Molecular aspects of autoimmunity

Immunology Today, VoL 10, No. 6, 1989

and tCealures C4d-region amino acid changes between residues 1101-1106 (pro C4 numbering); these are Pro-Cys-x-xLeu-Asp in C4A and Leu-Ser-x-x-IleHis in C4B. Construction of C4AC4B hybrid proteins and assay of their haemolytic and binding functions showed that the single substitution of Asp for His at position 1106 largely accounted for the determination of C4B-like or C4A-like functional activity, thus supporting the proposal 16 that the side chains of Asp or His at position 1106 are important in catalysing transacylation to acceptor nucleophiies. The availability of cDNA clones for most of the complement proteins should make it possible to carry out similar studies on them. However, unless sensitive and specific haemolytic or binding assays can bc used to assay the mutant proteins, it may be

334, 475-476 7 DiScipio,R.G., Chakravarti,D.N., M~iller-Eberhard,H.J.and Fey,G.H. (1988) J. BioL Chem. 263, 549-560 8 Kotwal, G.J. and Moss, B. (1988) Kenneth Reidar,d TonyDay are at the MRC Nature 335, 176-178 Irnmunochemistry Unit, Department of Bio- 9 Bevilacqua,M.P., Stengelin,S., chemistry, Universityof Oxford, South Parks Gimbrone, M.A. and Seed,B. (1989) Science243, 1160-1165 Road,Ox;ord0)(13QU, UK. 10 Johnston, G.I., Cook, R.G., McEver, R.P.(1989) Cell 56, 1033-1044 References 11 Siegelman,M.H., De Rijn, M.V. and 1 Klickstein,LB., Wong, W.W., Smith, Weissman, I.L. (1989) Science243, J.A. etal. (1987)J. Exp. Med. 165, 1165-1172 1095-1112 2 Morley, B.J.and Campbell, R.D.(1984) 12 Perkins,S.J., Haris, P.I., Sire, R.B.and Chapman, D. (1988) Biochembtry 27, EMBOJ. 3, 153-157 4004-4011 3 Patthy,L. (1988)J. MoL Biol. 198, 13 Lawler,J. and Hynes,RO. (1986) 567-577 J. CellBiol. 103, 1635-1648 4 Day,A.J. in Biochemistryand Molecular Biology of Complement (Sire, 14 Goundis, D. and Reid, K.B.M. (1988) Nature 335, 82-85 R.B., ed.), MTP PressLtd (in press) 15 Shinkai,Y., Takio, K. and Okumura, 5 rampbell, R.D., Law, S.K.A., Reid, K. (1988) Nature 334, 525-527 K.B.M. and Sim, R.B.(1988)Annu. Rev. 16 Dodds,A.W. and Law, S.K.A.(1988) Immunol. 6, 161-195 6 Stanley,K. and Luzio, P. (1988)Nature Complement 5, 89-97

Sustained humoral and~or cellular autoimmune responsesare currently thought to be the primary causes of a wide spectrum of systemic and organ-spo:ific human and animal diseases.Although a very good picture of the immunopathological characteristics of these diseases has emerged, their etiologies remain unknown. Studies initiated to define these diseases at the molecular geneffc level were the subject of a recent meeting* in Ville D'Esteret, Canada. Because these diseases are diverse and enormously complex, severalavenuesof investigation have been pursued. The primary focus of this meeting was the tripartite s~tem of immunoglobulin (Ig), T-cell antigen receptor (TCR) and major histocompatibility complex (MHC) genes.

Molecularaspeds of autoimmunity

The most extensively covered topic of this meeting was the genetic origin of human and murine humoral anti-self responses, including immunoglobulin (Ig) germline gene organization, the specific genes encoding autoantibodies, and the cellular origin and phenotypic characteristics of these molecules. Other areas covered included TCR

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*Themeetingon The MolecularAspectsof Autoimmunitywasheldin L'Esterel,Quebec,Canadaon 30October- 2 November,1988andwasorganized byN. Faridand C. Bona.

initially more rewarding to delete, insert or 'shuffle' whole domains to probe structure--function relationships.

finn ArgyriosN. ,olilopoulos and ReinhardKofler germline genes, and MHC molecules and their relationship to autoimmune diseases.

able by RFLP analysis (M. Zouali, P~ris); the human Ig VH gene complex has not been analyzed.

