Modlin et al., 1996). Despite the in uitro evidence, it is currently not clear whether B and T cells alone are sufficient for the SAg-driven immune stimulation to occur in uiuo. In particular the role of dendritic cells and macrophages in SAg presentation needs to be further evaluated. It is generally believed that B cells presenting antigen lead to an abortive response by naive T cells characterized by anergy or death, possibly due to lack of costimulation (Eynon and Parker, 1992; Finkelman et al., 1992; Fuchs and Matzinger, 1992). The exclusive presentation of MMTV SAgs by B cells might be a reason for clonal deletion of SAg-reactiveT cells. In support of this speculation, presentation of MMTV SAgs by vaccinia virus-infected macrophages leads to normal T cell stimulation and anergy induction but to a complete absence of clonal deletion (Krummenacher et al., 1996). Further experiments are required to determine the role of the different antigen presenting cells in induction of stimulation, clonal deletion, and anergy. Three to 6 days after MMTV infection, B cells differentiate in a T cell-dependent manner, suggesting that SAg-responsive CD4+ T cells are capable of providing competent help to B cells (Held et al., 1993a). Up to 30% of all B cells in the draining lymph node (or approximately 4 X lo6 B cells) differentiate into blasts with the phenotype of plasma blasts: B220'"w, MHC class II+, IgD-, IgM-, syndecan-1+, CD43+, CD86-, CD62L-, CD69-, CD44+,and CDllahid'(Ardavin and Acha-Orbea, unpublished). This phenotype was described in part by other groups for classic antigens (Lalor et al., 1992; Dustin et al., 1995; Smith et al., 1996). Most B blasts are found in the medullary cords and sinuses 3-8 days after infection (Luther et al., 1997). This site is typical for extrafollicular B cell differentiation leading to secretion of low-affinity antibodies and to death or emigration from the lymph node (MacLennan, 1994). Indeed, B cells staining for cytoplasmic IgM and IgG are detected in high numbers in the medullary cords (Luther et al., 1997). In addition, high levels of antibody can be detected in ELISPOT and ELISA assays between days 4 and 10. With all infectious MMTV strains analyzed so far the same pattern of B cell response was observed: On days 3 or 4 after MMTV injection, B cells secrete predominantly IgM antibodies. Peak numbers of antibody-secreting cells and antibodies in the serum are observed by day 6, when the response is dominated by IgG antibodies, in particular IgG2a (Held et al., 1993a; Luther et al., 1994). Virus infections generally induce the secretion of IFN--y and thereby the preferential switch of B cells to the IgG2a isotype. This subclass of antibody is known to be often of polyclonal nature (Coute-

180

SANJIV A. LUTHER AND HANS ACHA-ORBEA

lier et al., 1987, 1991). In the case of MMTV, large amounts of IFN-7secreting cells have been detected 5 days after infection, explaining the high titer of IgG2a serum antibodies (Palmer et al., 1996). This humoral response is to a large degree polyclonal (Held et al., 1993a), and was recently found to be in part directed against the viral envelope protein gp52 (Luther et al., 1997; see Section IV,B,5). Besides this extrafollicular B cell differentiation, infection with MMTV(SW) leads to the migration of B cells into the follicles at a time when the medullary cord reaction has already subsided (Luther et al., 1997). These follicular B cells start up a germinal center (GC) reaction that appears unusually late (day 12), stays limited in its extent, and is long lasting (several weeks; see Fig. 6). The following data suggest that this follicular B cell differentiation is strictly dependent on SAg-reactive T cells: (i) Mtu-7 congenic BALB.D2 mice do not form GCs in response to MMTV(SW)injection, and (ii) CD4' T cells present in these GCs belong almost exclusively to the SAg-reactive subset. The percentage of VP6+ T cells within the GC outnumbers by far the proportion of this subset in the paracortical T cell region. Currently, the specificity of the GC reaction is not known. The presence of viral env protein on the follicular dendritic cells as well as the long-lived virus-specific antibody response (Luther et al., 1997)suggest that MMTV-specific B cells are founding the GC reaction. A model for the T-B collaboration is shown in Fig. 6. The role of costimulatory molecules for T and B cell activation and differentiation as well as viral spread will be discussed in the following section. 4. Role of T-B Interaction for Viral Amplijkation and Spread Upon MMTV infection, viral intracellular DNA can be detected in the draining lymph node by PCR and therefore distinguished from viral RNA. PCR for the DNA of all MMTVs (endogenous and exogenous) can serve as an internal control due to the length polymorphism of the SAg sequence and it allows quantitation of the amount of viral DNA when more than 1%of cells are infected. For lower infection levels PCR specific for a particular SAg sequence has to be utilized (Held et al., 1993b). Using these techniques it has been shown that SAg DNA can be detected in the draining lymph node as early as 2 days (specific PCR) after infection with MMTV(SW) and peaks on day 6 (quantitative PCR). After 20 days, low levels of viral DNA are still present in the draining lymph node but not in the nondraining control (Held et al., 1993b).On Day 6 MMTV(SW) DNA and RNA were predominantly found in the B cells, whereas T cells, and in particular the SAg-reactive T cells, showed undetectable levels of MMTV(SW) DNA (Held et al., 1993b; 1994a) suggesting that infection of CD4+ and CD8+ T cells occurs later (Waanders et al., 1993b).

SUPERANTIGENS OF MMTV

181

It has been estimated that less than 1in 1000 B cells initially get infected by MMTV. By day 6 the infection level has increased at least 250-fold within the draining lymph node (Held et al., 1993b; 1994a). At this time point the infected B cells have on average less than four copies of integrated viral DNA (Held et al., 199313).Two phenomena could explain the amplification of the virus infection: reinfection of activated B cells or clonal expansion of few initially infected B cells. Reinfection can be blocked by AZT, a powerful inhibitor of reverse transcriptase because mice injected with MMTV(SW) during AZT treatment do not show viral infection nor SAg-mediated T cell stimulation. However, when the AZT treatment was started 2 days after infection with MMTV(SW),the mice showed a maximal T cell stimulation and virus amplification by day 6. This data would support the notion that few initially infected B cells amplify the virus infection by cell division rather than by reinfection. The partial inhibition of the SAg response when AZT was administered after 24 hr can be explained either by incomplete infection and reverse transcription after 24 hr or by a cell to cell spread of infectious particles during the first day of infection. The increase in numbers of infected cells in the draining lymph node coincides with the SAg-mediated increase of T and B cells, suggesting the direct implication of the SAg in the viral amplification. This hypothesis was confirmed in several experiments showing a close link between the absence of SAg-mediated lymphocyte responses and inefficient infection. These are (i) treatment with AZT or antibodies against the SAg of MMTV(SW) (Held et al., 1993b; 1994a), (ii) mice expressing a MHC haplotype unfavorable for efficient SAg presentation or mice lacking MHC class I1 expression (Held et al., 1994b; Beutner et al., 1996), and (iii)mice lacking the T cells reactive with the SAg of the injected MMTV, such as BALB.D2, TCR Vp-transgenic mice, or mice transgenic for the SAg of MMTV (Golovkina et al., 1992; Held et al., 1993b). Based on these findings a model has been proposed for the biological function of the SAg as illustrated in Fig. 6. In the absence of a SAg response, infection is strongly reduced and the virus is lost at the latest after several mouse generations (Golovkina et al., 1992, 1993; Held et al., 199313). Infection is not strictly dependent on the presence of a SAg but becomes highly inefficient. These more recent observations explain previous results on tumor induction with I-Edependent MMTVs. With such viruses I-E-negative mice were much more resistant to infection and tumor formation (Muhlbock and Dux, 1974; Pucillo et al., 1993; Held et al., 1994b). Similar results were obtained with nude mice, which lack T cells early in life (Squartini et al., 1970; Tsubura et al., 1988). None of these mice were entirely protected from tumor induction or virus transmission, but the percentage of chronically infected

182

SANJIV A. LUTHER AND HANS ACHA-ORBEA

mice was reduced and the time required for cancer induction increased. Similarly, MMTV particles with a stop codon in the SAg sequence quickly recombined with endogenous Mtvs to generate a functional SAg. This illustrates the important role of the SAg in MMTV infection (Golovkina et al., 1995). Recently, several gene-deficient and transgenic mice that are deficient in interactions between T helper cells and B cells have become available. Several of them have been analyzed for MMTV infection, transmission, and T cell tolerance, as summarized in Table 11. The results confirm the previously discussed observations that the successful T-B interaction in response to MMTV SAg relies on three principal players: on the B cells (see p M O ' O , Table 11), on the proper MHC haplotype expressed at normal levels (see MHC class II0/O,invariant chain"'", and mice expressing H-2s or lacking I-E expression; Table 11), and on the presence of SAg-reactive T cells (see nude mice, TCR transgenic mice, and BALB.D2 mice; Table 11).The success of conventional T-B interactions further depends on the interaction of costimulatory molecules and on the exchange of soluble factors such as cytokines. Several reports have emphasized the essential role of the CD40-CD40L (ligand)and CD80/CD86-CD28/CTLA4 receptor-ligand pairs in the dialogue between murine B and CD4+ T cells (reviewed in Clark and Ledbetter, 1994; Bluestone, 1995; Grewal and Flavell, 1996). CD40L is upregulated in activated T cells and delivers crucial activation signals from T cells to the B cells expressing CD40. It has been suggested that the interaction between CD40 and its ligand also transmits an important activation signal to the T cell (Grewal et al., 1995; van Essen et al., 1995).Overall, interference with the CD40 pathway leads to a profound and early block of the cognate T-B interaction, resulting in a complete loss of humoral responses to thymus-dependent antigens. Neonatal CD40LO'O mice exposed to infectious MMTV(C3H)show a barely detectable infection within the spleen that is not transmitted to the next generation. No VP-specific deletion occurs. CD40L-deficient T cells could only respond to SAg-expressing B cells when the latter were preactivated by CD40L-expressing membranes. Thus, the CD40-CD40L interaction plays a key role in the first steps of the MMTV life cycle (Chewonsky et al., 1995). B7 molecules (CD80 and CD86) are upregulated on activated B cells and interact with the constitutively expressed CD28 molecule on T cells. In this case the signal is delivered unidirectionally to the T cell and is thought to play an important role in the initiation and expansion of all kinds of T cell responses, such as proliferation, cytokine mRNA half-lives, cytokine secretion, and cytotoxicity (reviewed in Bluestone, 1995). Mice lacking CD28 or CD80 show greatly diminished T cell responses to most

SUPERANTIGENS OF MMTV

183

but not all antigens (Shahinian et al., 1993; Sharpe, 1995). Surprisingly, neonatal infection with MMTV(V) in the absence of CD28 did not impair TCR VP-specific clonal deletion or the virus transfer to the mammary gland. Unfortunately, the authors did not address the deletion kinetics. The adult immune response to MMTV(V) injection is characterized by reduced expansion of VP2-specific CD4+ T cells, a clearly lower number of IFN-y-producing cells in the draining lymph node, and a partial or no reduction in the number of IL-2- or IL-4-secreting cells, respectively. In addition, the number of antibody-secreting cells was substantially diminished, thereby affecting all isotype subclasses except IgG2b (Palmer et al., 1996). Thus, T and B cell responses to MMTV are clearly reduced in adult CD28-deficient mice. Nevertheless, the absence of CD28 does not interfere with the virus life cycle. In addition to CD28, the B7 molecules can interact with at least one other ligand on T cells, CTLA4 (reviewed in Linsley and Ledbetter, 1993; Thompson, 1995). This molecule has a 20-fold higher affinity for the B 7 ligands than CD28 and is induced only upon T cell activation. Mice lacking a functional CTLA4 molecule have a lymphoproliferative disease suggesting a role for CTLA4 in the downregulation of T cell responses (Waterhouse et al., 1995; Sharpe, 1995). Mice expressing large amounts of a soluble CTLA4-Ig construct in serum have a greatly reduced capacity to mount T-dependent antibody responses to conventional antigens supposedly due to the saturation of all ligands for CD28 and CTLA4 (Lane et al., 1994; Ronchese et al., 1994). When adult mice transgenic for CTLA4-Ig and MHC I-E were infected with MMTV(SW), they showed an almost normal SAg-mediated immune response up to day 6, including upregulation of activation markers on T and B cells, an increase in numbers of the SAgreactive T cell subset as well as B cells, and high numbers of IgM-secreting cells. However, further stages of the immune response as well as of the virus spread appeared to be defective. T and B cell stimulation arrested more rapidly than in controls. Only few B cells switched to IgG production and T cell deletion to levels below control was minimal. The dissemination of infected cells was strongly reduced, possibly explaining the low level of systemic T cell deletion. Similarly, neonatal infection of these mice with MMTV(SW) led to minimal T cell deletion and lack of virus spread (Champagne et al., 1996). Finally, the defects in the immune response were even more accentuated in the absence of I-E molecules. These results suggest that interactions via B7 are dispensable for T cell proliferation induced by high-affinity ligands but are essential for lymphocyte differentiation, spread, and transmission of the virus infection, similar to the CD40L-deficient mice. The discrepancy between the results obtained with neonatal infection of CD28-deficient and CTLA4-transgenic mice is hard to explain but might

TABLE I1 TRANSGENIC A N D KNOCKOUTMICE ANALYZED FOR Mtv S A g - M E D I A T E D

Deletion of T cells”

MHC class I1

Strain“

Conventional strains BALBIc B10.DZ C57BY6

129

Mice deficient in SAg presentation MHC IIom (C57BW6 X AKR/J)

DELETION

Infectious MMTV

Endogenous Mtu or SAg-tg

Mice

I M M U N E STIMULATION AND

MMTV strain

Infectionb/ transmission‘

CD4+ T cell siimulation (adultsId

CD4’ T cell deletion (neonates or adultsy

I-Ed, LAd I-Ed, I-Ad I-Ab

+++ +++

sw sw

+++I+++

+ + +/ND

+++ ++

+ (VP5)

C3H

+I-

ND

-

++ ( V P )

sw sw

+ +/ND

LAb

++I+

++ ++

++ ++

!& (no

-

C3H

+ (mam +,

- (in oitm,

ND

Beutner et d.( 1996)

MHC 11)

spl +, int

+++ +++

References

Held et d.(1993a) Held et d.(1994b) Pucillo et d.(1993), Beutner et d.(1996) Held et d.(1994b) I. Maillard et d. (unpublishedobservation)

Mtu7)

+++)Iliw (C57BU6 X

B1O.BR) Iim (BALBlc) BIO.G I-E tg (C57BW6 tg SJL)

I-Efi. I-A”

X

I-E tg (thymic epithelium) (C57BV 6tg X SJL) Bern” (C57BW6)

ND

ND

ND

V i d e et d.(1993)

ND

Luthi et al. (unpublished observation) Tomonari et d.(1991),Held etd.(1994a) Marrack d al. (1988). Pucillo et d.(1993). Held et d. (19944 Marrack et d.(1988),Pucillo ef al. (1993)

I-E~,I-A~

-

sw

ND

-

I-Aq

-

sw

+/ND

-

(+)

I-Eb, I-A”

+++

C3H

+ + +/ND + + +/ND

ND

sw

ND

I-E”, I-A”

++

C3H

-I-

ND

ND

I-Ah

+

sw

ND/+ + +

ND

ND

+++

+++

Raulet et d.(1994),Velan d d.(1996)

pMoP (C57BU6 X AKWJ) pMop and SAg tg (C57BU6 X C3H SAg low tg) @Momand MMTV tg (C57BU6 X C3H MMTV tg) Mice deficient in T cell response Nude (BALB/c, C3H. AKR)

BALB.DZ

Ei

Vp8.l tg (BALB/c X BALB.DZ) Vp8.2 tg (BALB/c) VpZ tg (C57BU6 X BALB/c) CD40m(B1O.BR) CD8- (B1O.BR) CD48- (BALB/c) Mice deficient in costimulation CD40Lop (C57BVS X B1O.BR) CD40Lom(C57BU6 X C3H MMTV tg) CDZS- (C57BU6 X B1O.BR) CTLA4Ig tg (C57BV6 I-E tg)

C3H,

+ (in uitro)

-

ND

Beutner et al. (1994). Chervonski et al. (1995) Chervonski et al. (1995)

+ (in nitro)

Chervonski et al. (1995)

I-E", I-A"

++++J

I-E", I-A"

+

I-E", LA"

+++

HYB/ PRO

I-Ed, LAd, IEL,I-A'

-

C3H

-

sw

(old +)/? -/ND

ND

-

ND ND

I-E~,I-A~

+++

sw

(+)I(+ 1

ND

-

+++I+++

ND

ND

I-E~,I-A~

+++

I-E~,I-A~ 1-Eud,I-Awd

ND

I-EL,I-A' I-Ek, I-Ak &Ed, I-Ad

I-E", I-A"

I-E", I-A"

sw

C3H

sw sw

-

(spl, inam)/-

+ ++/+ ++

+++

Fry et al. (1989). Hodes et al. (1989), Tsubura et al. (1988), A r d a ~ net al. (submitted) Held et al. (1993b) Pircher et d.(1989), Held et al. (1993b) Held et al. (1993b) wangetal. (1994)

(+)/(+)

+ +/+ +<

ND ND

ND

c4

ND/+ ND/ND ND/ND

ND ND

-h

+++e,

C3H C3H Mtu7

+ (in uitm)

ND

-

C3H

(+)

ND

-

(only S p w ND ND

Foy et d.(1995),Chewonsld et al. (1995)

(+) (in

ND

Chervonski et al. (1995)

++

+++

++

(+)

Shahinian et al. (1993). Palmer et d.(1996) Champagne et al. (1996)

+++' +++<

+++'

I-E", I-A"

+++'

I-E", I-Ab

+++

HYBI PRO BALB/ cv

sw

+

uitro)

+g/+

+/?

+e

+++ (iv)

++ (delay)

Penninger et al. (1994) Penninger et al. (1994) Penninger et al. (1995)

continues

TABLE I1 Continued Endogenous

Strain"

MHC class I1

Mice deficient in cytolcines or receptors IL-4- (C57BV6 X AKR/J) IFNa/B Rw (129)

I-EM, LALrn

IFNy Rw (129) Mice deficient in FadFas ligand lpr (MRL, C3H. AKR)

gld (C3H) Mto-free mice or mice transgenic for SAg Mtvw (BALB/c) SAg tg ( M t o SAg, C3W HeN)

Infectious MMTV

Mto or SAg-tg

Mice

Deletion of T cek

+++

MMTV strain

Infectionb/ transmission'

CD4' T cell stimulation (adults)d

C3H

+++V

ND

+++

Beutner et d.(1994) I. Maillard et d. (unpublished observation) I. Maillard et a[. (unpublished observation)

+++g

LAb

ND

sw

NDND

++

++

I-Ab

ND

sw

+ +/(+)

++

++

I-EL,I-A'

+++

sw

++vND

+++I

+++ (old +Y

I-EL,I-A'

+++

-

ND

ND

ND

I-Ed, I-Ad

-

sw

++ M D

+++

+++

I-EL.I-A'

+or+++'

C3H

+or

++' + or ++'

ND

- or ++

Mice transgenic for MMTVgenes MMTV tg (GR) (C57BU 6 X SJL X BALB/c) SAg tg (GR) (C57BV 6 X SJL X BALB/c)

References

Kotzin et al. (1989), Singer d d.(1989), Papiemik ei d. (199.5) Singer ei d.(1989)

Acha-Orbea el d. (unpublished observation) Golovldna et d.(1992. 1993)

Offspring

Mice Strain

CD4' T cell deletion (neonates or adultsId

MHC

Parents (deletion by tg)

Transmission

Deletion

MLR in oifro

References

I-Ed, I-Ad

++

ND

ND

ND

Acha-Orbea et d.(1991)

I-Ed, I-Ad

++

ND

ND

ND

Acha-Orbea d d.(1991). Lambert et nl. (1993)

SAg tg, 5' end of SAg

I-Ed, I-Ad

ND

ND

ND

Lambert et al. (1993)

I-Ed,I-A*

ND

ND

ND

Acha-Orbea et al. (1991)

(GR) (C57BW6 X

SJL X BALB/c) MMTV tg (CBH-K) (C57BU6 BALB/c)

w

m -l

X

SJL X

SAg tg (Mtu-SAg)

I-E', I-A'

SAg tg, stop in SAg (Mtv SAgCla tg) env and SAg tg (LEL tg) MMTV tg (HYB/PRO tg) MMTV tg, stop in SAg (HYEIPRO tg)

I-Ek,I-A' I-EL,I-A'

I-EL,I-A' I-EL,I-Ak

+ or + + + k +++

+++

+ (delayed)'

Golovkina et al. (1992. 1993. 1994a) Golovkina et d.(1995)

+++ +++I

+++ +++'

+

+++

Golovkina et d.( 1994a) Golovkina et d.(1994a, 1995) Golovkina et d.( 1995)

Note. Abbreviations used: + + +, strong and quick; + +, intermediate; +, weak; -, absent; ND, not determined: iv, intravenous;spl, spleen; mam, mammary gland; int. intestine. a Thymic deletion by endogenous Mni w as mostly measured in the peripheral blood of backcrosses from C57BU6 mice to I-E expressing mouse strains. It is likely that the deletion occurred already in the thymus, as shown for several lab strains. Because not all T cells expressing a reactive TCR VP react to the same degree, the result of the most responsive is shown. The mouse strain mentioned first was usually used for the experiments with infectious MMTV. * Detection of viral DNA by PCR in mice infected neonatally or as adults. Transmission of virus from virus-infected mothers to foster-nursed offspring (virus in milk or VP-specific T cell deletion in offspring measured). Measured i n u i w if not otherwise stated; stimulation usually measured in the draining popliteal lymph node after footpad injection (if not otherwise stated); deletion measured either in lymphoid organs or in blood after virus infection at birth or as adults. No kinetics. f Deletion of Mtu7-SAg-reactive T cells in the thymus and spleen was more efficient than in the mntrols, especially at early time points. g No precise data. No deletion among CD4-CD8' or CD4-CD8-T cells. ' Deletion was measured among CD4-CDS-Thyl.Z+V/36+T cells reactive with Mtu7-SAg. 1 Stimulation was seen among the SAg-reactive T cells independently of B220 expression and age of infection, but not among CD4-CDKCD3+ T cells. Deletion was also observed irrespective of B220 expression. Extent and speed of deletiodifectiodtransmissiondependent on the copies of tg. C3H-SAg with frameshift that recombined with Mtul.

' '

188

SANJIV A. LUTHER AND HANS ACHA-ORBEA

be due to the more complete block of all CD28/CTLA-4-B7 interactions

by the latter mouse model. The results obtained with the CD40L-deficient and with the CTLA4-Ig transgenic mice suggest that proper T cell activation leading to functional B cell differentiation might be a crucial event in the MMTV life cycle. In BALB/c mice cognate interaction between infected B cells and SAgreactive T cells leads to the progressive differentiation of the B cells. By day 6 after footpad injection of MMTV(SW), viral DNA is found particularly among the B blasts: namely, among the surface IgG+ population (Held et al., 1994a) and among the Ig-, BZZO'O'", MHC IIhighpopulation that is differentiating into plasma cells (Ardavin et al., 1997). It seems probable that the virus survives in differentiated B cells of memory phenotype. By in situ hybridization or PCR quantitation on sorted subpopulations, as many as 10-30% of all B cells, or 60% of all B blasts, were shown to express MMTV mRNA on day 6. Therefore, MMTV might make its way into two different B cell pools: the extrafollicular plasma blasts and the memory B cells. It is quite controversial as to whether extrafollicular B cells emigrate to other sites (Bachmann et al., 1994) or die in situ (Ho et al., 1986; Smith et al., 1996). Memory B cells, however, are by definition long lived. It is tempting to speculate that both B cell subsets are crucial for the maintainance of a critical number of infected cells as well as for spread of the infection. The migratory properties of both populations have been described (Tew et al., 1992; Gray, 1993; Bachmann et al., 1994; Ahmed and Gray, 1996). MMTV infection might spread via migrating B cells or possibly via infection of other lymphocyte subsets finally reaching the mammary gland. 5. Humoral Zmmune Response against M M T V The key features of a persisting virus infection are a sophisticated survival strategy of the virus and an insufficient immune response by the host (Oldstone, 1989; Gooding, 1992; zinkernagel, 1993; Smith, 1994; Sprigs, 1996). MMTV infects cells of the immune system and is propagated by them (Squartini et al., 1970;Tsuburaet al., 1988;Acha-Orbea and MacDonald, 1995).The immune response of mice to MMTV is very strong; however, it is mainly directed against the viral SAg and serves to amplify the virus infection rather than to eliminate it. The result is a persisting virus infection with no obvious pathology or viremia during several months. This is not surprising because the propagation of MMTV infection-by virus uptake in suckling mice-relies on the proper survival and reproduction of the host. The late appearance of mammary tumors does not interfere with this goal.

SUPERANTIGENS OF MMTV

189

The finding that inoculation with mammary tumor cells or extracts from one tumor immunized mice against transplantation with another tumor (Lavrinet al., 1966; Morton et al., 1969)spurred a phase of intense research on humoral and cellular immune responses against the MMTV and its tumors between 1965 and 1979. The earlier studies on humoral responses against MMTV are summarized in brief (reviewed in Bentvelzen and Hilgers, 1980; Bentvelzen et al., 1981). Specific antibodies were found in mice infected as adults or as neonates but not in uninfected mice. Feral mice were sometimes found to be positive (Fine et al., 1978). Equal levels were detected in males and nulliparous females infected neonatally with MMTV. The antibody titers progressively increased with age and were strongly enhanced upon lactation or tumor formation (Ihle et al., 1976; Arthur et al., 1978a,b;Arthur and Fine, 1978).There was a clear correlation between the presence of infectious virus and virus-specific antibodies as well as between virus dose and antibody titer (Ihle et al., 1976). It became obvious that mice are not tolerant to MMTV at the humoral level, even when they were infected as newborns or when they expressed the endogenous virus Mtvl (which retained infectious capacity)early in life. Therefore, MMTV breaks the rule that self-antigens are tolerogenic when encountered during the first days after birth. Similar observations have been made with other neonatal virus infections (Oldstone, 1989). With exogenous and endogenous MMTV the onset and titer of the antibody response displays a considerable variation. Most neonatally infected BALB/c, CBA, and C3H mice have anti-MMTV antibodies by 6 weeks of age (Muller and Zotter, 1973; Bentvelzen and Brinkhof, 1977; Arthur and Fine, 1978). In C3Hf mice [C3H mice lacking infectious MMTV(C3H) due to foster nursing], which cany the endogenous Mtul, a humoral response was measurable after 15weeks. Using radioimmunoassays the humoral response against MMTV(C3H)was found to be directed against the outer envelope glycoprotein, gp52 (Arthur et al., 1978a; Schochetman et al., 1979b). In vitro and in vivo assays showed that MMTV-infected mice developed neutralizing and cytotoxic antibodies (reviewed in Schochetman et al., 1980).Antisera against gp52, but not against the other four major viral proteins, were efficient in neutralization. In tumor-bearing mice this effect was IgG mediated (Massey et al., 1980a). Neutralization was measured in uivo as followed: Mammary tumor cells were incubated with immune serum, then inoculated into mice and the time and frequency of mammary tumors was taken as a readout (Blair, 1968). Alternatively, neutralization was measured in uitro by testing sera for their ability to prevent the formation of foci by the MMTV(C3H) pseudotype of Kirsten sarcoma virus containing MMTV gp52 (Schochetman et al., 1979b). Sera from MMTV(C3H)-but not from MMTV(RII1)-

190

SANJIV A. LUTHER AND HANS ACHA-ORBEA

or MMTV(GR)-infected mice could neutralize the pseudotype. Analysis of the gp52 RNA revealed differences in nucleotide sequence that could explain the lack of cross-reaction with sera from mice infected with MMTV(RII1) or MMTV(GR). The MMTV strains C3H, GR, CSHf, and GR were classified according to their serological cross-reactivity, to their recognition by monoclonal antibodies, and to their polymorphism in gp52 (Schochetman et al., 1980). Determinants found among all MMTV strains were called group specific, determinants common to some of them were termed class specific, and type-specific ones were unique to one virus strain. Based on these criteria MMTV(C3H) and MMTV(GR) as well as MMTV(RII1) and MMTV(C3Hf) were put in one class. Five functionally distinct antibody populations were found: (i) group-specific virus-precipitating antibodies, (ii,iii)class-specific neutralizing or cytotoxic antibodies, and (iv,v) type-specific neutralizing or cytotoxic antibodies (Schochetman et al., 1979a,b; Massey et al., 1980b). Evidence exists for lifelong protection from challenge by MMTV. BALB/c mice can be infected at any age with MMTV(CSH), although the efficiency decreases slightly with age. In contrast, mice carrying Mtvl cannot be infected after the age of 18 days (Nandi and DeOme, 1965). Most likely this is due to the appearance of MMTV-neutralizingantibodies. Similarly, mice congenic for a highly transcribed Mtu3 provirus cannot be infected with another MMTV strains when adult (Hainaut et al., 1990).This observation might be crucial in the analysis of the role of gene knockouts in MMTV infection. Many knockout mice were generated in stem cell lines derived from 129 mice that carry Mtu3. Neither cytotoxic nor neutralizing antibodies can prevent virus persistence, virus transmission, or mammary tumor formation; however, a delay in tumor formation was sometimes observed (see below). In some cases the sera of MMTV-infected or mammary tumor-bearing mice were shown to be cytotoxic to mammary tumor cells in vitro in a virus-specific manner (Stolfi et al., 1975; Fine et al., 1978). The cytotoxic effect could be even improved when the sera were preincubated with a rabbit antiserum to gp52, confirming previous reports on the presence of viral proteins in the serum (Ritzi et al., 1976; Fine et al., 1978; Bentvelzen et al., 1980). The shedding of uncleaved envelope protein gp73 either free or in vesicles has been described after incubation of tumor cells with antiserum to MMTV in uitro (Van Blitterswijk et al., 1975, 1979; Calafat et al., 1976). Several vaccination strategies have attempted to protect inice from MMTV infection and mammary tumor development (Charney and Moore, 1972; Charney et al., 1976; Sarkar and Moore, 1978; Cafruny et al., 1986; Dion et al., 1990). Injection of high doses of live virus results in lower levels of virus in milk (Charney and Moore, 1972; Moore et al., 1974),

SUPERANTIGENS OF MMTV

191

lower tumor incidence (Mooreet al., 1969,1974),and slower virus transmission (H. Acha-Orbea and W. Held, unpublished observations) possibly due to a good antibody response (immunity rather than infection). Similarly, immunization of MMTV-free mice with 1 pg of formalin-inactivated MMTV(RII1) or MMTV(A) in complete Freunds adjuvant protected on average 64-81% of mice against challenge with the same live MMTV, as measured by the presence of MMTV antigens in milk and by tumor formation (Charney et al., 1976).The effect of formalin-fixed virus was compared to that of purified gp52 and acid-treated (HCl) gp52. The first two were highly effective in a dose-dependent manner, whereas the latter had no effect (Sarkar and Moore, 1978).Purified gp52 (1-10 pg) was sufficient to induce 100%protection. Either sugar residues or conformational epitopes sensitive to acid treatment may be involved in protection. Attempts have been made to map the protective epitopes on gp52 based on predicted solvent-accessible peptide regions (Dion et al., 1990).All of the four tested peptides induced antibodies reactive with gp52. However, only one peptide (EP-3)provided good protection against M MTV-induced tumor formation. This protection was stronger than with purified gp52. Other mapping studies used monoclonal antibodies (Masseyand Schochetman, 1981).Two topographically distinct but overlapping sites on gp52 were defined, of which only one was neutralizing. Antibodies recognizing the other site could sterically block the binding of neutralizing antibodies. It has been proposed in explanation of a frequent finding that small fractions of the viral population escape neutralization even in the presence of excess antibody. Recently, our laboratory reinvestigated the quantity and quality of the humoral immune response after infection of adult mice with MMTV( SHN) and MMTV(SW) (Luther et al., submitted). By 5 or 6 days after footpad injection of MMTV(SHN) gp52-specific IgG antibodies are detected at near peak levels, This response remains consistent over months and confers life-long cross-protection against infection by other MMTV strains. In BALB/c mice, early protection around day 7 is mediated by neutralizing IgM and IgG antibodies, whereas the protective antibodies after 10 months were mainly of the IgGl isotype. The rapid appearance, the titer, and the longevity of this neutralizing response are dependent on the SAg-reactive T cells. Possibly, a T cell-independent response to MMTV exists during the first few days after infection, similar to other viruses (see Section IV,A). In contrast, injection of congenic splenocytes expressing M u 7 SAg induces a polyclonal antibody response similar to MMTV injection (Andersson and Acha-Orbea, 1994),but lacks any virus specificity. This is due to expression of Mtu7 SAg by all injected B cells and most likely due to preferential infection of virus-specific B cells after virus injection.

192

SANJIV A. LUTHER AND HANS ACHA-ORBEA

Currently, it is not clear why the antiviral antibody response induced in MMTV-infected animals is not capable of preventing mammary gland infection and virus transmission but rather leads to virus persistence. 6. Cellular lmmune Response against MMTV

A wealth of experimentaldata exists on cellular responses against MMTVinduced tumors but little evidence exists on cellular immune responses against virus-infected immune cells in vivo. Because no clear picture emerges from these early data, we will summarize the most prominent findings and refer the reader to more detailed reviews (Bentvelzen and Hilgers, 1980; Creemers and Bentvelzen, 1981). All data suggest that the cellular immune responses observed in tumorbearing mice are generally stronger than those seen in tumor-free mice. Cytotoxic responses directed against M MTV antigens were observed in vitro and in vivo (mammary tumor transplantation). Usually, no crossreactivity of the cellular immune response was observed suggesting that antigenic epitopes on specific mammary tumors were unique (Bentvelzen and Hilgers, 1980;Creemers and Bentvelzen, 1981).An inverse correlation was found between the size of the tumor and the cellular immune response (Creemers, 1977; Gillette and Lowery, 1977). Similarly, a higher reactivity against mammary tumor cells was found in mice with lower levels of MMTV infection than in mice with higher levels (Gillette and Lowery, 1977). In all cases, the anti-MMTV responses were not potent enough to prevent the development of mammary tumors. Using in vitro microcytotoxicity assays, Blair and co-workers could show that tumor cell proliferation could be inhibited by the addition of splenocytes from tumor-bearing mice and to a lesser degree by cells from tumorfree mice infected neonatally. Curiously, cells derived from uninfected BALB/c mice were more efficient in tumor growth inhibition than neonatally infected mice but only when they were older than 14 weeks, suggesting the expression of endogenous Mtu antigens (Blair and Lane, 1974). With spleen cells from all sources the reduction of tumor cell number in vitro was observed in two phases. The early phase of cytotoxicity (8-18 hr) was T cell independent and attributed to cells other than B or T cells. A step responsible for the induction of this cytotoxic response was B and T cell dependent and most likely involved the production of tumor-specific antibodies. The actual cytotoxic response was thought to be mediated by antibody-dependent cellular cytotoxicity (Blair and Lane, 1975; Blair et al., 1975; Gillette and Lowery, 1977). It is possible that antigen-antibody complexes are responsible for immune suppressiveeffects found frequently in mammary tumor-bearing mice, as described for other systems (Gorczin-

SUPERANTIGENS OF MMTV

193

ski et al., 1975).The second phase of cytotoxicity (36 hr later) was partially dependent on T cells (Lane et al., 1975; Roubinan et al., 1976). In another study, mammary tumors induced by MMTV(C3H) were transplanted into the footpad of syngeneic mice and CD8+T cells isolated 20-30 days later from the draining lymph node. The test of cell-mediated cytotoxicity was performed on syngeneic or allogeneic mammary tumor target cells. A biphasic cytotoxic response was found. The early peak of cell-mediated cytotoxicity was detectable at 6 hr, accounting for 20-30% of the total cell-mediated cytotoxicity and showing MHC restriction. The late peak appeared after 18-20 hr represented an additional 40-50% of cell-mediated cytotoxicity and was MHC unrestricted. The early and late phases of cell-mediated cytotoxicity could be abolished with antibodies against CD8+ T cells (anti-Ly-2.1). For the second phase CD4' T cells were required (Stutman, 1976; Stutman and Shen, 1978). Because the second part of the response is MHC unrestricted, it can be speculated that it is SAg mediated. A recent study identified potential Kd binding peptides from the published sequences of MMTV gag and env proteins by comparing these to published binding motifs. A total of 27 peptides were identified, out of which 6 efficiently bound to a soluble single-chain MHC class I fusion protein. Five of the six peptides induced CTLs in uivo, and 1was derived from gag, the other 4 were derived from env (Gillet al., 1994b).Immunization with a peptide derived from the sequence of gp36 of MMTV(C3H) induced CTLs in vivo that reacted to MMTV(C4)-induced tumors in vitro, suggestingpresentation of this peptide in tumors. Curiously, peptideimmunized mice were partially protected against the growth of tumors induced by MMTV(C4)but not by those induced by MMTV(C3H) (Wei et al., 1996). Evidence for NK cell activity was found among infiltrating cells from small MMTV(CSH)-inducedmammary tumors, whereas infiltrating cells from larger tumors decreased the NK activity of normal spleen cells (Gershon et al., 1981). Several studies characterized lymphocytes isolated from MMTV(C4)-inducedhyperplastic alveolar nodules. Nearly 30% of the infiltrating cells expressed the BALB/c NK marker ASGM (Wei et al., 1986). Neoplastic progression was slow with high NK activity and rapid in the reverse case. NK activitywas upregulated by treatment with poly(IC) and downregulated by treatment with antibodies anti-ASGM or by infiltrates from advanced C4 tumors (Wei and Heppner, 1987). Similarly, transfer of MMTV(C4)-infectedsplenocytes into naive BALBlc mice increased NK activity,which peaked on Day 7. Depletion of the SAg-reactive T cells before the transfer partially reduced the NK activity suggesting a SAg-dependent and a SAg-independent mechanism. In another approach,

194

SANJIV A. LUTHER AND HANS ACHA-ORBEA

injection of milk-purified MMTV(C4) resulted in elevated NK activity by day 3, which returned to normal levels after 2 weeks (Wei et al., 1986; Gill et al., 1994a).The current knowledge on cytokine production induced by MMTV infection supports a model in which the simultaneous presence of IFN-y and IL-4 promotes a Thl-dependent NK activation and a Th2dependent B cell activation, respectively. Together, the data suggest that NK and cytotoxic T cell responses occur in response to M MTV-induced tumors. Clearly, mice expressing endogenous Mtu do not display a strong tolerance to antigens from MMTV. It will be important to determine the role that cytotoxic T cells play during the whole course of MMTV infection, from the SAg-driven virus amplification to the induction and development of mammary tumors. C. POTENTIAL RECEPTORSFOR MMTV The first step of infection by retroviruses relies on the presence of one or several cellular membrane receptors (Weiss and Tailor, 1995). The expression pattern of such a receptor determines the host range and cell type infected. The cellular receptor for MMTV infection has not yet been identified. Various studies have aimed at identifying the MMTV receptor. Mouse cross-breeding experiments showed that the efficiency of virus infection and transmission is H-2 dependent (Bentvelzen and Hilgers, 1980), and can now be explained by the differential binding of the viral SAg with the various H-2 haplotypes (see Section 111,B). Binding studies with MMTV(C3H)showed good, dose-dependent binding to a rat embryo cell line (FRE) and to two mouse cell lines (SLP and NMuMG), one of which was derived from mammary epithelium (Altrocket al., 1981).By Scatchard analysis the binding affinities (KO) were determined for the interaction between tritium-labeled MMTV(C3H) and receptors on the three cell lines, all of which were above loQM-'.This could mean that the MMTV receptor is an evolutionary conserved surface component of murine and rat cells. In the same study investigations were performed on the focus formation by pseudoviruses consisting of VSV with the envelope glycoproteins of MMTV(C3H).MMTV(C3H)and MMTV(GR) could significantly inhibit the focus formation by the pseudovirus. By comparison, two other MMTV strains with a less related envelope protein (Schochetman et al., 1980),MMTV(C3Hf)and MMTV(RIII),did not show any inhibitory effect suggesting that MMTV strains are bound by different epitopes on the receptor or by different receptors altogether. In other studies using pseudotype viruses the infection of cell lines of various origin were tested with contradictory results (Zavadaet al., 1977; Schochetman et al., 197913; Hilkens et al., 1983).Interspecies hybrids were prepared between an infectable

SUPERANTIGENS OF MMTV

195

mouse and an uninfectable hamster cell line. A strong correlation between the loss of chromosome 16 and loss of infectability was observed in the subclones resulting from this fusion (Hilkens et al., 1983). Another study addressed the tissue distribution of the MMTV receptor in mice (Bolander and Blackstone, 1991).All tissues examined by Scatchard plot analysis bound MMTV but to varying degrees: lactating mammary gland % involuted mammary gland, spleen > salivary gland, ovary S adrenal gland, liver. Tissue distribution and hormone dependency of the virus-binding protein paralleled the known expression pattern of MMTV proteins. Moreover, the binding kinetics suggested a single high-affinity binding site for MMTV. Intact MMTV particles, but not isolated gp52, led to the redistribution of the virus receptor from the plasmalemma to the microsomes (Bolander, 1996). These results could indicate that internalization requires receptor aggregation by a multivalent viral envelope or the participation of another viral component besides gp52. A requirement for acidification for successful infection has been postulated (Redmond et al., 1984). Recently, PCR analysis was used to detect cells that have integrated viral DNA. It revealed the preferential infection of B cells and not T cells in the early phases after infection (Held et al., 1993a,b, 1994a). Dendritic cells and macrophages have not been analyzed so far. Moreover, quantitative PCR clearly showed that only a small proportion of B cells get initially infected, even when high virus doses are injected (Held et al., 1993b, 1994a). These results suggest that, for cell entry, MMTV uses a cell surface molecule that is expressed on B cells but poorly expressed on T cells. It argues for the infection of a few B cells that are either in a particular stage of activation (e.g., in cell cycle) or express a clonally distributed receptor such as immunoglobulin. Based on the recent observations of T cellindependent B cell activation by MMTV particles, one could imagine that immunoglobulins could be implicated in the process of infection and B cell activation. Cocross-linking of surface immunoglobulin with other B cell surface molecules could lead to B cell proliferation essential for the integration and amplification of this retrovirus. The early onset and the SAg dependence of the antiviral antibody response could even be explained by the preferential infection and amplification of virus-specific B cells. Besides the nature of the receptor, we also lack information about the mechanism of virus spread between lymphocyte subsets or to the mammary gland. It is assumed that MMTV is transmitted from the initially infected B cells to CD4+ and CD8+ T cells, the mammary epithelial cells, and other exocrine organs. When, where, and how this happens is not known. Possibly, the initially infected lymphocytes migrate throughout the body and thereby spread the infection. The transfer of MMTV to other cell