Ig genes

Ig genesencodingautoantibodies

Ig gerrnlinegeneorganization

A number of communications reported investigation of autoantibody primary structure, genetic restriction and 'proximal utilization': (use of the so-called 3' VH gene families). Autoantibodies whose variable (V) region primary structures have been reported include human anti-DNA (K. Siminovitch, Toronto), d,eurnatoid factors (RFs) (J.D. Capra, Dallas; P. Chen, La Jolla; F. Goni, New York), and anti-Sm (H. Dang, San Antonio) as well as murine anti-DNA, RFs,antihistone (R. Kofler), anti-bromelainetreated red blood cells (Br-RBC) (J-C. Jaton, Geneva), and anti-Sm (G. Haynes, London). In addition, sequence data for polyreactive human (T. Logtenberg, New York) and highconnectivity murine antibodies (D. Holmberg, Umer) were presented. These (auto)antibodies were encoded by a variety of V, diversity (D) and joining (J) gene segments, several of which had germline sequences (J.D. Capra), but evidence

The possible predisposition of particular Ig haplotypes to autoimmune disease and the possible etiologic role of Ig germline gene defects constitute central issues in autoimmunity. In the mouse, R. Kofler (La Jolla; Innsbruck) presented sequence and restriction fragment length polymorphism (RFLP) data clearly showing the lack of disease association with a particular Ig H and Ig K haplotype, and strongly arguing against Ig germline gene abnormalities being responsible for autoimmunity in all models investigated to date. The latter conclusion is, however, still controversial, since C. Bona (New York) reported V, gene abnormalities in MRL and C57BL/6 tsk autoimmune mice 1, and also suggested an important role of the unique NZB V~ haplotype for lupus in (NZB x NZW)F1 mice. In human lupus, V~ loci from patients and normal controls appear indistinguish-