196

SANJIV A. LUTHER AND HANS ACHA-ORBEA

types could either be due to virus release followed by receptor-mediated uptake or be due to cell-cell transfer. The transfer of proteins has been previously described to occur between interacting cells (Olsen et al., 1981; Abraham et al., 1988). The transfer of Mtv SAg protein has also been described in several studies (see Section V,A) (Pullen et al., 1988; Speiser et al., 1989; Modlin et al., 1995).Similarly, transfer of virus from cell to cell or by cell fusion has also been postulated to occur in other virus infections. The identification of the MMTV receptor(s) should be the primary goal of future studies. It would allow us to ask more precise questions about B cell infection, virus spread, cancerogenesis, and virus-host interaction. V. T and B Cell Response to Endogenous Miv

The majority of experiments with endogenous Mtv SAgs were done before the role of SAgs was known. The earlier results are only introduced briefly to help understand the previous nomenclatures. Thereafter, they are summarized based on the recent knowledge of the role of endogenous Mtv proviruses in this process. For more complete lists on the earlier literature see Festenstein (1973), Abe and Hodes (1989), and Janeway (1991). A. IMMUNE STIMULATION BY Mtu SAgs More than 20 years ago a striking observation was made in primary mixed lymphocyte cultures in uitro: When naive T cells from one inbred mouse strain were cultured with a genetically dissimilar, H-2-identical population of stimulator cells, up to 20% of all T cells were induced to proliferate in a mixed lymphocyte reaction (Festenstein, 1966). Further analysis revealed that mixed lymphocyte reactions were due to one single dominant gene ( M locus) that exists in a stimulatory and a null allele and is not linked to H-2 (Festenstein, 1973). To distinguish them from H-2 determinants, the M locus antigens were called Mls antigens. The breakthrough in understanding of Mls molecules came in 1988when monoclonal antibodies against various Vb domains of the TCR became available. The expression of Mls molecules in the thymus was shown to induce the deletion of T cell subsets expressing a particular TCR V/3 domain (Kappler et al., 1988; MacDonald et al., 1988b).It explained why the murine I-E molecule was initially described to induce thymic T cell deletion (Kappler et al., 1987a,b).This T cell modulation was found to be due to an I-E-dependent, Mtv-encoded SAg (Bill et al., 1989; Woodland et al., 1990). Because of its potent impact on the immune system, similar to the bacterial enterotoxins (White et al., 1989), the Mls antigen was termed SAg. Finally, the origin and effect of the Mls molecule was tracked down

SUPERANTIGENS OF MMTV

197

by genetic linkage analysis to the endogenous and exogenous forms of MMTV (Woodland et al., 1990, 1991a; Dyson et al., 1991; Frankel et al., 1991; Marrack et al., 1991) and in particular to the SAg encoded in the orf within the viral 3'LTR (Acha-Orbea et al., 1991; Choi et al., 1991). The properties of the Mtv loci and their SAgs are briefly summarized in Table 111. The Mtv loci are located on different chromosomes (Abe et al., 1987a,b; Pullen et al., 1989; Peters et al., 1992) and display a highly polymorphic COOH terminus (see Section III,A and Figs. 3 and 4) (Festenstein, 1973, 1974; Click et al., 1985, 1987; Abe et al., 1987a,b; Ryan et al., 1987;Acha-Orbea and Palmer, 1991).They always exist as stimulatory (termed, e.g., Mls-la in the older and Mtv7 SAg in the newer literature) or null alleles (Mls-lbin the older and no name in the newer literature), which was easily explained once it was shown that retroviral integrations and expression of their SAgs were responsible for this effect (see Table I11 for the old and new nomenclature). Different mouse strains express different Mtv proviruses (see Table 111) and therefore different SAgs. Once these facts were clear it was not difficult to explain the presence of such a strong mixed lymphocyte reaction between MHC-identical mouse strains. Contrary to classical peptide antigens, which can be presented by both MHC class I and class I1 molecules, MMTV SAgs can only be presented by MHC class I1 molecules. Therefore, it was not surprising that CD4+ T cells expressing the SAg-reactive TCR Vp element responded to SAgs presented by MHC class I1 molecules. It was more of a surprise that T cells usually interacting with MHC class I molecules (CD8' T cells) were also activated by SAgs associated with class I1 molecules. CD8+ T cells expressing the SAg-reactive VP, however, reacted less strongly with MHC class II-MMTV SAg complexes than CD4' T cells. In vitro and in vivo stimulation with Mtu7 congenic splenocytes resulted in an increase of SAgreactive CD4+ and CD8' T cells (MacDonald et al., 1990; Webb and Sprent, 1990b; Chvatchko and MacDonald, 1991). Similarly, after intravenous injection of SAg-presenting splenocytes into responsive recipients the SAg-reactive T cells initially decreased in numbers (up to 36 hr after injection) among the thoracic duct lymphocytes, but then numbers increased dramatically from days 2 or 3 in both the CD4' and CD8+ T cell subsets (Gao et al., 1989). This VP-specific expansion of CD8+ T cells in oitrn was accompanied by the secretion of IFN-y and completely lacked cytotoxic activity against Mtv7 expressing cells. Similarly, Mtv7-primed CD4+ T cells had no detectable cytolyhc activity (Herrmann et al., 1993). Although the presentation of SAgs is characterized by little MHC restriction compared to classical antigens, a clear difference in the efficiency of different MHC class I1 haplotypes to present SAgs of Mtv exists (Jones

TABLE I11 MTV DISTFUBUTION IN THE MOSTCOMMON LABORATORY MOUSESTRAINS _______

~~~

_____

~

Mtv

1

3

6

7

8

9

11

13

f. Etc-I. Dvbll.2 12

f. Dvbll.3

c. 2a

14

4

Mls phenotype

c, 4a

c

c, 3a

a. la

f,

Chromosome'

7

11

16

1

Dvbll.1 6

vp specifcity

3, l7a2?

MHC class IIb

E>A

3, 5, 17al E>A

3, 5. 17a2 ESA

6, 7, 5. 11, 12, 5.1. 5.2,11, 17al? 12, 17al? 8.1, 9 E>A E E

11, 12, 17

E

14

17

23

29

4

4

6

6

(11).(12)

7

31

43 Mk-lke

3. 17a2? -'

EPA

30

1 2 Y

16. (17a) 5?

?

6. 7. 8.1, 9

-

W Strain

H-2

I-E

haplot&

a

k

b

k

k

b

d

d

b

BALB.D2 Biozzi A B H B R6 BXSB

d

d

b

b

-

B1O.BR

k

k

C3HnleJ

k

k

V p deletione -

+

-

-

-

+

+

+

+

-

-

+

+

-

-

+

+

+

-

-

+

-

-

-

+

+ +

-

b

k

k

b b

-

b

k b

b

-

k

a

-

a

-

- b

+

+ -

C3H/Bi, Ki. Sm C57BU6J C57BU10 C57BWdJ c57y

+

+

+

+

-

+

-

3, 5, (7). (11). 12, (17) 5. 6. 7, 8.1. 9, 11, 12. (17al) 3, 5, 11, 12, (171 3.5.6, 7, 8.1, 9. 11, 12, (17)

+

+ +

5. 11, 12

-

-

3. 5. 11. 12,

+

+

+

+ + + +

+

+ + +

(17al)

-

-

? ?

-

17 3, 5, 6, 7. 8.1, 9. 11. 12. (17) None None (16). 17

I

I

I

I

I

I

I +

+

I

n. CL.

I +

I

I

I

I

I

I

I

I

I

I

I

I

I

l

I

l

I

I +

+

l

I

l

++

++

+ I

I +

+n.

I

I I + + + + +

I

1

I

I

I

+ I

I

l

I

+ I

l

*.+a.

I

I

I

l

l

1

l

l

l

I

l

l

I

I

I

+

l

l

l

l

I

1

I

1

+

+

1

l

I

I

I

+

l

I +

I

1

l

+ I

I

I

l

+ + + I

l

+ I +

+

+

l

l

I

I

I

l

+++++++

I

1

I

+

I

I

I

+

+

++

I

I

+ I +

I

I +

I

I

1

+ + I +

+ I

I

+

l

I

I

l

I

+ I

I

l

I

I + +

l

+

I

I

I

l

I

I +

I

l

I

I

I

1 + 1

I

l

I

I

I

I

I

+++++

+

I

I

l

I

I I

I

I

++

I

I

I

I I

I

I

+++ I

I

I

1

I

+

/

I

+ I

I

+++ + - I

I

I

I

+

+++

+

I

+

+

I

+ +

I

+

I

I

+ ++ + I

I +

I

I

+ +

+++

l

I

I

+

+

l

+ I

I

++

I

I

I

+

++ + I

I +

+ + + I 1

+

+ + I

++ ++

I +

1

I

I

+

+++

+ I

+

I

199

TABLE I11 Continued Note. This table with the strain distribution of the Mtv proviruses is an update of Scherer et al. (1993). It was determined by Southern blot by Frankel et (1991). Fairchild et al. (1991, 1992), and Scherer et al. (1993). Others are summarized in Kozak et al. (1987), Tomonari et al. (1993). Simpson et al. (1993). and WajJwauol et al. (1991, 1993). The chromosomal location of the Mtus is from Peters et 01. (1992). The Vp specificity is from papers listed in Table I. Data on additionalMtus: Mtu-MAZ is found in wild mice and deletes Vfl17a.2 in an I-E-independent manner (Jouvin-Marcheet al., 1992);Mtu2 is found on chromosome 18 of WXGP mice (Femck et al., 1992). ' In some cases the Mtv-ldzation on chromosomesvaries among mouse strains, e.g., Mto30 is found on Chromosome 12 in AKR/J mice but not in C57BU 6 or NZB/BlNJ mice. * MHC class I1 needed to see SAg effect; E :I-E, A : I-A; E>A: I-E more efficient than I-A. Mto14 makes a nonfunctional SAg (M.Braun and H. Acha-Orbea, unpublished data). Most inbred mouse strains can be categorized into three Vp haplotypes. The V@ haplotype shows a genomic deletion of the following Vp elements: 5,8,9,11,12, and 13 (Behlkeet al., 1986). The Vpbhaplotype typifies most strains which possess the fullcomplement of Vp genes and contain a V/317b pseudogene. The V p haplotype is similar to V@ strains but has additional genomic deletions of V/36,15,17a, and (3.2) (Haqqi et al., 1989). Furthermore, V/3 genes 1,3.1,6, and 10 from V@ haplotype strains differ from their counterparts in VP. and V p strains by few amino acid changes (Smith et al., 1990). To date, 20 Merent Vp subfamilies (V/31-Vp20) have been defined, most of which are single-member subfamilies. V/32l-V/325 are pseudogene subfamilies (Louie et al.. 1989; reviewed in Williams et al.. 1993). 'The V/3-specific deletion is deduced from the expression of I-E and the type of Mtu SAg. The deletion obsexved is mostly complete in adult mice. 'One Mtu locus has not yet been identified for 129 and Biozzi, STR; three Mtus have not yet been identified for NZW. c NZB mice also contain Mtu27 deleting Vp3' T cells (Tomonari et al., 1992), Mto28 (Tanaka and Matsuzawa, 1990), and Mtu44 deleting V/36+, 8.1+, and 9' T cells ( Fairchild et al., 1992). Some but not all NOD mice contain Mco45 (Fairchild et al., 1992). NZW mice also contain Mtu44 ( Fairchild et al., 1992). CS mice also contain Mco46, Mco48, Mto49, Mto50. and Mtv51 (Wajjwah et al., 1991). NC mice code in addition for Mto48, Mto.50, and Mtu.51 (Wajjwah et al., 1993). 01.

SUPERANTIGENS OF MMTV

201

and Janeway, 1982; DeKruyff et al., 1986; MacPhail and Stutman, 1986; Kappler et al., 1988; MacDonald et al., 1989a).For example, for Mtv7 gene products the following hierarchy of reactivity among H-2 determinants was observed: a, k, d > b S s S q identical to the previously described hierarchies in cancer susceptibility and SAg presentation or MMTV transmission (SectionsII,B,3 and 111,B).Although all SAgproteins are presented efficiently by class I1 I-E molecules, several of them are poorly presented by I-A molecules (Ryan et al., 1987; Abe et al., 1988) (see Table 111). Because most mouse strains code for approx. 20 different Vp elements, each Mtv SAg shows reactivity with 3-30% of all ap+CD4+T cells, which often belong to one to four different Vp subpopulations. For example, the SAg of Mtv7 reacts with most T cells bearing Vp6, -7, -8.1, and -9, a population that can be up to 30% of all CD4' T cells in a given mouse strain (Kappler et al., 1988; MacDonald et al., 198813; Happ et al., 1989; Kanagawa et al., 1989; Okada et al., 1990). Only a few wild mouse strains are devoid of endogenous Mtvs (Gallahan and Callahan, 1987; Jouvin-Marche et al., 1992). AU of the 19 or more tested laboratory mouse strains express one to eight endogenous Mtvs (Kozak et al., 1987; Scherer et al., 1993) (see Table 111). Practically all endogenous Mtvs show an intact SAg coding sequence suggesting that little evolutionary pressures exist to eliminate a functional SAg. A hierarchy of reactivity is also observed among the SAg-reactive Vp subsets upon injection of Mtv7 expressing cells in viva Only Vp6+ T cells get stimulated, whereas all T cells reactive with Mtv7 get deleted, although with different kinetics. The following hierachy of recognition was postulated for the SAg of Mtv7: Vp6>Vp9>Vp8.1>Vp7 (Waanders and MacDonald, 1992).A clear correlation is found between stimulatioddeletion and the expression level of SAg and MHC class I1 molecules (Waanders et al., 1995). Together, the existing data are in agreement with a model in which only high-affinity interactions lead to detectable stimulation prior to deletion, whereas interactions of medium or low affinity lead directly to rapid or slow deletion, respectively. The expression levels among different cell subsets could also explain the following findings in adoptive transfer systems: Transfer of Mtu7 expressing B cells induces T cell stimulation followed by deletion. Mtv7 expressing CD8+T cells are controversial in that only some reported T cell stimulation, whereas all reported T cell deletion. CD4' T cells were completely inactive in these transfer assays (Webb and Sprent, 1989; Webb et al., 1990; Waanders et al., 1993b; Modlin et al., 1996).The way CD8+T cells present the SAg remains elusive. CD8+ T cells express MHC class I1 molecules at a level sufficient for SAg presentation, acquire it through cell-cell transfer (Sharrow et al., 1981), or actively secrete a soluble form of the

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SAg that finally gets presented by MHC class I1 expressing cells. Although the current opinion is that mouse T cells do not synthesize MHC class I1 molecules de novo (Glimcher and Kara, 1992),low levels of these molecules have been detected by flow cytometry. The level of class I1 molecules on resting T cells (CD4' and CD8') was approx. 30-fold lower than that on resting B cells (Waanders et al., 1995).Evidence for a transfer of soluble SAg comes from studies using bone marrow chimeras, coinjection, or mouse strains expressingdifferent Ig allotypes (Pullen et al., 1988; Ramsdell et al., 1989; Speiser et al., 1989; Dannecker et al., 1991; Modlin et al., 1995, 1996).In this context it is noteworthy that injection of Mtv7 expressing cells into Mtu7-negative mice led to their rapid disappearance within days. One major problem with most of these studies is that the lymphocytes injected were matched only for MHC but not for multiple minor histocompatibility antigens. This might lead to rejection of the donor B cells. In agreement with this, several of these reports showed lack of long-term deletion and anergy of the SAg-reactive T cells (Rammensee et al., 1989; Webb et al., 1990;Webb and Sprent, 1993),which is readily found when Mtv congenic B cells are injected (Waanders et al., 1993b; Andersson and AchaOrbea, 1994). Other antigen presenting cells have also been retested for their capacity to present Mtv-encoded SAgs. Macrophages have never been reported to induce VP-specific T cell stimulation in vitro (Molina, 1989; Webb et al., 1989).The ability of dendritic cells to present Mtv SAg is still controversial, although they play a key role in clonal deletion in thymic organ cultures and upon intrathymic injection render T cells anergic (Inaba et al., 1991; Mazda et al., 1991). The B cell response in lymph nodes upon subcutaneous challenge with Mtv7 expressing cells is characterized by blastogenesis and a strong increase in local B cell numbers, peaking on day 3. The B blasts differentiate locally into plasma cells that secrete immunoglobulins (on day 4 predominantely of IgM and on day 6 of IgG isotype: IgG+1gG2b>IgM, IgG,, IgG,) (Andersson and Acha-Orbea, 1994).The generated antibodies are presumably of polyclonal origin because they are not directed against the host cells or self-antigens (Modlin et al., 1996). Interestingly, the increase in number of B cells and plasma cells is of host rather than donor origin (Bandeira et al., 1991; Modlin et al., 1995, 1996). In addition, purified host B cells from Mtv7-primed recipients efficiently present the passively acquired SAg to VP6' T cells in vitro (Modlin et al., 1995). Again, care must be taken with the interpretation because the injected B cells were not derived from Mtv congenic mice. Together, the data suggest that the SAg protein is transferred from donor cells to class I1 determinants on host B cells leading to T and B cell

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activation and differentiation. The mechanism of SAg transfer and host B cell differentiation is still very obscure.

B. ROLEOF Mtv SAgs IN TOLERANCE INDUCTION The in vivo consequences of expression of endogenous SAg are profound. The course of the immune response to the SAg depends on the time point of SAg exposure. Neonatal expression of the SAg leads to lifelong clonal deletion of the SAg-reactive TCR a/3' T cells in the thymus. In contrast, SAg exposure later in life leads to T cell expansion followed by the deletion or induction of anergy of the responding T cells. This latter form of deletion is not thymus dependent. 1. Negative Selection in the Thymus Studies with SAgs and TCR transgenic mice have been instrumental in demonstrating clonal deletion in the thymus, which is a major mechanism of lymphocyte tolerance. During this process, immature T cells with strong reactivity to self-antigens are eliminated from the repertoire by cell death in the thymus (for review see Sprent and Webb, 1987; Fowlkes and Pardoll, 1989; Blackman et al., 1990a; Ramsdell and Fowlkes, 1990; Sprent et al., 1990; Zinkernagel et al., 1991; Acha-Orbea, 1995; Kisielow and von Boehmer, 1995). Expression of endogenous Mtv SAgs leads to thymic deletion of SAgreactive T cells soon after birth (Kappler et al., 1987a,b, 1988; Abe et al., 1988; Fry and Matis, 1988; MacDonald et al., 1988b; Pullen et al., 1988; Kanagawa et al., 1989). Different kinetics of neonatal clonal deletion were observed. Complete deletion of the SAg-reactive T cells (>go%) is seen in I-E+ mice expressing Mtv6 3 days after birth, with Mtvl, Mtv7, and Mtv50 10 days after birth, and with MMTV(GR) Mtvl, Mtv8, Mtu9, Mtvl3, Mtv27, and Mtv44 several weeks after birth (Schneider et al., 1989; MacDonald et al., 1989a; Gollob and Palmer, 1991, 1992; Acha-Orbea et al., 1991; Simpson et al., 1993; Tomonari et al., 1993; Niimi et al., 1994; Morishima et al., 1994). The kinetics of deletion correlated well with the relative SAg RNA levels (Niimi et al., 1994; Waanders et al., 1995). Neonatal tolerance by clonal deletion of Mtv SAg-reactive T cells has been shown to occur during thymic maturation at the immature, doublepositive stage. Transgenic mice expressing a TCR reactive with both a peptide antigen and an Mtu SAg showed deletion of the former in the cortex and of the latter at the corticomedullary junction of the thymus (Hengartner et al., 1988; Kappler et al., 1988; MacDonald et al., 198813; Pircher et al., 1989). As a consequence, the SAg-reactive T cells in the thymus medulla (as well as in the peripheral lymphoid organs) are detected at strongly reduced levels for life. This thymic deletion of SAg-reactive T

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cells in Mtv expressing mice could be partially blocked by antibodies against the SAg, CD4, or MHC class I1 molecules (Fowlkes et al., 1988; MacDonald et al., 1988a; Jones et al., 1990; Acha-Orbea et al., 1992). Thymic deletion of VP6' T cells in Mtv7-positive strains affects equally the CD4' and CD8' T cells but the CD4 interaction is required for thymic deletion to occur (Fowlkes et al., 1988; Kappler et al., 1988; MacDonald et al., 1988b). Using Mtv congenic mouse lines, a clear difference in deletion kinetics was seen between Mtv8 and Mtv9, both of which interact with Vp5+ and+VPll+ T cells. MtuQ deletes faster in both the CD4' and CD8' subsets, whereas Mtv8 deletes more slowly and much more pronounced in the CD4' subset (Braun et al., 1995). These findings are supported by the higher levels of methylation of the Mtv8 locus indicating lower expression levels (Gunzburg et al., 1984). Transplantation of an Mtv7 expressing thymus into nude mice does not induce clonal deletion of the VP6+ peripheral T cells. In the presence of mature donor lymphocytes a transient partial deletion is seen (Yuuki et al., 1990). In the absence of a thymus from birth (nude mice) clonal deletion of SAg-reactive T cells was not observed (Fry et al., 1989; Hodes et al., 1989).In agreement with these studies, differences in clonal deletion between intraepithelial CD8' T cell subsets were observed. C D 8 4 T cells showed deletion, whereas CD8cra T cells were spared. The former are similar (or identical) to peripheral T cells, the latter most likely represent a thymus-independent intestinal population (Murosaki et al., 1991; Rocha et al., 1991; Poussier et al., 1992). Thymectomy soon after birth led to lifelong survival of potentially autoreactive T cells (Smith et al., 1989). Experiments with bone marrow chimeras and thymus grafts clearly show that bone marrow-derived cells are responsible for clonal deletion in the thymus (Marrack et al., 1988; for review see Blackman et al., 1990a; Ramsdell and Fowlkes, 1990; Sprent et al., 1990; Kisielow and von Boehmer, 1995). Surprisingly,neonatal injection of CD8' T cells expressing Mtv7 within the first 24 hr after birth requires 100-fold less cells for induction of neonatal tolerance, but not tolerance induced in adult mice, than injection of B cells or CD4' T cells (Webb and Sprent, 1990a,b). In addition, mice deficient in lymphocyte populations (B, CD4', and CDS'), costimulatory molecules (CD28 but not CD40L), orfm-mediated apoptosis (Zpdlpr and gld/gld ) usually delete the SAg-reactiveT cells (see Table I1 and references therein). In most cases the kinetics of deletion have not been addressed.

2. Positive Selection in the Thymus T cells that can recognize self-MHC molecules with low affinity are thought to be positively selected in the thymus (for review see Blackman

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et al., 1990; Sprent et al., 1990; von Boehemer and Kisielow, 1995). A higher percentage of T cells bearing particular TCR V/3 regions were found in mice expressing I-E molecules (MacDonaldet al., 1988a).Genes outside the MHC complex are important for influencing the amount of positive selection (Kappler et al., 1987a,b, 1989; Blackman et al., 1989, 1990b). For transgenic mice expressing I-E molecules in different thymic compartments, a role for expression in the thymic cortex was shown in positive selection (Marrack et al., 1988; Speiser et al., 1989; Benoist and Mathis, 1989; Berg et al., 1989b; Bill and Palmer, 1989). It has been speculated that Mtv SAgs can also play a role in positive selection of populations of T cells with weak affinity for the SAg (Liao and Raulet, 1992; Simpson et al., 1993; Tomonari et al., 1993; Braun et al., 1995; Scherer et al., 1995). In these studies, deletion of SAg-reactive T cells by endogenous Mtvs resulted in uneven compensation by certain TCR V/3 expressing T cell populations. Whether this uneven compensation was due to positive selection or to other unknown mechanisms is still a matter of debate.

3. Peripheral Tolerance: Deletion and Anergy Mtv SAg-reactive CD4' T cells are clonally deleted in thymectomized mice indicating the existence of peripheral tolerance mechanisms affecting mature T cells (Jones et al., 1990; Webb et al., 1990; Abromson-Leeman and Dorf, 1991; Dannecker et al., 1991). This lifelong T cell deletion in thymectomized mice is preceded by a dose-dependent clonal expansion phase and blastogenesis of SAg-reactive CD4' T cells exclusively at the site draining the injection (Webb et al., 1990; Bandeira et al., 1991; Dannecker et al., 1991; Andersson and Acha-Orbea, 1994). It suggests that tolerance is the end result of a powerful immune response. T cells taken ex vivo at the peak of the response are clearly Mtv7 reactive. The T cell expansion is a mixture of specific T cell trapping and T cell proliferation (Webb et al., 1994; Hayden et al., 1996; Le Bon et al., 1996). Usually a direct correlation between the intensity of the initial T cell proliferative response and the degree of subsequent T cell elimination was observed (Webb et al., 1994).Ex vivo cultures suggest that the SAg-reactive T cells undergo apoptosis (Webb and Sprent, 1993), similar to SEBstimulated T cells (Kawabe and Ochi, 1991; MacDonald et al., 1991). However, in the in vivo environment no evidence for T cell apoptosis after Mtv SAg challenge exists. Clearly, many specific T blasts leave the site of the local immune response because they are found a few days later in the thoracic duct (Webb et al., 1994). It has been observed that the SAgstimulated blast cells home to the gut-associated immune system (Webb and Sprent, 1993).The actual mechanism of T cell deletion is still unclear.

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Recent evidence implicates Fas-mediated cytotoxicity in SEB-induced clonal deletion of the SAg-reactive T cells (Renno et al., 1996).A role for Fas was not seen in MMTV-induced clonal deletion (Kotzin et al., 1988: Singer et al., 1989; Papiernik et al., 1995). Some SAg-reactiveT cells usually survive but exhibit a state of unresponsiveness (anergy) to TCR cross-linking or Mtu reactivity (Lilliehook et al., 1975; Rammensee et al., 1989; Webb et al., 1990; Jones et al., 1990; Bandeira et al., 1991; Dannecker et al., 1991; Todd et al., 1994). Anergy was defined as lack of proliferation and IL-2 secretion after specific stimulation in vitro (Lamb et al., 1983). Low expression of IL-2 mRNA was observed in anergic T cells after restimulation with SAg expressing B cells (Acha-Orbea et al., 1993). The extent of Vp-specific T cell deletion and anergy shows great variation between the different studies. The induction and maintainance of anergy is still controversial and poorly defined at the molecular level (reviewed in Schwartz, 1996). In vivo labeling of cells with BrdU allows following their fate. Animals can be pulsed with BrdU and the cells that divided during this pulse can be analyzed later. The surviving SAg-reactive T cells display a mixed phenotype. Among the long-term surviving BrdU+, SAg-reactive T cells only a small proportion has acquired and retained an activated/memory phenotype (CD62Llow,CD44h$, and CD45RB1"), whereas a significant proportion cannot be distinguished from naive cells. Curiously, these BrdU+VpG+T cells from long-term Mtu7-primed mice cannot be restimulated in vivo, despite lacking anergy in vitro upon stimulation with antiVp6 antibodies (Dannecker et al., 1991; Hayden et al., 1996), It has been suggested that the encounter with SAg tends to promote different forms of tolerance rather than memory (Webb and Sprent, 1993).Alternatively, this proliferative anergy could be due to terminal differentiation of T cells, as discussed below. More conclusive experiments are needed to further clarify the true nature of tolerance induced by Mtv antigens, The transfer of Mtu7 expressing cells does not only lead to the activation of T helper cells and B cells but also is accompanied by cytokine secretion (IFN-y) by CD8+ T cells. The cytokine pattern observed was strongly dependent on the route of cell transfer. Intravenous injection of Mtv7 expressing cells leads to a rapid and short-lived T cell response in the spleen that is accom aqied by a rapid downregulation of IL-2 and IFNy production. In the ong term, B cells secreting lgGl persisted, indicating the presence of a long-lived THz-dependentimmune response. In contrast, sc injection induced a longer lived T cell expansion with secretion of significant amount of IL-2 and IFN-y by already activated cells at approximately Day 4 (hdersson and Acha-Orbea, 1994). This response showed IgG2a production (TH1?) that disappeared after 10 days and was not

P

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replaced by a significant IgGl response. Most studies in transgenic and other systems are consistent with the notion that antigen challenge by systemic antigen delivery induces tolerance rather than immunity, whereas local antigen challenge leads to immunity, presumably due to the entry of specificT cells into the B cell follicles (Kearney et al., 1994, and references therein). The primed CD4+ T cells develop efficiently into effector cells as assessed by B cell activation and immunoglobulin secretion (Bandeiraet al., 1991;Andersson and Acha-Orbea, 1994;Modlin et al., 1995).Moreover, secondary challenge of primed T cells leads to a lack of T cell proliferation, on the one hand, and clearly enhanced antibody responses (in vivo and in limited dilution cultures in vitro) and shorter rejection times of the injected Mtv expressing cells on the other hand (Bandeira et al., 1991; Dannecker et al., 1991).Similarly, there is accelerated skin graft rejection across an Mtv7 incompatibility after in vivo priming (Berumen et al., 1984). In this study Mtu7 congenic mouse strains were used. These observations argue against functional unresponsiveness or in vivo tolerance and rather for terminal differentiation of the recently activated T cells, bringing about arrest in proliferation and short life spans. Alternatively, it could reflect a memory T cell population with qualitative differences to naive T cells. In support of this interpretation it has been shown that memory phenotype CD4' CD45B'"T cells isolated from control or KLH-primed mice cannot be stimulated with the staphylococcal SAg SEB. KLH-primed memory cells, however, are stimulated by KLH (Lee and Vitetta, 1992). For naive T cells, B cells represent poor antigen presenting cells (Eynon and Parker, 1992; Fuchs and Matzinger, 1992). A plausible mechanism for this effect is a lack of costimulation. In agreement with these observations, an important role for B cells in the Mtu-induced deletion process has been shown with anti-IgM treatment from birth. Such B cell-deficient mice show reduced clonal deletion induced by MMTV SAgs. Following clonal deletion patterns of SAgs that delete fast, such as Mtv7 SAg, deletion is partially inhibited early after birth, whereas with weaker SAgs, such as Mtv9 SAg, more efficient and long-lasting inhibition of deletion is observed (Webb and Sprent, 1989; Gollob and Palmer, 1991, 1993). Contradictory results were obtained with pMdo mice (Beutner et al., 1996). These mice had at least as efficient clonal deletion patterns as control mice. These discrepancies are still unexplained. AND AUTOIMMUNITY? C. ROLEIN TOLERANCE The polyclonal activation of T and B cells by SAgs has led to many speculations about their role in autoimmunity. These interactions could break tolerance in both the T and the B cell compartment. Concomitant injection of bacterial SAg with classical antigens can lead to an immune

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response rather than deletion of the SAg-reactive T cells that cross-react with the antigen. Such responses have been observed in autoimmune models as well as in classical peptide antigen responses (Brocke et al., 1993; Schiffenbauer et al., 1993; McCormack et al., 1994). Similar observations were made after LPS injection (Vella et al., 1995). Therefore, SAgreactive T cells can be rescued from deletion if they cross-react with SAg and a classical antigen. So far no experimental evidence for or against such a rescue of MMTV SAg-reactiveT cells exists. Neonatal thymectomywithin 3 days after birth leads to multiple autoimmune symptoms. Such mice also show a lack of clonal deletion of SAg-reactive T cells (Smith et al., 1989; Jones et al., 1990). So far, however, no direct experimental evidence for or against such a rescue of MMTV SAg-reactive T cells exists. More experiments are required to understand the influence of SAgs in autoimmunity. VI. Comparison with Other SAgs

SAgs have been described in a variety of microorganisms. Some of their features are summarized in Table IV.Many bacterial strains produce SAgs. The best known are the staphylococcalenterotoxins, which are the causative agents of food poisoning (SEA, SEB,SEC1-3, SED, and SEE) and the toxic shock syndrome (TSST-1) (Fleischer and Schrezenmeier, 1988; Janeway, 1989; White et al., 1989; Marrack and Kappler, 1990). Other bacterial SAgs are found in streptococci (SPE-A, -B, and -C) (Imanishi et al., 1990; Tomai et al., 1990), mycobacteria (Ohmen et al., 1994), mycoplasma (Cole et al., 1989,1996; Tumang et al., 1990), and yersinia (Stuartand Woodward, 1992; Yoshino et al., 1994). Care must be taken with the Vp specificities in Table IV. Bacterial SAgs are incredibly potent and often small contaminations of a protein preparation can be responsible for the effects (Gerlach et al., 1994; Fleischer et aE., 1995). SAgs have also been described in several viruses such as rabies (Lafon et al., 1992), Epstein-Barr virus (Sutkowski et al., 1996), cytomegalovirus (Dobrescu et al., 1995), a defective strain of murine leukemia virus (Hugin et al., 1991),and MMTV. The results on murine leukemia virus have recently been challenged (Doyon et al., 1996). The authors observed that MuLV-infected strains overexpressed Mtu SAgs, which could induce indirectly the observed SAg effect. More experiments using mouse strains lacking endogenous Mtu sequences are required to settle this point. More surprising was the description of SAg activity in protozoans and plants. The T o x ~ l a s m agondii SAg, tachyzoite antigen, preferentially activated CD8+ T cells, an observation so far not made with other SAgs (Denkers et al., 1994). Oligoclonal peptide-specific expansions of CD8+ T cells that could be mistaken for SAg responses have repeatedly

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TABLE IV V/3 SPECIFICITIES OF SOMEVIRALAND BACTERIAL SUPERANTIGENS V/3 Specificity

Bacterial SAgs SEA SEB

SECl SEC2 SEC3 SED SEE

TSST-1 ExFT SPE-A SPE-B SPE-C MAM

Yersiniu enterolitica Mycobacterium tuberculosis

Responding T cells

Human

Mouse

1.1, 5, 6’s, 7.3-7.4, 9.1 3, 12, 14, 15, 17, 20 3, 6.4, 6.9, 12, 15 12, 13.2, 14, 15, 17, 20 3, 5, 12, 13.2 5, 12 5.1, 6 s , 8, 18 2 2 8, 12, 14, 15 2, 8 1, 2, 5.1, 10 3, 17 3, 12, 14, 17 8

1, 3, 10, 11, 17 7, 8.1-8.3 3, 8.2, 8.3, 11 3, 8.2, 10, 17 7, 8.2 3, 11, 17 11, 15, 17 15, 16 10, 11, 15 ND ND ND 5.1, 6, 8.1-8.3

CD4, CD8 CD4, CD8

6, 7, 8.1, 9 6, 7

CD4 S CD8 CD4 S CD8

CD4,CD8

Viral SAgs

Mt07 Rabies

EBV

8 13

5

MuLV

CMV

12

CD4

Protozoa

Toxoplasm gondii

5

CD8

8.3, (8.2)

CD4, CD8

Plant SAg

Urticu dioica aggl. __

Note. This table represents an updated and modified version of the table by Kotzin et al. (1993). Data for this table were taken from Marrack and Kappler (1990). Callahan et al. (1990),Kappler et al. (1989a), Choietal. (1989),Coleetal. (1990),Abeetol.(1991).Friedmanetd. (1991),B d a e t d . (1992),Hudson et nl. (1993). Tomai et d.(1992), Mollick et d.(1993). Stuart and Woodward (1992), MacDonald et al. (1988a), Lafon et al. (1992, 1994). Sutkowski et d.(1996). Dobrescu et al. (1995), Hugin et d.(1991), Galelli andTruffa-Bachi (1993),Ohmenetd.(1994),andDenkersdal. (1994).For many oftheexogenous SAgs the complete responding human V a repertoire has not been defined. Thus, it is likely that other Vp populations will be added to this list. Discrepant results between different reports can be due to crosscontaminations with other toxins or to the existence of different subtypes that are serologically indistinguishable. No reactivity was seen for Vp3,11,17Withrecornbinant SEB, forVB3 with recombinant SEC3, and for Vp8.2with recombinant SED (Herman et ol., 1991).ND, not determined.

been found but the presented results argued for a polyclonal expansion (MacDonald et al., 1993; Pantaleo et al., 1994). The plant SAg Ur-ticu dioica agglutinin had an activity of a small lectin that recognizes sugar

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residues on TCR Vp8.3- and to a lesser degree TCR Vp8.2-bearing cells, which are not found in other TCRs (Gale& and Truffa-Bachi, 1993; Galelli et al., 1995). Many pathogens are able to produce SAgs that are often very different proteins by sequence analysis and are defined functionally to cross-link MHC class I1 molecules with TCR Vp elements. The role of the MMTV SAg has been clarified to be important for the productive infection as reviewed previously. Observations that could be interpreted in a similar way were made with the rabies SAg. Mice injected with this virus or its nucleocapsid protein showed a strong SAg response. This virus is highly neurotropic and causes fatal encephalitis and can induce flaccid paralytic symptoms. After virus injection, mortality was very high in mice expressing rabies SAg-reactive T cells, whereas under the experimental conditions used no mortahty was detected in mice lacking rabies SAg-reactive T cells due to expression of endogenous Mtvs. Similarly, paralysis was observed only in mice expressing SAg-reactive T cells (Lafon et al., 1994; Lafon and Galelli, 1996)-Interestingly, the SAg response decreased the virus-specific antibody response, whereas immune responses to coinjected antigens were enhanced (Astoul et al., 1996; Lafon and Galelli, 1996). These results clearly indicated a possible role as an adjuvants for the SAg. The role of bacterial SAgs is less well understood. It is a general belief that the SAg response induces immune paralysis by mechanisms such as secretion of large amounts of cytokines and cytotoxicity. Such immune suppression and also adjuvant-like effects have been repeatedly found (Pinto et al., 1978; Brocke et al., 1993; Schiffenbauer et al., 1993; McCormack et al., 1994). Surprisingly, injection of mice with bacteria containing or lacking the ability to secrete SAg only led to small differences in duration and intensity of infection (Rott and Fleischer, 1994). Similarities, but also clear differences, in the in vivo and in vitro effects between soluble SAgs and MMTV SAgs exist. Bacterial SAgs, such as SEA and SEB, stimulate CD4' and CD8' T cells equally (Kawabe and Ochi, 1991; Herrmann et al., 1992). The clonal deletion is observed for CD4+ T cells but CD8' T cells rarely reach levels below control levels (Kawabe and Ochi, 1990; MacDonald et al., 1991; Ochi and Kawabi, 1992). Injection of soluble SAgs leads to a faster kinetics of stimulation and deletion than injection of MMTV particles. The different kinetics of stimulation is easily explained by the requirement for infection, integration, and SAg expression by MMTV. A hint to the difference in deletion kinetics might come from the observations that inhibition of Fas-mediated mechanisms of apoptosis inhibit clonal deletion with bacterial SAgs but not with MMTV SAgs (Papiemik et al., 1995; Renno et al., 1996). Therefore, it is possible that the observed difference in kinetics of deletion is due to alternative mecha-

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nisms of clonal deletion. The differences in deletion are more difficult to explain. A short time after injection of bacterial SAgs its concentration decreases rapidly. Therefore, normal percentages as well as responsiveness of the SAg-reactive T cells are regained after 1 or 2 months in normal or thymectomized mice (Kawabe and Ochi, 1991; Ochi and Kawabi, 1992). These bacterial SAgs also induce very strong SAg-specific cytotoxic responses. Target cells incubated with SAg are lysed by cells obtained ex vivo (Herrmann and MacDonald, 1993),a response never seenwith MMTV SAg-induced CD8’ T cells (Herrmann et al., 1993). Most likely these differences are due to the SAg expression levels on the different presenting cells. VII. Conclusions

A. LIFECYCLEOF MMTV The life cycle of mouse mammary tumor virus is characterized by an unusual dependence on the immune system. This dependence starts right after uptake of the virus-containing milk by the neonatal mice: Infection is exclusively detected among the B cells of the Peyer’s patches, and in the absence of B cells no productive infection is seen. Binding of virus particles to B cells by themselves possibly induces the B lymphocytes to enter cell cycle and allows the integration of the viral DNA into the host genome. This step is required to achieve transcription of the viral genome and is followed by surface expression of the SAg. Because 5-30% of all local CD4’ T cells express the SAg-reactive TCR Vp determinants, the infected B cells get unlimited help by the activated T cells. This “cognate” T-B interaction is completely dependent on the viral SAg and leads to the proliferation and differentiation of the infected B cells, thereby amplieing the virus infection several hundred-fold. The SAg-dependent differentiation of the infected B cells into long-lived memory cells most likely guarantees virus persistence until the mammary gland gets receptive for MMTV infection. During this process no viremia is detectable. Despite (or because of) the early onset of the virus-neutralizing antibody response, the virus spreads systemically and reaches the mammary gland epithelium, possibly by cell-cell transfer between migrating lymphocyte subsets. It is the hormone responsiveness of the MMTV promotor that leads to the production of large amounts of infectious particles in the mammary gland of lactating females and finally to the infection of the next generation. Late in life, insertional mutagenesis can lead to mammary carcinomas.

B. FUTURE DIRECTIONS The life cycle of MMTV has become more transparent since the identification and characterization of the SAg molecule. The SAg response can

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be easily followed in vivo and in vitro. The recent progress allows us to design experiments to further dissect the virus-host interaction or, alternatively, to use MMTV as a tool to further dissect the immune response. Several aspects of the virus-host interaction are still unclear. The cellular receptor allowing MMTV infection of B cells is unknown as is the mechanism of virus transfer between lymphocytes and mammary gland epithelium. In addition, future studies need to address the way MMTV maintains a critical number of infected cells and avoids recognition by the host immune system. It is still not clear whether the SAg only plays a role in the initial steps of virus amplification and B cell differentiation or whether it is also important later. Despite the strong effects exerted by the SAg, our knowledge of the structure of the active protein is poor. Evidence points toward the existence of a soluble form of this protein. What could be the role of a soluble SAg in MMTV infection? Which cell type besides the B cells could present the SAg to T cells? Could the cell type and costimulatory molecules decide on the fate of the T cells? How do the SAg-reactive T cells get eliminated in the periphery? Mouse mammary tumor virus has proven to be a powerful tool to investigate important aspects of immune responses, such as antigen presentation, T-B interaction, lymphocyte activation and differentiation, as well as tolerance induction. The immune response to the SAg of MMTV is very similar to a classical immune response. Due to the TCR specificity and the strength of the response, it allows us to identify and follow the fate of interacting cells in vivo in adult as well as in neonatal mice. Unlike TCR transgenic mice, this retrovirus can be used to study immune responses against a natural pathogen in immunologically fully competent animals as well as in many of the now available gene-deficient animals. ACKNOWLEDGMENTS We thank Anne Wilson, Ulrich Beutner, and Daniela Finke for the suggestions and the critical reading of the manuscript.

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ADVANCES IN IMMUNOLOGY.VOL. 65

IgA Deficiency PETER D. BURROWS' AND MAX D. COOPERt 'Division of hvabpmnial and Clinical hnmunalagy, DepclmmMt d Micmbidogy, University of Alabama at Birmingham and ~oivisionof h d q n m n ~ and ~ l C/inkal /mmuna/agy, -rhnentr of Medicine, hdi&cs and Micmbiobgy, l/nivwsity d Alabama at Birmingham and he ffowad Hughes Medical Inslihrlb, Birmingham, Alabama 35294

1. introduction

An inability to produce antibodies of the IgA subclassesoccurs in approximately 1in 600 individuals of Northern European ancestry, a much higher incidence than is seen for other primary immunodeficiencies (Rosen et al., 1995a,b).Individuals with IgA deficiency (IgAD) are often asymptomatic but the other end of the spectrum includes IgAD patients with frequent sinopulmonary infections, allergies, gastrointestinal disorders, and autoimmune diseases (West et al., 1962; Ammann and Hong, 1971; Buckley, 1975).This biological diversity has both genetic and environmental components (Hanson et al., 1992). IgAD may be acquired after congenital viral infections (Lawton et al., 1973; Saulsbury, 1989) or drug therapy for epilepsy (Sorrell et al., 1971; Seager et al., 1975; Aarli, 1976). It may also be inherited with a disease susceptibility gene mapping to the major histocompatibility complex (Ambrus et al., 1977; Wilton et al., 1985). The existence of IgAD in only one of identical twins reinforces the idea of unknown environmental factors in the pathogenesis of IgAD (Huntley and Stephenson, 1968; Lewkonia et al., 1976). Associated T cell defects have been described in some cases (Waldmann et al., 1976; Atwater and Tomasi, 1978; King et al., 1979; Klemola et al., 1988), and approximately onefourth of the individuals diagnosed as having isolated IgAD prove to have unsuspected IgG subclass deficiencies (Oxelius et al., 1981; Preud 'homme and Hanson, 1990). Other individuals manifest only transient immunodeficiency in which the initially low IgA concentrations may increase to normal values with time (Plebani et al., 1986). This disease heterogeneity poses a major difficulty in understanding the pathogenesis of IgAD. The past 5 years have witnessed remarkable progress in identifylng many of the genes mutated in the primary immunodeficiencies (Rosen et al., 1995a,b; Fischer, 1996). This achievement has been facilitated by a rapid increase in our understanding of the molecular basis for the cellular interactions that must operate in concert to generate an effective immune response. Naturally occurring immunodeficiencies have provided important insight into the normal workings of the immune system, and the ability to 245

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create gene defects through homologous recombination has hastened the rate of this progress (Pfeffer and Mak, 1994; Loffert et al., 1994). The increased understanding of normal events in immune system development and function sometimes allows reasonable prediction of gene defects that could produce the phenotype of a particular immunodeficiency disease. For the moment, however, the pathogenesis of IgAD remains a mystery that has not yielded to this predictive type of analysis. Here we review this disorder and some of the clues that may ultimately lead to its resolution. 11. Clinical Manifestations of IgA Deficiency

IgA deficiency is usually defined by a serum IgA concentration of less than 5 mg % (50 pg/ml) and affected individuals are also deficient in secretory IgA antibodies as a rule. The clinical manifestations associated with IgA deficiency are highly variable. Individuals with profound IgA deficiency often appear perfectly healthy. Their IgAD may be identified when they serve as blood donors (Koistinen, 1975) or when they receive blood for an apparently unrelated illness only to undergo anaphylactic shock (Vyas et al., 1968; Branigan et al., 1983; Burks et al., 1986). This unanticipated consequence of IgA deficiency occurs because IgAD individuals often produce anti-IgA antibodies of IgE or IgG isotypes that can potentially elicit anaphylactic reactions (Sandler et al., 1994, and references therein). In other instances, IgA deficiency is identified during medical evaluation for a wide array of clinical problems, including recurrent infections, autoimmune disorders, allergic diseases, and malignancy, or during evaluation of an immunodeficient relative. One-half or more of the IgAD individuals may experience increased numbers of infections or unusual types of infections (West et al., 1962; Ammann and Hong, 1971; Burgio et al., 1980; Burks and Steele, 1986; Klemola, 1987; Koskinen, 1996). These range from recurrent infections of the mucosal surfaces to fulminating systemic infections, such as meningitis, septicemia, and fatal viral hepatitis. IgAD individuals frequently experience recurrent upper and lower respiratory tract infections including ear infections, sinusitis, recurrent bronchitis, and acute pneumonias usually caused by viruses and bacteria. Gastrointestinal infections in IgAD individuals include acute diarrheal illnesses caused by pathogenic bacteria and viruses and chronic diarrhea due to Giardia larnblia infestation. Infections of the biliary tract may lead to chronic gallbladder inflammation and gallstone formation (Danon et al., 1983). Chronic infections of the skin and mucous membranes with the fungal agent, Candida albicans, may occur in IgAD. IgAD individuals are prone to produce autoantibodies against a variety of tissue-related antigens. Perhaps as a consequence, they have a higher

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incidence of autoimmune syndromes than the general population (Ammann and Hong, 1971; Klemola, 1987; Burks and Steele, 1986; Koskinen, 1996). Systemic lupus erythematosus and rheumatoid arthritis have been reported in up to 5 % of IgAD individuals (Cassidy et al., 1969; Pelkonen et al., 1983).Other autoimmune disorders associated with IgAD include acute hemolytic anemias, chronic nephritis, thyroiditis, psoriasis, dermatomyositis, Sjogren’s syndrome, hypoadrenalism, idiopathic thrombocytopenia purpura, and pulmonary hemosiderosis (for review see Schaffer et al., 1991).Insulin-dependent diabetes mellitus (IDDM)and coeliac disease are familial autoimmune syndromes, the susceptibility to which may be inherited together with IgAD (Savilahti et al., 1971; Collin et al., 1992). All three conditions have been linked to particular major histocompatibility complex (MHC)haplotypes (Wilton et al., 1985; Todd et al., 1987; Schaffer et al., 1989; Volanakis et al., 1992; Olerup et al., 1990, 1992). The fact that the inheritance of coeliac disease and IDDM susceptibility may be dissociated from IgA deficiency in some population groups suggests that these autoimmune disorders may not reflect the IgAD but rather represent genetically linked susceptibilities in certain populations. Allergic diseases, including allergic rhinitis, asthma, and eczema, may occur in higher incidences in IgAD individuals (Oxelius et al., 1981; Dstergaard, 1980; Burks and Steele, 1986),although the relatively high incidence of these allergic disorders in the general population makes this association difficult to assess (Van Asperen et al., 1985; Plebani et al., 1987). The inability to produce mucosal antibodies of IgA isotype may favor the production of allergic or reagenic (skin-sensitizing)IgE antibodies to ingested or inhaled antigens. IgAD individuals have indeed been shown to produce relatively high titers of antibodies to many food antigens (Buckley and Dees, 1969). More than 20% of IgAD individuals produce antibodies to IgA (Sandler et al., 1994), thus rendering them susceptible to anaphylactic reactions to IgA-containing blood products (Vyas et al., 1968; Branigan et al., 1983; Burks et al., 1986). A variety of unusual skin disorders have been associated with IgAD. These include pyoderma gangrenosa (Bundino and Zina, 1984), trachyonychia (Leong et al., 1982), patchy depigmentation of the skin (vitiligo) (Wolf and Wolf, 1982), and hemorrhagic purpura (Martini et al., 1985). Malignancies, most commonly adenocarcinomas of the intestinal tract (Spector et al., 1978; Cunningham-Rundles et al., 1980; Filipovich et al., 1994), have also been associated with IgA deficiency. Less frequently encountered neoplasms in IgAD individuals include hepatoma, acute lymphoblastic leukemia, lymphosarcoma, melanoma, ovarian carcinoma, multiple myeloma, squamous cell carcinoma, and thymoma (Spector et

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al., 1978; Cunningham-Rundles et al., 1980; Hong and Ammann, 1989; Chevailler et al., 1990; Filipovich et al., 1994). An accurate picture of the clinical consequences of IgAD could only be envisioned by lifelong study of affected individuals. The closest approximation to this ideal has been a monitoring of 204 healthy young adults whose IgAD was identified when they served as blood donors (Koskinen, 1996). This study indicated that 80% of these IgAD individuals experienced episodes of infections, drug allergy, autoimmune disorders, or atopic disease during the next 20 years of their life in Finland. They had an increased susceptibility to pneumonia, recurrent episodes of respiratory infections, and a higher incidence of autoimmune diseases, including vitiligo, autoimmune thyroiditis, and possible rheumatoid arthritis. 111. IgA Stnrcture, Production, and Function

IgA monomers, like the other monomeric immunoglobulins, are composed of two identical light chains ( K or A) and two identical a heavy chains (-60 kDa) organized into a variable domain and three constant (CH) domains. However, IgA antibodies in humans come in two isoforms with unique biochemical and biological features that distinguish them from other classes of immunoglobulins. IgA antibodies are more heavily glycosylated than IgG, the major class of immunoglobulin in serum, and more negatively charged, resulting in /3 rather than y globulin electrophoretic mobility (reviewed by Kerr, 1990). The two IgA subclasses, IgAl and IgA2 (Kunkel and Prendergast, 1966), are encoded by separate CH genes (Flanagan and Rabbitts, 1982), but they differ by only 22 amino acids (Mestecky and McGhee, 1987; Kerr, 1990). Much of this difference is due to the absence in IgA2 of 13 amino acids found in the Pro/Ser/Thr-rich hinge region of IgA1, a structural difference that has significant functional consequences. Two allelic forms of IgA2, IgA2m(l) and IgA2m(2), have been described (Grey et al., 1968; Tsuzukida et al., 1979). IgA2m(l) is unique among human immunoglobulins in that the light chains are disulfide bonded to each other rather than to this a heavy chain that lacks the necessary cysteine residue (Kerr, 1990). Both IgAl and IgA2 antibodies exist as monomers or higher molecular weight polymers, usually dimers, that are disulfide linked to a 1SkDa joining ( J) chain (Mestecky and McGhee, 1987; Kerr, 1990). The J chain is synthesized by plasma cells and is also associated with the other polymeric immunoglobulin, IgM. Polymerization of immunoglobulins had been thought to require J chain, but studies of J chain-deficient cell lines indicate that this is not the case; IgM secreted by the mutant cells readily polymerizes but tends to form hexamers rather than the physiologic pentameric

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structures (Cattaneo and Nueberger, 1987; Randall et al., 1992; Niles et al., 1995).J chain thus regulates the degree of polymerization, the stability of the polymers secreted by plasma cells, and IgA transport into the mucous secretions. IgA antibodies are found both in the blood and in mucosal secretions, and these systemic and secretory IgA immune systems represent separate entities in many respects. Although appropriately administered antigens can engage both compartments, the resultant pools of serum and secretory IgA are largely nonoverlapping (Conley and Delacroix, 1987). The IgA in human serum is primarily monomeric, with 90-9596 being of the IgAl subclass. Humans differ in this respect from mice, which have only one IgA class that occurs as a dimer in serum (Kerr, 1990). Derived mainly from bone marrow plasma cells, serum IgA in humans is present in lower levels (mean, -1500 pg/ml) than IgG (mean, -10,000 pg/ml). However, direct comparison of these values is misleading because serum IgA is catabolized four times faster than IgG. In fact, synthetic rates for the systemic pools of IgG and IgA are similar: 20-30 and 30 mg/kg/day, respectively (Conley and Delacroix, 1987; Mestecky and McGhee, 1987; Kerr, 1990). Most of the IgA in mucous secretions is polymeric and the concentration of IgA2 is increased relative to serum (Brandtzaeg, 1995). Secretory IgA (S-IgA) represents the compound product of two cell types, submucosal plasma cells, such as those in the lamina propria of the intestine, and the epithelial cells that line mucosal surfaces (Brandtzaeg, 1995). Epithelial cells of the mucous membranes synthesize a polymeric immunoglobulin receptor (pIgR), a 100-kDa transmembrane protein that is targeted to the basolateral surface (Mostov, 1994). It binds polymeric IgA and, with lower affinity, IgM antibodies secreted by submucosal plasma cells. The IgApIgR complex is then transported through the cell by a process termed transcytosis, during which disulfide bond formation further stabilizes the high-affinity IgA-pIgR interaction (Lindh and Bjork, 1974; Kuhn and Krahenbuhl, 1979; Chintalacharuvu et al., 1994). When the complex reaches the apical surface about 30 min later, the pIgR is proteolytically cleaved near the transmembrane segment. This releases the secretory IgA antibodies attached to the pIgR cleavage component, referred to as secretory component (SC), onto the mucous surface. The remaining 20-kDa pIgR transmembrane fragment is internalized and degraded. The SC serves to protect the IgA antibodies from proteolytic degradation in the lumen of mucosal tissues such as intestinal and respiratory tracts (Lindh, 1975). Although rodents express the pIgR on hepatocytes, allowing polymeric IgA to be transcytosed from blood to enter the mucosal immune system via the bile duct (Schiff et al., 1986; Phillips et al., 1988),this route

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of IgA egress is unavailable in humans, whose hepatocytes do not express the pIgR. Because the pIgR is specific for polymeric immunoglobulins, it was predicted that Ig transport into mucosal secretions requires an associated J chain. Consistent with this hypothesis, J chain-deficient mice were found to have decreased IgA levels in bile and feces (Hendrickson et nl., 1995). However, the IgA levels in local mucosal and glandular secretions such as breast milk, intestinal and colonic fluids, and bronchodveolar and nasal lavages were normal or elevated (Hendrickson et al., 1996). The IgA in these secretions was nonpolymeric and, interestingly, not associated with secretory component. These findings indicate that J chain is required for the stable association of dimeric IgA with SC and suggest that the transport of J chain-deficient IgA may utilize an alternative receptor not present on hepatocytes. These findings also highlight the importance of the SC in preventing degradation of IgA in the intestine. Secretory IgA antibodies play an important role in protection from microorganisms invading via the mucosal surfaces of gastrointestinal, respiratory, and genitourinary tracts. Toxin neutralization in the lumen of these internal organs and prevention of bacterial adherence to the mucosal surfaces are important functions of S-IgA antibodies (Brandtzaeg, 1995; Lamm et al., 1995). These functions are mediated primarily by the variable portion of the antibody molecule and are complemented by non-antigenspecific functions mediated by the constant region. The heavily glycosylated S-IgA can bind, for example, to the mannose-specific lectins on the surface of a wide range of enterobacteria, causing their agglutination and preventing attachment to host cells (Kilian et al., 1988; Wold et al., 1988). An unusual biological feature of the mucosal immune system is the potential for intracellular neutralization of viruses that infect epithelial cells (Mazanec et al., 1993). The pIgR-IgA antibody complex colocalizes with the viral invader during its transit through the cell, although the exact cellular compartment where this defensive interaction takes place has not been elucidated. The IgA transport system also provides a mechanism to eliminate antigenIgA antibody complexes formed beneath epithelial cells, via their transcytosis and secretion into the luminal exterior (Mazanec et al., 1993). Mixed complexes containing IgG and IgA antibodies to the same antigen are also efficientlyremoved by this mechanism (Kaetzel et al., 1994).This excretory immune system has been postulated to be an important avenue for removal of antigen-antibody complexes that might otherwise provoke a local inflammatory reaction or systemic immune complex disease. The absence of this mechanism in IgAD individuals may account in part for their increased susceptibility to autoimmune and immune complex diseases. The fate of antigen-IgA antibody complexes is entirely different in the systemic immune system, wherein the IgA complexes bind to Fca receptors