(~) 1989. Elsevier Science Publishers Ltd. UK. 0167-4919/89/$02.00

Immunology Today, Vol. 10, No. 6, 1989

for somatic mutations was also pre- natural and other autoantibodies (C. sented (T. Logtenberg). The primary Bona). structure did not suggest abnormalities in the molecular generation of Autoantibodiesand Ly-1÷ Bcells Ly-1 ÷ B cells are a long-lived these autoantibodies, and did not provide any clues as to why these self-renewing B-cell subset that primolecules escape tolerization, or in marily produce (but may not readily certain cases, may be pathogenic. secrete) polyreactive IgM antibodies Definite conclusions will have to with little or no somatic mutation 3.4. await three-dimensional structure A.L. Notkins (Bethesda) identified determinations or computer predic- EBV-transformed human Ly-1+ B tions, as shown for anti-carbo- cells from normal donors as the main hydrate antibodies (E. Kabat, New source of 'natural' autoantibodies. This population is increased and York). While the overall disease- hyperactive in rheumatoid arthritis associated anti-self response in mice, (RA), but apparently normal in sysand possibly also in humans, may be temic lupus erythematosus (SLE).Furessentially unrestricted, autoanti- thermore, EBV-transformed CD5 + B bodies with particular specificities cells from RA patients produced two (and shared idiotypes) can be quite types of RF with discrete reactivities: biased in their V gene usage, for the first is polyreactive, binds to IgG example, clonally unrelated anti- Fc with low affinity, and is similar to Br-RBC autoantibodies were encoded RFs produced by CD5 + B cells from by identical VH/D/JH and V,IJ, gene healthy subjects; the second is segments (J-C. Jaton), and human monoreactive, binds with high afpolyspecific and RF-like paraproteins, finity to IgG Fc, and is produced by although encoded by a variety of VH CD5 + cells from RA patients only. genes, preferentially use VJIIb genes The pathogenic role of this Leu-1+ (F. Goni; P. Chen). The correspond- B-cell subset in autoimmune dising germline genes have now been eases, particularly RA, remains cloned (P. Chen). Other anti-self re- controversial 5. Data concerning sponses, such as the anti-DNA re- murine Ly-1+ B cells were discussed sponse in human lupus (K. Simino- by C. Bona. The vast majority of vitch; K.J. Barrett, Boston), and the hybridomas from viable motheaten anti-DNA and RF responses in lupus mice (mainly Ly-1+ B cells) did not mice (R. Kofler), use a large number secrete Ig, but more than 50% of of different Ig gene segments (see secretors produced poiyreactive antibodies. In contrast to human CD5+ B Ref. 2 for review). Several reports discussed biased cells, these hybrids expressed VH use by autoantibodies of 3' VH gene genes randomly with only a slight families. T. Logtenberg observed bias toward the proximal 3' VH gene random VHgene use in approximate- families, but showed overreprely 1000 Epstein-Barr virus (EBV)- sentation of certain V, gene families transformed B-cell clones from nor- (V,1, V,4, V,9, VK10). mal adults and, interestingly, from neonates as well. However, Leu-1+ Phenotypiccharacteristicsof autoantibndies (CD5 +) B cells (perhaps the main andautoantigens Fine specificity and idiotypy of source of natural polyreactive (auto)antibodies, paraproteins, and B-cell murine anti-histone antibodies, and malignancies), preferentially use structural analysis and homo!ogy be3'VH genes. A bias for such VH genes tween retroviral gag sequences and was also reported by D. Ho!mberg the autoantigen recognized by for fetal- and adult normal mice- mouse and human anti-RNP autoderived natural, high connectivity antibodies were reported by M. hybridorna antibodies (frequently Monestier (Newark) and I. Pettersson poly- and self-reactive). However, (Stockholm), respectively. D. Holmproximal VH gene use may not apply berg reported high connectivil~ to autoantibodies associated with, among 'natural', often poly- and ancl possibly causing, autoimmune self-reactive, antibodies from fetal or disorders, at least in mice (R. Kofler). neonatal normal mice, while hybriInterestingly, proximal V gene use doma antibodies from adults lack may not occur in the kappa light this phenotype; this suggests an imchain locus: the most 3' murine V~ portant role for this network in the gene family (V, 21) is not over- formation of the immune repertoire expressed in the fetal repertoire or in in physiological and autoimmune

states. The importance of Tyr95 in the complementarity-determining region (CDR) 3 of an antithyroglobulin antibody for anti-idiotypic recognition was determined by sitespecific mutagenesis experiments by M. Zanetti (San Diego). This promising approach, combined with X-ray crystallography, will hopefully shed more light on the structural basis of (auto)antibody-anti-idiotype interactions.

Generallg genetics The above studies led to several observations that, while not directly related to autoirnmunity, are significant to general Ig genetics. Perhaps most important was the observation by J.D. Capra of D segment inversions and duplications as a novel mechanism generating additional diversity in Ig heavy chains. J-C. Jaton described a new, small VH gene family (VH11), associated with anti-BrRBC autoantibodies that is, along with VH10 (defined as an anti-DNA autoantibody; Ref. 6), the second murine VH gene family discovered in the course of analyzing autoantibodies. J-C. Jaton, M. Zanetti, and E. Sercarz (Los Angeles) reported the astounding observation of identical V/D/J rearrangements in clonallyunrelated hybridomas, which implies that the sequences at the V/D and D/J boundary are not, as previously thought, randomly generated. Controversial data concerning polymorphism of the human VH gone complex were also reported. Based on repeated isolation of identical VH gene sequences from members of different ethnic groups, J.D. Capra suggested that this locus may be essentially monomorphic in man; this conclusion was challenged by K.J. Barrett, who provided evidence for polymorphism in the population using CDR oligonucleotide probes. TCRgenes

TCRgermlinegenes Relevant findings from autoimmune animal models were discussed by A.N. Theofilopoulos (La Jolla), who stressed the potential significance of unique, deleted, or allelic TCR genes as well as tolerancerelated repertoire modifications, as predisposing factors in the development of autoimmune syndromes. Data were presented documenting: (1) a deletion of the CI31, DI32 and Ji32 elements in NZW mic& (also