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expressed on neutrophils and monocyte/macrophages ( Fanger et al., 1983; Albrechtsen et al., 1988; Chevailler et al., 1989). This interaction results in phagocytosis and metabolic activation of the phagocytic cells (Shen et al., 1989; Kerr, 1990; Monteiro et al., 1990). As a means of eliminating microorganisms that evoke an IgA response, this may provide an important avenue of systemic host defense. This also provides an efficient mechanism for clearance of potentially toxic IgA immune complexes from the vasculature and may play an important general role in the relatively rapid catabolism of serum IgA. The asialoglycoprotein receptor expressed on hepatocytes is the gatekeeper for another major catabolic route for IgA and other serum proteins that have become desialyated (Daniels et al., 1979; Stockert et al., 1982; Moldoveanu et al., 1988; Mestecky et al., 1989). Pathogens have evolved their own strategies to evade IgA-mediated immunity. Some streptococcal bacteria express a cell surface receptor that recognizes the Fc portion of IgA (Burova et al., 1983; Lammler et al., 1988). This receptor serves as a virulence factor, presumably because of its antiphagocytic activity. Another tactic employed by mucosal pathogens, including Neisseria gonorrhoeae, N . meningitidis, Haemophilus influenxae, and Streptococcus pneumniae, is the production of IgAl-specific proteases (Kilian et al., 1988; Plaut, 1983). These bacterial enzymes cleave IgA in the proline-rich hinge region to yield F(ab’)2and Fc fragments. IgA2 antibodies lack this hinge region, thus rendering them protease resistant. This advantage may account for the relative enrichment of the IgA2 subclass at mucosal surfaces, particularly in the lower intestines where bacterial growth is abundant. IV. IgA Deficiency Viewed in the Context of the Genesis of IgA-Producing Cells

Secretion of IgAl and IgA2 by plasma cells is the terminal event in differentiation of the progeny of IgM B cell clones generated within the bone marrow. The bone marrow-derived B cells that are ultimately responsible for IgA production migrate preferentially to mucosal lymphoid tissues such as the intestinal Peyer’s patches, the appendix, and bronchial epithelial-associated lymphoid aggregates (Mestecky and McGhee, 1987; Brandtzaeg, 1995). These mucosal-associated lymphoepithelial tissues are collectively referred to as MALT. Specialized follicle-associated epithelial cells (Bockman and Cooper, 1973),known as M cells (Neutra, et al., 1996), overlay the lymphoid structures in MALT and serve as a conduit for viruses, bacteria, and other intralumenal immunogenic elements to reach the underlying T, B, and antigen presenting cells (Neutra et al., 1996). The migration and homing of lymphocytes to these strategically located structures have been reviewed elsewhere (Shaw and Brenner, 1995; Butcher

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and Picker, 1996; Mayer et al., 1996). Relatively little is known about the functional integrity of this pathway in IgAD patients. IgAD can follow the transplantation of bone marrow from an IgAD donor into a histocompatible sibling not previously deficient in IgA (Hammerstrom et al., 1985b). This implicates the cells of hemopoietic origin in the pathogenesis of IgAD but does not incriminate the particular cell type(s). Abnormalities that could lead to IgAD include defects in (i) a chain constant region genes, (ii)isotype switching, or (iii) terminal differentiation of IgA B cells. The immunoglobulin heavy chain locus on chromosome 14q32 contains clusters of VH, D, and JH gene segments that are somatically recombined in developing pro-B cells to generate a complete variable region exon (Sleckman et al., 1996). A string of exons encoding the constant regions of the human immunoglobulins, p-Sy3-yl-@-alrCry-y2-y4-~-a2,is located downstream of the JH segments in a region spanning more than 250 kb of DNA (Honjo and Matsuda, 1995). The locus downstream of CS appears duplicated, perhaps having evolved from a primordial Y - Y - E - ~ unit (Flanagan and Rabbitts, 1982). This CH gene organization makes it unlikely that C a gene deletions would cause IgAD because a simple deletion to remove both C a l and Ca2 would show an unambiguous recessive mode of inheritance and would invariably result in associated IgE, IgG2, and IgG4 deficiencies. In a study of more than 100 cases of immunoglobulin class and subclass deficiencies, including IgAD, no such homozygous gene deletions of the Ig CH locus were observed (Hammerstrom et al., 1985a). Rare cases of either IgAl or IgA2 subclass deficiency due to CH gene deletions have been described in consanguineous populations (Lefranc et al., 1982, 1991; Engstrom et al., 1990; Plebani et al., 1993). These deficiencies have included other Ig isotypes because the deletions span multiple CH genes, for example, C y l + Cy4 and C a l + CE. Notably, the inability to produce these immunoglobulin isotypes rarely predisposes the affected individuals to increased numbers of infections. Combined C a l and Ca2 gene defects thus rarely, if ever, account for IgAD. Reappearance of the silent IgA allotype has also been noted in the offspring of IgAD individuals (Hammarstrom et al., 1987),thereby excluding subtle defects in &-acting regulatory elements in the IgH locus as the cause for IgAD in these kindreds. Other structural abnormalities in the IgH locus remain candidates for IgAD gene defects. An enhancer located in the region 3’ of the murine C a gene has been postulated to form part of an Ig heavy chain locus control region (Pettersson et al., 1990; Lieberson et al., 1991; Dariavach et al., 1991). Replacement of the 3’ enhancer with a neomycin gene leads to isotypespecific serum immunodeficiency in IgGSa, IgG3, and IgA to a much lesser extent, and to multiple defects in isotype switchingin vitm (Cogn6et d.,1994).

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Analogous enhancer elements have not yet been identified in humans, but theoretically they could play a role in the pathogenesis of IgAD or common variable immunodeficiency (CVID). However, the observed heterogeneity and inheritance patterns of both diseases make a simple, all or nothing deletion of this element an unlikely possibility. Moreover, analysis of a large number of IgAD and CVID patients did not reveal a correlation with Ig heavy chain haplotypes (Truedsson et al., 1995). Newly formed B cells express IgM only, but may later express non-IgM isotypes through the use of two types of switch mechanisms. During their maturation, virtually all B lymphocytes utilize a differential RNA splicing/ polyadenylation mechanism to express both IgM and IgD (Moore et al., 1981).In this case RNA transcription initiates upstream of the productively rearranged variable region exon and terminates downstream of CS yielding two types of spliced transcripts, .5’-VHCp-3’ and 5’-VHC63’, resulting in IgM and IgD products with identical VH domains. The second switching mechanism is irreversible in that it involves DNA deletion mediated by highly repetitive switch (S) regions located upstream of all the CH genes except C6, which has only a very primitive, inefficient switch sequence (Zhanget al., 199.5).In the switch from IgM to IgA1, for example, recombination occurs between the Sp and S a l switch regions to reorganize the heavy chain locus as follows: 5’-VH-Sp/sa-Cal-r-cy2-cy4-c€-ca2-3’. The deleted Cp-CSCy3-Cyl-+ segment can be ligated end to end to form a deletion circle (Iwasato et al., 1990; Matsuoka et al., 1990; von Schwedler et al., 1990). Lacking an origin of DNA replication, the DNA deletion circles are diluted by cell division and eventually may be degraded. The switch to IgA2, the most 3‘ CH gene, generates an even larger DNA deletion circle of about 250 kb containing the entire upstream CH locus. Isotype switching by DNA deletion was originally demonstrated in plasma cell tumors and in pre-B and B cell lines. An alternative hypothesis held that normal B cells may use the same RNA splicing mechanism that results in IgM and IgD coexpression to express IgG, IgA, or IgE together with IgM on their surface. This would require a very large primary transcript, -2.50 kb in the case of IgA2, and the existence of isotype-specific splicing factors to generate VHCpand VHCa2 messages while ignoring the intervening CH genes in the transcript. This hypothesis has been difficult to test directly because of technical difficulties arising from the low frequency of normal IgG- and IgA-bearing B cells, coupled with the existence of Fc receptors on B cells. Succeeding in isolating IgAl+ human blood B cells in sufficient purity for molecular analysis, Irsch et al. (1994) found that deletional recombination, often occurring on both homologs of chromosome 14, is the primary mechanism employed by B cells to switch to

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IgA. Hence, defects in IgA-specific RNA splicing factors can be excluded as a factor in IgAD. Differential regulation of isotype switching is the first step in determining differences in the serum concentrations of the immunoglobulin isotypes (IgGl > IgG2 > IgG3 >> IgG4; IgAl >> IgA2). Studies of T celldeficient mice and humans initially suggested that antigen-induced isotype switching by B cells was T cell dependent, and recent studies have defined a central role for direct T-B cell interaction in initiating isotype switching. This encounter includes a crucial interaction between the CD40 molecule on the B cell and the CD40 ligand expressed by the activated T cell within the germinal centers created by the antigen response (Callard et al., 1993; Fuleihan et al., 1993; Banchereau et al., 1994; MacLennan, 1994). The responding germinal center B cells also undergo somatic hypermutation of their variable region genes (Liu et al., 1996). T cell-derived cytokines participate in this process by directing the isotype switch to particular CH genes (Esser and Radbruch 1990; Coffman et nl., 1993; Harriman et al., 1993; Zhang et al., 1995). Finally, B cells may be heritably predisposed to a particular isotype switch pattern (Burrows et al., 1981; Stavnezer et al., 1988; Lutzker and Alt, 1988). The commitment to switch to a particular isotype in vitro is reflected by molecular changes in the targeted CH gene prior to switch recombination. The changes that herald isotype switching include demethylation of the 5’ flanking region, appearance of DNAse hypersensitive sites, and transcriptional activation of the unrearranged CH gene (Lutzker and Alt, 1988; Stavnezer et al., 1988; Burger and Radbruch, 1992; Gaff et al., 1992; Esser and Radbruch 1990; Coffman et al., 1993; Harriman et al., 1993; Zhang et al., 1995). The germline transcription initiates several kb upstream of the switch region at TATA-less promoters and continues through one or more short, intervening “I” exons and the downstream CH exons. The I exons contain multiple stop codons in all three reading frames and, hence, the transcripts are “sterile,” being incapable of translation into proteins of any significant size. Sterile transcription ceases after isotype switchingwhen the I exon is deleted from the DNA. Although the sterile transcripts alone do not induce isotype switching, the importance of germline transcription is indicated by gene targeting studies. Deletion of the Iyl, Iy2b, or IE exons in mice severely impairs switch recombination to IgGl, IgGzb, or IgE, respectively (Jung et al., 1993; Xu et al., 1993; Zhang et al., 1993; Bottaro et al., 1994).In an interesting exception, mutant mice in which the I a exon has been replaced by a human hypoxanthine phosphoribosyl transferase minigene have normal levels of serum and secretory IgA and their B cells can be induced to switch to IgA in vitro (Hamman et al., 1996b).Whether this exception is a feature of IgA switching or the targeting strategy still needs to be resolved. The spliced sterile

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transcripts or the splicing process, and not just the transcription per se, appear to be required for the switch. Gene targeting strategies that preserve transcription, but abolish the spliced transcripts, prevent isotype switching to the mutated CH gene (Lorenz et al., 1995). Helper T cell-derived cytokines regulate sterile transcription and isotype switching by mitogen-activated B cells (Banchereau et al., 1993). IFN-y is a highly pleiotropic cytokine that can induce sterile Cy2a transcripts and switching to IgG2a in mice (Snapper and Paul, 1987; Finkelman et al., 1988; Snapper et al., 1988; Collins and Dunnick, 1993). Also in mice, IL-4 induces formation of C y l and, at higher concentrations, CEtranscripts (Coffman et al., 1986; Snapper and Paul, 1987; Berton et al., 1989). In humans IL-4 was originally shown to target Cy4 and CE (Gauchat et al., 1990; Zhang et al., 1991; Jabara et al., 1990; Rousset et al., 1991; Gascan et al., 1991; Kitani and Strober, 1993; Wakatsuki and Strober, 1993). This difference in isotype preference between mice and humans was curious, because IgG4 is the least abundant human serum immunoglobulin, whereas IgGl is the most abundant in both species. Recent studies have demonstrated IL-UCD40 ligand-induced sterile transcription and switching to C y l and Cy3 (Kotowicz and Callard, 1993; Fujieda et al., 1995), but possibly not Cy2 (Fujieda et al., 1995). The lack of a Cy2 response to T cell-derived IL-4 may reflect the fact that anticarbohydrate responses in humans are mainly of the IgG2 isotype. IL-13 also supports IgG4 and IgE isotype switching by human B cells in vitro (Aversa et al., 1993; Punnonen et al., 1993). TGF-P1 appears to be an important switch cytokine for IgA in mice (Sonoda et al., 1989, 1992; Coffman et al., 1989; Kim and Kagnoff, 1990a,b; Gaff et al., 1992) and in humans (Defrance et al., 1992; van Vlasselaer et al., 1992), in whom it induces sterile transcription of both C a l and Ca2 genes (Islam et al., 1991; Nilsson et al., 1991). Nevertheless, the effects of TGF-P1 on immune cells are complex. When simply added to mitogen activated B cells, TGF-P1 is a much less robust switch cytokine for IgA than is IL-4 for IgG1. This may relate to the fact that TGF-P1, at the concentrations required to induce switching in vitro,is antiproliferative and inhibits the production of the secretory form of the immunoglobulin heavy chain (Briskin et al., 1988; Kehrl et al., 1991). These effects are seemingly contradictory to the needs anticipated for generation of an effective IgA response, but in vivo the switch recombination and subsequent proliferation and differentiation to IgA-secreting plasma cells are probably temporally and spatially separable events. The relatively meager IgA response induced by TGF-01 in vitro can be boosted dramatically with the appropriate combination of activators and cytokines. Anti-Sdextran plus a CD40L-CD8a fusion protein in combination with IL-4 and

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IL-5 induces very high frequencies (10-20%) of sIgA' cells (McIntyre et

al., 1995) compared with the IgA precursor cell frequency ( ~ 2 5 0 0seen ) when TGF-P1 is combined with IL-2 and LPS stimulation (Kim and Kagnoff, 1990b; Ehrhardt et al., 1992). In view of its importance in IgA switching, a possible role for TGF-P must be considered in IgAD. The physiologic importance of cytokine signalinghas been demonstrated in uiuo. Loss of function mutations of the IL-4 gene (Kuhn et al., 1991) severely impair IgGl and, especially, IgE antibody responses. Conversely, transgenic mice that overexpress IL-4 have elevated IgGl and IgE levels and depressed IgG3, IgG2a, and IgG2b (Burstein et al., 1991). TGF-P1 knockout mice have been created (Shull et al., 1992; Kulkarni et al., 1993; Christ et al., 1994) but the analysis of IgA isotype switching has been complicated by the fact that the mice die at -3 weeks of age from a wasting syndrome combined with an overwhelming inflammatory reaction of unknown etiology. V. Relationship of lgAD with Common Variable Immunodeficiency

The clinical features of IgAD clearly attest to the value of IgA antibodies in body defense, but it is important to note that IgA deficiency is frequently accompanied by additional defects in the immune response. Deficits in IgG subclasses, most commonly IgG2 and IgG4, are seen in 20-30% of IgAD patients (Oxelius et al., 1981; Preudhomme and Hanson, 1990), and recurrent infections are seen more frequently in this subgroup of IgAD individuals (French et al., 1995). The defects in antibody production in IgAD patients represent a continuum with those seen in CVID, which is defined as either combined IgA and IgG deficiency or, more commonly, pan-isotype deficiency (Schaffer et al., 1989; Rosen et al., 1995a,b). An arrest in terminal plasma cell differentiation is the central feature in both IgAD and CVID (Fig. 1A) (Cooper and Lawton, 1972; Schaffer et al., 1989,1991),the principal difference being the extent of the immunoglobulin isotype deficiencies and attendant clinical severity. Moreover, IgAD or CVID often occur in members of the same family (Schaffer, 1989;Volanakis et al., 1992; Olerup et al., 1992). These observations suggest that IgAD and CVID represent polar ends of a clinical spectrum reflecting a single underlying genetic defect. VI. Genetic Susceptibility for lgAD and WID

The incidence of IgAD is highly variable in different population groups. IgAD is found in one of every 500-700 Europeans (Bachmann, 1965; Johansson et al., 1968; Frommel et al., 1973; Koistinen, 1975; Gudmunds-

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son and Jensson, 1977; Holt et al., 1977; Burgio et al., 1980) and North Americans (Huntley and Stephenson, 1968; Collins-Williams et al., 1972; Cassidy and Nordby, 1975; Vyas et al., 1975; Clark et al., 1983), but the incidence is approximately 1 in 18,5000 Japanese (Kanoh et al., 1986). A relatively low incidence of IgAD among African Americans is suggested by the greater than 20 to 1 ratio of white to black IgAD patients seen in two large referral centers in the southeastern United States, where the population is nearly 50% African American (Lawton et al., 1972; Buckley, 1975).These data indicate that genetic background is a major contributing factor in the pathogenesis of IgAD. The frequent occurrence of IgAD and CVID in members of the same family is also indicative of a susceptibility gene(s) for Ig deficiency (Fig. 1B). The pattern of inheritance may vary from one suggesting autosomal dominant transmission to one consistent with autosomal recessive inheritance. Assessment of a total of 88 families with multiple cases of IgAD led Vorechovsky et al. (1995) to favor autosomal dominant inheritance. Studies of IgAD kindreds suggest the existence of disease susceptibility gene(s) within the MHC, which spans nearly 4 mb of DNA and contains more than 50 genes, many of which are important in immune responses (Campbell and Trowsdale, 1993; Trowsdale, 1993). Presentation of antigenic peptides by the highly polymorphic class I and I1 cell surface glycoproteins is essential for the development of CD8' and CD4' T cells, respectively, and for their subsequent function in class I-restricted cytotoxicity, in class II-restricted help for B cells, and in the activation of macrophages (Cardell et al., 1994; Raulet, 1994; Grusby and Glimcher, 1995). Other class I and I1 genes in the MHC are much less polymorphic and some show tissue-restricted expression (Beckman and Brenner, 1995; Stroynowski and Forman, 1995). A large family of class IB genes found in mice present antigenic N-formylated peptides generated from prokaryotic proteins, which use N-formyl methionine to initiate translation. The class II-like DM molecule enhances the peptide binding to the classical class I1 molecule (Roche, 1995). The LMP and TAP genes, which generate peptides from cytosolic proteins and translocate them into the ER to associatewith assembling class I heterodimers (Heemels and Ploegh, 1995), are also found in the class I1 region of the MHC. The class I11 region contains genes encoding the complement components, C2, C4 (two isotypes, C4A and C4B), and factor B, and the 21-hydroxylase gene involved in steroid biosynthesis. Other class I11 region genes encode TNF-a, lymphotoxin a (also known as TNF-P) and lymphotoxin /3 (LT-p), and two hsp70 heat shock protein genes (Campbell and Trowsdale, 1993;Trowsdale, 1993; Browning et al., 1993).

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CVID

FIG.1. Related phenotypic and inheritance patterns of IgAD and CVID suggest continuum of a disease spectrum. (A) Cellular differentiation defect in IgAD and CVID. Capital letters indicate major classes of immunoglobulins and numbers indicate subclasses. Curved arrows indicate secreted immunoglobulins. (Top) Model illustrates the decreased frequency of switching to IgA and the inability of the IgA-bearing B cells to differentiate into IgAsecreting plasma cells. In some cases the Ig deficiencyalso includes IgG2 and IgG4, although the level of the defect is unknown. (Bottom) Model illustrates the inability of the CVID B cells of most or all isotypes to differentiate into antibody-secreting plasma cells. The broken lines indicate that differentiation into IgM-secreting plasma cells occurs in B cells from some individuals with CVID (adapted from Schaffer et al., 1991). (B) Occurrence of IgAD and CVID in a single family. Individuals with CVID are shown as solid symbols and those with IgAD as hatched symbols. Generations are numbered with roman numerals and individuals with arabic numbers. Lowercase letters are used to trace the inheritance of the extended MHC haplotype I (see text). The designation a/c in generation 111 indicates that haplotype I could be either a or c (for further details see Volanakis et a!., 1992).

259

IgA DEFICIENCY

B

m I

a,cb

b

a

b

1 2 3 4 alc alc alc alc

FIG.1. Continued

The presence of the lymphotoxin Q! (LT-a) and LT-P genes in a region of the MHC associated with IgAD may be significant because these genes are important for normal immunity. Targeted disruption of the LT-a gene results in mice that lack lymph nodes and Peyer’s patches (De Togni et al., 1994; Banks et al., 1995). Although the serum IgG levels in these mice are relatively normal, they are unable to mount an IgG response to deliberate immunization. Intriguingly, the LT-a knockout mice are also IgA deficient. Activated T and B cells normally express a heterotrimeric complex of LT-CUlP chains on their cell surface (Browning et al., 1993) with which they may interact with dendritic cells bearing the LT-P receptor (for review, see Ware et al., 1995; Liu and Banchereau, 1996) Treatment of mice with a LT-PR-Ig fusion protein that blocks this interaction disrupts lymph node development and splenic architecture (Rennert et al., 1996). The importance of the LT-CUlP gene products in lymphocyte interactions that normally occur in germinal center responses makes them interesting candidates for the genetic defect in IgADKVID patients. Studies in Europe, North America, and Australia have indicated an increased frequency of particular MHC haplotypes in patients with IgAD (Ambrus et al., 1977; Hammerstrom and Smith, 1983; Hammerstrom et al., 1984; Strothman et al., 1986; French et al., 1991). In addition, the same MHC haplotypes have been shown to occur with increased frequency in patients with CVID (Ashman et al., 1992; Volanakis et al., 1992). The large number of MHC genes coupled with the relative infrequency of crossovers complicates the search for the Ig deficiency gene(s) in this region of the genome.

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Because the MHC class I1 genes and their products are central to T helper cell recognition and subsequent collaboration with B cells to achieve an isotypically diverse antibody response, one logical possibility is that certain class I1 alleles contribute to the Ig deficiencies in IgAD and CVID patients. In support of this idea, IgAD has been linked to class I1 genes, being especially likely to occur in individuals having MHC haplotypes that contain HLA-DQ alleles encoding a neutral amino acid at residue 57 of the DQ-P chain (Olerup et al., 1990). However, other studies of IgAD and CVID patients and their families suggested that the MHC class I11 region represented the most conserved portion of the susceptibility haplotypes. Attention has been drawn particularly to this region by the frequent association of Ig deficiencywith haplotypes having deletions or duplications of the C4A gene (French and Dawkins, 1990; Volanakis et al., 1992) and by the observation that mice deficient in the C4 complement component have impaired antibody responses, particularly affecting the switched isotypes (Bottger and Bitter-Suermann, 1987). One of the extended MHC haplotypes that has been associated with IgAD/CVID in family studies is TAPlA/TAP2A, HLA-DQPl'0201, -DR3, C4B-S' C4A-0, G l l - 1 5 , Bf-0.4, C2-a, HSP-7.5, TNF-a-5, HLA-B8, and -A1 (Haplotype I; Fig. 1B). This haplotype is also associated with a high incidence of IDDM and celiac disease, and approximately 15% of individuals selected for homozygosity for this haplotype have IgAD (Volanalaset al., 1992).In order to determine whether IgAD is associated with the class I1 or class I11 regions, investigators have examined Sardinian individuals who have the class I1 region of this haplotype but, due to an ancestral crossover, differ distally in the class I11 and class I regions starting from the HLA-DR-P3 locus. This Sardinian DR3 haplotype is associated with a high incidence of IDDM and celiac disease but was found not to be associated with IgAD (Cucca et al., 1996). These data are consistent with the hypothesis that the IgAD susceptibility gene lies within the class I11 region of this haplotype, perhaps in the neighborhood of the TNFILT-cU/LT-P gene family. VII. Pathogenesis of IgA Deficiency

IgAD is due to an arrest in the normal differentiation of IgM B cells into IgA-secreting plasma cells, but the precise point at which the differentiation arrest occurs is a subject of controversy. IgAD individuals have normal numbers of sIgM/sIgD+B cells and, in most cases, have normal serum levels of the other immunoglobulin isotypes. This phenotypic pattern precludes a generalized defect in isotype switching or in terminal plasma cell differentiation as the cause of IgAD. Deficiency in TGF-P or other cytokines or

IgA DEFICIENCY

26 1

their receptors that favor IgA switching and terminal differentiation would be reasonable candidates (Scott et al., 1994). However, studies of TGFPl knockout mice (Shull et al., 1992; Kulkarni et al., 1993; Christ et al., 1994) suggest that the absence of TGF-P1 is an unlikely cause of IgAD because the dramatic effects on regulation of inflammatory responses result in much more severe manifestations than seen even in the worst cases of IgAD. Normal levels of TGF-P1 mRNA were seen in blood mononuclear cells from IgAD patients (Islam et al., 1994b), but decreased serum levels of TGF-P have been reported in some IgAD individuals (Muller et al., 1995).The physiologically relevant source of TGF-P1 for isotype switching is unknown. Many cell types synthesize TGF-Pl and have receptors for it, but the regulation of this cytokine is complicated by the fact that it may be secreted in a latent form that must be activated by proteolytic cleavage (Derynck, 1994; Kingsley, 1994; Massaguk, 1996). The effects of TGFPl are known to be highly concentration dependent (Stavnezer, 1995). Moreover, although there is usually good concordance between induction of sterile transcripts and switching in mitogen-activated B cell, this is not always the case. Given this complexity, a cell-specific deficiency in TGFPl, defective activation, or TGF-P receptor expression remain theoretical pathogenesis factors for IgAD. The presence of sIgA' B cells in IgAD would appear to place the defect downstream of the switch and implicate problems in triggering of the IgA' B cell. IL-5 and IL-6 are important cytokines for the induction of terminal differentiation of sIgA' B cells into IgA-secreting plasma cells in mice (Murray et al., 1987; Beagley et al., 1989, 1995), but this IL-5 function does not appear to exist in humans. Moreover, IL-5-deficient mice have normal IgA levels, indicating a redundancy in this pathway (Kopf et al., 1996). IL-6 knockout mice have reduced numbers of mucosal IgA plasma cells and are deficient in antibody responses to mucosal challenge (Ramsay et al., 1994). The remaining IgA plasma cells in the mucosa of the IL-6 knockout mice are postulated to reflect their IL-6-independent derivation from peritoneal B1 cells (Beagley et al., 1995). Reduced numbers of circulating IgA' B cells in IgAD (Conley and Cooper, 1981) suggest a switching defect. In keeping with this possibility, decreased levels of sterile a transcripts were seen in IgAD (Smith et al., 1993; Islam et al., 1994a), and analysis of sp/sa junctions created during IgA switching indicated a significant decrease in their numbers (Islam et al., 199413).However, the same studies demonstrated that in vitro treatment with TGF-P1 and PMA induced sterile transcription of the C a locus in the IgAD B cells. In other in vitro studies, IL-10 and immobilized CD40L induced IgAD B cells to synthesize and secrete IgA (Brikre et al., 1994). These results indicate that a switch regions in IgAD B cells can be rendered

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TABLE I EXPERIMENTAL MODELSOF IgA DEFICIENCY’ Catagory T cell deficiency Neonatal thymectomy Thymic aplasia (nude mice) TCR-P-I- mice TCR-K- mice B cell deficiency Early bursectomy

B cell defects Ca-l- mice CD19-’- mice 1yn -/- mice 3‘ Enhancer-’- mice NF-KB p6O-l- mice

Comment Impaired antibody responses, especially IgA whn mutation No IgA plasma cells Impaired mucosal IgA responses

Reference

Nehls et al. (1994) Segre et al. (1995) J. McGhee et al. (unpublished results) Fujihashi et al. (1996)

IgAD, IgG and IgAD, or agammaglobulinemia, depending on time of bursectomy

Kincade and Cooper (1973)

IgAD Reduced IgA Elevated IgA Slightly reduced IgA Defective IgA switching in

Harriman d al. (1996a) Engel et al. (1995) Nishizumi et al. (1995) Cogn6 et al. (1994) Snapper et al. (1996)

uivo

J chain+ mice OBF-l/OCA-B/Bob 1-’mice Cytokine deficiencies IL-6-/- mice

T-B interaction defects CD40/CD40L mutations

CD4

Decreased IgA in bile and feces Reduced IgA

Hendrickson et al. (1995)

Severe IgA deficiency, IgA responses to some but not all antigens Absent LN and Peyer’s patches, IgA deficient Partial IgA deficiency Impaired mucosal IgA responses

Ramsay et al. (1994) Bromander et al. (1996)

IgM antibodies produced but blocked in isotype switching and somatic mutation Normal IgA levels but defective IgA responses

Kim et al. (1996) Schubart et al. (1996)

De Tongi et al. (1994) Banks et al. (1995) Okahashi et al. (1996) Fujihashi et al. (manuscript in preparation) Xu et al. (1994) Renshaw et al. (1994) Castigli et al. (1994) Hornquist et al. (1995)

“ Models in which IgAD is a prominent, but necessarily exclusive, feature of the immunodeficiency.

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accessible and switch competent, when exposed to the appropriate cytokines and signals in vitro, but do not explain why they are unable to switch in uivo. Mice with a targeted inactivation of the p50 subunit of the NFKB transcription factor have normal levels of sterile a transcripts but are unable to switch to IgA, even when stimulated with the potent combination of CD40L, IL-4, IL-5, anti-Sdextran, and TGF-P1 (Snapper et al., 1996). VIII. Conclusions

It deserves reemphasis that the defect in IgAD patients and their relatives often extends to compromise other antibody isotypes, most frequently IgG, but sometimes to all of the immunoglobulins. Viewed in this broader context, Table I lists some of the existing animal models in which a deficiency of IgA is a feature of the genetic or experimentally induced defect. Many of these models have been discussed, and some excluded as a likely cause of human IgAD, but the clear message is that many types of immune system defects may dysregulate the production of IgA. Although throughout the review we have emphasized the heterogeneity of IgAD, the hope is that a much shorter table of gene defects responsible for the human disease may soon be a reality. Currently there is no satisfactory treatment for isolated IgAD because it is difficult, and even hazardous, to replace the missing IgA antibodies. In patients with broader immunoglobulin deficiency-that is, those individuals in whom IgG production is also severely compromised-monthly infusions of intravenous immunoglobulins preparations (largely IgG) may reduce the infectious complications. The real future for these patients is an understanding of the pathogenesis which will allow a more direct attack on the basic lesion.

ACKNOWLEDGMENTS We thank Drs. Jerry McGhee, Harry Schroeder, Jr., and John Volanakis for helpful discussions. The skilled secretarial assistance of Ms. Ann Brookshire and the proofreading by Ms. Dottie Lang are gratefully acknowledged. This work was supported in part by NIH Grants A139816 and AI34568. MDC is a Howard Hughes Medical Institute Investigator.

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Sander, S. G., Eckrich, R., Malamut, D., and Mdory, D. (1994). Hamagglutination assays for the diagnosis and prevention of IgA anaphylactictransfusion reactions. Blood 84,20312035. Saulsbury, F. T. (1989). Selective IgA deficiency temporarily associated with Epstein-Barr virus infection. j . Pediatr. 115, 268-270. Savilahti, E., Pelkonen, P., and Visakorpi, J. K. (1971). IgA deficiency in children. A clinical study with special reference to intestinal findings. Arch. Dis. Child 46, 665-670. Schaffer, F. M., Palermos, J., Zhu, Z. B., Barger, B. O., Cooper, M. D., and Volanakis, J. E. (1989). Individuals with IgA deficiency and common variable immunodeficiency share polymorphisms of major histocompatibility complex class 111 genes. Proc. Natl. Acad. Sci. USA 86,8016-8019. Schaffer, F. M., Monteiro, R. C., Volanakis, J. E., and Cooper, M. D. (1991).IgA deficiency. Immuno&&iency Reu. 3, 15-44. Schiff, J. M., Fisher, M. M., Jones, A. L., and Underdown, B. J. (1986). Human IgA as a heterovalent ligand: Switching from the asialoglycoprotein receptor to secretory component during transport across the rat hepatocyte. 1. Cell. Biol. 102, 920-931. Schmitz, I., and Radbruch, A. (1989). An interleukin $-induced DNase I hypersensitive site indicates opening of the y l switch region prior to switch recombination. Int. Immunol. 1,570-575. Schubart, D. B., Rolink, A., Kosco-Vilbois, M. H., Botteri, F., and Matthias, P. (1996). Bcell-specific coactivator OBF-l/OCA-B/Bobl required for immune respose and germinal centre formation. Nature 383, 538-542. Scott, L. J., Bryant, A., Webster, A. D. B., and Farrant, J. (1994). Failure in IgA secretion by surface IgA-positive B cells in common variable immunodeficiency (CVID). Clin. Erp. Immunol. 95, 10-13. Seager, J., Wilson, J., Jamison, D. L., Hayward, A. R., and Soothill, J. F. (1975). IgA deficiency, epilepsy, and phenytoin treatment. Lancet 2, 632-635. Segre, J. A., Nemhauser, J. L., Taylor, B. A,, Nadeau, J. H., and Lander, E. S. (1995). Positional cloning of the nude locus: Genetic, physical, and transcription maps of the region and mutations in the mouse and rat. Genomics 28,549-550. Shaw, S. K., and Brenner, M. B. (1995). The beta 7 integrins in mucosal homing and retention. Sem. Immunol. 7 , 335-342. Shen, L., Lasser, R., and Fanger, M. W. (1989). My43, a monoclonal antibody that reacts with human myeloid cells inhibits monocyte IgA binding and triggers functi0n.j. Immunol. 143,4117-4122. Shull, M. M., Ormsby, I., Kier, A. B., Pawlowski, S., Diebold, R. J., Yin, M.,Allen, R., Sidman, C., Proetzel, G., Calvin, D., Annunziata, N., and Doetschman, T. (1992).Targeted disruption of the mouse transforming growth factor-01 gene results in multifocal inflammatory disease. Nature 359, 693. Sideras, P., Mizuta, T. R., Kanamori, H., et al. (1989). Production of sterile transcripts of Cy genes in an IgM-producing human neoplastic B cell line that switchesto IgG-producing cells. Znt. Zmmunol. 1, 631-642. Sleckman, B. P., Alt, F. W., and Gorman, J. R. (1996). Accessibility control of antigen receptor variable region gene assembly: Role of cis-acting elements. Annu. Reu. Immunol. 14,459-482. Smith, C. I., Bakin, B., Christensson, B., Sideras, P., Hammarstrom, L., and Islam, K. B. (1993). Human immunoglobulin germline alpha ( IgA) transcripts are expressed in vivo and their level correlates to class switching. Immunodeficiency 4, 231-235. Snapper, C. M., and Paul, W. E. (1987). Interferon-gamma and B cell stimulatory factor1 reciprocally regulate Ig isotype production. Science 236,944-947.

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ADVANCES IN IMMUNOLOGY,VOL. 6 .5

Role of Cellular Immunity in Protection against HIV Infection SARAH ROWLAND-JONES, RUSUNG TAN, AND ANDREW McMlCHAEL Mokcukr immunobgy Group, lnstihtte of Mdeculor Medicine, John RadcI& Hospital, Wiwfun, Oxford OX3 OW, United Kingdom

1. Introduction

The cellular immune response is mediated by T lymphocytes that release cytokines and lyse target cells expressing foreign antigens. It generally occurs in parallel with the humoral (antibody) response, although the two can be separated in certain circumstances. Infection with viruses usually evokes both arms of the immune response, which broadly differ in their function: The cellular immune response controls the infection and the humoral response prevents further infection with the same agent. Protection of infants by transfer of maternal antibody is an important component of immune protection against infection in children (reviewed in Zinkernagel, 1996). Cytotoxic T lymphocytes (CTLs) are major contributors to the antiviral T cell immune response. This T cell population carries the CD8 cell surface glycoprotein and recognizes peptide antigens presented by class I major histocompatibility (MHC) molecules of the immune system (Zinkernagel and Doherty, 1974, 1975). When these cells make contact with antigen through their specific T cell receptor (TCR),provided this is accompanied by certain important cosignals, the T cell is activated to divide, differentiate, and mediate lysis of infected cells. The Iyhc process is caused both by release of perforin and through fas ligand triggering programmed cell death infas expressing cells (reviewed in Kagi et al., 1995a). CTLs can also release tumor necrosis factor alpha (TNF-a), which can potentiate killing, and interferon-? ( IFN-y), which has activities against a number of pathogens. The CTLs that initially expand on antigen contact can persist as memory cells: The number of memory cells specific for a particular antigen is usually higher than that found in unexposed animals. CD4positive T cells, the T helper (Th) cells, also have antipathogen activities. Th cells can be broadly grouped into three categories depending on the cytokines they are programmed to release: Thl cells produce IFN-.)I and IL-2, Th2 cells secrete IL-4, IL-5, and IL-10, and Tho clones make a mixture of cytokines including IFN-y, IL-2, and IL-4 (Mosmann, 1994; Mosmann et al., 1986). Thl cells play a particular role in defense against pathogens through direct cytotoxicity and by providing help for CTLs, and by means of the cytokines they produce, particularly IFN-y. 277

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CTLs recognize small peptides derived from intracellular proteins, including those expressed by intracellular pathogens, bound to class I MHC molecules (Townsend and Bodmer, 1989). Foreign proteins are broken down in the cytosol of infected cells by the proteases of the proteasome complex (Goldberg and Rock, 1992). Peptide fragments are translocated to the endoplasmic reticulum (ER) by a transporter (TAP), where they bind to newly synthesized class I molecules in a groove formed by the Q helices of the aland a2domains (Townsend and Trowsdale, 1993). The MHC molecules, HLA in humans, are extremely polymorphic and most of the polymorphism occurs in the peptide-binding groove. The consequence of this is that different MHC allotypes bind different kinds of peptides. Th cells recognize peptides presented in a similar way by class I1 molecules of the MHC. Class II-associated peptides are normally derived from extracellular proteins taken into the cell and digested in lysosomes, which then meet the class I1 molecules in special endosomal compartments before export of the complexes to the cell surface (reviewed in Germain, 1991). This form of antigen processing normally involves specialized antigen presenting cells, the most potent of which is the dendritic cell (Steinman, 1991). 11. Cellular Immunity in the Control of Other Viruses

There is an extensive body of evidence that MHC class I-restricted CTLs play a central role in the control of intracellular microbial infections. CTLs were first demonstrated to be of importance in virus infections in lymphocyhc choriomeningitis (LCMV) infection in mice (Zinkernagel and Doherty, 1974, 1975). However, it is worth noting that their principal role in this infection is to mediate chronic immunopathology because the virus is not cytopathic (Buchmeier et al., 1980). Subsequently, CTLs were detected in murine influenza virus infection (Zweerink et al., 1977), and it was demonstrated that CTL clones transferred into infected mice had potent antiviral effects, which were largely mediated by killing virusinfected cells (Lin and Askonas, 1981; Lukacher et al., 1984). Similar observationswere made for respiratory syncybal virus (Cannon et al., 1988) and herpes simplex infection in mice (Bonneau and Jennings, 1990). CTLs have been demonstrated in many human virus infections (Bangham and McMichael, 1989). Evidence for a protective role in these infections has been harder to obtain. In influenza, CTL levels correlated with protection from deliberate infection ofvolunteers (McMichael et al., 1983). In immunosuppressed patients following bone marrow transplantation, CTL levels correlate with protection from cytomegalovirus (CMV) infec-

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tion (Reusser et al., 1991) and CMV-specific CTL transfer seems to be an effective immunotherapy in this situtation (Walter et al., 1995a). Epstein-Barr virus (EBV) is an excellent model for human CTL-virus dynamics, providing lessons for the study of HIV. The virus causes acute infectious mononucleosis with high levels of CTL activity and huge expansions of oligoclonal CTLs in the blood (Callan et d., 1996). After clinical recovery, the virus persists in B cells but expresses a very limited range of viral gene products, probably only EBNA-1 in B cells (Rowe et aZ., 1987; Young et al., 1989). The virus has thus evolved a strategy for evading CTL responses and the one remaining gene product inhibits its own proteol p c degradation for presentation to CTLs (Levitskayaet al., 1995). Breakthrough expression of viral genes and transformation of lymphoid cells is controlled by a strong lifelong CTL response. However, immunosuppression, iatrogenic or during HIV infection, can result in the development of lymphomas, transformed by EBV (Beral et al., 1991; Rowe et al., 1991). The role of CTLs in preventing uncontrolled lymphoproliferation has been strongly supported by recent reports that it has been possible to treat some EBV-related lymphomas by cell transfer of EBV-specific CTLs (Rooney et al., 1995). Further support for the importance of CTLs in infections comes from the extreme polymorphism of the HLA class I system, particularly at the HLA-A and -B loci (Bodmer, 1972).The function of HLA class I molecules is to present peptides to T cells (Townsend et al., 1986); viruses are a major source of foreign peptides and it is likely that the polymorphism of the HLA class I system reflects evolutionary selection by intracellular pathogens (Hill, 1992). The best example is the protective effect of HLAB53 against severe life-threatening malaria in children in West Africa (Hill et al., 1991, 1992); the frequency of this allele is very high in this part of the world (0.25 compared with 0.01 in Europe). Conversely, in Southeast Asia HLA-A11 appears to have selected a variant of EBV that has a mutation in the immunodominant epitope that it presents to CTLs (de Campos Lima et al., 1993, 1994). Another indication of the importance of class I MHC-restricted T cell responses in control of virus infections is the finding that several viruses have evolved strategies to evade CTL recognition (Hill and Ploegh, 1995). Thus, adenovirus produces E19 proteins that retain class I MHC molecules in the ER (Andersson et al., 1985);herpes simplex virus expresses ICP47, which interferes with TAP-mediated peptide transport into the ER (Fruh et al., 1995; Hill et al., 1995); EBV EBNA 1 blocks its own proteolytic degradation by the proteasome (Levitskaya et al., 1995); and human CMV recycles nascent class I molecules into the cytosol, where they are degraded (Wiertz et al., 1996).All of these processes decrease class I MHC expression

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on the surface of infected cells and facilitate evasion of CTL recognition. Indeed, these findings imply that persistent viruses have to develop some means of evading the CTL response. 111. CTL Effector Mechanisms

CTLs have two antiviral activities that can be measured in vitro. They can kill virus-infected cells, and this can be demonstrated in vivo as well (Kagi et al., 1994,1995b;Zinkernagel et al., 1986; Klenerman et al., 1996). They can release cytokines (Morris et al., 1982) and chemokines (Cocchi et al., 1995) with antiviral activity. These activities are not mutually exclusive, and all could play a part in anti-HIV activity in viuo. LYSIS OF HIV-INFECTED CELLS A. CTL-MEDIATED The lysis of HIV-infected cells as a means of controlling HIV infection has been assumed but little examined. Klenerman et al. (1996) and Yang et al. (1996) have studied the rate at which CTLs kill virus-infected cells in uitro. Klenerman and colleagues have argued that, by using CTLs taken ex vivo and testing antiviral lytic activity immediately (Walker et al., 1987), the rates could be close to those that are operative in viuo. Thus, at a ratio of peripheral blood mononuclear cells [approximately 1% of which are CTLs (Moss et al., 1995)] to target cells of 50: 1, i.e., 2% infected cells (Pantaleo et al., 1991, 1993), the half-life of infected cells in a patient with a strong ex vivo CTL response was about 12 hr; the range in different patients was found to be from 6 hr to 4 days. The average half-life of HIVinfected cells in vivo is close to 2 days and is independent of CD4+ cell counts (Ho et al., 1995; Wei et al., 1995). Zn vitro, infected cells start producing virus particles around 24-48 hr after infection (Yang et al., 1996). Thus, strong CTL responses could kill many virus-infected cells before production of new virus particles: Even allowing for the lag time of 24 hr before infected cells become targets for CTLs (Yang et al., 1996), a slightly less strong CTL response might kill many infected cells before they had produced their full complement of virions. If CTLs reduce virus production, the effect on the measured half-life (t3)of infected cells would be minimal because what is actually measured after antiretroviral drug therapy is the half-life of plasma virus, which is weighted toward the infected cells that produce the most virus; this could explain why the & of infected cells appears to vary so little over a range of CD4 T cell counts, and presumably also of CTL activities. These estimates assume that the frequency of CTLs in blood is similar to that in lymphoid organs, a reasonable assumption from the limited data available, showing similar levels in blood and lymph nodes (Hadida et al., 1995). According to this model,

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the CTL would control the level of virus replication according on the strength of the CTL response; quite small changes in CTL activity could have large effects on virus production and virus load (Klenerman et al., 1996).These estimates, which are based on ex vivo lysis data and reasonable assumptions about the life cycle of infected cells, show that in the aymptomatic mid-phase of HIV infection, CTLs could kill most virus-infected cells, as has been independently suggested by Cheynier and Wain-Hobson (Cheynier et al., 1994; Wain Hobson, 1995). OF HIV REPLICATION BY CD8' CELLS B. SUPPRESSION There is considerable evidence that CD8+ cells play an important role in the control of HIV infection by a direct effect on viral replication. This was first demonstrated by Walker et al. (1986), who showed that HIV could readily be cultured from CD8+-depleted peripheral blood mononuclear cells (PBMCs) taken from healthy seropositive subjects, but that adding back the CD8+ cells suppressed virus production in a dosedependent manner. Further studies demonstrated that this anti-HIV activity is closely correlated with the clinical state and CD4' cell count of the infected individual (Mackewicz et al., 1991). Particularly vigorous activity has been described in a group of long-term nonprogressors (Cao et al., 1995), fuelling speculation that this may be one of the more important mechanisms controlling the length of the asymptomatic period in HIV infection (Levy, 1995). Potent suppression of HIV replication can occur across a semipermeable membrane or by transfer of the supernatant from CD8' cells, suggesting that it is mediated by release of soluble factors. There is no consensus as to whether or not this effect is primarily a property of class I-restricted CTLs, although most of the evidence suggests that these two effector functions are distinct. Although optimal suppression of viral replication was initially observed in HLA-matched CD4' cells, cytotoxicity is not involved: The infected cells are not eliminated from the culture, and removal of the CD8+ cells leads to resumption of HIV replication (Walker et al., 1991).In many studies potent suppression has been observed without any HLA matching (Brinchmann et al., 1990; Levy et al., 1996; Toso et al., 1995),but in other studies MHC class I matching provides maximal antiviral activity and the cells that mediate the suppression have the typical phenotype of CTLs (Tsubota et al., 1989). Some CTL clones have been shown to have potent antiviral activity (Koenig et al. 1995; 0. Yang and B. Walker, unpublished data; D. Nixon, T. Dong, and S. Rowland-Jones, unpublished data; Klenerman et al., 1996). However, the secretion of TNFa by other CTL clones in an antigen-specific manner can actually enhance viral replication in chronically infected T cell lines (Bollinger et al., 1993;

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Harrer et al. 1993).At the clonal level, up to 20% of the CD8+ T cell clones generated from an infected donor exhibit the phenomenon (Hsueh et al., 1994).Detailed analysis of a panel of CD8' clones from asymptomatic HIV-infected donors showed that the majority of suppressing clones did not exhibit HIV-specific cytolytic activity, and that some specific CTL clones showed no evidence of viral suppression-although a few clones had both properties (Toso et al., 1995). HIV-suppressing CD8+ cells have been shown to express certain cell surface markers: DR+,CDllb- (Mackewicz and Levy, 1992), CD28+ (Landay et al., 1993), CD29+, CD45RA-, LFA-1' (Tsubota et al., 1989), CD45ROt, and CD38+, but a diversity of other markers has been observed among suppressing clones, suggesting that CD8+ clones with antiviral activity are phenotypically heterogeneous (Toso et al., 1995). Maintenance of the CD8+ antiviral response in HIVinfected people appears to be dependent on the presence of Thl cytokines, particularly IL-2 (Barker et al., 1995). Suppression of many different strains of HIV-1, HIV-2, and SIV has been observed, both laboratory-adapted strains grown in transformed cell lines and patient isolates grown in primary T cells, although the conditions for suppression show some differences between different systems (Levy, 1993).It has been demonstrated that CD8' cells suppress HIV replication at the level of LTR-driven transcription (Levy et al., 1996),and this activity can extend to the LTRs of other viruses, such as HTLV-1 and Rous sarcoma virus (Copeland et al., 1995). Some T cell clones from uninfected people may also have this property (Hsueh et al., 1994), particularly activated T cells such as allostimulated CD8' cells (Bruhl et al., 1996), but these generally suppress HIV replication only in the "endogenous system," using CD8+-depleted cultures from infected donors (Mackewicz and Levy, 1992). The CD8' antiviral factor (CAF) effect could not be assigned to any known cytokine, although some reversal of the activity was seen with antibodies to IL-8 (Mackewicz et al., 1994a). Although IFN-.)I has some viral-suppressive activity (Emilie et al., 1992) and is produced by CTLs after antigen-specific contact, it does not have the properties attributed to CAF. However, recent reports have identified the CC chemokines MIPla, MIP-1/3, and RANTES (Cocchi et al., 1995) as potent suppressive factors produced by CD8' T cells. In addition, IL-16 has some suppressive activity when produced by CD8' cells from SIV-infected African green monkeys, but with much lower potency than the CC chemokines (Baier et al., 1995). The HIV-suppressive activity of human IL-16 has been questioned (Mackewiczet al., 1996), and it is also disputed whether the chemokines account for all of the CAF activity (Levy et aZ., 1996). Initial observations showed that the activity of the chemokines is greatest against