181

e ,s nd eatures identified by others 8) that does not appear to correlate with autoimmune disease expression; (2) lack of new V13 genomic polymorphisms among several normal and autoimmune strains other than the two previously described hap!otypes 9, one termed V~a, encompassing strains (SWR, SJL, C57L, C57BR) that have deleted approximately 50% of their V~ repertoire, and another, termed V~b (or non-deleted), encompassing al~ other tested murine strains; (3) coding sequence polymorphisms, determined by RNAse protection analysis, in the V~I, 3, 6 and 10 gene products between these two V~ haplotypes (preliminary studies by C. David and associates ~° suggest that the V~6 allele of the non-deleted V~ haplotype is required for responses to collagen type II and ~rthritis development in appropriate H-2q mice); and (4) a rather extensive polymorphism in murine V~ genes ~, with four major haplotypes identified (V,,a, V~b, V~c, V~d). No unique TCR haplotype associated with autoimmune disease predisposition has been identified.

and gld homozygous mice, combined with the demon~boted p3~irip#tion of CD4 (Refs 17, 18) and CD8 (Ref. 19} in anti-self MHC class II or antiself MHC class I clonal deletions, respectively, and the inhibitory effects of anti-CD4 antibody in the lymphoid enlargement of these mice 2o, suggest that the Iprlgld cells have passed through the double-positive stage. Using anti-V~-specific antibodies, Kotzin et al. 21 have recently drawn a similar conclusion. It was suggested that double-negative, TCR e~+ Iprlgld lymph node cells (and a subset of normal double-negative TCR al 3+ cells) are probably derived through a CD4/CD8 loss pathway involving a novel negative selection mechanism acting on thymocytes with moderately autoreactive TCRs that escape conventional clonal elimination, but down-regulate their accessory molecules. This suggestion is compatible with the reported autoimmune behavior of doublenegative, TCR al~ +, cells22-24. Thus, export of such cells in large numbers to the periphery may be an important contributor to the induction of systemic autoimmunity.

Tolerance-relatedT-celldonal deletions

Applying a multiprobe RNAse protection assay, Theofilopoulos reported the confirmation, at the RNA level, of the previously described I-E-rela~ed V1317a (Ref. 12) and I-FJMIsa-related VI~6 (Ref. 13) and Vi38.1 (Ref. 14) clonal deletions in peripheral T cells of appropriate mice. He further described the identification of several new tolerance-related clonal deletions, including V1~3.1 in I-E+IMIs ~ mice (also identified by othersl5,16 he interim), VI~7 and V69 in iV/Isa mice, and V~5.1, 11, 12 ancl 16 in I-E+ mice. T-cell subset analyses show that although, as previously suggested 13.~7.~8, many of these deletions occur at the doublepositive (CD4+CD8 +) stage of T-cell differentiation, some (Vl311, Vl312) appear to take place aTter the conversion of these cells to the singlepositive CD4+CD8 - subset. Dependmg on [heir Mis and i-E hapiotypes, all lupus mouse strains were found to exhibit the corresponding major clonal deletions outlined above, including mice congenic for the Ipr or gld autoimmunity inducing/ enhancing genes. As discussed by P.A. Singer and A.N. Theofilopouios (submitted) this latter finding in Ipr

T cellsin autoimrnune diseases

The primary role of T cells in the rat model of HgCI2-induced autoimmunity was addressed by P. Druet (Paris), who indicated that this disease requires, and can be transferred by, T cells (primarily anti-self MHC class II reactive) of afflicted donors as well as by naive T cells incubated in vitro with HgCl2. Cytotoxic, MHC class I-restricted, T-cell clones specific for thyroglobulin epitopes, and corresponding anti-idiotypic Tcell clones were also described (J. Charreire, Paris). In addition, an expansion of V~8+ T cells was identified in peripheral blood and draining lymph nodes of patients with Crohn's disease in addition to autoantibodies cross-reacting with V~8 products and epithelial cells (D.N. Posnett, New York). This increase did not appear to correlate with disease activity, and was also found in healthy family members; similar increases of VI38+ cells were seen in peripheral blood and lungs of sarcoidosis patients. Finally, MHC class II-expressing intestinal epithelial cells from normal indMduals were reported (L. Mayer, New York) to preferentially induce CD8 + suppressor T cells, a finding compatible with

Immunology Today, Vol. 10, No. 6, 1989

the dominant presence of CD8 + cells in murine ana human intestinal intraepidlulium ~,26 Importantly, such cells from patients with inflammatory bowel diseases (Crohn's disease, ulcerative colitis) instead induce CD4 + T cells; it was suggested that normal intestinal epithelial cells express a novel CD8 ligand that is reduced or lost during these inflammatory processes, and thus suppressor T-cell engagement is defective. iVlHCgenes Insulin-dependent diabetesmellitus

J. Todd (Stanford, Oxford) reported his collaborative work with H.O. McDevitt (Stanford) on the relationship of MHC structures to autoimmune disease susceptibility, particularly insulin-dependent diabetes mellitus (IDDM), where a good correlation between DR3 and/or DR4 and, in particular, certain DQ molecules (mostly DQw3.2 and DQw2) has been identified. Gene segments encoding the amino-terminal polymorphic domains of such molecules were sequenced and found to be similar in IDDM patients and normal controls, that is disease susceptibility is not caused by mutant class II alleles found exclusively in IDDM patients. However, close scrutiny indicated that nearly all DQ~ chains commonly (>90%) found in diabetics have a neutral residue (Ala, Val or Ser) at position 57. In contrast, DQi3 chains, rarely (<10%) found in IDDM Caucasian patients, have Asp at this position. DQ-mediated IDDM susceptibility (non-Asp at position 57) was found to be recessive, and DQ-mediated resistance (Asp at position 57) was found to be dominant. Remarkably, this correlation extends to the NOD (non-obese diabetic) mouse, whereby its DQI3homoiogue, that is, I-AI3 is unique at codon 57 (Ser in NOD, Asp in non-diabetic strains). Other studies (reviewed in Ref. 27) suggest, however, that this rule is not absolute, since there are several haplotypes with a neutral residue at position 57 that do not confer high susceptibility (for example DR7), and some with Asp57 that seem to be disease-associated (for example DR4 and DRw9 in Japanese). The increased risk for DR3/4 individuals relative to DR3/3 or DR4/4 is also difficult to reconcile with the simple residue-57 model. Nevertheless, whether the DQ structure affected

Immunology Today, VoL I0, No. 6, 1989

fe i.res-

g

by non-charged or charged amino acids provides susceptibility or resistance, respectively, by affecting the binding of a putative diabetes autoantigen, or through positive and negative T-cell repertoire selection, remains speculative. Of interest in this regard is the observation 28 that introduction of an I-Ec~ gene into the I-E- NOD mouse results in inhibition of diabetes. Thus, instead of, or in addition to, the I-AI~ structure, the absence of I-E, and possibly the resultant defects in the I-EIMIsdependent T-cell negative selection processes outlined above, may play an important role in IDDM susceptibility. Repertoire analysis of NOD mice and of appropriate crosses and transgenic mice will provide some answers. Other genes and environmental or triggering factors may also play a role. Viruses may be prime candidates among the variety of triggering factors, as outlined by J-W. Yoon (Calgary), who showed activation of endogenous retroviruses in islet 13 cells of NOD mice. Rheumatoidarthritis The structural correlates of MHC with susceptibility to RA were addressed by R. Winchester (New York), who emphasized the importance of a 'shared epitope' as a unifying principle in explaining correlations of RA with serologically diverse MHC alleles such as DR4 (Dw4, 14, 15), DR1 (Dwl), DRwlO and DRw53, but not DR4-DwlO or DR4-Dw13. Nucieic acid sequencing studies indicate that these diverse haplotypes associated with susceptibility exhibit cis-encoded sequence homologies (al!owing for some conservative substitutions) in their DRI]I third variability region (residue 67 to 74). These homologies, attributed to the gene conversion events, might result in a conformationally-equivalent oLhelical structure in these otherwise serologically and, overall, structurally different molecules, that dominantly confer susceptibility to RA. It was further suggested that two different DR alleles may trans-complement each other to recreate this same set of susceptibility confering third diversity region conformations. Despite the concerted efforts outlined above, the molecular basis of autoimmune diseases remains elusive. It is very clear, however, that important advances have been made

in several directions that may soon lead to the basic finding that will have the quantum effect in unraveling the mysteries of these disorders. This is Publication Number 57691MM from the Department of Immunology, Research Institute of Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, CA 92037, USA. Argyrios Theofilopoulos and R. Kofler are at the Department of Immunology, ScrippsClinic and ResearchFoundation, North Torrev Pines Road, La Jolla, CA 92037, USA; and Reinhard Kofler is also at the I,~stitute for General and Experimental Pathology, University of Innsbruck, Austria. References