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macrophage-tropic and primary HIV isolates, and they have little effect against laboratory-adapted strains such as HIV-IIIB/LAI (Cocchi et al., 1995).These observations can be explained by the recent identification of members of the chemokine receptor family as coreceptors for HIV. Different isolates of HIV use different coreceptors for cell entry, and coreceptor usage is the principal basis for cellular tropism. HIV-IIIB and other T cell-tropic strains of HIV-1 use the C-X-C chemokine receptor CXCR4 (LESTWfusin) (Feng et al., 1996), the ligand for which (SDF-1) has recently been identified and blocks entry of T-tropic HIV isolates (Bleul et al., 1996;Oberlin et al., 1996).Macrophage-tropicisolates predominantly use CCR-5, a CC chemokine receptor that binds RANTES, MIP-la, and MIP-1P (Alkhatib et al., 1996; Deng et al., 1996; Dragic et al., 1996), which blocks entry of these isolates. Unusually, HIV strains can use other members of the CC chemokine receptor family, namely, CCR-3 (Choe et al., 1996) and CCR2b (Doranz et al., 1996). These findings can account for CD8+ T cell-mediated HIV suppression by the CC chemokines and explain why there has not always been consistency between past studies of HIV suppression using strains of HIV that differ in their tropism and presumably also in their coreceptor usage. However, competition for a viral coreceptor does not account for all the properties of CAF, such as the ability to suppress HIV replication by an effect on LTR-driven transcription (Levy et al., 1996). Such factors, if powerful in vivo, could have interesting implications for CTL surveillance in that infected cells might be pushed into a state of viral latency in which they cease to be targets for CTL recognition. These latently infected cells could then be reactivated later so that virus control is actually not achieved. This process has not been fully explored in these terms. The relative contribution of CTL-mediated killing and the antiviral effect of chemokines and other factors produced by CD8' cells to control HIV infection has yet to be determined. Antiviral factors do not affect the life span of infected cells; if there was no T cell-mediated cytolysis and the virus was cytopathic with a ti of 2 days, the measured half-lives of infected cells would be as described (Ho et al., 1995; Wei et al., 1995). If the only antiviral activity of CD8' T cells was antigen-stimulated chemokine production, viral escape by mutation of CTL epitopes (as described below) would probably be unlikely because mutant and wild-type viruses released from adjacent cells would be equally susceptible to the chemokines. However, mutant virus might stimulate the release of chemokines less effectively so there could be weaker responses in the environment of mutant virions. Similarly, antagonism could inhibit release of chemokines by cells exposed to adjacent wild-type infected cells (Klenermanet al., 1995; P. Klenennan et al., unpublished results) giving benefit to both mutant and wild-type

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viruses, although no additional advantage to the mutant. The increasing evidence for viral escape to fixation (see below) implies that lysis is important in control of HIV. On the other hand the chemokine effects are strong and specific; therefore, both probably contribute to control of the virus in vivo. Thus, in real people, there is probably a combination of these activities going on, and the balance between them could determine the efficiency of control. High-level chemokine production might be very effective but may only inhibit entry to T cells and not macrophages (Schmidtmayerova et al., 1996). It is interesting that this antiviral activity should be uniquely available against HIV infection, so far the only virus known to use the chemokine receptors for cell entry, but control still fails. Lysis of infected cells could contribute substantially to the reduction and control of virus load but at the price of killing CD4+T cells and macrophages. The secretion of viral transcription inhibitors would reduce virus load but enhance latency. N. HLA and HN Infection

If CTLs are important in the control of HIV infection, HLA class I type should play a major role in determining disease progression. Selection of epitopes is almost entirely determined by HLA type, and selection of conserved compared to variable epitopes as targets for CTL responses could be a major contributing factor to the rate of disease progression. Furthermore, unlike most other major infectious diseases, HIV has newly arrived as a human pathogen so there could have been no selection over the past millenia to select for resistant HLA haplotypes, as has happened, for example, with malaria in West Africa (Hill et al., 1991). Studies of HLA associations with infectious diseases are, however, often confounded by the extreme polymorphism of the HLA complex. There are currently more than 200 alleles described at the A, B, and C loci and more continue to be identified. Thus, a calculated probability value has to be multiplied by the number of variables (alleles) studied, and few studies survive this statisticalcorrection. This can be countered by performing more than one study, particularly if the second is focused on an HLA type identified as a candidate in the first (Hill et al., 1991).If two independent studies come up with the same result, this can also solve the problem. A related issue is the problem of rarity: Few alleles reach a gene frequency as high as 0.1 so very large studies are needed to show a significant decrease in frequency in patients, i.e., resistance to infection or progression. In the classic study of its kind, Hill et al. (1991) needed to study nearly 2000

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children to show a decrease in antigen frequency from 25 to 15% in order to demonstrate that HLA-B53 offered protection against severe malaria. Against this background, there have been several attempts to demonstrate HLA associations with slow or rapid progression to AIDS in HIV infection. A few HLA types or haplotypes give consistent findings. HLAB35 has been shown in five studies to be associated with rapid progression to AIDS (Cameron et al., 1990; Itescu et al., 1992, 1995; Jeannet et al., 1989; Plum et al., 1990; Sahmoud et al., 1993). However, in a study of apparent resistance to infection in West African prostitutes, Rowland-Jones et al. (1995) showed that HLA-B35 might have some advantages because of its ability to present epitopes that are conserved between HIV-1 and HIV-2. This could be an example in which results in one population cannot be transferred to another not only because of differences in prevalent HLA types but also because of differences in prevalent viruses, i.e., HIV1 or -2 and the different HIV-1 subtypes. The HLA haplotype HLA Al-BB-DR3 has also been found to be associated with rapid progression in four studies (Cameron et al., 1990; Kaplan et al., 1990; Kaslow et al., 1990; Steel et al., 1988). It has been shown that HLA-B8 selects epitopes that vary considerably and that B8 may be particularly susceptible to such epitope variation (Klenerman et al., 1994, 1995; McAdam et al., 1995; Phillips et al., 1991). It is also noticeable that despite the extensive characterization of epitopes made by several groups, no epitope has been described that is presented by HLA-A1, a relatively common HLA ty-pe (McMichael and Walker, 1994). Thus, the A1-B8 combination could be particularly unfavorable. This leads to the prediction that homozygotes should be even more susceptible; there is no information on this point, probably because homozygotes are rare. Recently, the 12th International Histocompatibility Workshop analyzed HLA types in 363 HIV-l-infected patients. The protective effects of HLAB27 were confirmed and similar effects were seen for HLA-A32. Associations with progression were found for HLA-B35, Cw4, B39, and A24 (Thorsby, 1996). Kaslow et al. (1996)have devised a novel way to address the complexities of demonstrating HLA associations with HIV infection. Taking a cohort of over 100 HN-infected men, they ranked them by HLA type and clinical course and calculated an odds ratio for each allele. These were summated in individuals and the results were compared with a second cohort of similar size. Highest ranked of the HLA class I types associated with protection were HLA-B27 and HLA-B57, both of which tend to select conserved epitopes (McMichael and Walker, 1994; Goulder et al., 1997). The TAP2.3 allele in association with HLA-A25, -26, and -32, and B18 also appeared to offer protection. HLA types associated with rapid progres-

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sion were B37, B49, and certain combinations of TAP alleles and class I types including B8-TAP2.1 and the B-C combination B35-C4. This method needs to be confirmed in other laboratories, particularly the involvement of TAP polymorphism in contributing to susceptibility or resistance; no other study has been able to identify functional differences between allelic products of TAP (Rowland-Jones et al., 1993b). In a cohort of prostitutes in Nairobi, a small number of women have been identified who show resistance to HIV infection despite repeated exposure (Fowke et al., 1996). In this group, HLA-A"6802 and HLAB18 appear to be more frequent than expected (F. Plummer, personal communication). Some associations of HIV infection with class I1 HLA types have been described, although the published literature is confusing and there are few consistent findings. HLA-DR13 and DR2 were found in one study to reduce transmission from mother to baby (Winchester et al., 1995). HLADR5 has been associated with the sicca-CD8' lymphocytosis syndrome in which progression to AIDS is delayed (Itescu et al., 1989, 1994). Several studies (Cruse et al., 1991; Donald et al., 1992; Fabio et al., 1990; Just et al., 1995) have described more rapid progression associated with the HLADR3-DQ2 haplotype which frequently occurs with HLA-B8 in linkage disequilibrium. In the 12th International Histocompatibility Workshop, protective effects were shown for HLA-DR13 and DQ6 and deleterious effects for DR3-DQ2 (Thorsby, 1996). It has been demonstrated for Epstein-Barr virus that a predominant HLA type in the population can select variants of the virus that fail to elicit strong responses through that HLA molecule (De Campos Lima et al., 1993, 1996). This has not yet been described for HIV, but might be anticipated in populations in which there are predominant HLA types, such as HLA-A2 in most populations or HLA-B35 in West Africa. However, there is currently no suggestion that different HIV clades are in any way selected by prevalent HLA types. Overall there are now sufficient consistent data to be sure that HLA type does play a role in determining susceptiblity to HIV infection and progression to immunodeficiency. This is still a field worth exploring because HLA typing techniques become more sophisticated and applicable to large numbers of DNA samples (Bunce et al., 1995). V. The Nature of HN-Specific CTls

Although most of the HIV-specific CTLs that have been characterized are classical CD8' class I-restricted CTLs; there is some evidence that at least two components exist among CTLs recognizing the envelope protein (Mc-

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Chesneyet al., 1990).Clonalexpansion ofenv-specific CTLs generates CD8' class I-restricted CTLs, but peripheral blood cells can be detected that lyse both matched and mismatched env-bearing targets (Koeniget al., 1988)and that are not blocked by antibodies to CD3 or CD8 (Riviereet al., 1989a).In some cases this may represent antibody-mediated cytotoxicity (Weinholdet al., 1988).In addition, MHC-unrestricted lysis of HIV env-expressing cells has been described by unfractionated PBMC from both infected and uninfected people: This appears to be a property of CD4+cells and is not antigenspecific (Heinkelein, 1996). Characteristically, circulating HIV-specific CTLs also express CD38 and HLA-DR (Ho et al., 1993)and CD45RO and S6F1 (Watret et al., 1993). MHC class 11-restrictedCD4+CTLs have been described in the blood of infected patients (Littauaet al., 1992),but their role in HIV infection is not clear. Studies in envelope vaccine recipients have demonstrated both classical class I-restricted CTLs and CD4+ class 11-restricted CTLs (Hammond et al., 1992),and CD4+ CTLs have also been generated in vitro from the cells of seronegative donors that have been repeatedly stimulatedwithgpl20 (Lanzavecchiaet al., 1988; Orentas et al., 1990; Siliciano et al., 1988).The importance of these findings is that in theory CD4' CTLs have the potential to lyse uninfected, activated CD4' lymphoblaststhat have bound free gpl20 through the high-affinity interaction between gpl20 and the CD4 molecule (Lanzavecchiaet al., 1988;Siliciano et al., 1988).If such a mechanism operated in vivo, then the activation of memory T cells by other pathogens (for example, during opportunistic infections) could lead to CTL-mediated destruction.However,class 11-restrictedenv-specificCTLs have not been demonstrated in fresh tissues of HIV-seropositivedonors. Despite the theoretical possibility of harm, there is no evidence that immunization with recombinant gpl20 or gp160 damages CD4' T cells (Redfieldet al., 1991). W. Measurement of HN-Specific CTLs

Several different methods for assaying HIV-specific CTLs have been used, some unique to this virus. It is worth considering how they interrelate, especially quantitatively, because they do not all measure the same thing. The first assay used was the simplest-direct measurement of CTLs ex vivo without any culture in uitro (Plata et al., 1987; Walker et al., 1987), which is later referred to as CTLe or "fresh" CTL. Nixon et al. (1988) devised a restimulation technique where CTLs are stimulated in vitro by culture with autologous PHA-activated T cells; because some of the latter are infected CD4' T cells, activation should lead to expression of viral antigens. Walker et al. (1988, 1989) have used direct cloning, initiating the culture with anti-CD3 antibody and then cloning by limiting dilution.

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Others have cloned from bulk cultures initiated by the “Nixon method” and then maintained by addition of IL-2 before cloning by limiting dilution (McAdam et al., 1995; Rowland-Jones et al., 1992). In macaques, Letvin et al. (Tsubota et al., 1989; Yamamoto et al., 1990) have detected CTLs from cultures set up with Con A as the stimulating agent; this has worked in both SIV-infected animals and, surprisingly, vaccinated animals (Shen et al., 1991). In vaccinees and exposed uninfected humans, cultures can be stimulated with autologous PHA blasts infected with SIV or HIV (Gallimore et al., 1995; Gotch et al., 1991). In the study by Gallimore et al. the CTLp frequency measured by limiting dilution analysis correlated with the lysis observed from these “bulk’ cultures. Stimulator cells infected with recombinant vaccinia expressing HIV genes and then inactivated with either paraformaldehyde (van Baalen et al., 1993) or psoralen and ultraviolet light (Lubaki et al., 1994) are also effective in generating specific CTL cultures. Rowland-Jones et al. (1995) have shown that stimulation of PBMC with epitope peptides plus IL-2 is effective; the kinetics of the response were compatible with a secondary in vitro response rather than a primary response. Addition of IL-7 to peptide-stimulated cultures improves CTL generation (Lalvaniet al., submitted): IL-7 is also effective in enhancing the generation of CTLs from HIV- (Carini and Essex, 1994)or vaccinia(Ferrari et al., 1995) stimulated cultures. It is widely assumed that these assays measure the same thing, but in fact there are probably important differences. This is most apparent when responses are quantified (Fig. 1). Estimates of CTL precursor frequencies by limiting dilution assays range from 1in lo4for enu-specific CTL precursors (Hoffenbach et al., 1989) to 1 in 5 X lo3for gag-specific CTLs (Gotch et al., 1990), figures that are only marginally higher than those estimated for other persistent virus infections such as EBV and CMV (Borysiewiczetal., 1988a).Recent exhaustive studies of CTL precursor numbers in HIV-infected people have demonstrated figures of up to 1/2000against gag, with somewhat lower numbers of precursors (up to 1/10,000)against env and pol: These figures were highest in healthy asymptomatic donors with a CD4’ T cell count of more than 400 p1 and appeared to be much lower inpatients with HIV-related disease (Carmichael et al., 1993). Many subsequent studies support these estimates (Klein et al., 1995; Koup et al., 1994; Moore et al., 1994).The frequencies in mid-phase infection are higher than those previouslyreported for EBV (upto 1/10,000), CMV (l/ZO,OOO), andvaricellazoster virus (1/100,000)(Alpet al., 1990;Borysiewicz et al., 1988a,b). However, these values would not be enough to give fresh CTL responses; experiments with CTL clones indicate that an effector :target ratio of between 0.125 : 1and 0.5 : 1is needed to give measurable lysis in a 5-hr chromium release assay (Gotch et al., 1990; S. Rowland-

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FIG.1. Measurement of CTLs. CTL clones probably exist in three developmental stages, indicated as naive CTL (nCTL), memory CTL (mCTL), and effector CTL (eCTL). The figure indicates estimated frequencies of these populations in uninfected individuals (nCTL) and HIV-infected persons (mCTL and eCTL). The number of cell divisions needed to detect each of these populations in vioo together with the essential cofactors, cytokines and Th help, are indicated. Also shown are cell types that are detected by the commonly used assays (bulk culture, limiting dilution assay, and fresh assay).

Jones, unpublished results). This means that there would have to be a frequency of CTLs of around M O O PBMC to account for the lysis observed in blood samples from many HIV-infected donors. Support for a higher CTL frequency comes from measurements of T cell receptor mRNA transcript frequencies of dominant clones by Moss et al. (1995) and Kalams et al. (1994). Using nucleotide probes based on the TCR-j3 chain CDR3 sequence of a dominant clone, frequencies between 1 and 5% were found; active CTLs may express more TCR mRNA than resting T cells so these could be slight overestimates. However, because the actual responses are not normally monoclonal, it is more likely that the actual CTL frequency is underestimated. Altman et al. (1996) used tetrameric peptide-HLA complexes as a direct method of staining CTLs specific for HLA-A2 and a gag or pol epitope. Frequencies of between 0.2 and 1.2%of CD8’ T cells were found in patients, none of whom had detectable fresh CTL assays in a 5-hr chromium release assay. The highest frequency so far obtained by this assay was 1.6%in a sample that did show “fresh” CTL activity (P. Moss and G. Ogg, unpublished data). All these results can be reconciled, given that the limiting dilution assay requires division of CTL precursors. In order to lyse 10% (the usual cutoff

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point) of 5 X lo3 target cells, one cloned T cell must grow to at least 1 X lo3cells, i.e., 10 divisions. If the CTLs measured by fresh lytic assays, mRNA transcripts, and direct staining include a substantial population that cannot grow in uitro, the differences would be explained. This would be in line with the finding in acute HIV (Fauci et al., 1996; Pantaleo et al., 1994a) and EBV infection (Callan et al., 1996) of huge expansions of antigen-specific CTLs (over 30% of all CD8’ T cells), most of which die by apoptosis in vitro and probably also in vivo. Following this line of argument, limiting ddution assays measure memory cells and the information gained should be seen as such. Actual CTL activity in patients may be more acurately reflected by assays in which CTLs are directly measured by lysis ex viuo or counted by staining or mRNA transcript quantitation. This difference becomes important in late infection when precursor numbers may be low or may appear to be low because of inefficient CD4’ T cell help, while fresh CTL activity is maintained (Rinaldo et al., 199513). This distinction is also of importance in considering whether CTL killing in vivo is important in controlling HIV and reducing CD4+ T cell numbers (P. Klenerman et al., 1996). MI. Role of HN-Specific CTLs in the Nahrral History of H N Infection

Although immunological abnormalities, particularly of CD4’ cell function, can be detected from the earliest stages of HIV infection, there is nevertheless a vigorous immune response to the virus. Antibodies are generated against all the structural and nonstructural gene products, some of which are able to neutralize heterologous isolates of HIV, whereas others can initiate antibody-dependent cellular cytotoxicity in uitro. A similarly vigorous cellular response against HIV is also observed. The detection of HIV-specific CTLs was first described in 1987 and was remarkable in that HLA-restricted CTLs specific for both gag and env gene products could be readily detected in freshly separated peripheral blood mononuclear cells (Walker et al., 1987) or in alveolar lymphocytes from patients with HIV-related pneumonitis (Plata et al., 1987), without the need for any in vitro culture or restimulation.This level of CTL activitywas unprecedented for virus infections and has subsequently only been described in infection with HTLV-1, another retroviral infection (Jacobson et al., 1990; Parker et al., 1992).Subsequent studies have estimated that between 15 and 88% of HIV-infected subjects have “fresh”or primary HIV-specificCTLs (Grant et al., 1992; Koup et al., 1989; Riviere et al., 198913; Walker et al., 1987). This is clearly a large range and may depend on different assay conditions in different labs-fresh responses are more readily detectable if a longer incubation time (e.g., 16 hr rather than 4 hr) is used (Klenerman et al.,

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1996). As indicated previously, this response may be the most relevant to antiviral activity. CD8' class I-restricted CTLs have been demonstrated against most of the HIV gene products, predominantly directed against gag, pol and enu, but also targetting the regulatory proteins such as nef, tat, and vif (McMichael and Walker, 1994). CTLs have usually been studied using peripheral blood lymphocytes, but they have also been isolated from infected organs, such as the lungs (Plata, Autran et al., 1987), lymph nodes (Hadida et ul., 1995; Hoffenbach et al., 1989), spleen (Cheynier et al., 1994), central nervous system ( Jassoy et al., 1992; Kalams and Walker, 1995),and from the vaginal mucosa of simian immunodeficiency virus (S1V)-infectedmacaques (Lohman et al., 1995). Although most of the descriptions of HIV-specific CTLs have been in adults, it is clear that perinatally infected children can also mount an HIV-specific CTL response, even in the first year of life (Luzuriaga et al., 1995). The first identified peptide epitope was an HLAB27-restricted 15-mer in gag (Nixon et al., 1988), now known to be the decamer KRWIILGLNK (Rowland-Jonesand McMichael, 1993),and subsequently a large number of epitope peptides have been identified (reviewed in McMichael and Walker, 1994), which are now recorded in the Los Alamos HIV Molecular Immunology database (available on-line at the WWW site, http://hiv-web,lanl.gov/immuno/) (Korber et al., 1995). A striking feature of HIV infection is that the HIV-specific CTLs of an infected person are directed toward multiple epitopes: This is different from most virus infections previously studied, in which a dominant CTL response has been identified for a given restriction element and most people (or mice) with that MHC type respond through that allele to a single epitope. In some cases, the whole of the virus-specificCTL response is directed against a single peptide epitope-for example, mice of the H2bhaplotype focus their CTL response to vesicular stomatitis virus entirely on a nine amino acid stretch of the nucleocapsid protein (Van Bleek and Nathenson, 1990). People infected with HIV, in the mid-phase of their infection, usually make CTL responses against multiple epitopes through one or several of their HLA molecules, even though one may be the dominant response: For example, at least three peptides from gag are restricted by HLA-B8, and CTLs against all three epitopes can be detected in a single donor (Phillips et al., 1991) and one of these donors also makes a strong CTL response to an A2-restricted peptide in reverse transcriptase (McAdam et al., 1995; Moss et al., 1995).Another healthy donor has been found to make CTL responses to at least six different peptides from an assortment of HIV proteins (T. Harrer et al., 1996a), and we have studied many similar donors (P. Goulder, G. Ogg, and S. Rowland-Jones, unpublished observations). However, we have also found HIV-infected hemophil-

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iacs whose entire CTL response has been directed toward a single epitope in gag over several years (Nixon et al., 1988; Goulder et al., 1997). When the positions of the epitopes are mapped (Fig. 2), it is apparent that there are clusters of epitopes in certain regions of the viral proteins, for instance, in nef (Culmann et al., 1991). The reasons are unclear but may relate to access of the intracellular processing machinery to these regions. On the other hand, epitopes are more evenly distributed through gag. There are several instances of the same epitope being presented by more than one class molecule-for example, the nef peptide 73-82 contains epitopes presented by HLA-AS, - A l l , and -B35 (the latter is an

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FIG.2. Map of HIV epitopes recognized by CTLs. The immunodominant HIV proteins that contribute the majority of the epitopes recognized by CTLs are represented linearly with the amino terminus on the left. Under each epitope the presenting HLA molecule is indicated. Many epitopes are clustered in the same regions and overlap. The details of these epitopes are derived from the Molecular Immunology Database (Korber et al., 1995) and published and unpublished observations from our own and other groups.

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octamer, 74-81, but the peptides presented by A3 and A l l are identical) (Culmann et al., 1989; Koenig et al., 1990); another nef peptide, 190-198, is presented by three HLA-A2 subtypes as well as HLA-B52 (Hadida et al., 1995). Although generally donors respond to the epitopes in a predictable manner, indicating the strong selective influence of the HLA type, there are examples in which all donors with a particular class I molecule do not respond to the same epitope: For example, donors with HLA-A"201 usually respond either to an epitope in p17 gag or to one in pol, but rarely to both (McMichael and Walker, 1994; P. Goulder et al., 1997). These epitopes are present in different amounts at the cell surface of HIV-infected A2expressing cells, with the p17 peptide being more abundant (Tsomides et al., 1994), but this does not explain why the pol response is immunodominant for some donors. Responses to a third HLA-A2 epitope, in a highly conserved region of reverse transcriptase (which should therefore be a particularly valuable target for CTLs), are observed only rarely (E. Harrer et al., 1996; P. Goulder and S. Rowland-Jones, unpublished results). In a study of CTL responses to the gag protein, HLA type alone did not always predict the target of the response (Buseyne et al., 1994). It is possible that mutations in the flanking regions of CTL epitopes may lead to different rates of processing, or that immune response genes other than HLA may influence immunodominant responses, but these mechanisms have yet to be demonstrated in humans. These studies underline the extent and complexity of the CTL response to HIV. Much of the evidence for the role of HIV-specific CTLs in controlling HIV infection has come from observations of CTL activity in HIV-infected people at different stages of disease (Fig. 3). Although correlation of CTL activity and disease state provides strong circumstantial evidence of their importance, it remains to be fully proven that CTLs are directly responsible for control of virus load rather than a marker of immunological good health. These studies have also attempted to be quantitative, whereas qualitative differences in CTL activity (e.g., to epitopes that cannot vary without damaging the virus) may be more important. Direct evidence that CTLs are important for control of HIV infection requires uneqivocal proof that escape mutations are selected in uiuo, which is forthcoming (discussed below), demonstration that adoptive transfer of CTLs reduces virus load, and demonstration that vaccine induction of CTLs alone can protect against infection with HIV (or SIV as a surrogate).

A. ACUTEINFECTION The high levels of viremia that characterize primary infection with HIV-1 generally occur 3-6 weeks after HIV exposure and are frequently accompanied by clinical symptoms that include fever, malaise, rash,

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FIG.3. The relationship between CTL response, virus load, and CD4' T cell count during HIV infection. The plots are based on data from several reports and do not represent an actual patient. Shown are virus load as RNA copies per milliliter plasma, CD4+ T cell count as cells/mm3 blood, effector CTL (eCTL) as percentage of CD8 T cells in the peripheral blood and precursor T cell frequencies (pCTL). Data for CTLs are based on published measurements (see text).

lymphadenopathy and, less commonly, neurological problems such as meningoencephalitis and transverse myelitis (Cooper et al., 1985). At this time, plasma viral RNA levels may be as high as 10 million copies per milliliter (Mellors et al., 1995; Piatak et al., 1993),and the CD4 count is low-occasionally it may be sufficiently depressed to allow the development of opportunistic infections (Gupta, 1993). CD4+ T cell function is also markedly abnormal (Pedersen et al., 1990).There is usually a profound CD8' T cell lymphocytosis, with huge (up to 40% of all T cells) oligoclonal expansions that express CD38, CD27, and DR but are CD28 negative (Roos et al., 1994). In culture these CD8+CD28- cells are primed for apoptosis (Brugnoni et al., 1996) and probably represent terminally differentiated effector CTLs (Pantaleo et al., 1994a). Over the next few weeks, the plasma virus load falls by several orders of magnitude, although antibodies with the capacity to neutralize the virus are rarely detected at this stage (Ariyoshi et al., 1992; Koup et al., 1994). Virus-specific CTLp have been described as early as 2 days after clinical presentation and within 3 weeks of the onset of symptoms in four of five patients in one study (Koup et al., 1994); these CTLs had specificity for the env, gag, and pol proteins. In another study HIV-specific CTLs were

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detected in four of five patients as early as 6-8 days after the development of symptoms; CTLs recognizing env, gag, and tat were generated, although the predominant response appeared to be toward gp160 (Borrow et al., 1994). In each of these studies one donor failed to make a detectable CTL response and exhibited a rapidly progressive course of HIV infection without control of virus levels, suggesting that the early generation of a vigorous HIV-specific CTL response may not only be responsible for the initial control of viremia but also influence the subsequent disease course. A third study examining the specificity of HIV-specific CTL responses in acute infection found that seven of nine donors had detectable CTL activity in the first 4 weeks after seroconversion (Lamhamedi-Cherradi et al., 1995). These CTLs were predominantly directed toward env, gag, and pol (particularly the integrase component of pol); only three patients responded to nef and none to rev, vif, or tat. The two patients with weak or undetectable responses in this study reported no clinical symptoms of acute infection and did not show a rapid disease progression. In macaques Letvin et al. (Yasutomi et al., 1993b) have detected CTL precursors as early as 4 days after infection, peaking with the viremia at around 2 weeks; Gallimore et al. (1995) also found the peak of CTLs at 14 days after infection. Studies of the TCR repertoire in primary HIV infection have shown that the CD8' response is represented by large but transient oligoclonal expansions in many patients (Pantaleo et al., 1994a). The expansions are identified by the overrepresentation of CD8' T cells with particular VP chains that have restricted amino acid sequences in the third complementarity determining (CDR3) region. The latter is the most variable part of the /3 chain and the limited variability is typical of an antiviral peptide CTL response (Bowness et al., 1993; Moss et al., 1991), implying that the expanded T cells are antigen-specific, Similar findings have been made in acute SIV infection (Chen et al., 1995,1996).These findings are not unique to HIV and SIV infection because similar huge expansions, up to 40% of all T cells in the blood, have been described in acute infectious mononucleosis (Callan et al., 1996) and may be found in all acute virus infections (Tripp et al., 1995). In the HIV-infected patients, poor prognosis was associated with a particularly narrow repertoire of CD8+ expansions, and it was suggested that a relatively limited CD8+ response may facilitate viral escape from the immune system or lead to more rapid immune exhaustion (Pantaleo et al. 1994a; Safrit and Koup, 1995). More detailed study of the fine specificity of the acute HN-specific CTL responses of two patients mapped them to epitopes in gp41; clones from these patients recognized their autologous virus sequences and continued to do so for up to 15 weeks after presentation (Safrit et al., 1994a). However, in two other patients

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with evidence of rapid progression, virus variants with changes in the epitopes recognized by their dominant acute CTL response, sufficient to abrogate CTL recognition, emerged duringthe first few months of infection (Borrow et al., 1997; Price et al., 1996). These last findings (discussed below in the context of immune escape) provide strong evidence for the importance of CTLs in the control of the initial viremia. In addition to cytolytic responses, CD8+ cell-mediated suppression of HIV replication has been described early in HIV infection, before the development of a neutralizing antibody response (Mackewiczet al., 1994b). In these studies, CD8+ suppressive activity was most marked before seroconversion and showed an inverse correlation with plasma viral load in three of seven subjects. Further evidence to support this has come from the simian model of SIVmac infection, in which CD8’ cells capable of inhibiting SIV replication were detected within 13-60 days of experimental infection (Tsubota et al., 1989). Taken together, these studies demonstrate that most people with acute HIV infection develop a broadly reactive HIV-specific CTL response soon after exposure, and that the resolution of acute viremia is in parallel with both the cytotoxic and noncytolytic CD8’ activity. The very high level of CTL response in the acute phase may give the T cells the chance to kill a high proportion of virus-producing cells before they generate large amounts of virus and so bring the infection under a degree of control; in other infections it is complete. There may be a correlation between both the extent of CTL activity and the breadth of the response at the clonal level, and clinical outcome, but this remains to be established in larger studies. It has also been proposed that the dynamics of viremia in acute infection are consistent with a model in which immune responses play no part at all (Phillips, 1996), but the rapid progression in people with weak or no detectable CTL responses and the acquisition of CTL “escape” variants in epitopes recognized by the dominant CTL response early after infection argue strongly against this simple hypothesis. B. ASYMPTOMATIC PERIOD OF HIV INFECTION As indicated previously, in the asymptomatic mid-phase of HIV infection, as many as 1%of peripheral blood mononuclear cells (PBMCs) can be effector CTLs (Gotch et al., 1990), whereas estimates of memory CTLs range from 1 in lo3 to 1 in lo4. The discrepancy between effector and memory CTL numbers is consistent with some degree of terminal differentiation of the effector CTLs, possibly as a result of overstimulation: This could leave the CTLs vulnerable to depletion from clonal exhaustion (Moskophidis et al., 1993).

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This vigorous CTL response against HIV is likely to result from continuous antigen stimulation by a virus that is constantly turning over in multiple sites (Ho et al., 1995). Infection of dendritic cells may also contribute (Cameron et al., 1992; Knight and Macatonia, 1991; Steinman, 1991) CTLes have been detected in long-term nonprogressors with low levels of plasma viremia (T. Harrer et al., 1996b). It has been suggested by Now& and Bangham (1996) that low antigen loads might stimulate strong CTL responses if helper T cell function is good and that the same level of CTLs might require far more antigen when helper T cell functions (or other accessory factors) are impaired. Generally, the anti-HIV CTL response is considered to be stable throughout the symptomatic period. However, a stable total CTL response may conceal an unstable pattern of shifting immunodominant responses in response to variation in dominant virus mutants (Nowak et al., 1995; Phillips et al., 1991). Although this hypothesis is not universally accepted (Miedema and Klein, 1996; Wolinsky et al., 1996), and longitudinal data analyzing immunodominant epitopes and their variation are not abundant, some data from other groups (Autran, et al., 1996b) are consistent with this idea. The implication is that the stability might be an illusion, at least in some patients. It is also likely, based on the arguments presented previously on the role of T cell-mediated killing, that good control of HIV in this phase might be achieved at the cost of a gradual decline in CD4' T cells. C. HIV-SPECIFIC CTL AND DISEASE PROGRESSION There have been several attemps to correlate either the specificity or the magnitude of the HIV-specific CTL response with clinical outcome. Studies in the Amsterdam cohort of HIV-infected donors recruited since 1984 compared the gag-specific CTLp frequency in long-term asymptomatic (LTA) donors and rapid progressors (Klein et al., 1995). All the LTA subjects had detectable CTLp against gag, estimated at between 1/300 and 1/21,000,that were maintained over several years of follow-up, during which CD4' cell numbers and function remained stable and virus load was low. In contrast, one of the six rapid progressors had no detectable gag-specific CTLs, and in four others it was either transient or declined to undetectable levels with disease progression. These studies demonstrated that long-term asymptomatic HIV infection is characterized by sustained gag-specific CTL responses, although the rapid progressors were not protected from disease despite initially high levels of circulating CTLs. Studies in the MACS cohort of homosexual men in Pittsburgh examined the presence of fresh CTLe in healthy HIV-infected subjects and detected CTLes against at least one of gag, pol, and env in 83% of the men during

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the first 8 years after seroconversion (Rinaldo et al., 199513). There was no correlation between the levels of CTLe activity and the CD4+ or CD8+ count, or between the duration of infection or the use of antiretrovirals, nor did the presence or absence of CTLes predict the disease course. These findings contrast with those in a French study, in which fresh effector gag-specific CTLs were elicited in 18 of 38 patients. The risk of progression to CDC stage IV disease was estimated to be 1.89in those without CTLes to gag compared with those with a detectable response (Riviere et al., 1995). There was no significant difference in the risk of progression for those with or without CTLes toward env. These studies may be adversely affected by the insensitivity of the current CTLe assay; detectable lysis at 4 hr needs a CTLe frequency of around 1%;lower levels down to 0.1%, which are still high in conventional terms, may be missed unless the assay is prolonged or a novel method is employed (Altman et al., 1996). Few studies have examined the response to HIV-2, which is endemic in West Africa and is distinguished from HIV-1 by lower rates of transmission and slower disease progression (Markovitz, 1993). A study of 20 HIV2-infected people in The Gambia demonstrated HIV-2-specific CTLs to gag in 90% and to pol in 70%,but to nef in only 25%. An estimate of “total” HIV-specific activity, combining responses against all three proteins, showed an inverse correlation with HIV proviral load: This relationship was strongest for CTLs against gag (Ariyoshi et al., 1995). The determinants of disease progression are thus still poorly defined. Loss of CD4 T cells is likely to be a contributing factor (see below) and the rate of CD4+ cell loss may well be determined by virus load, which is in turn controlled by the CTL response. However, the interrelationship between these parameters is complex (Nowak and Bangham, 1996).Both virus and CTLs destroy HIV-infected cells, but strong CTL responses could substantially reduce virus replication and hence the rate of disease progression. D. LONG-TERM NONPROGRESSORS Other studies have focused on the immune responses of those strictly categorized as “long-term nonprogressors” (LTNPs). This is currently understood to refer to people with at least 8 years of asymptomatic H N infection on no antiretrovirals, whose CD4+ count is more than 500/mm3, and who show no significant “slope” in a plot of their CD4+ cell numbers (Schrager et al., 1994). In general, these people have a broad range of immune responses to the virus, consistent with a largely undamaged immune system, making it hard to determine which is cause and which is effect. Cellular responses in these cohorts include both noncytolytic (Cao et al., 1995) and cytotoxic activity (Pantaleo et al., 1994b). A characteristic

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feature of long-term nonprogressors is a high absolute CD8’ lymphocyte count (Pantaleo et al., 1996) Even in cohorts initially fulfilling the LTNP definition, disease progression can still occur after more than 15years of stable infection. It therefore seems likely that LTNPs constitute a heterogeneous group, and that “absolute nonprogressors” are extremely rare, if they exist at all. More detailed analysis of the subset of subjects with the lowest viral burden and persistent nonprogression from the San Francisco city clinic showed CTL responses were strong, with four of seven subjects demonstratingfresh CTLes against gag and six of seven with restimulated CTLs against several HIV proteins (T. Harrer et al., 1996b). In contrast, neutralizing antibody activity was weak or absent. This study indicates that a detectable plasma viral load, consistentwith active virus replication, is not necessary for the maintenance of circulating activated or fresh CTLes, and implicates CTLs rather than humoral responses as important for long-term nonprogression. More detailed studies of the CTL specificities detected in one donor from this group of LTNPs demonstrated recognition of recombinant vaccinia viruses expressing p17, p24, reverse transcriptase, gp120, gp41, and nef, and characterized six epitopes in detail (T. Harrer et d., 1996a).Interestingly, there was very little variation in the sequences of these epitopes from the donor’s own virus, despite the persistent high levels of CTL activity directed against them. Further studies in the MACS cohort showed that a subset of LTNPs had uniformly low levels of plasma viral RNA, and that this coincided with much higher CTLp frequencies than those found in intermediate or rapid progressors (Rinaldo et al., 1995a). As previously described, there was no correlation with CTLe activity. There was no particular protein targeted by the CTLs of the nonprogressors, but responses to gag, pol, and env were most frequent in the responders. There is little information about whether or not qualitative differences in the responses of progressors and nonprogressors can be found. Another donor from the San Francisco cohort, who has been seropositive since 1978 but remains well with a normal CD4’ count, has been shown to make an immunodominant CTL response to a highly conserved HLAA2-restricted epitope in the active site of reverse transcriptase, with the following sequence: VIYQYMDDL (E. Harrer et al., 1996). The authors speculate that responses to such critical parts of the virus may be particularly valuable in containing virus replication. This epitope is not commonly targeted by donors with HLA-A2, but we have seen responses to it in an exposed but persistently seronegative Nairobi prostitute and to the equivalent HIV-2 sequence in an HIV-2-infected Gambian LTNP (S. Rowland-Jones, unpublished observations).

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The same group has studied the relative efficiency of CTL clones in killing HIV-infected CD4+ cell lines transfected with the appropriate HLA type. They find that maximal lysis is higher with gag- and env-specific clones (similar to peptide-sensitized targets) than with pol-specific clones (Yang et al., 1996). This difference could not be explained by differences in CTL sensitivity for the cognate epitopes and may be due to different levels of expression of gag and pol. Whether this is reflected in lower efficiency of pol-specific CTLs in vivo is not known.

E. DECLINE IN HIV-SPECIFIC CTL ACTIVITYIN LATEDISEASE Most investigators agree that HIV-specific CTL activity becomes progressively harder to detect as disease progresses (Carmichael et d., 1993; McMichael and Walker, 1994; Rinaldo et al., 1995b; Klein et al., 1995; Wolinsky et al., 1996). If CTLs are primarily responsible for keeping virus load at low levels this would lead to escape of virus and ultimately more rapid progression. The reason for the loss of inducible CTLs is not fully known, but there are a number of possible reasons that are discussed. 1 . Is the Decline in CTL Activity Secondary to Loss of CD4’ T Cell Help? The most important potential mechanism is that the loss of CTL activity is secondary to the loss of CD4’ T cell numbers and impairment of their function. It is well established that HIV infection depletes CD4’ T cells (reviewed in Fauci, 1993; Fauci et al., 1996; Pantaleo and Fauci, 1995). If the CTL response is dependent on T helper activity, then the decline in CTL activity is inevitable if the virus is not completely controlled and is likely to accelerate as CD4+ T cell function deteriorates. If, as argued previously, the CTLs are largely responsible for the loss of CD4’ T cells in slowly progressing patients, the CTLs effectively “cut off their own blood supply.” In conventional antiviral CTL assays in vitro, the initial reactivation of human memory CD8+T cells requires antigen and CD4+T cells (Biddison et al., 1981) and is facilitated by addition of IL-2 and IL-7 (Dong et al., 1996; Lalvani et al., 1994). In limiting dilution assays, the benefits of these additions are evident (Carmichael et al., 1993).The long-term maintenance of CTLs in vitro requires, as a minimum, IL-2 and peptide antigen (De Vries and Spits, 1984; McMichael et al., 1986, 1988; Wallace, et al., 1982a,b). Although human CTL clones can be grown in the presence of recombinant IL-2 as the only added cytokine (McMichael et al., 1988), supernatants of activated T cells are better and imply that other cytokines are needed (Wallace et al., 1982b).Although these are most likely to come

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from CD4' T cells, there are other possible sources, including the CTLs themselves, B lymphocytes, and dendritic cells. Although it might be expected that primary CTL responses in vivo would be more dependent on T cell help, some experiments suggest otherwise. Lightman et al. (1987) showed that when mice were depleted of CD4' T cells by infusion of an anti-CD4 antibody and infected with influenza virus, humoral responses were abolished but CTL responses remained, and the mice could clear the acute infection. In LCMV infection in mice, depletion of CD4+ T cells by antibody treatment did not impair the primary CTL response, but the mice failed to maintain CTL memory during persistent infection (Matloubian et al., 1994). However, under more rigorous conditions, Ewing et al. (1994) showed that in mice transgenic for an irrelevant TCR Vfi chain, although depletion of CD4' T cells in vivo greatly impaired the anti-Sendai CTL response, it had less effect on an anti-influenza CTL response. Additional support for the helper T cell independence of the primary CTL response comes from experiments in CD4-'- (Battegay et al., 1994; von Herrath et al., 1996) and MHC II-'- mice (Hou et al., 1995; Rock and Clark, 1996). A different story emerges, however, when epitope peptides are used to prime CTL responses. A consistent finding is that it is very difficult to prime with peptides that are purely class I-restricted epitopes: Epitopes that are recognized by CD4+ T cells have to be added (Sauzet et al., 1995; Shirai et al., 1994,1996). Furthermore, peptide priming of CTL responses in mice can be blocked by anti-CD4 treatment in vivo (Fayolle et al., 1991; Gao et al., 1991). A possible explanation of the contradiction comes from experiments on the roles of antigen dose and of dendritic cells in priming of CTLs. Rock and Clark (1996) primed mice with particulate ovalbumin; MHC class I1 presentation was required at low antigen doses but not at higher doses. Therefore, viruses might appear CD4+ T cell independent because the amount of antigen is usually high, whereas antigens such as minor transplantation antigens (Hurme et al., 1978) and some allo-MHC responses (Lee et al., 1994) might depend more on CD4+ T cell helper for induction of CTL responses. It is probably relevent that dendritic cells, which are capable of presenting particulate antigens by the class I pathway, are sufficient to induce primary CTL responses in vitro without T cell help (Bhardwaj et al., 1994; Young and Steinman, 1990). In summary, the experiments in mice indicate that priming with peptides or low-dose antigen requires T cell help, but that priming by virus infection may not. However, it has been shown repeatedly that the maintenance of CTL memory is dependent on the presence of CD4+ T cells; this could be crucial to our understanding of AIDS pathogenesis.

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In humans most of the data on the role of CD4+ T cells in generating and maintaining CTL responses in vivo have come from studies in HIVinfected patients with depletion of CD4+ T cells. Carmichael et al. (1993) showed that the precursor frequencies of HIV-specific CTLs were low in late HIV infection when CD4' T cell counts were <200/pl. EBV-specific CTL numbers were not impaired, so the effect may be antigen specific. However, it is possible that the slightly different culture conditions in the limiting dilution assay for the HIV- and EBV-specific T cells may have contributed to the difference. Klein et al. (1995) also showed a drop in CTL precursor numbers late in HIV infection; similarly, CTL responses in bulk cultures have also been shown to fall as AIDS develops (Rinaldo et al., 1995b). However, in these experiments it is not clear to what extent there is a fall in actual CTL precursor numbers or whether much of the perceived loss is due to impaired CD4' T cell function. Clerici et al. (1990) showed that the weak anti-influenza CTLs in HIV-infected humans could be restored by addition of alloantigenic cells to the culture, except in patients in which the alloreactive T cell response was also lost. This implicates CD4+T cells in the maintenance of the human memory CTL response and suggests that loss of CD4+ T cells in HIV infection is the primary immunopathogenic event. Thus, the failure to find CTLs does not necessarily mean that they are not there; for instance, Rinaldo et al. (1995b) found that fresh blood CTL activity, unlike restimulated CTL responses, did not fall as AIDS developed. However, if CTL precursors are maintained, how does this square with the findings in mice that CTL memory requires continuous CD4+ T cell help? Antigen dose, which is increasingly high in progressive HIV infection and very low in the mouse experiments, may account for the difference. Alternatively, and perhaps more likely,there may be a true decline in CTL numbers as HIV infection progresses, but this is accentuated by the more profound decline in CD4' T cell numbers and function.

2. Is There a Thl to Th2 Switch? A switch in the phenotype of CD4+ cells may also account for loss of helper function. In 1986, Mossman et al. showed that a panel of murine CD4+T cell clones could be classified according to their cytokine secretion profiles. Thl cells were characterized by the production of IL-2, IFN-y, and TNF-P, whereas Th2 cells predominantly secreted IL-4, IL-5, IL-10, and IL-13. These secretion patterns were subsequently termed type 1and type 2 responses (Clerici and Shearer, 1994). Not all T cell clones exhibit such a clear dichotomy of cytokine production, and lymphocytes that produced a mixture of type 1 and type 2 responses are referred to as Tho (Street et al., 1990). Subsequent reports have confirmed the presence of

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T h l and Th2 phenotypes in human cells (but with a strong preponderance of Tho over Th2) and defined their responses to many natural infections (reviewed in Romagnani, 1994). Type 1responses promote cellular immunity, whereas type 2 responses bias the immune response toward antibody production. Infection with HIV provokes both cellular and humoral responses in viva However, neutralizing antibodies appear relatively late in the infection (Moore et al., 1994) and seem to be relatively ineffective because of virus envelope variation. Antibodies to other virus proteins that do not neutralize are made earlier but play an ill-defined antiviral role. As discussed previously, CTLs are probably the key factor in control of disease. Clerici and Shearer (1993) first suggested that a switch from a predominantly type 1 response to a type 2 response, resulting in decreased cellular immunity, may underlie the immune dysfunction in AIDS. Their hypothesis was followed by some evidence that over the course of HIV infection PHAstimulated peripheral blood lymphocytes produced increasing amounts of IL-4 and IL-10 (Clerici et al., 1993a, 1994c; Meyaard et al., 1994). Moreover, the anti-HIV suppressive factor produced by the CD8+cells of longterm survivors as described by Levy et al. (Levy, 1993; Mackewicz et al., 1991) appears to be dependent on a type 1 cytokine environment (Barker et al., 1995). It is not known whether production of C-C chemokines is Thl or Th2 dependent, though it is clear that CD4+ clones can make them (T. Dong et al., unpublished observations). The Thl-2 cytokine switch has not been easily found by other groups. In particular, Graziosi et al. (1994) looked at cytokine mRNA expression in the lymph nodes of HIV-seropositive patients at differing stages of disease and found little change in secretion patterns over the course of disease. Similarly, Maggi et al. were unable to show an increase in type 2 responses in infected versus uninfected patients using PHA or PMA plus anti-CD3-stimulated PBMC (Maggi et al., 1994b).They did, however, find a shift toward IL-4 secretion among CD8' T cell clones derived from the skin (Maggi et al., 1994a), although the relevance of this is unknown. Maggi et al. (1994) and Vyakarnam et al. (1995) have demonstrated that HIV preferentially replicates in the Th2 or Tho cells, whereas T h l cells appear relatively resistant to infection. This correlates with evidence that virus-induced cell death is primarily of Th2 cells, whereas the T h l subset appears protected (Clerici et al., 1994b; Estaquier et al., 1995). This could mean that a switch to an increased number of Th2 cells during infection could result in upregulated virus production in CD4' lymphocytes and subsequent apoptosis. This hypothesis is supported by the finding that HIV-infected patients with high circulating IgE levels, presumably as a result of Th2 activity, appear to have a worse prognosis (Israel Biet et al.,

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1992; Lucey et al., 1990), although in some cases this does not correlate with increased IL-4 levels (Vigano et al., 1995). Thus, patients whose immune systems are activated by atopic conditions or infections that upregulate Th2 responses may cope poorly with HIV infection, although further evidence is needed. A complicating factor is that in vitro experiments do not take into account the complex cellular milieu in vivo. A number of other cells, principally B cells and macrophages, also secrete cytokines that modify lymphocyte responses. Macrophages are an important reservoir of HIV infection and, whereas IL-4 activates T cells and upregulates lymphocyte virus production, the same cytokine has a virostatic effect upon macrophages, as do IL-10 and IL-13 (Montaner and Gordon, 1995). Th2 cytokines therefore might not be wholly bad in HIV infection. If the T h m h O subset of cells is preferentially infected and killed, the loss would result in lower concentrations of IL-4 in the lymphatic microenvironment and the reactivation and secretion of new virus from macrophages to renew the cycle (Montaner and Gordon, 1995). It is currently difficult to interpret whether infection and death of subsets of CD4' lymphocytes is a result of, or the reason for, a T h l to Th2 switch-if indeed such a switch is real. Experiments done to date appear to present conflicting data with respect to a cytokine switch during the course of HIV infection. These disagreements illustrate the complexity of the field, which is highly dependent on the sensitivity and reproducibility of different protocols, on the different sampling times and specimens, and perhaps even on statistical error (reviewed in Romagnani et al., 1994). More convincing is the evidence that HIV replicates preferentially in T h y 0 cells and that the Th2 subset is more susceptible to apoptosis than T h l cells. However, the relevance of this phenomenon in vivo is still unknown.

3. Is There CTL Exhaustion? Another possible reason for the progressive decline in CTL activity is that these activated effector cells become exhausted as a consequence of prolonged high-level stimulation. An analagous situation has been described in an animal model transgenic for an LCMV-specific T cell receptor challenged with high doses of LCMV (Moskophidis et al., 1993a), but this was an acute phase response and there is no evidence that it occurs in humans. We have detected high levels of cells in peripheral blood expressing the same HIV-specific TCR over several years in two hemophiliac donors (Moss et al., 1995) but have not yet analyzed the frequency of these CTLs during progression to AIDS. Recent studies of telomere length as an indication of replicative potential have shown that the CD8+ cells of HIV-infected people have significant telomere shortening (Effros et al.,

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1996): This is in contrast to the CD4’ subset (F. Miedema, personal communication), suggesting that there is much greater CD8+ cell turnover and hence potential for exhaustion. If CTL exhaustion does occur, then failure to generate new CD8’ CTLs could be a result of dendritic cell infection or infection of CD4+ CD8’ CTL precursors in the thymus. However, in a recent trial of adoptive immunotherapy using HIV-specific CTL clones transduced with marker and “suicide” genes, HIV-infected patients with CD4’ counts between 200 and 400/p1 were able to generate CTLs specific for the foreign genes and eliminate the infused CTLs (Riddell et al., 1996); therefore, at this level of immi~nosuppressioneffective CTL responses to new antigens can still be made. It would be instructive to know whether recipients with very low CD4+ T cell numbers would still make such a vigorous immune response. 4. HlV lnfection of CTL

Despite the lack of CD4 expression, there have been occasional reports of HIV infection of CD8’ cells: For example, in long-term SIV-macspecific CTL cultures (Tsubota et al., 1989) and, recently, in as many as 400/million CD8’ cells taken from HIV-infected people in the late stages of disease (Livingstone et al., 1996). It is possible that HIV-specific CTLs become infected at the time of lysis of infected target cells (De Maria et al., 1991). It has also been shown in human fetal thymic explants into SCID mice that CD4’8’ thymocytes can be infected with HIV and are then depleted; because these cells are precursors of CD8+ T cells, it is possible that CTLs could be infected by this route. However, this seems unlikely to be a major mechanism because multiple cell divisions are involved, and infected cells would activate virus expression and be killed by virus cytopathic effects or by the immune response.

5. Suppression of HN-Specific CTL Activity by Other Cells A population of CD8’ HNK1’ CD4- CD16- T cells was identified among the alveolar lymphocytes of AIDS patients (Joly et al., 1989) that were able to inhibit the activity of HIV-specific CTLs as well as CTLs against HLA-alloantigens in a non-MHC-restricted manner. Further studies from this group have shown that these alveolar lymphocytes are characterized by expression of CD57, and that suppression of CTL activity is mediated by a lectin-binding soluble factor (Sadat-Sowtiet al., 1991,1994). “Suppressor” lymphocytes have not yet been convincingly demonstrated in peripheral blood, although a CDS’CD11’ population of PBMC caused some reduction in cytotoxicity mediated by other CD8’ or CD16+ (NK) cells from the same donor (Kundu and Merigan, 1992).

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6. Apoptosis of HN-SpeciLfc CTLS The proportion of CD8' cells expressing fas increases during HIV infection, which makes them vulnerable to depletion by fasL-induced apoptosis (Gehri et al., 1996). There is some evidence that CD8' cells taken from people with late-stage HIV infection are prone to apoptosis in the presence of HIV antigens in vitro, which reduces the detection of HIV-specific CTL activity (Chia et al., 1995). This is associated with reduced expression of the Bcl-2 protein, which protects cells from apoptosis (Boudet et al., 1996), and expression of a CD45RO' DR' CD38' CD28- phenotype. Apoptosis has also been seen in the SIV models; Xu et al. (1997) have found that many CD8' T cells in macaques infected with a clone of SIV-mac 251 showed spontaneous apoptosis, probably related to increased expression of fas. This lysis masks the CTL response because the responding CTLs die in culture. It is not yet known what cell mediates the lysis.

7. Speajic Cytokine Defects In addition to the need for IL-2, a bone marrow stroma-derived cytokine, IL-7, has been shown to be important for the generation of CTLs in uitro. Addition of IL-7 to CTL cultures from HIV-infected people can greatly enhance the detection of HIV-specific CTLs (Carini and Essex, 1994): This effect is also seen in generating CTLs from vaccine recipients (Ferrari et al., 1995) and exposed seronegative individuals (Lalvani et al., 1997). However, both CD4' and CD8' cells from some HIV-infected people lack IL-7 receptor expression, and it is difficult to grow CTLs from them, even with the addition of IL-7 (Carini et al., 1994). The basis for the dysregulation of IL-7R expression is not clear. 8. Antagonism by HN Variants A further important possibility, which is discussed in detail below, is that variants of HIV that escape CTL recognition may be generated during the course of infection, and some of these variants may not only evade recognition but may affect CTL responses to the original sequence through TcR antagonism. Thus, as mutant viruses accumulate, the overall CTL response might be impaired, although the CTL precursors are still present. These mechanisms (Sections VII,E,l-VII,E,8) are not mutually exclusive and all could contribute to the repeatedly made observation that the CTL response that can be demonstrated in vitro fails as the CD4' T cell level falls below 2OOlpl. However, the relative contribution of each of these processes is important to understand because the implications for the treatment may be quite different. If the impairment of CTL activity in vitro is due to loss of CTL precursors, perhaps due to clonal exhaustion (Moskophidis et al., 1993b; Pantaleo et al., 1994a; Pantaleo and Fauci,

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1995), an effective treatment might be to replace the CTLs by adoptive transfer (Koenig et al., 1995; Riddell et al., 1996; Riddell and Greenberg, 1995b). On the other hand, if it is secondary to loss of CD4+T cell activity, possibly including a switch from Thl to Th2 responses, treatment would be replacement of CD4+ T cells-assuming that a suitable donor was available (Walker et al., 1993). Some support for this view comes from Greenberg et al., who have treated bone marrow transplant recipients with CMV-specific CD8' CTL clones (Riddell and Greenberg, 1995a,b; Walter et al., 1995a). They found much better survival of the infused clones when there was an ongoing anti-CMV Th response (Walter et al., 1995a).

F. HIV-SPECIFIC CTLs IN EXPOSED PERSISTENT SERONEGATIVES There is now a considerable body of evidence that people exposed to H N who do not appear to be infected have markers of cellular immunity to the virus. The largest studies have demonstrated CD4' cell responsesproliferation and IL-2 secretion-in response to a panel of HIV envelope peptides in up to 75%of seronegative people with a history of HIV exposure by a variety of routes (Clerici et al., 1992, 1993b, 1994a). These responses do not prove that the subject was exposed to replicating virus rather than viral proteins or replication-incompetent HIV, but they do raise the intriguing question of how a cellular response can be generated in the absence of detectable antibody to HIV. The authors of these studies have proposed a model in which low-dose virus exposure preferentially elicits a T h l type of immune response (which could include a CTL response), which may be associated with protection, whereas high-dose exposure stimulates a Th2 response, with antibody production and persistent infection (Clerici and Shearer, 1993). In response to these observations, it was suggested that the detection of MHC class I-restricted CTLs might be a more reliable indication of exposure to infectious virus after HIV exposure (Miedema et al., 1993). HIV-specific CTL responses were first reported in 3 children born to infected mothers (Cheynier et al., 1992); these children lost maternal antibody and remained virus negative by PCR, but HIV-specific CTLs could be detected up to 3 years of age. Subsequently, CTLs to H N were described in 3 more children (Aldhous et al., 1994; Rowland-Jones d al., 1993a) but these responses were transient and observed only in the first year of life: This might suggest that persisting antigen is needed to maintain a CTL response. However, a recent study of 23 uninfected children of HIV-positive mothers, aged 12-50 months, showed that 6 (26%) had fresh HIV-specific CTLe activity (De Maria et al., 1994), implying a high frequency of circulating effector cells (Moss et al., 1995), which is hard to explain in the absence of detectable replicating virus.

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In repeatedly HIV-exposed adults, Langlade-Demoyen et al. (1994) described an increased frequency of HLA-A2-restricted HIV-nef-specific CTL precursors (measured by limiting ddution analysis, stimulating the cultures with PHA, and testing on P815-A2 cells transfected’ with HIV genes as targets) in the sexual partners of HIV-l-infected adults: In this study there was a surprisingly high frequency of CTL precursors measured against other HIV products (env and gag) in both exposed and unexposed subjects, which because of the assay conditions could represent crossreactive alloresponses from parous women. We have described the finding of CTLs specific for several HLA-B35restricted HIV peptides (from gag, pol, and nef) in three of six seronegative and apparently uninfected female prostitutes with HLA-B35 in The Gambia, West Africa (Rowland-Jones et al., 1995), where HIV seroprevalence among sex workers is approximately 35%. These CTLs were elicited by stimulation of PBMCs with peptides representing epitopes from HIV-1 and HIV-2, which are recognized by CTLs from infected Gambians with HLA-B35, the most common class I molecule in that population. Initially HIV-2 was the dominant virus in The Gambia but most new infections are with HIV-1; these epitopes are cross-reactive between HIV-1 and HIV2 in that CTLs from a donor infected with one HIV strain recognize the corresponding peptide from the other strain. The CTLs generated from the uninfected women are also cross-reactive and kill target cells infected with recombinant vaccinia virus expressing both HIV-1 and HIV-2 proteins. HIV-specific CTLs have been elicited on four occasions over the past 2 years and have been recently detected in a further two of the original six women-a total of five of six-although these women have been persistently seronegative for at least 7 years. CTLs could not be elicited by the same protocol in a large panel of unexposed controls with HLA-B35. One possible explanation for our findings is that the CTLs were initially primed by exposure to HIV-2, which appears to be both less transmissible (Adjorlolo-Johnson et al., 1994; Kanki et al., 1994) and less pathogenic (Del Mistro et al., 1992; Pepin et al., 1991; Whittle et al., 1994) than HIV1, leading to protection against HIV-1 in women making a CTL response that is cross-reactive between the two viruses. If so, the high frequency of CTL responses in exposed seronegatives with HLA-B35 might be because B35 presents several cross-reactive epitopes, whereas the majority of class I molecules so far studied do not. A larger, prospective study is needed to study the incidence of HIV infection in repeatedly exposed women with and without detectable CTL responses to determine whether these responses are actually associated with protection. We have also looked for HIV-l-specific CTLs in the Nairobi cohort of highly exposed but apparently HIV “resistant” female prostitutes (Fowke

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et al., 1996), using epitope peptides selected for class I molecules, which are common in this group. We find that several of these women also have HIV-specific CTLs that recognize epitopes that are highly conserved between HIV-1 clades, which intuitively may be more likely to be associated with protection (S. Rowland-Jones and Dong, manuscript in preparation). CTLs have also been detected in this group using autologous PHA blasts infected with HIV-IIIB (a clade B virus) (K. Fowke et al, manuscript in preparation), which again suggest that cross-clade CTLs are an important feature of this cohort because virus exposure in Nairobi is mostly to HIV clades A and D. Neither the Gambian prostitutes nor the women in the Nairobi cohort with CTLs have the CCRS receptor defect described in exposed uninfected men in New York (T. Dong and S. Rowland-Jones, manuscript in preparation). A recent report describes the transient appearance of CTLs specific for HIV envelope peptides in 7 of 21 health care workers exposed to HIVinfected blood who did not seroconvert: CTLs were not detected by the same protocol in 20 workers with HIV-negative exposures (Pinto et al., 1995). The rate of actual infection in exposed health care workers is extremely low, but these observations, together with the high rate of HIV-specific proliferative responses observed in this group, suggest that significant exposure to HIV antigens occurs in a majority of people with needle-stick injuries. The transient nature of these responses, like those observed in many of the babies born to infected mothers, suggests that a single exposure does not lead to detectable memory CTLs. This is in contrast to the prostitute cohorts in which exposure may occur on a daily basis and in whom HIV-specific CTLs have been maintained over months and years of study (S. Rowland-Jones, unpublished observations). Thus HIV-specific CTLs without antibody have now been observed in a number of different exposed seronegative groups, but it remains to be established whether or not they are actually associated with protection from future infection rather than simply markers of past exposure. The detection of CTLs in the Nairobi cohort, for whom there is the most convincing evidence of resistance to HIV infection in the face of intense exposures (Fowke et al., 1996), may point toward a protective role. G. POTENTIAL ADVERSEEFFECTS OF HIV-SPECIFIC CTL ACTIVITY HIV-specific CTLs have been isolated from the lungs of infected people and their activity correlates with measured abnormalities in pulmonary gas exchange and alterations in the alveolar-capillary barrier (Autran et al., 1990). It is possible that CTL activity against HIV-infected alveolar macrophages leads to injury to the pulmonary epithelium by some as yet undefined mechanism. Similarly,the presence of HIV-specific CTLs in the CSF

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of patients with neurological disease (Sethi et al., 1988) led to speculation that they may be contributing to neuropathology. More vigorous HIVspecific CTL activity may be found in the CSF of some patients with AIDSrelated dementia rather than in their peripheral blood, which suggests that the recruitment of CTLs into the CSF could play a part in the pathogenesis of dementia (Jassoy et al., 1992). The CD8+lymphocytotosis syndrome is characterized by increased numbers of circulating CD8' cells that infiltrate salivary glands, lung, gut, and kidneys of a few HIV-infected people with particular HLA types (as described previously). Although the infiltrating cells have not been definitely shown to be HIV-specific CTLs, they have the phenotype of antigendriven expansions. Surprisingly, people with this syndrome have a relatively good prognosis, which implies that an excess of CD8' antigen-specific cells is beneficial rather than harmful (Itescu et al., 1993). Some HIV-specific CTLs release cytokines on contact with HIV-infected cells such as TNFs (Jassoy et al., 1993), which have the potential to upregulate HIV replication (Harrer et al., 1993). TNF-a secretion from some CTL clones produced in response to rgpl20 vaccination could be shown to increase HIV production from chronically infected T cell lines (Bollinger et al., 1993). Lysis of uninfected activated CD4' lymphoblasts by CD8+ cells has been observed in humans (but not chimpanzees) infected with HIV (Zarling et al., 1990). Further studies have shown that these CTLs recognize an activation-dependent, nonpolymorphic molecule on uninfected CD4' lymphocytes (Bienzle et al., 1996); however, HIV-specific CTLs do not have this activity. If such a mechanism operates widely, then this could contribute to the decline in CD4+cell numbers during the course of HIV infection. Similarly, if CD4' env-specific CTLs are really operating in vivo, then lysis of uninfected CD4+ cells that have adsorbed gpl20 onto their surfaces could contribute to immune depletion (Lanzavecchia et al., 1988; Siliciano et al., 1988). It remains to be established if these actually are important mechanisms for immunopathology. The role of CTLs in destroying HIV-infected CD4+ cells has been discussed previously. Zinkernagel (1995) has argued that if HIV is not cytopathic in vivo, there will be a critical balance between virus dynamics and the immune response: If there are few HIV-infected cells and an effective immune response, then the virus will be controlled or even eliminated. However, if the balance is in favor of the virus, then CTLs could be responsible for immunopathology. This model predicts that individuals with high viral loads but a poor CTL response would do well clinically; recent results in sooty mangabeys, which are frequently infected with SIV but do not get sick, suggest that this situation is present because the virus

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is not cytopathic in this species (M. Feinberg and P. Johnson, personal communication). In humans the evidence is compelling that HIV is cytopathic in vivo (Ho et al., 1995; Wei et al., 1995). In this situation HIV infection in the absence of an immune response is rapidly progressive and fatal (Pantaleo and Fauci, 1995); CTLs can slow this process but will gradually erode CD4' cell numbers. WII. Does H N Escape from the Cn Response?

Given the importance of CTLs in controlling HIV infection and the variability of the virus it must be virtually inevitable that escape mutation will occur. The virus genome is lo4 nucleotides, the mutation rate is estimated to be per generation (Coffin, 1995), and lo9 or 10" virions are generated every day (Ho et al., 1995; Perelson et al., 1996). Thus, there are 10' or lo9 point mutations made each day, with every possible point mutation occurring multiple times and in many combinations, although the vast majority of mutations result in defective virus. Recent evidence on the sequences of the proteases supports the view that the virus quasispecies contains vast numbers of mutants; escape mutations are there before any drug is given (Kozalet al., 1996). However, the database of HIV amino acid sequences shows that there are clear consensus sequences (Korber et al., 1995) and nearly all patients examined have predominant virus sequences that are close to the consensus sequence (Holmes et al., 1992; Meyerhans et al., 1991; Phillips et al., 1991). This implies that conserved sequences are selected at or around the time of transmission in the absence of any immune response, presumably for their transmission characteristics. There are different consensus sequences in different geographical areas that constitute the different HIV-1 clades (Louwagie et al., 1993). Examination of envelope amino acid sequences at seroconversion demonstrates conservation even in the most variable part of the molecule, the V3 loop (Zhang et al., 1993; Zhu et al., 1993; Wolfs et al., 1990). Once the immune response begins to control the virus there is a diversification in the envelope sequence that must result largely if not entirely from selection by neutralizing antibody (McKeating et al., 1989; Wolfs et al., 1990). Evidence for this comes from measurement of nonsynonymous to synonymous or dN/dS (coding to noncoding) nucleotide changes in envelope sequences in virus in macaques infected with molecular clones of SIV (Bums and Desrosiers, 1994; Shaper and Mullins, 1993). Certain amino acid changes were clearly selected. Although antibody is almost certainly responsible, it has been suggested that there could be another force acting on the envelope (Weiss, 1996). The envelope and probably

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the V3 region binds to a coreceptor as well as CD4 on T cells, CCR5 (or more unusually CCR2b or CCR3), or CXCR4 (Alkhatib et al., 1996; Choe et al., 1996; Doranz et al., 1996; Dragic et al., 1996; Feng et al., 1996), and the virus could change its envelope to bind to different coreceptors and so change its tropism. It is possible that an increase in target cell range could also act as a selecting force on the envelope sequence. However, it is very likely that the high titer of neutralizing antibody that is often present has a selective effect. Both antibody selection and alteration in tropism could combine as a potent force in the pathogenesis of HIV infection. The initial viremia is controlled by the CTL response, as described previously: The appearance of the latter occurs just as the viremia falls (Koup et al., 1994). The high levels of expanded CTLs seen (Pantaleo et al., 1994a; Pantaleo and Fauci, 1995) are almost certainly sufficient to kill most virus-infected cells before new virions are produced (Klenerman et al., 1997). Selection of escape mutants can occur at this time. Borrow et al. (1997) and Price et al. (1996) have both described acutely infected patients in whom there were strong CTL responses to single dominant epitopes. In both, escape mutants were selected and completely dominated the virus quasispecies within 8-10 weeks. For both examples, the mutant epitopes, one in env and one in nef, failed to bind to the presenting HLA molecule, HLA B44 and B8, respectively. The apparent association between oligoclonalityin the primary response and poor outcome (Pantaleo and Fauci, 1995) could reflect selection of such escape variants and/or overstimulation and exhaustion of responding T cell clones. Unlike escape from antibody, in which the escape mutant is dominant and penetrates the barrier of neutralizing antibody to infect target cells, a mutant within an infected cell is recessive if there are multiple virus genomes within the cell: When the cell is killed all virus variants within that cell die. However, an HIV-infected cell only integrates a single copy of HIV cDNA and the mutations arise preintegration so that all viral products in the cell have the same mutations. In these circumstances selection by CTLs can occur. In the previous examples of escape from CTLs, there was a CTL response that was directed toward one immunodominant epitope. This could be crucial to the ability of HIV to escape from CTLs. In the original description of virus escape from CTLs, the infected mice were transgenic for an antiviral CTL TCR so that there was a monospecific selection force acting on the virus, LCMV (Aebischer et al., 1991;Pircher et al., 1990). Similarly, Koenig et al. (1995) described a patient who was treated by infusion of very large numbers of a single CTL clone specific for a nef epitope presented by HLA-A3. This resulted in the appearance of virus with deletions in nef that eliminated the epitope. It is likely that the transfer of such a large

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number of a single clone made it the dominant selection force. In these circumstances a single mutation can evade the whole CTL response. In established infection with HIV, the picture appears more complex. In a study of HIV-infected patients with HLA-B8, it was found that they tended to make immunodominant CTL responses to an epitope in gag p17, 24-31, which is variable and can escape from CTL recognition (McAdam et al., 1995; Phillips et al., 1991). However, the same patients also respond to a second epitope in gag, p24 258-267, which can also vary. Nowak et al. (1995) showed that in one of these patients the response was unstable over time and that the dominant CTLs switched from one epitope to the other. At the same time, there was a fluctuation in the predominant virus sequence with escape variants for each epitope increasing in frequency when the CTL response was strong and decreasing when it was weak. In a theoretical model, Nowak et al. (1995; Nowak and McMichael, 1995) argued that as escape mutations were selected in the immunodominant epitope, the T cell response switched to a second epitope in an immunodominance hierarchy and this changed the selective force acting on the virus so that different mutants were selected. Thus, there were shifts in immunodominant epitopes and in the predominant virus species. A mathematical model was based on the simple premises that immunodominant CTL clones outgrew other reacting clones (not only to the same epitope but also to other epitopes in the virus) and that once the antigen changed they would decline, to be replaced by rapidly reacting clones to the next epitope. This model appeared to describe what was seen in the patients with HLA-B8 when studied over several months. Many patients have been described who have concurrent CTL responses to multiple epitopes (Autran et al., 1996a; Autran and Letvin, 1991; Chenciner et al., 1989; Hadida et al., 1992; Harrer et al., 1994, 1996; T. Harrer et al., 199613; Johnson et al., 1991, 1992, 1993; McMichael and Walker, 1994; Safrit et al., 1994a,b; Tsomides et al., 1994; Venet and Walker, 1993; Walker et al., 1987,1988,1989).When examined, almost all have mutations in these epitopes in provirus that would affect the CTL response (Couillin et al., 1994, 1995; Klenerman et al., 1994, 1995; Meier et al., 1995; Meyerhans et al., 1989, 1991; Nowak et al., 1995; Phillips et al., 1991; RowlandJones et al., 1992). The argument has been made that it would not be possible for virus to escape from CTLs because simultaneous muations would have to occur for the escape to be possible (Miedema and Klein, 1996); even at the rate of accumulation of mutations observed (Coffin, 1995), simultaneous mutation at more than two epitope sites would be extremely rare. There are three counters to this argument. One is that the CTL responses are rarely equal so that one may account for as much as 90% of the CTL activity (Goulder et al., 1997); the selective pressure

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would be much greater at this epitope. Careful quantitation of the strengths of the component CTL responses may be needed to identify where the selection forces are strongest. Second, even if some responses are equally strong, a small reduction in the total CTL response may give some mutant virus variants sufficient edge to gain advantage. Third, and most likely, the multiple CTL responses seen in mid-infection may have resulted from progressive escape of the type described previously (Nowak et al., 1995; Nowak and McMichael, 1995).The CTL response may start as monospecific and then diversify as epitope escape mutants are selected (Borrow et al., 1997; Price et al., 1996).Thus, the heterogeneous CTL responses that seem typical of HIV-infected individuals may reflect escape and selection that has already occurred. In a number of studies, analysis of mutations in major epitopes and comparison with nonepitope regions suggests escape and selection. This was clearly shown by Price et al. (1996) in the acute anti-nef response in an acutely infected patient, where dN/dS ratios increased over time at the epitope compared to adjacent regions. Phillips et al. (1991) showed increased variability in the p17 24-32 region of gag in patients with HLAB8, and similar observations have been made by Couillin et al. (1994, 1995) on an HLA-All-restricted epitope in gag and by Wolinsky et al. (1996) on an HLA-B7-restricted epitope in env. These are strong arguments for prior selection even where the changes in CTL response and virus were not directly observed. Sometimes the CTL response remains specific for a small number of epitopes over several years and in some cases only 1. Patients with HLAB27 all respond to an immunodominant epitope in gag 263-272 that is highly conserved in all clades (Phillips et al., 1991; Goulder et al., 1997). In three of six patients this was the only response consistently obtained. In two patients followed over 6 years the response was strong and stable, but as CD4+ counts fell to very low levels both individuals selected a mutant virus with a lysine instead of arginine at the second anchor position. HLA-B27 has a near absolute requirement for arginine at position 2 in the epitope peptide (Jardetzky et al., 1991; Rammensee et al., 1995). If this is replaced with lysine the peptide binds only poorly to HLA-B27 and has a greatly increased off-rate (Colbert et al., 1994; Goulder et al., 1997). Thus, CTLs fail to recognize cells infected with this mutant virus. It is not clear whether the change in virus preceded or followed the accelerated fall in CD4+ counts to very low levels in these patients. It is also puzzhng that the escape should have happened so late, after 12 years in both cases. The mutant virus must have been present earlier, although it is possible that the lysine mutation has to be compensated for by one or more other mutations in the gag p24 so that a viable escape virus would be very rare.

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Other immune responses may have contributed to the control of HIV in these patients so that the virus could only escape from the CTLs when these responses failed; a contribution from Thl T cells that failed when the CD4' count fell to very low levels might also explain the findings. It is possible that a very strong CTL response might control the lysine escape virus if the epitope is briefly exposed at the surface of infected cells so that the virus would only escape when the CTL response fell below a threshold. There are several ways by which a mutant epitope might escape recognition by CTLs. These can be mapped according to the step in the class antigen processing pathway that is affected (Koup, 1994). Mutation in the regions flanking epitopes might interfere with selection of the epitope by the cytoplasmic proteases (Couillin et al., 1994). Currently, there is little understanding of what amino acid sequences might be involved; the proteases select the C-terminal residue of the peptide (reviewed in Elliott et al., 1993) so alterations here could affect their action, as well as affecting HLA binding if the peptide is generated. Cerundolo et al. (1991) showed that presentation of an influenza virus epitope in NP by HLA A"6801 was abrogated by changes outside the epitope. Couillin et al. (1994) have described a patient with HLA-A11 who failed to respond to the expected dominant epitope in HIV nef; the epitope sequence itself was unaltered but there were differences in the flanking regions, which might have interfered with processing. This type of escape is hard to identifybut could be common. Some mutant peptides might not be transported into the ER; for instance, prolines at the second and third positions of the transported peptide seem to interfere with transport by TAP (Neefjes et al., 1993). The clearest escape mutations are those where the altered peptide fails to bind to the presenting HLA molecule; there are now several examples in which complete escape to fixation occurs (Borrow et al., 1997; Nowak et al., 1995; Price et al., 1996; Goulder et al., 1997); for nef in particular, deletions that remove the whole epitope are described (Koeniget al., 1995; Price et al., 1996), leaving the virus apparently stdl viable and virulent. Mutations that affect interactions with the TCR are the most common in a given epitope (Reid et al., 1996). Such changes, which need not necessarilybe in residues in direct contract with the TCR, are likely to affect only some clones (McAdam et al., 1995). However, the T cell response is often oligoclonal (Kalams et al., 1994; Moss et al., 1991, 1995), so it may be susceptible to this type of change; one possible consequence might be a diversification of the T cell response. Apart from nonrecognition, antagonism is also possible (Klenerman et al., 1994, 1995; Meier et al., 1995). The altered peptide ligand has a partial interaction with the TCR and prevents the CTLs from lysing cells infected with wild-type virus.

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Cytokine or chemokine release may also be affected so that CTLs fail to inhibit virus replication (P. Klenerman, unpublished results). Meier et al. (1995) have shown that such effects can occur when the antagonist and agonist are on different cells. However, inhibition of CTL lysis of cells infected with wild-type virus was seen only when the antagonist cells were in excess. Thus, mutant virus may gain an advantage at a focal site until they are in local excess, then the response to the wild-type virus is also impaired so that the mutant may not reach fixation. Also, the wild-type virus may act as an antagonist for the new CTL response to the mutant virus, impairing CTL responses to the new variant. The descriptions of escape mutants underline the importance of CTLs in controlling the virus. They further suggest that lysis is an important in vivo mechanism for control of thls virus infection; escape is very simple if the alternative is death. CD8+ T cells that release chemokines could also select escape mutants, but if a mutant virus-infected cell was next to a cell infected with wild-type virus, virus released from both might be equally inhibited from infecting new cells. However, this could be counteracted by antagonism. Now the most important issue to resolve is the contribution of escape mutation to the progression of HIV infection to AIDS. In one model (Nowak and McMichael, 1995), escape as seen in the two acutely infected patients described previously would be typical and would be the first of a continuing series of escape mutations changing the response to different epitopes in a hierarchy of imunodominance. If the CTL responses faded to eliminate virus variants completely, the result would be a broadening of CTL specificity with shifting patterns of immunodominance constantly altering predominant virus variants (Nowak et al., 1995). In a few patients the HLA type may fortuitously select parts of the virus that do not vary very much because the sequence cannot change significantly without impairing the structure and function of the protein. In the previous example, HLA-B27 dominantly selects a conserved epitope in which escape occurs late, for reasons that are as yet unclear. In this model the CTL response gradually becomes more heterogeneous in response to successive rounds of virus escape and this results in a gradually increasing virus load. If the immunodominance hierarchy is inversely related to the amount of virus antigen needed to maintain a virus-suppressive response, this would be inevitable. Higher virus loads would hasten the decline of CD4+ T cells. A point might be reached when the impaired CD4+ T cell activity fails to maintain CTL function and immune control would collapse (Nowak and McMichael, 1995). The alternative view is that, although escape cannot be denied, it is rare and not a major contributor to the development of AIDS (Meyerhans et

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al., 1991). Thus, in the patients with HLA-B27, AIDS had already developed before escape occurred and so had little to do with the decline in CD4' T cells. However, the strongest proponent of this view argues that the CTLs are actually responsible for the loss of CD4+ T cells (Cheynier et al., 1994). If this is so then there must be pressure on the virus to escape. The jury is still out in this case and further longitudinal studies on the relationship between immunodominant CTL responses and the escape variants should resolve the argument. IX. Therapeutic Implications of the Importance of HN-Specific CTLs

A. ADOPTIVEIMMUNOTHERAPY The adoptive transfer of autologous class I MHC-restricted CTL clones or lines to patients infected with HIV has been based on the success of this therapy in the control of other viral infections in humans (Riddell and Greenberg, 1995b). Apart from the obvious intention to treat patients, this type of study directly addresses the role of CTLs in HIV infection. In uncontrolled trials, donor-derived CMV-specific CTL clone infusions protected bone marrow recipients from CMV disease until reconstitution of immune responses (Riddell et al., 1992; Walter et al., 1995b). These studies also demonstrated the relative safety of lymphocyte infusions, the ability of transferred cells to survive up to 12 weeks in most patients, and the apparent dependence of infused CTLs on CD4+ lymphocytes for survival and proliferation. Infusion of donor leukocytes or donor-derived CTL lines also appears to be effective for the treatment of EBV proliferative disorders in bone marrow recipients (Papadopoulos et al., 1994; Rooney et al., 1995) and confirms the relative safety of infusions and the relative long-lived survival of infused cells. Early experiments with adoptive transfer of lymphocytes for HIV infection have been discouraging. Koenig et al. (1995) transferred large numbers (around 10" cells) of a nef-specific clone to an HIV-infected patient (CD4 count approximately400/pl) twice over a period of 14 months. As expected, the number of CD8' cells was increased posttransfusion but surprisingly a matching rise in CTL-specific lysis was not seen. This is probably because the patient still possessed high baseline activity against the nef epitope or, less likely, because the infused cells rapidly distributed to the lymphatic system with the displacement of non-nef-specific cells into the periphery. Unexpectedly, there were transient increases in virus load following both infusions and an apparent concomitant decline in CD4' cell counts. The first infusion was given with a massive dose of IL-2 that probably contributed to the virus replication by activating CD4' T cells in viva However, a similar rise in virus load was observed after the second infusion without

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IL-2. There was one direct effect of the infusion: Sequencing of viral clones from the patient revealed a selection for viruses deleting part or all of the targeted nef epitope. This implies that the infused CTLs were active against the virus but-possibly because they were monoclonal-they were ultimately ineffective. The patient subsequently died of AIDS. Riddell et al. (1996) have transferred gag-specific clones to six patients with HIV. As a safety measure cells were genetically modified to carry both the hygromycin phosphotransferase gene and the Herpes simplex virus thymidine kinase (HSV-TK)gene, which could, if required, efficiently phosphorylate gancyclovir and eliminate the transfused cells. Unfortunately, the infused cells appeared to express HSV-TK resulting in a class I HLA-restricted CTL response and elimination of the foreign cells following the second infusion given 2 weeks after the first. At least the study showed that patients with CD4+counts of around 2OOlp1 can make primary CTL responses to a novel antigen. Data on gag-specific CTL activity or virus loads after the first infusion were not published. From the limited data currently available, therefore, adoptive transfer of CTLs does not appear to provide significant benefit. Administration of two or more clones may decrease the likelihood that viral mutants arise and large doses of simultaneous IL-2 may be harmful. This raises the problem of survival of the infused CTLs in the recipient; data from CMVspecific CTL transfer shows that they last longer in the presence of Th activity. This is unlikely to be present in recipients with low CD4+ T cell counts, the group with low CTL activity who might benefit from the transfer. The success or failure of CTL infusions will depend on the critical mechanisms of viral control. Comparison of adoptive transfer between HIV and other non-HIV viral infections may not be valid. In the case of CMV or EBV, cellular immunity, which normally controls latent virus, is absent because of bone marrow ablation and CTL infusions replace this function until host immunity is restored. In the case of HIV, host immunity is rarely if ever restored and the primary pathology is of the immune system. Thus, long-lasting effects will be needed, making the role of accessory cells and factors critical. Other mechanisms of viral control may also render CTL infusion therapy ineffective. For example, if a population of CD8+CD28- cells are primarily selected by ex vim expansion methods, these cells when infused may be susceptible to apoptosis in vivo or fail to function adequately (Levine et al., 1996). Moreover, there may remain a pool of latently infected cells that are sequestered from CTL lysis (Chun et al., 1995) or that express viral antigens defectively (Tsomides et al., 1994). In these cases CTL infusions are unlikely to stem the course of disease. Finally, the mechanism by which CTLs inhibit viral replication may represent a combination of

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antigen-triggered CTL lysis as well as antigen-triggered chemokine secretion. Therefore, it may be necessary to determine whether antigen-specific clones are those that produce inhibitory chemokines. Immune reconstitution of HIV-infected patients by adoptive transfer of syngeneic whole lymphocytes or fractionated class I1 MHC-restricted CD4' lymphocytes has also been attempted. In a report from Bex et al. (1994), an uninfected twin was first vaccinated with recombinant envelope protein from HIV and, followingdemonstration of envelope-specific CTLs, his peripheral blood lymphocyte population was obtained by leukopheresis and transfused into the infected twin who had an undetectable CD4+count. No changes in the clinical, immunological, or lymphocyte parameters of the recipient were noted following the first infusion. A second infusion was performed using lymphocytes preincubated with HIV gp160. After the second transfer there was a increase in total lymphocyte counts (both CD4' and CD8+ cells) and an improvement in functional proliferation assays. However, there was also an increase in viral load following both transfer. The infected twin subsequently died of unrelated causes. In a similar infusion of lymphocytes obtained by leukopheresis from an uninfected identical twin to his HIV-infected sibling,we have demonstrated partial changes in the TCR repertoire of CD8' cells and a temporary in vivo expansion of CD4+and CD8+ cells up to 4 weeks after transfer (R. Tan and S. Rowland-Jones, unpublished observations). In a study from Lane et al. (1990), 16 HIV seropositive twins were treated with zidovudine combined with six infusions of peripheral blood lymphocytes obtained from the seronegative twin and bone marrow transplantation at the end of the study. After lymphocyte infusions, there was an increase in the percentage of CD4+cells present and an increase in the number of patients with delayed-type hypersensitivity reaction to tetanus toxoid. However, there was no change in the clinical status of the patients and the immunologic changes were transient. A second study that has recently begun is an ongoing phase I/II trial designed to examine the safety and efficacy of repeated infusions of activated, ex vivo expanded syngeneicT lymphocytes in HIV-infected twins. Some data on five patients have been collected. In one patient there was a marked rise in plasma virus coincident with each infusion. No further data are available. Restoration of T cell repertoire may be possible but this has yet to be demonstrated. It seems likely that transfer of activated CD4' lymphocytes would result in the infection of infused cells and a subsequent rise in viral load. Direct transfer of lymphocytes without IL-2 or anti-CD3 activation may represent a better alternative. Overall, immunotherapy, either of anti-HIV CTL clones or more general attempts to restore the immune system, is currently unproven, expensive, and laborious to carry out. Currently, it cannot compete with the promising

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reductions in virus load achieved by the newer antiretroviral drugs. However, it is quite likely that, although the drugs may control the virus in the short or medium term, there could be a place for immunotherapy in attempting to repair greatly damaged immune systems to facilitate longer term management of patients.

B. VACCINESTHAT INDUCE A CELLULAR RESPONSE The search for an effective vaccine for HIV has progressed on an empirical basis because the protective immunological correlates of infection remain unknown. It is assumed, however, that host immune response to infection (in addition to host genetic and viral factors) is at least partly responsible for the varying rates of progression to AIDS and the phenomena of both exposed and uninfected patients and long-term nonprogressors (Haynes et a!., 1996). Neutralizing antibodies are readily produced following infection and most appear to be directed toward portions of the envelope protein (Barin et al., 1985; Putney et al., 1986; Steimer et al., 1991; Von Gegerfelt et al., 1991; Weiss et al., 1985). Furthermore, antibodies to gp120 have been correlated with a decreased incidence of maternofetal transmission of HIV in some studies (Devash et al., 1990; Goedert et al., 1989). Unfortunately, the expression of new HIV variants during the long time course of natural infection results in relatively rapid loss of neutralizing activity (Albert et al., 1990; Groopman et al., 1987; Montefiori et al., 1991; Moore et al., 1994; Nara et al., 1990; Reitz et al., 1988). Moreover, a strong correlation between neutrahzing antibody activity and progression of disease has not been demonstrated. Similarly, vaccine-induced antibody responses alone do not appear sufficient to protect monkeys or humans from HIV infection, and in some cases antibody responses may even enhance virus replication (Mascola et al., 1993). It therefore appears unreasonable to assume that even a large and broad antibody response to the envelope region could provide the sterilizing immunity that is thought to be required to prevent host infection and progression to disease (Graham and Wright, 1995; Hilleman, 1995). However, a recent study in which macaques were protected from SIV challenge by a recombinant vaccinia expressing SIV env or gag/ pol showed that the protected animals developed CTL to SIV antigens in the challenge but not the vaccine preparation, suggesting that sufficient infection had occurred on challenge to elicit these new responses (Kent et al., 1996).Thus, sterilizing immunity may not be essential for protection. Given the increasing evidence that the very strong CTL response in the primary phase of infection plays a role in reducing the very high initial viremia, it is reasonable to ask whether prior induction of this response by vaccines could terminate an HIV infection or contain it more effectively.

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Recent vaccine attempts have therefore focused on generating CTL responses. Even if there are low levels of circulating effector CTLs when HIV exposure occurs, good levels of memory cells should ensure a stronger and more rapid CTL response. This may not necessarily produce sterilizing immunity but could result in lower virus loads and better long-term survival. In vivo virushost systems for investigating HIV vaccines have included higher primates (SIV/cynomolgusand rhesus macaques and HIVkhimpanzees) and humans (live viral vector or subunit preparations). Most primate studies have been hampered by small numbers of vaccinees and inconsistent results. In addition, a potentially important difference between these models is the relative pathogenicity of each virus in a given system (Hoth et al., 1994). SIV produces relatively rapid disease in monkeys and HIV generally produces an indolent, nonfatal infection in chimpanzees (Fultzet d,1986);the pathogenic course of HIV in humans falls between these two models. The best evidence to date that a retroviral vaccine could provide protective immunity comes from the use of an attenuated strain of SIV. Macaques immunized with a nef-deficient virus were able to resist subsequent challenge with large doses of wild-type (heterologous) virus (Almondet al., 1995;Danieletal., 1992).The basis ofprotection has been shown not to involve antibodies ( J. Stott, personal communication), so cellular immune responses, which are vigorous in animals infected with the attenuated virus (Gallimore et al., 1995),are candidates, but by default. The attenuatedvirus itselfis an unlikely vaccine candidate because nef-deficient SIV appears to be highly pathogenic to neonatal rhesus monkeys despite its apparent safety in adults (Baba et al., 1995),raising doubts as to its suitability for human trials. Although the mechanism has not been defined, it suggeststhat an immature or incomplete immune system may be at risk from attenuated viruses. Attenuated HIV vaccines are unlikely to be tested in humans because of these concerns. A natural experiment exists, however, in the case of a nef-deficient virus that was isolated from a long-survivingAustralian cohort of patients and that appeared to lack pathogenicity (Deacon et al., 1995),although it has been argued that this may not have been the case with one of the patients who died from Pneumocystis carinii infection (Ruprecht et al., 1996). In an earlier series of experiments inactivated whole SIV also conferred protection to macaques (Carlson et nl., 1990; Desrosiers et al., 1989; Murphey-Corb et al., 1989; Stott et al., 1990)but only under highly specific conditions in which both infectious challenge and vaccine virus were grown in human rather than monkey cell lines. The mechanism of this protection now appears to be mediated through neutralizing antibodies generated against class I and class I1 molecules that were incorporated into the viral envelope during virus culture (Stott et al., 1991). A correlation between CTL responses and protection from SIV has been lacking in most studies. However, Gallimore et al. (1995) have shown a