1 Bona, C.A. (1988)Annu. Rev. Immunol. 6, 327-358 2 Kofler, R., Dixon, F.J. and Theofilopoulos, P,.,~'.(1987)!mmunol. Today 8, 374-380 3 Hayakawa, K. and Hardy, R.R.(1988) Annu. Rev. Immunol. 6, 197-218 4 Foerster, I., Gu, H. and Rajewsky, K. (1988) EMBOJ. 7, 3693-3703 5 Kipps, T.J. and Vaughan, J.H. (1987) J. Immunol. 139, 1060-1064 6 Kofler, R. (1988)J. Immunol. 140, 4031-4034 7 Noonan, D.J., Kofler, R., Singer, P.A. et al. (1986)J. Exp. Med. 163, 644-653 8 Kotzin, B.L., Barr, V.L. and Palmer, E. (1985) Science 229, 167-171 9 Behike, M.A., Chori, H.S., Huppi, K. and Loh, D.Y. (1986)Proc. NatlAcad. Sci. USA 83, 767-771 10 Haggi, R.M., Banerjee, S., Behlke, M.A. eta/. (1988) FASEBJ. 2, A661 11 Singer, P.A., McEviily, R.J., Balderas, R.S., Dixon, F.J.and Theofilopoulos, A.N.

(1986) Proc. Natl Acad. Sci. USA 85, 7729-7733 12 Kappler, J.W., Roehm, N. and Marrack, P. (1987) Cell 49, 273-280 13 MacDonald, H.R., Schneider, R, Lees, R.K. etai. (1988) Nature 332, 40-45 14 Kappler, J.W., Staerz, U., White, J. and Marrack, P. (1988) Nature 332, 35-40 15 Pullen, A.M., Marrack, P. and Kappler, J.W. (1988) Nature 335, 796-801 16 Abe, R., Vacchio, M.S., Fox, B. and Hodes, R.J. (1988) Nature 335, 827-830 17 Fowlkes, B.J., Schwartz, R.H. and Pardoll, D.M. (1988) Nature 334, 620--623 18 MacDonald, H.R., Hengartner, H. and Pedrazzini, T. (1988) Nature 335, 174-176 19 Kisielow, P., Bluthmann, H., Staerz, U.D., Steinmetz, M. and von Boehmer, H. (1988) Nature 333, 742-746 20 Santoro, T.J., Portanova, J.P. and Kotzin, B.L. (1988) J. Exp. Med. 167, 1713-1718 21 Kotzin, B.L., Babcock, S.K. and Herron, L.R. (1988)J. Exp. M,ed. 168, 2221-2229 22 Datta, S.K., Patel, H. and Berry, D. (1987)J. Exp. Med. 165, 1252-1268 23 deTalance, A., Regnier, D., Spinella, S., Morisset, J. and Seman, M. (1986) J. Imrnunol. 137, 1101-1108 24 Sakaguchi, S., Fukuma, K., Kuribayashi, K. and Masuda, T. (1985) J. Exp. Med. 161, 72-87 25 Goodman, T. and Lefrancois, L. (1988) Nature 333, 85_~-857 26 Carter, L., denHollander: N., Ramaswamy, K., Riley, D. and Befus, D. (1988) Immunol. Today9, 368-369 27 Erlich, H.A. (1989) Nature 337, 415 28 Nishimoto, H., Kikutami, H., Yamamura, K. and Kishimoto, T. (1987) Nature 328, 432-434

ImmunologyTodaySpecial issue To coincide with the forthcoming International Congress of Immunology in Berlin, Immunology Todaywill be bringing out a special August issue, containing a bound-in supplement on the major areas of immunological research in the Federal Republic of Germany. 183