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link between high precursor levels of vaccinia nef-induced CTLs and protection from intravenous challenge of SIV. Gallimore et al. made the point that to achieve protection, vaccines had to induce high levels of memory CTLs, >1 in lo4PBMC, which is not often attained by currently available vaccine candidates. Therefore, more studies of this type are needed. Subunit vaccines have also had limited success, restricted primarily to protecting monkeys from homologous challenge with SIV (Hu et al., 1992). One problem with recombinant vaccines is that the uptake of peptide or protein by endocytosis biases cells toward a class 11-mediated response with the production of CD4' CTLs (Orentas et al., 1990). The role of these cells in natural protection is unknown. However, evidence that a subunit vaccine could produce a CD8+restricted CTL response in humans was presented by Hammond et al. (1992), who vaccinated seronegative volunteers with a vaccinia-env construct followed by a booster consisting of soluble env protein. Class I-restricted envelope-specific lysis was subsequently detected in two patients. Other modified live-virus vector vaccines in trial include vaccinia-env constructs with or without gag p24 (Hu et al., 1993; Perales et al., 1995),canary avipox vectors (Pialow et al., 1995) expressingenv and gag proteins, and similar constructs in BCG (Yasutomiet al., 1993a).In a phase 1study of recombinant canary pox vectors expressing gp160, 7 of 18 vaccinated volunteers developed some envelope-specific CTLs, the majority of which exhibited a CD3'CDB' phenotype (Pialow et al., 1995). Recently, SIV-specific memory CTL responses were found in monkeys vaccinated with a DNA vaccine encoding SIV env and gag (Johnson et al., 1992; Yasutomi et al., 1996),but, as with previous studies, these responses were not associated with protection from disease (Yasutomi et al., 1996).These viral vector and DNA vaccines may provide the balance required between the potential efficacy of attenuated vaccines and their potential danger. X. Conclusions

The importance of the CTL response in controlling HIV infection rests on six lines of evidence. The temporal inverse relationship between CTL responses and virus loads is compelling but indirect. Similarly, the demonstration of very strong CTL responses in infected patients and their presence at the sites of infection (Cheynier et al., 1994) is very suggestive of an important role. The evidence that CTLs can inhibit virus replication in vitro is solid and rests on both cytotoxic mechanisms and the potent effects of the chemokines and other secreted factors, but the studies are in vitro or at best ex uivo. Selection of escape mutants in instances in

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which the CTL response focuses on a single epitope and the subsequent fixation of those variants is very strong evidence, but how far can it be generalized? Our own view is that this is a very important component of the whole pathogenesis of HIV infection but this still needs more proof. Vaccine induction of strong CTL responses is now possible, e.g., by DNA vaccines, and this should allow direct testing of the proposal that CTLs alone can control SIV in macaques and, by implication, HIV in humans. Further studies in the highly exposed resistant cohorts, a few of whom have already revealed the importance of the CCR5 receptor in primary infection, could cement the hypothesis that at least some of them are protected by their cellular immune response. Together these arguments make a strong case for the CTL response being one of the major elements in understanding HIV infection and AIDS pathogenesis. There are already initiatives to develop vaccines that elicit CTL responses and some early human trials are in progress. There remains, however, a requirement to probe and test the hypotheses proposed in this review. There could still be some surprises.

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Yamamoto, H., Miller, M. D., Tsubota, H., Watkins, D. I., Mazzara, G. P., Stallard, V., Panicali, D. L., Aldovini, A., Young, R. A., and Letvin, N. L. (1990). Studies of cloned simian immunodeficiency virus-specific T lymphocytes. gag-Specific cytotoxic T lymphocytes exhibit a restricted epitope specificity. ] Immunol. 144(9),3385-3391. Yang, O., Kalams, S., Rosenweig, M., Trocha, A., Jones, N., Koziel, M., Walker, B. D., and Johnson, R. P. (1996).Efficient lysis of HIV-1-infected cells by cytotoxic T lymphocytes. J. Vtrol. 70, 5799-5806. Yasutomi, Y., Koenig, S.,Haun, S. S., Stover, C. K., Jackson, R. K., Conard, P., Conley, A. J.,Emini, E. A., Fuerst, T. R., and Letvin, N. L. (1993a).Immunization with recombinant BCG-SIV elicits SIV-specific cytotoxic T lymphocytes in rhesus monkeys. 1. Immunol. 150(7), 3101-3107. Yasutomi, Y.,Reimann, K. A., Lord, C. I., Miller, M. D., and Letvin, N. L. (1993b).Simian immunodeficiency virus-specific CD8+ lymphocyte response in acutely infected rhesus monkeys. I. Virol. 67(3),1707-1711. Yasutomi, Y., Robinson, H. L., Lu, S., Mustafa, F., Lekutis, C., Arthos, J., Mullins, J. I., Voss, G., Manson, K., Wyand, M., and Letvin, N. L. (1996). Simian immunodeficiency virus-specific cytotoxic T-lymphocyte induction through DNA vaccination of rhesus monkeys. 1.Virol. 70(l),678-681. Young, J. W., and Steinman, R. M. (1990).Dendritic cells stimulate primary human cytolytic lymphocyteresponses in the absence of CD4+ helperT cells.]. Exp.Med. 171(4),1315-1332. Young, L., Alfieri, C., Henessey, K., Evans, H., OHara, C., Anderson, K. C., Ritz, J., Shapiro, R. S., Rickinson, A.. Kieff, E., and Cohen, J. I. (1989).Expression of Epstein-Barr virus transformation associated genes in tissues of patients with EBV lymphoproliferative disease. N . Engl. I. Med. 321, 1080. Zarling, J. M., Ledbetter, J. A., Sias, J., Fultz, P., Eichberg, J., Gjerset, G., and Moran, P. A. (1990). HIV-infected humans, but not chimpanzees, have circulating cytotoxic T lymphocytes that lyse uninfected CD4+ cells. 1.Immunol. 144(8), 2992-2998. Zhang, L. Q., MacKenzie, P., Cleland, A., Holmes, E. C., Leigh Brown, A. J., and Simmonds, P. (1993). Selection for specific sequences in the external envelope protein of HIV-1 upon primary infection.1.Virol. 67, 3345-3356. Zhu, T., Mo, H., Wang, N., Nam, D. S., Cao, Y., Koup, R. A., and Ho, D. D. (1993). Genotypic and phenotypic characterization of HIV-1 in patients with primary infection. Science. 261, 1179-1181. Zinkemagel, R. (1995). Are HIV-specific CTL responses salutary or pathogenic? Curt-. Opin. Immunol. 7,462-470. Zinkemagel, R. M. (1996).Immunology taught by viruses. Science 271(5246), 173-178. Zinkemagel, R. M., and Doherty, P. C. (1974). Restriction of an in vitro T cell-mediated cytotoxicity in lymphocytic choriomeningitiswithin asyngeneic or semiallogeneicsystem. Nature 248,701-702. Zinkemagel, R. M., and Doherty, P. C. (1975). H-2 compatibility requirement for T-cell mediated lysis of target cells infected with lymphocytic choriomeningitis virus. 1. Exp. Med. 141, 1427-1436. Zinkemagel, R. M., Haenseler, E., Leist, T., Cemy, A., Hengartner, H., and Althage, A. (1986). T cell-mediated hepatitis in mice infected with lymphocytic choriomeningitis virus. Liver cell destruction by H-2 class I-restricted virus-specific cytotoxic T cells as a physiological correlate of the 51Cr-release assay?]. Exp. Med. 164(4), 1075-1092. Zweerink, H. J.. Coutneidge, S. A., Skehel, J. J., Crumpton, M. J., and Askonas, B. A. (1977).Cytotoxic T cells kill influenza virus infected cells but do not distinguish between serologically distinct type A viruses. Nature 267, 354-356. This article was accepted for publication on 31 October 1996.

ADVANCES IN IMMUNOLOGY, VOL. 65

High Endothelial Venules: Lymphocyte Traffic Conhl and Controlled Traffic

1. lnhoduction

Lymphocytes continuously recirculate from blood to lymphoid organs and back to the blood via the lymphatics. This way the body allows the immune system to effectively survey all parts of the body in search of foreign antigens or altered body cells. In this process, high endothelial venules are crucial because they are the sites in lymphoid tissue where lymphocytes are able to leave the bloodstream and enter the tissue. High endothelial venules (HEVs) in lymphatic tissues have been recognized and described for almost a century. In 1898 Thome (1) described them for the first time as specialized microvascular structures in the lymph node of the macaque. Interestingly, they were regarded as phagocytic structures based on the presence of numerous lymphocytes in the wall of these vessels. No doubt this assumption was strongly influenced by the then recent discoveries and descriptions of phagocytic cells by Metchnikoff and his ardent advocacy that phagocytes were central cells in the body’s defense against infectious diseases. Despite of Metchnikoff s zealous attempts immunologists at the beginning of the 20th century turned away from the cellular concepts of immunology and devoted their time to the study of noncellular components of the immune system. Also, although over the years high endothelial venules were described and even suggestions for a function in lymphocyte exchange were proposed (2-5), it was only in the 1960s, coinciding with a renewed interest in the cellular concepts of the immune system, that the precise function of the high endothelial venule was revealed. In the classic paper of Sir James Gowans (6)it was described for the first time, in an experimental setting using radiolabeled cells, that lymphocytes are able to leave the bloodstream via the high endothelial venules in lymphoid organs. Since then, high endothelial venuIes and the processes of lymphocyte recirculation and the associated adhesion phenomena have become a central focus of interest in immunology. It has given us insight into the concepts of immune surveillance and in the events of cell adhesion and extravasation as seen under steady-state conditions in high endothelial venules, but also at sites of inflammation. The discovery and description of adhesion molecules and adhesion cascades has led to 347

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fascinatingideas on cellular interaction and cross-talk and may prove useful in the design of therapeutic interventions based on adhesive interactions. In this review we will focus on the structure and function of the high endothelial venule as it is found in lymphoid structures. Its unique features in comparison with venules at sites of inflammation will be described and emphasis will be put on its role in immunological surveillance and the way its function is regulated by the surrounding tissue. It. Structure of High Endothelial Venules

HEVs are typically composed of an endothelial wall, a thick basement membrane, and a prominent perivascular sheath. They can be identified by the presence of large numbers of lymphocytes in the lumen and within the various layers of the wall. HEVs are present in lymph nodes, tonsils, Peyer’s patches, appendix, bronchus, and nasal-associated lymphoid tissue (7).They are absent as such from thymus and spleen. In rodents, which have a relative immature immune system at birth, the development of HEVs takes place during the neonatal period, coinciding with the development and population of lympoid organs. In man the development of the immune system and the occurrence of HEVs is an earlier event, taking place before birth (8). Their location in the lymphoid organs seems to be restricted to the T cell-dependent areas, such as the paracortical areas and interfollicular regions of lymph nodes. They cannot be found in germinal centers or medullary regions (9, 10). In lymph nodes capillary networks are located in the follicular regions. The postcapillary venules therefore start in this region, but it is only after intervening segments of flat-walled venules have reached the paracortex that the HEVs develop. In most mammals HEVs are composed of modified endothelial cells with a cuboidal or columnar morphology. They are differentiated from the normal, flat endothelial cells that line all other vessels. Although in most species high endothelial venules are characterized by their plump appearance, this is not an absolute prerequisite because in sheep no high endothelid venules can be found at all (ll),and in other species the “highness” may vary considerably. Nevertheless, the functional capacity to sustain lymphocyte transmigration in lymphoid organs can be demonstrated. The plumpness of the endothelial cells, often described as necessary to prevent leakage of fluids during the transmigration of lymphocytes, may therefore be a secondary phenomenon, resulting from the activation of the cells. Typically, endothelial cells of HEVs have abundant cytoplasm, pale nuclei and a prominent, dense nucleus, and enhanced nonspecific esterase activity (12-15). The cells show signs of active biosynthesis and secretion

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not found in normal, flat-walled endothelia. Unique for these cells, as described in earlier studies, was their selective incorporation of sulfate (15-17), which was later found to be an essential feature of their adhesion molecules (see below). One obvious advantage of the cuboidal structure is the fact that the more irregular lining of high endothelial venules will result in increased turbulence of the passing bloodstream, leading to enhanced chances of lymphocytes to bump onto the endothelium and start the adherence and transmigration process. Much of our knowledge on the structure of HEVs and the accompanying connective tissue in relationship with the structure of the lymph node has come from the work of Anderson and Anderson in their detailed studies on rat HEVs (12, 18).Using histochemical and ultrastructural techniques they described the remarkable structure of the rat HEVs. The endothelial lining is ensheathed by several layers of reticular cell plates and connective tissue. In turn, these plates are linked to the reticular meshwork of the lymph node by collagen fibers and anchoring filaments. These filaments are inserted into the external limiting membrane of the plate (Fig. 1). It was suggested that these individual plates are able to move with respect to each other, thereby limiting or increasing the space between them. Tissue or intravascular pressure could be a determining factor in this

FIG.1. Schematic representation of a high endothelial venule. Reconstruction drawing based on the original drawing by B. Gould [from Ref. (IS)], showing the cuboidal endothelium and sequential stages of adhering and transmigrating lymphocytes. The endothelial lining is surrounded by a multilaminated reticular sheath formed by reticular cells and anchored to the reticulum of the lymph node by collagen fibers.

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process. Extravasating lymphocytes have to infiltrate between these plates, thereby minimizing vascular leakage. In addition, adjacent endothelial cells seem to overlap in such a way that gaps between the cells are closed by virtue of the blood flow. This seal could effectively prevent intraluminal tracers from reaching the basement membrane, although small amounts of tracer can be found accompanying transmigrating lymphocytes. 111. Role of HEVs and Lymphocyte Migration

The migration of lymphocytes through the body is an essential element of the immune system, whereby the various lymphoid organs are connected with each other. In this process high endothelial venules play a crucial and unique role. This is obvious when one determines the rate of lymphocyte migration in lymphoid organs compared to nonlymphoid organs. In a lymph node it is estimated that one of four lymphocytes passing through the node in the bloodstream will migrate into the node. Only in lymphoid tissue does such a large-scale migration take place (19-22). That this migration is part of a larger recirculation scheme that is now known to be so essential for the functioning of the immune system was inferred from the observations of Gowans (6, 9, 23-25). Intrigued by the “mystery of the disappearing lymphocytes,” Gowans set out to study the observation that from the lymphatic ducts lymphocytes enter the blood in such quantities as to replace all those in the blood 10 times daily. He showed that when in rats the thoracic duct was cannulated over a longer time, this would lead to a severe depletion of lymphocytes entering the lymph. This reduction in cell number could be prevented by reinfusion of the thoracic duct lymphocytes intravenously. Furthermore, it was shown that the cells that entered the thoracic duct were not newly formed and that when these cells were radiolabeled and reinfused into the animal they reappeared in the lymph as well as in the lymphoid organs. In this way the principle of lymphocyte recirculation was established. Now it is well recognized that large numbers of especially naive B and T cells continuously migrate from blood into lymph and back and that in this process all HEVs bearing lymphoid organs are engaged. As expected from the absence of HEVs, the thymus is not involved, and also the involvement of the bone marrow is limited and seems to be restricted to one-way traffic. In contrast, the spleen is an organ with major involvement in lymphocyte recirculation, although no HEVs exist in this organ. From the early studies on lymphocyte recirculation it became apparent that this process of going back and forth between organs did not lead to a uniform distribution of lymphocytes in lymph or blood. From studies in sheep, which are big enough to cannulate individual lymph

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nodes, it was learned that the composition of cells entering the efferent lymph depended on the organs studied. In fact, three main populations of cells were discriminated: lymphocytes from intestinal lymph, which had a preference to recirculate through the intestines, lymphocytes from efferent lymph from peripheral lymph nodes, with a preference for peripheral sites; and cells from sites of inflammation, which seemed to have an increased tendency to circulate through inflamed tissue (26-29). IV. In viho HEV Binding Assay

It became apparent that lymphocytes were not recirculating randomly, but showed specific lodging, which seemed to be determined by the site they had visited before. Studies to determine the mechanisms underlying this specificitywere now set in motion. A fortuitous finding that has helped enormously to elucidate the function of HEVs was the fact that the adherence of lymphocytes to HEVs could be mimicked in vitro. This was originally reported by Stamper and Woodruff (30, 31) for rat HEVs in lymph nodes and could be observed in many species. This in essence extremely simple method, using frozen sections from lymphoid tissues, allows one to look at one important step in the recirculation process, the adherence to HEVs, and many basic questions as to what cells would adhere to which HEVs and what the requirements of this adhesion were could now be resolved. By comparing binding capacities of various cell types to HEVs in different organs, it was found that the dual specificity reported for lymphocytes entering lymphoid organs correlated completely with the binding specificity of HEVs. In other words, other cells would bind to HEVs in peripheral lymphoid organs than to HEVs in, e.g., Peyer’s patches. T lymphocytes would show a preference to adhere to HEVs in peripheral lymph nodes, B lymphocytes would adhere more prominently to Peyer’s patch HEVs, and adherence to mesenteric lymph node HEVs showed an intermediate pattern. Furthermore, it was found that certain lymphomas would bind with almost absolute specificity to one type of HEV and not to the other (32,33).From these studies a genetic polymorphism controlling the extent with which HEVs were recognized by adhering cells was detected (34). That the adherence of lymphocytes reflected the actual immigration into the tissue was demonstrated by comparing the adherence ratios of different lymphocyte mixtures on HEVs in vitro with the actual immigration of such mixtures after in vivo transfer. It was found that binding to the HEVs must have been the determining step in the observed selectivityof immigration (35, 36).

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These experiments clearly showed the importance of HEV-lymphocyte interactions in the dissemination of lymphocytes through the body and opened the way to study the molecular interactions involved in these processes. In addition, when it was realized that lymphocyte homing into lymphoid tissue and also into sites of inflammation was determined at the level of leukocyte entry, it led to investigations into directing leukocyte traffic for therapeutic applications. V. Molecules Determining HEV-Lymphocyte Interactions

From studies into the nature of the receptors and ligands involved in the interaction of lymphocytes and endothelial cells of the high endothelial venules it has become apparent that members of several families of adhesion molecules are involved. These include the selectins of which the L selectin is the major contributant. Ligands for the L selectin are found within a variety of molecules, all characterized by the expression of carbohydrate structures. Within the integrins, the LFA-1 (CDlldCDlS)is important for general adhesion of the lymphocytes, interacting with members of the Ig superfamily (Fig.2). In addition, it has been found that the a4p7 integrin dimer is important in conferring specific adhesion to HEVs by interacting with a special member of the Ig superfamily, mucosal addressin cell adhesion molecule-1 (MAdCAM-1). In the next sections the identification and characteristics of the various molecules, both on high endothelial cells and on lymphocytes, will be described. Next, their role in the physiology of the lymphocyte-HEV interaction will be specified. VI. 1Selectin

With the aid of the in vitro binding assay the search was started to identify molecules on the surface of lymphocytes that would govern the interactions with HEVs. By developing monoclonal antibodies against the 38C-13 lymphoma, showing a strong preference to bind to peripheral lymph node HEVs but not to Peyer’s patch HEVs, the MEL-14 antibody was generated, recognizing a 90-kDa glycoprotein, which was able to interfere with the binding of lymphocytes to HEVs in a specific way (37). Lymphocytes pretreated with the antibody were incapable of binding to peripheral node HEVs but showed unaltered binding to Peyer’s patch HEVs. The expression of MEL-14 on the surface of lymphocyte populations completely correlated with the cells’ ability to bind to HEVs of peripheral lymph nodes as demonstrated for thymocytes, germinal center cells, and a series of lymphomas (36-41). Cloning of the molecule and

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FIG.2. Scanning electron microscopic images of rat HEVs. (A) The luminal side of the HEVs is shown with an adhering and transmigrating lymphocyte (arrows) (X4300). (B) At higher magnification, the attachment of a lymphocyte (Ly) with a microvillus (arrow) to the microfolds of the endothelial HEV cell is shown. The white dots on the endothelid microfolds are gold particles indicating the presence of ICAM-1 (X105,OOO)[microphotographs by Dr. K. Sasaki, from Ref. 263. Sasaki, K., Okouchi, Y., Rothkotter, H. J., and Pabst, R. (1996). Ultrastructural localization of the intercellular adhesion molecules (ICAM1) on the cell surface of high endothelial venules in lymph nodes. Anat. Rec. 244, 105. Copyright 0 1996 Wiley-Liss, Inc. Reprinted by permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.].

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further structure analysis revealed that the MEL-14 antibody recognized a unique transmembrane molecule composed of a carbohydrate-recognizing domain (CRD) at its extracellular amino terminus, an epidermal growth factor-like domain, and two exact repeats of a complement regulatory domain, making up the remainder of the extracellular portion (42-45). A. STRUCTURE AND FUNCTION OF L SELECTIN It is now well established that the molecule recognized by the MEL14 antibody is a member of a group of proteins, the selectins (46, 47). Within the selectins three members can be discerned; L selectin (CD62L), E selectin (CD62E), and P selectin (CD62P). All three molecules consist of the typical carbohydrate binding site, an epidermal growth factor domain, and two (L selectin), six (E selectin), or eight or nine (P selectin) complement regulatory domains. There is a close homology of 60-70% between the three molecules in the CRD and epidermal growth factor domain. Furthermore, their genes are closely linked on chromosome 1, in close evolutionary relationship with complement-receptor genes. All three molecules are specifically involved in recognition events between endothelium and leukocytes. L selectin is expressed on lymphocytes and myeloid cells, such as granulocytes and monocytes, whereas E selectin is expressed on activated endothelial cells. P selectin was originally described on platelets but is present on activated endothelium as well (46, 47). Selectins as a group are included in the C-type CRD superfamily (48, 49). This superfamily is divided into six main groups on the basis of amino acid sequence comparisons of their carbohydrate-recognition domains, and the members are involved in a wide range of functions and include groups of molecules such as collectins, proteoglycans, and macrophage mannose receptors. Within the groups the proteins all have the same overall structure. The general property to combine them in a superfamily lies in the CRDs, which have a characteristic consensus sequence, largely based on hydrophobic residues. The CRD is able to interact with carbohydrate structures in a Ca2+-dependentmanner. Although this CRD lectin domain is important for the ligand recognition and interaction, the EGF domain is also involved in binding. In the mouse it was demonstrated that an antibody against the Ly-22 surface marker recognized the EGF domain of L selectin (50), and that binding of lymphocytes to HEVs could be inhibited by the antibody. Also, for P selectin a role of the EGF domain in cell adhesion has been observed: By creating chimeric selectin molecules consisting of exchanged domains between P and L selectin it was demonstrated that the CRD lectin domain was conferring the ligand recognition function. However, chimeric molecules composed of the L selectin CRD domain and the P selectin EGF domain showed dual binding specificity.

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It is therefore assumed that the combination of the two most extracellular domains forms a functional recognition unit (51). With relation to the interaction with HEVs of the selectins, only L selectin is of major importance (Fig. 3). E and P selectin can be found on endothelium in lymph nodes but only under inflammatory conditions (52,53), and it has to be assumed that under normal noninflamed situations, in HEV-bearing organs E and P selectin do not play a role in the interaction with lymphocytes. L selectin, on the other hand, is not restricted to a function in lymphoid organs. This can be inferred from its widespread distribution on leukocytes and it has become clear that L selectin also plays a role in the entry into inflammatory sites of both lymphocytes and neutrophils (53-58). Furthermore, the selectivity of the L selectin for HEVs in peripheral lymphoid organs was not found to be absolute, and involvement of L selectin in the homing into mucosal Peyer’s patches was described and was found to correlate with the expression of the ligands for L selectin on HEVs at these sites (59).

FIG.3. The expression of L selectin ligands. Cryostat sections incubated with human L selectin-IgG chimera molecules showing the expression of L selectin ligands on an HEV in a human tonsil.

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B. REGULATIONOF L SELECTIN EXPRESSION The expression of L selectin on the surface of the lymphocyte is regulated and varies depending on the activation state of the cells. The general view is that naive, not activated, cells express high levels of L selectin, corresponding with active recirculation involving HEVs. Once cells become activated they may downregulate L selectin expression in favor of other adhesion molecules. These other molecules will allow them to differentiate at certain sites, as is the case for germinal center cells, or enable the cells to enter other sites of the body such as the skin or sites of inflammation, as seen with differentiated memory cells (60-63). However, the situation of L selectin expression and memory phenotype is not entirely clear. Several reports claim that memory cells do express L selectin, probably dependent on tissue-specific activation (64-68). Interestingly, downregulation after activation may be preceded by a transient upregulation of L selectin expression on the cell surface. This was shown for human T cells stimulated by mitogens or IL-2. An increase, followed by a marked decrease of L selectin, was observed, with parallel changes in specific mRNA (69). In addition, regulation of L selectin expression can also occur at the posttranslational level. Activation of T lymphocytes by cross-linking the T cell receptor leads to changes in the affinity of L selectin on the cell surface. This was tested using phosphomannan polysaccharide (PPME), a phosphomannan that resembles the carbohydrate structure of the natural ligand of L selectin (70). This suggests that cell activation can lead to conformationalchanges in L selectin, increasing the affinity of the molecule for its ligands. Of course, the most apparent posttranslational change in L selectin expression is the active shedding of the molecule from the cell surface. Initially observed in granulocytes (71) and thought to play a role as a downregulatory mechanism of granulocyte extravasation, it has since been observed in lymphocytes as well (72-74). Shedding of L selectin can be induced after activation of the cell by phorbol esters, IL-8 (71),or activation via the T cell receptor by superantigens (75). Cross-linking of the molecule itself, without further activation of the cell (76), also leads to cleavage of the molecule. The shedding process needs the activation of protein kinase C (69,77).In human serum the soluble form of L selectin is readily detectable, and it is still functional in terms of ligand binding. Nevertheless, there is indication that the shed molecule has changed its molecular conformation (78). Attempts to link the concentration of the cleaved molecule in plasma to states of disease have been made (77-79). High levels of the shed form of L selectin have

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been found in patients with acute and chronic leukemia, which seemed to correlate with the levels of leukocytes in the blood (78, 80). Studies on the cleavage process itself have revealed that it is mediated by an unusual protease activity in that it is resistant to a variety of protease inhibitors (81). The molecule is cleaved in a short region between the second complement domain and the transmembrane domain (72). Point mutation studies in this region have indicated that the cleavage site is determined by the physical length of the site or other secondary structural characteristics rather than the strict sequence of the site (82). Although the importance of the L selectin receptor for lymphocyte homing via HEVs is well established and many aspects of its regulation have been elucidated, the precise role of the shedding process itself is not clear. The kinetics of the cleavage and shedding process, occurring very rapidly after activation of the cell, implies a regulatory role in downregulating the cell's ability to bind to endothelial cells and thus influencing its capacity to recirculate.

C. ACTIVATION AND SIGNALING VIA L SELECTIN It is evident that adhesion molecules not only function as connecting bridges between cells or cells and stromal elements but also serve as signaling molecules. This property has also been described for L selectin. Cross-linking of L selectin on the surface of neutrophils leads to increased Ca2+levels and upregulation of the oxidative burst in these cells, with concomitant tyrosine phosphorylation (83). Activation via L selectin also resulted in the upregulation of the Mac-1 p2 integrin expression on neutrophils and increased adhesion and transmigration across endothelial cells in vitro (84,85). In lymphocytes, binding of antibodies against L selectin can lead to homotypic aggregation of the cell. This process does not involve Ca2' mobilization, but activation of protein kinase C was required (86,87).This aggregation was independent of the presence of cations and could not be blocked with antibodies against integrins. Furthermore, it was found that activationvia the L selectin molecule would enhance the binding of lymphocytes to antigen presenting dendritic cells but not to macrophages (V. S. Swarte et al, manuscript in preparation). Together, it can be stated that the L selectin molecule is extremely important in the initial recognition of HEVs in peripheral lymphoid organs, that its expression on the cell surface is dependent on the activation state of the cell, thereby dictating the ability to recirculate, and that via activation signals cellular changes can occur that are important for the functioning of the cells either at transmigration or after the cell has passed the endothelial layer and enters the underlying tissues.

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VII. lntegrins and Their Role in Lymphocyte-HEV Interactions

The integrin family of cell surface receptors includes receptors for both extracellular matrix proteins as well as receptors involved in many aspects of cell-cell interactions (88). Integrins are heterodimeric transmembrane molecules composed of an a subunit and a noncovalently associated subunit. Based on differences in the p chain, different subfamilies can be discerned within the integrin family. Thus far, eight different p and 16 different a subunits have been identified (89). When we focus on the interaction of leukocytes with endothelium three major groups of integrins are important: the pl, the p2, and the P7 integrin subfamilies. In the Pl family the a 4 p l integrin is an important adhesion molecule based on its interaction with VCAM-1 on activated endothelium at sites of inflammation (90-92). Other members of the pl family are mainly involved in interaction with ligands on extracellular matrix proteins. Within the p2 (CD18) integrin subfamily four members are present that are all expressed on leukocytes and differ in the use of the a chain. These leukointegrins are critical to many immune functions. To date they include CDlldCD18 (aLp2; LFA-l), CDllb/CDl8 (aMP2; Mac-1), CDllc/ CD18 (p150,95), and CDlld/CDlS (aDP2). Of these the LFA-1 integrin (CDlldCD18)has the broadest distribution on cells of hemopoietic origin. It is involved in many cell adhesion processes via interaction with ICAM1, ICAM-2, and ICAM-3 (93). Also, in the attachment of lymphocytes to high endothelial venules LFA-1 plays a role, as was originally inferred from in vitro binding studies (94, 95). It strengthens the bond between endothelium and leukocyte after the cells have made contact by more selective mechanisms such as via selectin interaction. Therefore, LFA-1 is an important molecule for lymphocyte-HEV interaction, but it does not confer specificity to the interaction.

A. THEa4P7 INTEGRIN: THEPEYERS PATCHHOMING RECEPTOR The third subfamily of p integrins involved in lymphocyte-endothelium interactions includes one important member, a4p7, which, in contrast to the other integrin subfamilies, does confer specificity to the interaction of lymphocytes and high endothelial venules. Its identification was started in a very similar manner as described for the discovery of the L selectin molecule. The findings with L selectin confirmed the original observations made by many researchers in the field of lymphocyte traffic on the discrepancy between peripheral and mucosal homing, and could now attribute a molecular base for the phenomenon of organ-specific homing. Inspired by L selectin and its role in peripheral homing, the quest for molecules involved in mucosal homing processes was started. By raising antibodies against the TK1 T cell lymphoma, which selectively interacted with HEVs

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in mouse Peyer’s patches, a new integrin molecule was identified that was coined LPAM-1 (lymphocyte Peyer’s patch HEV adhesion molecule-1). It consisted of the a4chain linked to a new P chain (96, 97). Further analysis of the /? chain revealed that it was the murine homolog of P7 (98). In vivo and in vitro studies made it clear that the a4P7 integrin played a major role in the traffic of lymphocytes to mucosal sites via HEVs found in Peyer’s patches and mesenteric lymph nodes (99). Phenotypic analysis of lymphocytes in man and mice lend further support for the identification of a4P7 as a adhesion molecule specific for the lamina propria (100-105). In line with reports on other integrin molecules, it was found that the the a4p7 complex uses different epitopes on the molecule to bind to its respective ligands MAdCAM-1, VCAM-1, and fibronectin (102). Interestingly, the a4P7 molecule itself was able to bind to a 4 chains, thereby creating an additional adhesion mechanism that could be involved in lymphocyte interaction (106). The a 4 chain is also able to combine with the Pl chain, and this complex (VLA-4)interacts with VCAM-1 and fibronectin. VCAM-1 can be induced on endothelium after activation under the influence of cytokines but is not constitutively expressed on HEVs. Anti-VCAM-1 antibodies fail to interfere with HEV interactions (99). Although both a 4 P l and a4P7 molecules can interact with VCAM-1, on cells expressing both complexes the a 4 P l integrin seems to dominate the binding to VCAM-1 (107-109). Binding of a4P7 to fibronectin has been suggested, especially based on blocking experiments using the CS-1 peptide. This 25-amino acid peptide from the alternatively spliced fibronectin region can inhibit the a4mediated binding to fibronectin (110-112). However, based on in vivo homing experiments it was concluded that CS-1 plays a minor role, if any, in the adhesion to mucosal HEVs (99). The third ligand for a407 was identified as MAdCAM-1, which was selectively expressed on HEVs in Peyer’s patches and mesenteric lymph nodes (see below), and the organ specificity of mucosal homing is now predominantly attributed to the interaction between a4P7 and MAdCAMl(113, 114). In addition to the a 4 chain, the p 7 chain is found in combination with a unique a chain forming the aEP7. This molecule is found on several cell types, including intraepithelial lymphocytes in the gut, but is thought not to be involved in homing but rather in retention of cells (115-117). As a ligand for this complex the E-cadherin has been described (118). VIII. CD44 and Lymphocyte Homing

A role of CD44 in lymphocyte-HEV interactions was initially deducted from observations on frozen sections of human lymph nodes. Monoclonal

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antibodies raised against the putative human homolog of L selectin turned out to be directed against CD44. In the frozen section assay one of these antibodies, HERMES-3, could block the binding to HEVs in Peyer’s patches but not to HEVs in peripheral lymph nodes (119, 120). CD44 is a widely distributed glycoprotein found on most hemopoietic cells but also on many other cells and tissue components, displaying a great molecular heterogeneity due to posttranslational modification and alternative exon use. Based on molecular characteristics CD44 shows homology to proteoglycan and cartilage link proteins, all of which interact with glycosaminoglycan hyaluronate, the major constituent of the extracellular matrix (121). Although involved in many aspects of cellular interactions, ranging from metastasis to lymphopoiesis, the precise contribution of CD44 to lymphocyte homing remained unclear. From in vivo homing studies it seemed that CD44 would be more important in extravasation into sites of inflammation than in entry into lymphoid tissue (122), correlating with enhanced expression of CD44 on activated and memory/effector lymphocytes (123, 124). In vitro CD44 can mediate binding of lymphocyes to cultured endothelial cells via its ligand hyaluronate (125-127). Recently, it was found that CD44 can also function as a primary adhesion mechanism of activated lymphocytes on endothelial cells of smaller venules, whereby the cells display rolling activity which is supported by hyaluronate on the endothelium (128). Taken together, the data imply a role of CD44 in extravasation at sites of inflammation and a small, if any, role in organ-specific lymphocyteHEV interaction. IX. Homing Receptor Ligands on High Endothelial Cells

A. SUGARS AND MUCINS Concomitant but quite independent from the discovery and structural analysis of the L selectin as a sugar-binding molecule, evidence for a role of sugar moieties in the interaction of lymphocytes and HEVs was demonstrated. That carbohydrates would play a role in lymphocyte traffic had already been suggested in the early 1960s by studying the effects of glucosidases (129). Using the in vitro frozen section assay Stoolman and Rosen were able to demonstrate that the attachment of lymphocytes to HEVs in mouse peripheral lymph nodes could be inhibited by several sugars, ranging from the simple monosaccharides L-fucose, D-mannose, and mannose-6-phosphate, to the sulfated polysaccharide fucoidin and PPME (130-132). Using PPME coupled to beads, it was established that the phosphomannan interacted with the L selectin molecule on lymphocytes (133). Furthermore, experiments in which frozen sections were

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treated with neuraminidase also showed that sialic acid was an important constituent of the interaction between lymphocytesand HEV ligands (134). Interestingly, the treatment with neuraminidase affected the binding of lymphocytes to peripheral lymph node HEVs, but not Peyer’s patch HEVs, indicating that the organ specificity of the interaction could partly be defined by sialic acid. This was confirmed in vivo by injection of neuraminidase prior to lymphocyte homing assays in mice (135).These findings made it clear that the ligand structures on the endothelial cells of the HEVs should contain complex sugar structures. At the same time attempts to identify HEV-specific ligands were started by raising monoclonal antibodies. The antibody MECA79 was identified, which predominantly recognized HEVs in mouse peripheral lymph nodes and which was able to interfere with the in vivo homing of lymphocytes to peripheral nodes, but not to Peyer’s patches (136). An antibody with somewhat reciprocal distribution and function, MECA367, was simultaneously identified and could block binding to HEVs in Peyer’s patches (137). These antibodies elegantly confirmed the organ specificity of HEVlymphocyte interaction and were therefore coined vascular addressins. The search for the molecules began.

B. PERIPHERAL NODE VASCULAR ADDRESSINS 1. GZyCAM-1 Using a soluble recombinant form of mouse L selectin coupled to the Fc portion of an Ig molecule (138), putative ligands for L selectin could be precipitated from peripheral and mesenteric lymph nodes but not from Peyer’s patches. Two moieties of 50 and 90 kDa, respectively, were identified. They interacted with L selectin in a Ca-dependent and carbohydrate-inhibitableway, as would be expected, and could furthermore be precipitated by the MECA79 antibody (139, 140).The molecules were sulfated, fucosylated, and sialylated. Cloning of the smaller, 50-kDa glycoprotein (Sgp50), revealed a novel, mucin-like structure containing two serinehhreonine-richdomains, which was called GlyCAM-1(glycosylationdependent cell adhesion molecule-1) (141, 142). Based on its structure with an amphipathic helix it was predicted that the molecule could be secreted, Making use of the extremely specific sulfate incorporation of HEVs (143),it was found that the GlyCAM-1molecule was indeed secreted from HEVs and could be found in serum (144). GlyCAM-1 has been identified and cloned in mouse and rat but not in man, although indications for its presence in human HEVs have been given (140). Antisera raised against predicted peptide sequences of the GlyCAM-1 molecule confirmed its localization in HEVs of peripheral and mesenteric

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lymph nodes and its absence in Peyer’s patches. Interestingly, the antipeptide sera also revealed activity in lactating mammary glands, confirmed by mRNA detection and genomic analysis (145-147). However, in milk the GlyCAM-1 molecule lacks the sulfate-modified carbohydrate required for its interaction with L selectin. It is therefore assumed that the GlyCAM1 protein backbone can be differentially glycosylated based on its origin from either epithelial gland cells or endothelial HEV cells and serves as a scaffold on which carbohydrate structures dictate the function.

2. CD34 Cloning of the Sgp90 fraction that could be precipitated from lymph nodes revealed that this molecule was CD34 (148). Hitherto this heavily 0-glycosylated molecule had no clear function but was used succesfully to purify human stem cells from bone marrow (149). Its expression on endothelial cells had been appreciated but there seemed to be no restriction to expression on endothelial cells of HEVs. It was therefore concluded that the role of CD34 as a ligand for L selectin was determined by posttranslational modification of the molecule, whereby the carbohydrate structure was site specific and resulted from selective glycosylation capacities of the endothelial cells of HEVs and also at certain vessels in inflammatory lesions (150, 151). It is assumed that the precipitation of the CD34 molecule by the MECA79 antibody lies in the recognition of the HEV-specific carbohydrate ligand of the molecule and that the structure of the ligand is identical on GlyCAM-1 and HEV-CD34. Interestingly, when CD34deficient mutant mice were generated, it turned out that part of the band, which precipitates with the MECA79 antibody at 90 kDa, was still present. This suggests that yet another molecule is present in the 90-kDa fraction that may form an additional ligand for L selectin (152).

3. Sgp2OO Based on sulfate incorporation and interaction with chimeric L selectin and MECA79, a 200-kDa molecule can also be identified in lymph node preparations. The nature of this L selectin ligand is so far unknown (153). C. MUCOSALVASCULAR ADDRESSINMAdCAM-1 The search for the mucosal vascular addressin was continued using the MECA367 antibody, which was found to selectively recognize HEVs in Peyer’s patches and mesenteric lymph nodes and was able to interfere with lymphocyte homing into these organs. Affinity isolation of a molecular fraction from murine mesenteric lymph nodes yielded a fraction that was able to support lymphocyte binding, with the characteristics and specificity expected from a putative mucosal vascular addressin (154). Upon further

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cloning of the 58 to 66-kDa glycoprotein fraction, the mucosal addresin, designated MAdCAM-1, was found to be a novel immunoglobulin family member (155). It consists of three domains, of which the two aminoterminal domains display strong homology with ICAM-1 and VCAM-1. The third, membrane-proximal domain shows homology with the third constant domain of immunoglobulin A l . Strikingly, the molecule contains a serinehhreonine-rich region, with many sites for addition of O-linked sugars, between the membrane-proximal domain and the other two domains, indicating the existence of a mucin-like part. This region was found to contain carbohydrate structures that could be recognized by the MECA79 antibody (156). This antibody recognizes the carbohydrates on the peripheral vascular addressins, the ligands for L selectin. Expression cloning of human and macaque MAdCAM-1 revealed that the mucin part had great variability between species and seemed to be less well conserved than the two amino-terminal domains (157, 158). The latter part is the region that determines the mucosal vascular addressin specificity because here the a4p7 integrin molecule binds, preferably with the first domain, but sequences within the second domain support the interaction (159). The related a 4 p l integrin molecule does not bind to MAdCAM-1 (114) OF THE L SELECTIN LIGAND D. SUGARSTRUCTURE The discovery of GlyCAM-1 has led to more insight into the structural requirements of the L selectin ligand. Biochemical analysis performed by Rosen and co-workers (160-163) made it clear that the O-linked oligosaccharides displayed by the ligand molecules and that were essential as terminal recognition structures were in fact sulfated variants of the sialyl Lewis X structure. As major capping carbohydrate moieties of GlyCAM1,6’-sulfo sialyl Lewis X and 6-sulfo sialyl Lewis X were proposed (Fig.4). All three members of the selectin family, L-, E-, and P-selectin, recognize the tetrasaccharide sialyl Lewis X (sLex) and related structures (164, 165). However, recognition is determined by the presentation of the carbohydrate structures on protein backbones. Optimal P selectin adhesion is found when sLeXis displayed on serine and threonine-linked oligosaccharides residing on P selectin glycoprotein-1 (166, 167). This molecule also seems to represent a high-affinity counterreceptor for E selectin (168, 169). In addition, E selectin ligand has been described, as well as other glycoprotein molecules, critically depending on the posttranslational presence of sLeX(170). Although the importance of the carbohydrate group in providing ligand binding is obvious, the high-affinity binding observed cannot be generated by the single oligosaccharide structures. Therefore, the overall structure of combined carbohydrate groups on a protein backbone will generate

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.e

sialyl Lewis X

u-sulfo sialyl Lewis x

6-sulfo sialyl Lewis X

FIG.4. Carbohydratestructure of the L selectin ligand. The major carbohydrate capping groups of the L selectin ligand, consisting of differentially sulfated sialyl Lewis X molecules [based on the data in Refs. (162) and (163)l. D, fucose; sialic acid; 0, galactose; W, GlcNac; 0, SO4.

+,

high-affinity ligands only when the 0-linked oligosaccarides are combined in uniquely spaced or clustered chains (171). It has to be assumed that for the L selectin ligands this is dictated by the serine/threonine mucin regions in the GlyCAM-1, CD34, and MAdCAM-1 protein cores. The generation of the carbohydrate groups involves posttranslational modification of the protein backbone by glycosyltransferases. This large group of enzymes functions in the pre-Golgi and Golgi network to modify glycoproteinsby adding sugars (172),and the expression of certain glycosyltransferases in a cell type will determine the cell’s ability to produce carbohydrate structures. It has therefore been envisaged that the apparently unique structural combinations of sugar moieties as found on the L selectin ligand are represented by a likewise unique combination of glycosyltransferases in the high endothelial cell producing the ligand. Studies on the different steps leading to the sulfated, sialylated, and fucosylated GlyCAM-1 component indicated that sialylation of the molecule preceded fucosylation and sulfation (173).Expression of s k Xis determined by cellspecific expression of one or more a(1,3)fucosyltransferases(174).Further analysis ofthese enzymes showed that one of them, a(1,3)fucosyltransferase VII ( Fuc-TVII), was selectively expressed in high endothelial cells of peripheral lymph nodes, mesenteric nodes, and Peyer’s patches (175). In

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addition, it was demonstrated that in mice made deficient for the FucTVII there was a deficient E and P selectin-mediated adhesion and a deficient lymphocyte homing. High endothelial venules of these Fuc-TVII knockout mice were devoid of L selectin ligand expression as was demonstrated using an L selectin chimera molecule as an immunohistochemical probe, in correlation with a strongly reduced number of lymphocytes in peripheral nodes (176). Interestingly, the epitope recognized by the MECA79 antibody, and thought to represent the L-selectin ligand, could still be determined. This indicated that the MECA79 epitope does not require Fuc-TVII for its synthesis and that this epitope alone is not enough to confer L selectin ligand specificity. X. Additional Molecules on High Endothelial Venules Involved in Lymphocyte Migration

A. VAP-1

VAP-1 is a heavily sialylated glycoprotein of 170 kDa that was originally described on the lumenal side of high endothelial venules of human peripheral lymph nodes and tonsils, and that the presence of which can be upregulated on vessels at sites of inflammation such as found in synovium and inflamed gut tissue (177-179). Its sialylation seems to be indispensable for its function in mediating binding of lymphocytes to the vessel wall, and anti-VAP-1 antibodies can block binding of lymphocytes in frozen section assays. In later studies the distribution of VAP-1 seemed to be more ubiquitous on various vessels outside lymphoid organs (180, 181). To date the nature of the ligands on the lymphocyte reacting with VAP1is unknown. L selectin is not involved in binding to the molecule (182). VAP-1 interaction may form an additonal adhesive bond between lymphocytes and endothelial cells, adding to the regulatory mechanisms that govern the entry of lymphocytes into lymphoid tissues. XI. Adhesion and Exinwasation

Recent studies on the physiology of lymphocyte recruitment through endothelium have led to a concept of multistep cascades to explain the complete process of extravasation. It is thought that the initial attachment under high shear forces in the bloodstream, the stabilization of the binding of the lymphocyte with the endothelial wall, and the subsequent diapedesis are mediated by adhesion and activation cascades that each can confer specificity, and although operating seemingly independent, each is indis-

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pensable for the whole process (Fig. 5 ) . Essentially, four steps can be discerned [see Refs. (183) and (184)for reviews]. Step 1involves the initial contact of the lymphocyte with the endothelium. This is a transient step, characterized by rolling processes. In step 2, rapid activation (triggering) of the cell takes place, in which chemokines and chemokine receptors are

Peripheral lymph node HEV

L selectin - GlyCAM-I L selectin - CD34 L selectin - sgp200

G protein linked receptors

LFA-1 - ICAM-1

CD31 chemoattractants ?

Peyer's patch HEV

a487 - MAdCAM-1 L selectin MAdCAM-1

G protein linked receptors

LFA-1 - ICAM-I a4p7 MAdCAM-1

CD31 chemoattractants ?

-

~

Venules at inflammation site

~~

a4pl - VCAM-1 CD44 - hyaluronate E selectin - ESL-1 P selectin - PSGL-1 L selectin - sLex? VAP-1 - ?

G protein linked receptors

-

I

LFA-1 - ICAM-1 a4pl -VCAM-1

I

CD31 chemoattractants ?

~

FIG.5. The transmigration process. Schematic representation of the rolling, adhesion, and diapedesis events occuning during lymphocyte migration. The width of the conus indicates the importance of a given process and its reversability over time. The major molecular interactions involved in the various processes in peripheral lymph nodes, Peyer's patches, and at sites of inflammation are listed.

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involved. The activation leads to arrest of the cell and stable binding involving activated integrin molecules (step 3), after which the cell may start the diapedesis (step 4). Both the initial attachment step and the stable adhesion phase are potentially reversible and therefore critical in determining the migratory behavior of various cell populations. A. ROLLING A N D TETHERING How do these processes function in high endothelial venules? Studies using intravital microscopy of exposed blood vessels and ffow chambers in which receptor ligand interactions with purified molecules could be studied have made it clear that the “tethering” and rolling of cells is predominantly mediated by the selectin molecules and their respective ligands (186-189). The tethering refers to loose and transient adhesion of lymphocytes to endothelium resulting in rolling of the cells along the surface of the endothelial wall. This rolling at sites of inflammation is P and E selectin dependent, but in high endothelial venules in lymphoid organs L selectin is the major selectin involved. It has been shown that the expression of L selectin on leukocytes is predominantly found at the tips of microvilli (190),which are the sites of initial attachment under flow. Such concentration of receptors of course enhances the efficiency of interaction. L selectin ligands in the mouse are expressed on many molecules as discussed previously including MAdCAM-1. However, rolling is not restricted to selectin-ligand interaction: Rolling on MAdCAM-1 can also be accomplished via interaction with a4p7 (114, 156). In addition, rolling via a 4 P l on VCAM-1 has been described, as well as a function of CD44 in the rolling of activated lymphocytes (128, 190-192), but an important contribution of these molecules to rolling in HEVs is not likely. Because the ligands for L selectin are predominantly expressed in peripheral lymph nodes, rolling in these organs will be accomplished by L selectin-ligand interaction, whereas the rolling in Peyer’s patches and mesenteric lymph nodes will rely on the rolling activity of a4p7 integrin molecules on MAdCAM-1. Although MAdCAM1 in Peyer’s patches does express L selectin ligands on its mucin part, the contribution of L selectin-mediated rolling may be less outspoken, based on receptor density. The first step of organ-specific homing therefore lies in the different requirements of rolling and tethering molecules (185). The rolling leads to slowing down of the cell and can prepare the cell for the next step, the activation cascade, which in turn is essential for the firm adhesion mediated by activated integrins.

B. ROLEOF CHEMOKINES AND CHEMOKINE RECEPTORS Looking at the function of chemokines during adhesion and migration, a distinction can be made between so-called proadhesive activity, leading

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to integrin acivation and arrest of the cell on endothelium, and the classical chemotactic activity by which cells show locomotion. In the case of rolling and adhesion of myeloid cells to endothelium it has been demonstrated that several chemoattractant receptors can mediate proadhesive activity by agonist-induced integrin-dependent arrest of the cells. Especially the rhodopsin-related Gal-linked 7 transmembrane “serpentine” receptors of the chemoattractant subfamily are implicated in adhesion and also in locomotion and crawling of leukocytes into tissue (193-196).For example, neutrophils show rapid activation of integrin molecules by chemokines leading to firm adhesion. Although it has been demonstrated that lymphocytes can also be stimulated through pertussis toxin-sensitive receptors to adhere rapidly to a4 and p2 integrin ligands, indicating a role for G proteinmediated activation (197-199), the precise role for chemokine activation of lymphocytes is less clear. Chemotactic activity for lymphocytes has been described for several members of both the a and p subfamilies of chemokines (198-201), and chemokines can also affect uropod formation and redistribution of adhesion molecules on lymphocytes (202).However, these chemokines do not show proadhesive activity with lymphocytes and are unable to induce rapid binding of lymphocytes to integrin substrates, as seen with myeloid cells (197-199, 203). This suggests that the chemokines and chemokine receptors on lymphocytes involved in the proadhesive activity may be quite distinct from myeloid cells. Recent reports in which chemokine receptors were transfected in lymphoid cells showed that rapid integrin activation could occur, but that there are differences in the requirements for chemokines to evoke proadhesive and chemotactic activity in these cells (204, 205). This allows for even further refinements on the regulatory mechanisms that govern the adhesion and the subsequent migration into tissues. Together, there is strong evidence that when we look at recirculation of naive lymphocytes through high endothelial venules, they will become activated to induce firm integrin-mediated adhesion to the wall of the HEVs through G protein-coupled activation via chemokine receptors. However, there is little knowledge of the chemokines or the chemokine receptors involved in this process. Some of the orphan receptors, which are related to the known chemokine receptors highly expressed on B and T cells (206, 207), may be likely candidates. C. INTEGRIN-MEDIATED ADHESION The integrins involved in the firm adhesion of lymphoid cells are a4P1, a4P7, and the P2 integrin LFA-1 (aLP2).That the a4 integrins do not only confer strong adhesion but are involved in rolling can be inferred from the finding that these integrin molecules are also expressed on the

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microtips of lymphocytes, in contrast to P2 integrins, which are predominantly excluded from the microtips (189, 208). The integrin activation is transient and reverses spontaneously in minutes. This allows the cell to either start the diapedesis process or, alternatively, to resume rolling or enter the blood flow.

D. THEPROCESS OF DIAPEDESIS The decisive steps to start the diapedesis, as well as the exact mechanism of this process in lymph nodes, are not known. Indications for a role of CD31 (platelet-endothelial cell adhesion molecule-1, PECAM-1) in the transmigration of leukocytes through the endothelial wall have been presented (209). CD31 is expressed on platelets and most leukocytes but is also constitutively present on endothelial cells. On cultured endothelial cells it is concentrated at cell-cell junctions (210, 211). The molecule is a six-domain member of the Ig superfamily and it has been demonstrated in several models of in vivo inflammation that antibodies against CD31 could block emigration of neutrophils (212,213).In a recent report it was shown that treatment with antibody led to accumulaton of leukocytes in the vessel wall, where they seemed unable to pass the basement membrane (214).Ligation of CD31 can lead to activation of Pl and P2 integrins (215, 216) and it can be envisaged that the interaction of leukocytes with CD31 gives rise to an additional activation step whereby the cells are now able to interact with constituents of the extracellular matrix, such as laminin and fibronectin,via Pl integrins. A role for the Pl integrins a4Pl and a5Pl in adhesion of lymphocytes to high endothelial cells has been established in an in vitro model (110, 111). That CD31 is also of importance in HEV transmigration in organized lymphoid tissue is not clear, although in activated lymph nodes lymphocytes expressing CD31 are found adhering to HEVs (217).This seemed to occur only at the peak of the induced immune response when inflammatory factors may have induced additional adhesive capacities to the cells of the HEVs. MI. Adhesion Cascade and Specificity of Lymphocyte Homing

That the adhesion and transmigration of lymphocytes through the walls of high endothelial venules is governed by several steps that can each lead to decisions as to whether to continue the adhesion process or stop adhering and return to the blood flow made it clear that the specificity of homing characteristics are not dictated by simple ligand receptor interactions, but instead are based on multiple events. The three major events taking place during the interaction of the lymphocyte with the endothelial cell are transient processes and dependent on the next step to take over. Therefore,

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when one regards the homing of a naive lymphocye into the wall of a peripheral lymph node HEV, it is obvious that this cell has to comply with a large set of conditions to be able to enter the parenchyma of the peripheral lymph node. For rolling, a lymphocyte will need an appropriate density of L selectin molecules, positioned at the tips of its microvilli, to be able to adhere to extending mucin threads that extrude into the lumen of the HEVs. During the initial arrest of the cell on the endothelium, it is important that the cell rapidly screens the endothelium for the presence of chemokines, which are presented by proteoglycans at the luminal side of the endothelial cell. Only when there is a correct match between the chemotactic factors and the receptors on the lymphocyte surface will the cell start signal transduction, whereby surface integrin molecules are activated by inside-out signaling, leading to firm adhesion of the cells. If there is no activation, the cell will leave for the bloodstream again. Lack of activation can occur for two reasons: (i) HEVs are only limited stretches of endothelium, and once the cell has rolled past it will become loose again because of lack of appropriate receptors; and (ii) the rolling itself may affect the behavior of the cell by stimuli generated through selectin-ligand interaction or interaction with cytoskeletal proteins (218).This may change the microvilli or lead to shedding of selectin molecules, resulting in deadhesion. When the cell has received the appropriate stimuli and integrin activation has taken place, firm adhesion will require the presence of appropriate ligand structures of the immunoglobulin superfamily. The integrin activation is a transient conformation change of the integrin dimers leading to rapid changes in the morphology of the cell because of the intimate connections of the integrin molecules with the cytoskeleton (219). The cell is then ready to extravasate. At all steps decisions to stop adhesion can be made and only with all the right configurations does the cell seem able to extravasate and enter the lymphoid tissue.

A. ORGAN SPECIFICITY OF LYMPHOCYTE MIGRATION The major distinction in organ specificity at the level of high endothelial venules is between peripheral lymph nodes and Peyer’s patches, which can be explained on the basis of L selectin-ligand and a4P7-MAdCAM1 interactions (185). At the level of chemokine activation it is unknown whether differences exist in local chemokines as to make distictions between peripheral and mucosal sites. At the level of the integrin-Ig superfamily interaction it is obvious that MAdCAM-1 plays a unique role because it can interact with both a4P7 and LFA-1 molecules (159), whereas in peripheral nodes that lack MAdCAM-1, integrin interaction will predominantly rely on LFA-1 binding to ICAM-1.

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Naive, newly formed lymphocytes that have not been stimulated by antigen express high levels of L selectin and have been shown to recirculate efficiently through peripheral lymph nodes and also through Peyer’s patches. Subtle differences in homing, as found for the major classes of lymphocytes, CD4, CD8, and B lymphocytes (35, 36, 220, 221), may be the result of small differences in the balance between L selectin and a4P7 expression. Although the data with naive cells have been questioned and been attributed to artefacts in the experimental system (222, 223), it is dear that the regulation of homing dramatically changes when lymphocytes have undergone antigenic stimulation. It is generally accepted that antigenic stimulation of naive lymphocytes has to take place within the microenvironment of organized lymphoid tissues. Here the appropriate antigen presenting cells, dendritic cells in particular, can stimulate lymphocytes. Lymph nodes are optimally suited to fulfill this function based on the “counterflow” by which naive lymphocytes enter through HEVs into the paracortical area. In this region they encounter dendritic cells that are continuously immigrating from the drainage region of the lymph node by virtue of the afferent lymphatics (224, 225). The drainage region can be a region of the skin or any major organ. In essence, dendritic cells present antigens that they have processed at the sites where they have come from as passenger leukocytes. Lymphocytes will screen the antigen presenting cells for the right MHC-peptide complexes, for which adhesive interactions between APC and lymphocyte and lymphocyte and stroma have to take place. These interactions also have a transient characteristic, and when no activation takes place the lymphocyte leaves the lymph node, probably passively with the lymph flow, and may try its luck in the next lymph node (Fig. 6). However, once the cell becomes activated and changes into memory/ effector lymphocyte its homing potential will allow for extravasation at extralymphoid sites, such as skin, the lamina propria of the intestines, inflamed joints, and so on. This is reflected in the upregulation of new adhesion molecules, such as the skin-homing receptor, or the preferential up- or downregulation of existing receptors, such as a4P7 and L selectin (61-68, 109, 226, 227). From the many studies on memory cell migration it has become clear that there is extreme heterogeneity in behavior, which may partly be explained by the difficulty in identifylng memory cell populations as such (228).The extreme tissue selectivity observed in recirculation of memory cells is often explained as a way to target cells back into those tissues where they are most likely to reencounter the antigen for which they were activated (63, 229-232). This would argue for a site-specific change of otherwise identical naive lymphocytes of their repertoire of adhesion mole-

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blood

thoracic duct lymph

FIG.6. Where, whence, whether. Scheme of the recirculation pathways of naive and memory lymphocytes and the involvement of HEVs and postcapillary venules. Adapted from Ref. (184).

cules (and chemoldne receptors) under the influence of local stimulation. In other words, lymphocytes activated in peripheral lymph nodes will have a predisposition as memory/effector cells to selectively migrate into inflamed skin and other peripheral tissues, whereas after activation at a mucosal site, such as Peyer’s patches or mesenteric lymph nodes, preferential migration into, e.g., the lamina propria is seen. The mechanisms that direct these changes are unclear but the interaction with APC, the costimulatory signals, and local cytokines seem to be instrumental. In this respect it is interesting that another major dichotomy found in the immune system, the distinction between Thl and Th2 lymphocytes, is also largely reflected in a division between peripheral and mucosal sites (233, 234). Whether the expression and regulation of adressins is under control of the same mechanisms that may control T h l and Th2 induction is a matter of speculation (235). XIII. Regulation of the Unique Feahrres of the High Endothelial Venule

It is apparent that high endothelial venules found in secondary lymphoid organs are unique structures. Although when compared to activated small

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vessels at sites of inflammation many similarities in the processes of adhering and transmigration occur and overlap in the use of interactive molecules can be observed, the HEV is a distinct, integral component of the immune system. As such, it is constitutively active and it is clear that without its functioning the immune system will be greatly impaired. To maintain its function in the dynamic environment of a lymphoid organ in which a continuous turmoil of cell activation and cytokine production take place, HEVs will have to be tightly regulated. From many experimental data a role of the afferent lymph has been inferred. By cutting of the afferent lymphatics of rat lymph nodes the HEVs in these nodes became flattened and dimished production of sulfated components was observed. Despite an intact blood supply, lymphocyte migration into the nodes was halted and the overall structure of the rat lymph nodes degenerated (236, 237). Afferent lymph occlusion was found to affect the presence of macrophages in the lymph nodes and a role for these cells in the integrity of HEVs was suggested. When the the afferent lymphatics were allowed to reconnect, HEVs in the lymph node regained their normal structure and function before the reappearance of subcapsular sinus macrophages, excluding a major role of these cells in HEV function. Nevertheless, in lymph node organ culture experiments a (possibly intermediate) function of the subcapsular sinus macrophages could not be excluded (238). Also, the effects of deafferentiation in the mouse on the expression of vascular addressins and HEV-specific markers, such as those recognized by the MECA325 antibody (239), were investigated. Coinciding with the downregulation of lymphocyte homing in vivo and lymphocyte binding to HEVs in uitro,the expression of the peripheral node vascular addressin changed from luminal to abluminal (240). Within the time span of 7 days the lymph node had changed dramatically, with an apparent loss of macrophages and also of interdigitating dendritic cells and a loss of compartmentalization in T and B cell areas, In this time period mRNA for GlyCAM-1 was rapidy lost from the lymph nodes as was the GlyCAM-1 protein (241). Staining the interrupted lymph node with a chimeric recombinant L selectin molecule revealed the complete absence of the ligands for L selectin, and the lymphocytes that could be found in these lymph nodes were predominantly L selectin negative (Fig. 7). Flattening of HEVs has also been observed in irradiated animals in which virtually no lymphocyte recirculation takes place (242),but in irradiated lymph nodes GlyCAM-1 expression is normal, as it is in SCID and nude mice (241). The loss of L selectin ligands is also reflected in the loss of the Fuc-TVII fucosyltransferase from deafferentized lymph nodes, indicating that via downregulation of this enzyme the sugar ligands can no longer be made (V. S. Swarte et al., manuscript in preparation).

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FIG.7. The importance of afferent lymph in HEV functioning. Seven days after deafferentiation of the lymphatics of the popliteal lymph node, sections of the node were incubated with L selectin chimera molecules to detect the presence of L selectin ligands. (A) Control lymph node with clear expression of L selectin ligands; (B) absence of staining on flatwalled HEVs in operated lymph node.

This suggeststhat factors from the afferent lymph activate HEVs or maintain their functioning,either directly or indirectly, through intermediate cells such as the subcapsular macrophages. Although the macrophages in the subcapsular sinus do not make direct contact with HEVs, it has been suggested that the structure of the lymph node is such that factors that reach the subcapsular sinus or are being produced there can reach the HEV area immediately because of a conduit system formed by the reticular network of the organ (243,244).The existence of such a transport system was inferred from anatomical observations and from evidence showing rapid transport system of factors from afferent lymph to HEVs (Figs. 8 and 9).Cytokines administered in a drainage bed induced lymphocyte migration via HEVs into the draining lymph node in a matter of minutes (200, 243), and application of tracer in afferent lymphatics leads to rapid accumulation around HEVs with no trace in the interstitial tissue (18,245).Thus, it is possible that HEVs directlyreact to changes in the draining region, and enhanced lymphocyte influx could occur before any antigen-specific stimulation has taken place in the node itself. The fast Aux of cytokines and chemokines resulting from an inflam-

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FIG.8. The reticular conduit system of the lymph node. Factors arriving or produced in the subcapsular can be directly transported to the vicinity of the HEV by means of the reticular fiber system. Abbreviations used: AL, afferent lymph; ck, cytokine; CS, cortical sinus; FC, fenestrated capillary; HEV, high endothelid venule; IDC, interdigitating cell; m, macrophage; mc, mast cell; mcg, mast cell granules; SCS, subcapsular sinus. [From Ref. (243)l.

matory insult outside the node will superimpose inflammatorycharacteristics to the endothelial cells of the HEVs. On the one hand, these chemokines will activate the endothelial cells into expressionof additional adhesion molecules normally not present in HEVs, such as VCAM-1 and E and P selectins (52, 53), and upregulation of ICAM-1 (246). On the other hand, capture and presentation of chemokines in the glycocalyx of the endothelial cells (203, 247)will lead to the attraction ofcells from the bloodstream that are otherwise not able to enter the lymph node, such as monocytes and granulocytes (248,249).

A. EFFECTS OF AN ONGOING IMMUNE RESPONSEON HEV ACTIVITY Antigenic stimulation leads to apparent changes in the lymph nodes, with increasing cell numbers from local proliferation and increased reten-

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FIG.9. Tracer movement in the reticular network. Horseradish peroxidase was injected as tracer in the afferent lymphatics of a rat lymph node. One hour later the node was excised and stained for tracer activity. (A) The presence of tracer in the area directly beneath the subcapsular sinus (SCS) can be seen. (B) The tracer is found directly surrounding the HEV, illustrating the rapid transport of factors from lymph to the HEV area. Photographs by Dr. A. Anderson.

tion (250), but proliferation of structural elements, including HEVs, can also be seen (251). Morphometric analysis of the increase of HEVs in relationship with the bulk increase of the lymph node showed that expansion of the paracortical areas in which HEVs are located preceded HEV

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growth during an immune response, but that after a certain time the ratio of HEVs per tissue unit was again comparable to control values (252). This suggests that there is a fixed amount of HEVs necessary to serve a certain region of lymphoid tissue (252). In a study on the effects of a primary immune response on the expression of vascular addressins, it was found that in the course of the response there was a decrease in secretion and expression of GlyCAM-1 and also of secreted sgp200, but that CD34 and Sgp200 expression on HEVs remained unaffected (253). An increase of homing of lymphocytesand also of monocytes and neutrophilsto stimulated nodes was observed, leading to the suggestion that the secretion of both GlyCAM-1 and Sgp200 from the HEVs is involved in the regulation of cell influx, possibly by competing for adhesion receptors. Also, a role for platelets in altered homing behavior may be envisaged. Recently, an indirect role for P selectin during rolling on HEVs was found in which activated platelets could form an intermediate bridge between HEVs and Iymphocytes through P selectin on the platelets (254). The P selectin reacted with ligands on the T cells and recognized sugar ligands on the peripheral node addressins. That P and L selectin recognized common endothelial ligands had been shown before (255) and this mechanism may enable lymphocytes that have low amounts of L selectin to adhere to HEVs in peripheral nodes under certain inflammatory conditions.

B. EFFECTS OF THE LOCAL MICROENVIRONMENT ON THE DIFFERENTIATION OF HEVs The importance of incoming lymph in maintaining the integrity of the HEVs also posed the question whether the regional drainage area would be able to differentiallyaffect the expression of the various addressins. This question was addressed by transferring lymph nodes from their original peripheral location to a mucosal site and vice versa (256-258). When the environment of adult peripheral or mesenteric lymph nodes or Peyer’s patches was changed, the expression of vascular addressins on HEVs did not alter and preservation of the type of addressin composition of the original site was observed. Only when these experiments were performed with neonatal lymph nodes could a role for the environment on the induction of vascular addressin be observed (Fig. 10). Neonatal mesenteric lymph nodes transplanted into a peripheral site did not show an induction of MAdCAM-1, and this is in line with the finding that in the mouse the period around birth is critical for the development of HEVs and lymph nodes (259). In fact, during the first 24 hr after birth the dominant adhesion molecule on HEVs in lymph nodes is MAdCAM-1, the mucosal addressin (Fig. 11).Despite the fact that many L selectin-positiveleukocytes can be found before birth, fetal lymph nodes attract preferentially a4P7 expressing

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FIG. 10. The role of the local microenvironment on addressin expression. Neonatal peripheral lymph nodes were transplanted into the mesentery and 8 weeks later they were examined for the presence of the peripheral node addressin as identified by the MECA79 antibody. Sectionsof different lymph nodes show partial (A and B) or complete (C)expression of MECA79 on the endothelial cells of HEVs (X35) [from Ref. (256). by permission of Oxford University Press].

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-

E2 z

21

16 11

6 & 4:

7 4 1

1

1

I

I

I

I

I

I

1

I

I

80

60

40

20

0

20

40

60

80

100

Yucosal (TK1)

Peripheral(38C13)

Recognitionrpeclflclty of lymphomas bound to PLN venules (percent of total cells bound)

FIG.11. Ontogenetic development of HEV-binding specificity. Invitro binding of tissueselective lymphomas to HEVs in peripheral lymph nodes at various time points after birth. At 1 day after birth predominant binding of the mucosal lymphoma (TK1) to HEVs can be observed, which shifts over time to predominant binding of the peripheral lymphoma (38C13)at 3 or 4 weeks after birth [from Ref. (259)].

cells. After only 2 days after birth the peripheral addressins are upregulated in peripheral lymph nodes, coinciding with a slow decline and eventual disappearance of MAdCAM-1 from peripheral HEVs. It is suggested that this developmental switch is important in directing sequentially different cell types into the lymph nodes, which are supposed to be instrumental for its development (259, 260). XN. Concluding Remarks

High endothelial venules are important structures involved in the immunological surveillance of our body as essential traffic control sites in the recirculation of lymphocytes. Already critically important during the organogenesis of the lymph node, the unique anatomical position in the reticular organization of the lymphoid organs enables the high endothelial venule to rapidly adapt its function in adherence with changes imposed by the

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lymph node or its draining region. This adaptivity is a striking phenomenon and adds to the flexibility of the immune defense. During the past years many aspects of the function of HEVs have been elucidated: the fascinating molecular cascades, the structural requirements, and the control of HEV functioning by the microenvironment. Rapid progress has been made in our understandingof the molecular interactions, but not all questions are answered. The nature of the chemokines involved in lymphocyte activation during adherence, the factors that control HEV activity, and the precise role of unique and specific components, such as Hevin (261, 262) and MECA325 (238), are still largely unknown. The mystery of the disappearing lymphocytes has been resolved. Many mysteries are left to challenge us. ACKNOWLEDGMENTS Over the years many thanks are due to our friends and colleagues in the field of lymphocyte migration for their sharing of ideas, information, and reagents. Special thanks to Dr. A. Anderson (Medical Research Institute of Infectious Diseases, Frederick, MD) and Dr. K. Sasaki (Yamagata University, Japan) for contributing the wonderful photomicrographs and schemes from their work.

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INDEX

A

rapamycin and, 121 reactive oxygen intermediate-generating molecules and, 122-123 rheumatoid arthritis drugs and, 123-124 salicylates and, 121-122 spergudin, 125-126 steroids and, 118-120 Antioxidants, NF-KB and, 122-123 Anti-TNF-a antibodies, action of as rheumatoid arthritis treatment, 124 AP-1, interaction with NF-ILG, 18-19 AP-1 family, 29 Apigenin, action of, 123 Apoptosis, of HIV-specific CTLs, 306 APPs (acute phase proteins), 6 APRF (acute phase response factor), 19 ASFV, see African swine fever virus AspergiUus infections, ghotoxin, 126-127 ATP TAP peptide binding and, 84-85,87-88 TAP peptide transport and, 76-77 Autoimmune diseases NF-KB and, 117-118 superantigens and, 207 TAP and, 92-94

al-acid glycoprotein (ACP), 22 ABC transporters, 55, 56-57, 59 Acute phase proteins (APPs), 6 Acute phase response factor (APRF), 19 Addressins, lymphocyte-HEV interaction, 361-363, 377 Adenovirus, cytotoxic T lymphocytes and, 279 Adhesion, lymphocytes in high endothelial venules, 367-372 African swine fever virus (ASFV), 127-128 AGP (al-acid glycoprotein), 22 AGP gene, 22 AIDS CTL suppression in, 305 HIV progression to, 285, 299, 302, 304 AML1.24-25 ANK repeat, 14 Ankylosing spondylitis (AS), HLA-B27 and, 94 Antigens, see also Superantigens: speciic antigen

ABC transporters, 55, 56-57, 59 need for transporter, 47-49 superantigens, see Superantigens TAP, 47-96 Anti-inflammatory and immunosuppressive molecules NF-KB as target, 128-132 antioxidants and, 122-123 anti-TNF-a antibodies, 123-124 CAMP and, 124-125 cyclosporin A and FK506, 120 deoxyspergualin and, 125-126 gliotoxin and, 126-127 glucocorticoids and, 118-120 gold compounds and, 123-124

Bacterial superantigens, 14, 208-210 B cell response, to MMTV endogenous Mtv,196-208 neonatal, 171-175 T-B cell interaction, 180-188 T cell-dependent differentiation, 178- 180 T cell-independent activation, 168-171 bc2-3 gene, 14 397

398

INDEX

C CAF (CD8t antiviral factor), 282 Calcineurin, cyclosporin A, action of, 120 Calnexin, MHC Class I assembly and, 89,90 Calreticulin, 90, 91 CAMP,immunosuppressive activity of, 124-125 Cancer, TAP dysfunction, 95 @-caseingene, 20 CBF family, 24 CBFu proteins, 24 CD8t antiviral factor (CAF), 282 CD31, leukocyte transmigration, 369 CD34,lymphocyte-HEV interaction, 362, 377 CD44, lymphocyte-HEV interaction, 359-360 CD62E, 354 CD62L, 354 CD62P, 354 C/EBP, 3-7 knockout mice studies, 30 structure of, 1 C/EBPy, 18-19 C/EBP family, 3-8 Cellular immunity, 277-278, 322-323 adenovirus, 279 cytomegalovirus infection, 278-279 Epstein-Barr virus (EBV), 279 herpes simplex virus, 279 HIV infection, 280 adoptive immunotherapy, 317-320 CTL-mediated lysis, 280-281 CTLs and, 280-322 HLA and, 284-286 replication suppression, 281-284 in seronegatives, 287, 307-309 vaccines, 320-322, 323 HLA Class I system, 279 influenza virus, 278 Chemokine receptors, lymphocytes in high endothelial venules, 367-368 Chemokines, lymphocytes in high endothelial venules, 367-368 CHOP-10, 3-6 Ciliary neurotrophic factor (CNTF), 20 dm,48-49, 78 Class I MHC molecules, cytotoxic T lymphocytes and, 279-280

Class I modifiers, see cim Class I1 HLA types, HIV and, 286 Clonal deletion, of MTV superantigenreactive T cells, 203-204, 210 Common variable immunodeficiency (CVID), 256-260 CpG islands, 50, 51 CREB, interaction with NF-IL6, 19 C-REL knockout mice, cytokine induction, 31-32 CTLs, see Cytotoxic T lymphocytes Curcumin, action of, 123 CVID, see Common variable immunodeficiency CXCR4, 283 Cyclosporin A, NF-KB, 120 CW2D5 gene, 23 Cytochrome P450, 23 Cytokine response element (CyRE), 20 Cytokines gene regulation by NF-IL6 and NF-KB, 1-33 inflammatory process, 111 lymphocyte-HEV interaction, 374 rheumatoid arthritis and, 123-124 Cytomegalovirus (CMV), cytotoxic T lymphocytes and, 278-279 Cytotoxic T lymphocytes, 277-278 adenovirus, 279 cytomegdovirus infection, 278-279 Epstein-Barr virus, 279 herpes simplex virus, 279 HIV infection, 280 adoptive immunotherapy, 317-320 CTL-mediated lysis, 280-281 CTLs and, 280-322 hla and, 284-286 replication suppression, 281-284 in seronegatives, 287, 307-309 vaccines, 320-322, 323 HLA Class I system, 279 influenza virus, 278 transporters, need for, 47-48 tumor immunity and, 94.95

D Deoxyspergualin (DSG), 125-126 Dexamethasone, NF-KB and, 119 Diapedesis, 369

399

INDEX

Dithiocarbamates, action of, 123 DQ6, 286 DR2. 286 DSG (deoxyspergualin),125-126

E ElA, 11 ElB 19K, NF-KB activation and, 128 EBPy, 18 Epstein-Barr virus (EBV) cytotoxic T lymphocytes and, 279 HLA and, 286 E-selectin, 3,54, 355, 363, 367 Extravasation, lymphocytes, 360, 365-367

F Feline immunodeficiency virus (FIV), LTR and, 10 FK506, NF-KB, 120 Forskolin, action of, 124-125 FUC-TVII,364-365, 373

G-CSF gene, regulation, 28-29 Gene regulation, by cytokines, NF-IL6 and NF-KB, 1-33 GH (growth hormone), gene regulation by, 20 Gliotnxin, 126-127 Glucocorticoid hormones (GHs), 22 Glucocorticoid receptors (GRs) interaction with NF-IL6, 21-22, 23 NF-KB and, 118-120 Glucocorticoid response element (GRE), 22 Glucocorticoids, 21, 23 NF-KB and, 118-120 GlyCAM-1, lymphocyte-HEV interaction, 361-362, 363, 364, 373, 377 Gold compounds, action of as rheumatoid arthritis treatment, 124 GRE (glucocorticoid response element), 22 Growth hormone (GH), gene regulation by. 20 GRs, see Glucocorticoid recepton:

H HAM genes, 49 Hemophiliacs, HW-infected, 291-292 Hepatitis B virus (HBV), C/EBP and, 10 HERMES-3. 360 Herpes simplex virus, cytotoxic T lymphocytes and, 279 High endothelid venules (HEVs), 379-380 flattening, 373 history, 347-348 lymphocyte migration and, 350-351 lymphocytes and, 352 addressins, 361-363, 377-379 adhesion, 365-372 CD34,362,362-377, 377 CD44,359-360 extravasation, 360, 365-367 GlyCAM-1, 361-362, 363, 364, 373, 377 homing receptor ligands, 360-365 integrins, 352,358-359, 368-369, 370 in oitro binding assay, 351-352 MAdCAM-1,352,359, 362-363, 367, 370, 377, 379 migration, 350-352 mucins, 360-361 selectins, 352, 354-357, 363-365, 367 Sgp200, 362,377 sugars, 360-361, 363-365 VAP-1, 365 regulation, 372-375 differentiation. 377-379 immune response and, 375-377 rolling, 367, 368 structure of, 348-350 tethering, 367 Histocompatibility antigen modifier gene, 49 H IV CD8' antivird factor, 282-284 chemokine receptor family, 283 cytotoxic T lymphocytes, 286-287 acute infection, 293-296 adoptive immunotherapy, 317-320 adverse effects of CTL activity, 308-311 apoptosis, 306 asymptomatic period of infection, 296-297 CTL decline in late disease, 300 CTL exhaustion, 304-305 disease progression, 297-298

400

INDEX

HIV, cytotoxic T lymphocytes (continued) epitopes, 291-293, 301, 312, 313 escape mutation, 311-317, 322-323 infection, 290-307 long-term nonprogressors, 298-300 lysis of infected cells, 280-281 measurement of HIV-specific CTLs, 287-290 replication suppression, 281-284 in seronegatives, 287, 307-309 Thl to Th2 switch, 302-304 therapeutic implications, 317-322 vaccines, 320-322, 323 HLA Class I system, 284-286 lysis of infected cells, 280-281 progression to AIDS, 285, 299, 302, 304 replication suppression, 281-284 variants, 306-307 HN-1, LTR and, 10 H N infection CTL-mediated lysis, 280-281 in seronegatives, 287, 307-309 HLA Al-B8-DR3, H N progression to AIDS and, 285 HLA-B8, HIV and, 285 HLA-B27 HIV and, 285 spondyloarthriticdiseases, 94 HLA-B35, HIV progression to AIDS and, 285 HLA-B37, 286 HLA-B49, 286 HLA-B57, H N and, 285 HLA Class I system cytotoxic T lymphocytes and, 279 HIV infection and, 284-286 HLADR3-DQ2, 286 HLA-DR5,286 HLA-DR13, 286 hst-I gene, 155 HSV, see Herpes simplex virus Human papillomavirus (HPV), C/EBP and, 10 Humoral immune response, against MMTV, 188-192 Hypoxia, IL-6 expression, 27

I ICAM-1, synergistic activation of, 21 ICAM-1 gene, NF-KB and, 119

ICP47,279 IFN-7, 282 Ig/EBP, 3-5 IKB, deficiency effects, 32 I K B ~14 , IKBCI gene, 23 IKB family, 14-15 IKB knockout mice, cytokine induction, 32 IKB proteins, 117 a4Pl Integrin, 358, 368 a4P7 Integrin, lymphocyte-HEV interactions and, 352, 358-359, 368 aEP7 Integrin, 359 Interleukin-1 signaling events, 117 gene regulation, 25-26 Interleukin-2, H N and, 282 Interleukin-4, transcription induced by, 19 Interleu!&-4 promoter, 29 Interleukin-6 gene NF-KB and, 119 regulation 23,25,26-28 Interleukin-6 promoter, 26 Interleukin-7, cytotoxic T lymphocytes, 306 Interleukin-8 gene, regulation, 23, 28 Interleukin-16, 282 Immune response, 111 to H N , 290-293 acute infection, 293-296 adverse effects of CTL activity, 309-311 apoptosis, 306 asymptomatic period of infection, 296-297 CTL decline in late disease, 300 CTL exhaustion, 304-305 disease progression, 297-298 escape mutation, 311-317 long-term nonprogressors, 298-300 in seronegatives, 287, 307-309 Thl to Th2 switch, 302-304 therapeutic implications, 317-322 to MMTV (mouse mammary tumor), 167-168, 212 adult T-cell response, 175-178 cellular response, 192-194 humoral response, 188-192 neonatal response, 171-175, 203 receptors for, 194-196 superantigen dependent, 171-194

401

INDEX

superantigen and T cell-independent, 168-171 T-B interaction, 180-188 T cell-dependent B cell differentiation, 178-180 Immune system high endothelial venules, 373, 375-377 lymphocyte migration, HEVs and, 350-372 Immunodeficiency, IgA deficiency, 245-263 Immunoglobulin A function, 251 production, 248-251 structure, 248 Immunoglobulin A deficiency ( IgAD), 251-256, 263 clinical manifestations, 246-248, 256 common variable immunodeficiency (CVID) and, 256 genetic susceptibility, 256-260 pathogenesis, 260-263 treatment, 263 Immunosuppressive molecules, see Antiinflammatory and immunosuppressive molecules Immunotherapy. HIV, 317-320 Inflammatory diseases, NF-KBand, 117-118 Inflammatory reactions, 11, 122-123 Influenza virus, cytotoxic T lymphocytes and, 278 Insulin-dependent diabetes mellitus (IDDM), TAP and, 92-93 Integrins, lymphocyte-HEV interactions and, 352, 358-359, 368-369, 370 int genes, 154, 155 Intracellular localization, TAP protein, 62-63 IRAK, 117 Isotype switching, 253-254

J JAK2, activation of, 20-21 J chain, 248

Knockout studies, cytokine induction, 29-32

1 LAP (liver-enriched transcriptional activator protein), 2 LFA-1, lymphocyte-HEV interactions and, 352,358, 368, 370 LIP (liver inhibitory protein), 2 Lipoarabinomannan (LAM), 26 Lipoxygenases, action of, 122 Liver-enriched transcriptional activator protein (LAP),2 Liver inhibitory protein (LIP), 2 Long terminal repeat (LTR) enhancer, viral infection and, 9-10 Long-term nonprogressors, HIV, 298-300 LPAM-1, 359 LPS, 27 L-selectin, 352, 354, 367 activation and signaling via, 357 lymphocyte-HEV interaction, 355, 357, 358, 361 regulation of, 356-357 shedding of, 356 structure and function of, 354-356 sugar structure of, 363-365 LTR (long terminal repeat) enhancer, viral infection and, 9-10 Lymph nodes high endothelial venules, 373-377 reticular conduit system, 375 Lymphocyte migration diapedesis, 369 extravasation, 360, 365-367 GlyCAM-1, 361-362, 363, 364, 373, 377 CD34, 362, 377 Sgp200,362,377 high endothelial venules (HEVs) and, 350-351 addressins, 361-363, 377-379 adhesion, 365-372 CD44,369-370 extravasation, 360, 365-367 GlyCAM-1,361-362, 363,364,373,377 homing receptor ligands, 360-365 integrins, 352, 358-359, 368-369, 370 in oitro binding assay, 351-352 MAdCAM-1,352,359, 362-363, 367, 370,377,379 mucins, 360-361 selectins, 352, 354-357, 363-365, 367

402

INDEX

sugars, 360-361, 363-365 VAP-1, 365 organ specificity, 370-372 recirculation pathways, 371-372 Lymphocytic choriomeningitis (LCMV) infection, cytotoxic T lymphocytes and, 278 Lymphomas, induction by MMTV, 155-156 Lysis, of HIV-infected cells, 280-281

MAdCAM-1, lymphocyte-HEV interaction, 352, 359, 362-363, 367, 370, 377, 379 MALT, 251 Mammalian multidrug resistance (MDR) gene, 51 Mammary tumors, in mice, see MMTV; Mouse mammary tumors M cells, 251 MECA79, 361-363, 365 MECA367,361, 362 MEL-14, 352, 354 MGF/STAT5, 20 MHC Class I molecules, 48-49 TAP and, 87-92 MHC Class I1 molecules, MMTV superantigens and, 164-165 MHC locus, MMTV-induced tumors and, 156 mim-1 gene, 22 MIP-la, HIV and, 282 MIP-1/3, HIV and, 282 Mitochondrial enzymes, action of, 122 MIS antigen, 139, 196 MMTV (mouse mammary tumor virus), 139-141, 211-212 endogenous ( M u ) immune stimulation by superantigens, 196-203 superantigen expression, 152-153 tolerance induction, superantigens and, 203-208 virology, 141-146 exogenous adult T-cell response, 175-178 neonatal response, 171-175, 203 T cell-dependent B cell differentiation and, 178-180 virology, 141-146 immune response to, 167-168, 212 adult T-cell response, 175-178

cellular response, 192-194 humoral response, 188-192 neonatal response, 171-175, 203 receptors for, 194-196 superantigen-dependent, 171-194 superantigens and T cell-independent, 168-171 T-B interaction, 180-188 T cell-dependent B cell differentiation, 178-180 superantigens (SAgs), 140, 152-153 expression, 152-153 immune stimulation by, 140, 141, 171-194, 196-203 MHC Class I1 molecules, interaction with,164-165 protein structure, 157-164 TCR VP, interaction with, 165-167 tumor formation and, 152, 155 virology, 141-145 amplification and spread, 180-188 infection and transmission, 148-151 life cycle, 211 structure, 146-148 tissue distribution, 151-152 transcriptional regulation, 148 Mouse mammary tumors, 139 induction of, 153-155 virus, see MMTV mtp genes, 49 Mucins, lymphocyte-HEV interaction, 360-361 Mucosal vascular addressin, lymphocyte-HEV interaction, 362-363 Murine encephalomyocarditis (EMC) virus, resistance to infection, 31 Mutation, HIV genome, 311-317 Myb, intraction with NF-IL6, 22 myb gene, 22

NBD, see Nucleotide-binding domain NFAT family, 120 NF-IL6.3-8 activation of, 2-3 CYP2DG gene and, 23 gene regulation, 8-9 AP-1, interaction with,18-19 CREB, interaction with, 19

403

INDEX

cytokine induction, 29-30 glucocorticoid receptors, interaction with, 21-22 Myb, interaction with, 22 NF-KB, interaction with, 16-18 PU.l, interaction with, 21 STAT family, interaction with, 19-21 knockout mice studies, 29-30 structure and functin of, 1-2 in viral infection, 9-11 NF-IUP, 3-6 NF-ILG gene, 2 NF-KB, 1, 111 activation of, 16-18, 130 disease and, autoimmune and inflammatory, 117-118 gene regulation, 1, 26, 32, 111 cytokine induction, 30-32 interaction with NF-ILG, 16-18 glumrticoid-mediated repression, 23 induction, 111 endogenous, 114 exogenous, 112-113 inflammatory process, 111-118 knockout mice studies, 30-32 structure and function of, 11-12 target for anti-inflammatory and immunosuppressive molecules, 128-132 antioxidants, 122-123 anti-TNF-cu antibodies, 123-124 CAMP, 124-125 cyclosporin A and FK506, 120 deoxyspergualin. 125-126 gliotoxin, 126-126 glucocorticoids. 118-120 gold compounds, 123-124 rapamycin, 121 reactive oxygen intermediate-generating molecules, 122-123 rheumatoid arthritis drugs, 123-124 salicylates, 121-122 spergualin, 125-126 steroids, 118-120 target genes for, 115-116 viruses and, 112, 127-128 NF-KB box, TAP genes, 60 NF-M, 2-3, 7 Nuceotide binding, TAP, 86 Nucleotide-binding domain (NBD), 56

P p50 knockout mice, cytokine induction, 30-31 p50 protein, 15 p53 gene, regulation, 25 p105 protein, 15-16 Papillomavirus, see Human papillomavirus PEBP2 family, 24 Peptide binding, to TAP, 84-86 Peptide supply factor gene, 50 Peripheral node vascular addressins, lymphocyte-HEV interaction, 361-363 Phospholipases, action of, 122 Phosphomannan polysaccharide (PPME), 356 Phosphorylation activation of NF-IL6, 2-3 NF-KB, 15-16 Polymeric immunoglobulin -tor (pIgR), 249 Polymorphism, TAP, 71-75 PPME (phosphomannan polysaccharide), 356 PRE-I, 18-19 Prostaglandin E2, action of, 124-125 Proteosomes, 15 Proviruses, 143 Mtv, 142-146 P-selectin, 354, 355, 363, 367 psfl gene, 50, 51 PU.l,interaction with NF-ILG, 21 Pyrrolidine dithiocarbamate, 123

RANTES, HIV and, 282 Rapamycin, NF-KB and, 121 RB, 25 Reactive oxygen intermediates (ROIs), NFKB and, 122-123 Receptors, for MMTV, 194-196 Reiter’s syndrome, HLA-B27 and, 94 RelA/p65 knockout mice, cytokine induction, 31 RelB knockout mice, cytokine induction, 31-322 REL family, 12-13 Retroviruses, integration of viral DNA, 168 Rheumatoid arthritis (RA) anti-TNF-a antibodies and gold compounds and, 123-124 HLA-B27 and, 94

404

INDEX

RING4,50, 51 RING11 gene, 50.51 RIP, 117 Rolling, high endothelial venules, 367, 368 Runt domain, AML1, 24, 25

SAgs, see Superantigens Salicylates, NF-KB and, 121-122 SDF-1, 283 Secretory antibodies, 249, 250 Selectins, 352, 367; see also E-selectin; Lselectin; P-selectin Sialyl Lewis X,selectins and, 363 Sicca-CDB+lymphocytosis syndrome, 286 SIV, vaccines, 321-322 Spergudin, 125-126 Spondyloarthriticdiseases, TAP and, 93 STAT3, 19 STAT5, 20 STAT6, 20 STAT family, interaction with NF-IL6, 19-21 Steroid hormones, NF-KB and, 118-120 Subcellular localization, TAP protein, 63, 65 Sugars, lymphocyte-HEV interaction, 360-361.363-365 Superantigens (SAgs) bacterial, 14,208-210 MMTV (mouse mammary tumor), 140, 152-153 expression, 152-153 immune stimulation by, 140, 141, 171-194, 196-203 MHC Class I1 molecules, interaction with, 164-165 protein structure, 157-164 TCR Vp,interaction with, 165-167 tumor formation and, 155, 156

T Tachyzoite antigen, 208 TAP, 96 background, 47-56 disease and autoimmune disease and, 92-94 dysfunction in tumors, 94-95

polymorphism, 92-94 viral inhibitors, 95 gene structure, 58, 59-61 MHC Class I assembly and, 87-92 polymorphism, 71-75,92-94 protein structure as heterodimer, 61-62, 79, 81 intracellular localization, 62-63 subcellular localization, 63, 65 topology, 64, 65-71 tapasin, 91 TAP1,50,85 TAP1 gene, structure, 58, 60-61 TAP2, 51,85 TAP2 gene, structure, 58, 60-61 TAP complex, 75-76 length specificity, 78 peptide and nucleotide binding, 84-86 sequence specificity, 78-84 transport assays, 76-78 transport model, 86-87 Tapasin, 91 TATA box, TAP genes, 60 Tax protein, 29 T cell response cytotoxic T lymphocytes and, 279-280 to MMTV adult, 175-178 endogenous Mtv, 196-208 neonatal, 171-175 T-B cell interaction, 180-188 TCR Vp, interaction with MMTV superantigen, 165-167 Tethering, high endothelial venules, 367 Thymus, deletion of superantigen-reactive T cells, 203-204 Tocopherol, action of, 122 Tolerance induction, Mtv superantigens in, 203-208 Transcription factors, gene regulation, 1 CIEBP, 3-8 IKB family, 14-15 NF-ILG, 1-3, 8-11, 16-18 NF-KB family, 11-12, 15-22,30-32 REL family, 12-13 Transmembrane domain (TMD), 56 Transmembrane spanning segments (TMSs), TAP protein, 66-67 Transporter associated with antigen processing, see TAP

INDEX

Transporters, 47-56 ABC transporters, 55, 56-57, 59 antigen processing see ATP need for, 47-49 TSG-6, 18 Tumor necrosis factor, signaling events, 117 Tumors induction hy MMTV, 153-IS6 TAP dysfunction in, 94-95

Uhiquitin, conjugation of, 117 Upstream induction sequence (UIS), 25

v Vaccination, MMTV infection, 190-191 Vaccines, 323 HIV, 320-322 SIV, 321-322 VAP-1, lymphocyte-HEV interaction, 365

405

Vascular addressins, lymphocyte-HEV interaction, 361-363, 377 Vasoactive intestinal peptide (VIP) gene, 20 VCAM-1, 359 Viral infection cytotoxic T lymphocytes (CTLs) and adenovirus, 279 cytomegdovirus, 278-279 Epstein-Barr virus, 279 herpes simplex virus, 279 HIV, 280-322 influenza virus, 278 NF-IL6 in, 9-11 Viral replication, HIV, CD8' and, 281-282 Viruses NF-KB,strategies to control, 127-128 superantigens of, 208 Vitamin E, action of, 122, 123 VLA-4. 359

Wnt-I genes, 154, 155

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CONTENTS OF RECENT VOLUMES

Volume 61

Analysis of Gene Function in Lymphocytes by RAG-%Deficient Blastocyst Complementation JIANZHU CHEN

CD40-CD40 Ligand: A Multifunctional Receptor-Ligand Pair CEESVAN KOOTENAND JACQUES BANCHEREAU

Interferon-y: Biology and Role in Pathogenesis ALFONS BILLIAU

Antibody Class Switching JANET STAVNEZEH

Role of the CD28-B7 Costimdatory Pathways in T Cell-Dependent B Cell Responses KARENS. HATHCOCK AND RICHARDJ. HODES

Interleukin-2 Receptor Signaling Mechanisms LARRY M. KARNITZ AND ROBERTT. ABRAHAM

Control of the Complement System M. KATHRYN LISZEWSKI, TIMOTHY C. Prostaglandin Endoperoxide H Synthases-1 M. LUBLIN. ISABELLE and -2 FAHRIES. DOUGLAS A. ROONEY. A N D JOHN P. ATKINSON WILLIAM L. SMITHAND DAVIDL. DEWIIT V( D)J Recombination Pathology Human Tumor Antigens Are Ready to Fly KLAUSSCHWARZ AND CLAUS R. BARTRAM ROBERTA. HENDERSON AND OLIVERA J. FINN Major Histoconipatibility Complex Class I1 Deficiency: A Disease of Gene Regulation VIKTORSTEIMLE. WALTER REITH.AND Inflammatory Mediators, Cytokines, and BERNARDMACH Adhesion Molecules in Pulmonary Inflammation and Injury TH1-TH2 Cells in Allergic Responses: At the NICHOLASW. LUKACS AND PETERA. Limits of a Concept WARD AEBISCHERAND BEDAM. STADLER IWAN

INDEX

INDEX

Volume 62

Volume 63

Organization of the Human Immunoglobulin Heavy-Chain Locus FLIMIIIIKO MATSUDAAND TASUKU HONJO

Surrogate Light Chain in B Cell Development

407

408

CONTENTS OF RECENT VOLUMES

HAJIME G R A S U Y A M A , ANTONIUSROLINK, FRITZMELCHERS

Volume 64

AND

CD40 and Its Ligand TERESA M. FOY,A N D LISAB. CLARK, RANDOLPH J. NOELLE Human ImmunodeficiencyVirus Infection of Human Cells Transplanted to Severe Combined Immunodeficient Mice DONALD E. MOSIER Lessons from Immunological, Biochemical, and Molecular Pathways of the Activation Mediated by IL-2 and IL-4 ANGELITAREBOLLO,JAVIER G6ME2, AND CARLOS MART~NEZ-A.

B Lymphocyte Development and Transcription Regulation in Vi m DAVINA OPSTELTEN

Proteasomes and Antigen Processing KEIJI TANAKA, NOBUYUKI TANAHASHI, TSURUMI, KIN-YA YOKOTA, AND CHIZUKO NAOKISHIMBARA Recent Advances in Understanding V(D)J Recombination MARTINGELLERT The Role of Ets Transcription Factors in the Development and Function of the Mammalian Immune System ALEXANDERG. BASSUK AND JEFFREY M. LEIDEN Mechanism of Class I Assembly with P2Microglobinand Loading with Peptide TEDH. HANSEN AND DAVID R. LEE

Soluble Cytokine Receptors: Their Roles in Immunoregulation. Disease, and Therapy RAFAEL FERNANDEZ-BOTRAN, PAULA M. AND YUHE MA CHILTON,

How Do Lymphocytes Know Where to Go?: Current Concepts and Enigmas of Lymphocyte Homing MARKOSALMI AND SIRPA JALKANEN

Cytokine Expression and Cell Activation in Inflammatory Arthritis LIONEL B. IVASHKIV

Plasma Cell Dyscrasias NORIHIRONISHIMOTO, SACHIKO SUEMATSU, AND TADAMITSU KISHIMOTO

Prolactin, Growth Hormone, and Insulin-like Growth Factor I in the Immune System RONKOOIJMAN,ELISABETH L. HOOGHEPETERS, AND ROBERTHOOCHE

Anti-Tumor Necrosis Factor-cY MARCFELDMANN, MICHAEL J. ELLIOTT, N. WOODY, AND RAVINDER N. JAMES MAINI

INDEX

INDEX