Multiple sclerosis: From immunogenetics to immunotherapy

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Journal of the Neutvlogical Sciences, 115 (Suppl.) (1993) $29- $37 ~ 1993 Elsevier Science Publishers B.V. All rights reserved 0022-510X/93/$06.00

JNS 04013

Multiple sclerosis: from immunogenetics to immunotherapy Jorge R. Oksenberg, Michael A. Panzara and Lawrence Steinman D~g)artment of Neurology and Neurological Sciences, &anJbrd UniversiO, School of Medicine, &anJb,d, Calilornia, ~'),:4

Key words'. Multiple sclerosis; lmmunogenetics; Immunotherapy

Introduction Among demyelinating diseases of the central nervous system, the most common is multiple sclerosis (MS). Despite intensive scientific and clinical research on MS, at present nearly all manuscripts on the subject state: "...The etiology and pathogenesis of multiple sclerosis are unknown, but it is probable that a combination of environmental factors in a susceptible host are manifested as an autoreactive phenomenon". A number of candidate infectious agents, possibly viruses, may mimic self-antigens or may unmask sequestered self-antigens. These auto-antigens are recognized by products of complex genetic systems like the immunoglobulin supergene family, and may activate genes encoding cytokines. In the brain, at the site of demyelination, there is an accmnulation of macrophages, plasma cells, major histocompatibility complex (MHC) class 11 positive antigen presenting cells and activated T cells secreting various cytokines. This is in tact the foundation for the general recognition of MS as an autoimmune disorder. Based on the hypothesis that MS is mediated by an aberrant inmmne response, many forms of imnmnotherapy have been attempted in MS in the past decade. In spite of some encouraging preliminary results, there is still no definitive evidence of the therapeutic value of such treatments as plasmapheresis, levamisole, transfer factor, gamma-interferon (y-IFN), retinoic acid, and others. Some of these approaches, like "y-IFN actually exacerbate MS. Some immunosuppression protocols in MS (Rosen 1979; ttauser et al. 1983; Ellison et al. 1989; The Multiple Sclerosis Study Group 1990; The Canadian Cooperative Multiple Sclerosis Study Group 1991) must be considered as experimental treatments at this time, while in others benefits do not outweigh the risks (Goodin 1991). A comprehensive review of nearly 50 clinical trials currently in progress for MS, is beyond the scope of this paper. The reader is referred

Correspondence to." Dr. J.R. Oksenberg, Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA 94305-5235, USA.

to the Clinical Trials Report compiled by' the National (USA) MS Society, April 1991. We will examine some recent clinical and experimental reports that might advance our understanding of demyelination in general and MS in particular.

Competitive binding of peptides to the major histocompatibility complex In order to stinmlate a T cell response, protein antigens must be processed by antigen presenting cells into peplides that bind to MHC class I and class I1 molecules (Allen 1987; Bjorkman 1987). Similarly, MHC molecules must bind selfpeptides in order to activate T cells responsible for the autoimmune process. Therefore, by inhibition of this binding it should be possible to block the stimulation of autoaggressive inmmno-pathogenic T cells. Exogenous peptides have been proposed as potential candidates to mediate such inhibition. In experimental allergic encephalomyelitis (EAE), a prototype for antigen-specific T cell-mediated autoimnmne disease and the putative animal model of MS, the detailed study of the molecular interactions between MHC-T cell receptor (TCR) and the myelin basic protein (MBP) epitope Ac (acetylated) l 11 lead to attempts to use in vivo competition between selfpeptides in order to modulate the induction of autoreactivity (Zamvil et al. 1990) (Tables 1 and 2). AcN1 11 is a strong pathogenic peptide for PL/J and (PLSJ)F1 mice, and T cells that can recognize this self-antigen mediate encephalomyelitis in these strains of mice. Non-encephalitogenic, but inmnmogenic, MBP peptides 1 20 and Ac9 20 suppressed proliferation to the encephalitogenic peptide AcN1 l l both in viw~ and in vitro. The peptides p l 20 and At9 20 significantly prevented EAE induction at a 3:1 coimmunization ratio with AcN 1 11 [9,10]. In reviewing representative sections of 20 mice treated with competitors (N 1 20 and AcN9 20), which did not show any clinical signs of EAE, no perivascular cuffs or submeningeal cell infiltrates were e,,ident (Steinman 1991). Additional studies have revealed that a single amino acid

$30 TABLE 1 DISCRETE T-CELL EPITOPES OF MBP IN MICE Peptide

Encephalitogenicity

Class II restriction

TCR V usage

Acl-9

+

I-ActUAl3u

Va4.3 and VI38 predominantly

Acl-ll

+

I-ActUAl~u

Vot4.3 and VI38 predominantly

Acl-20

+

I-AaUAI3 u

VI~8 preedominantly

1-20

+

I-AGtUAI3u

VI38 preedominantly

5-16

-

I-Ao~UAI3u

VI38 preedominantly

I-ActSAl3u 17-27

?

35-47

+

I.AotSA~ s

Not determined

I-EctUEI3u

Not VI~8

l-EctUEI3s 89-100

-

I.ActSAl3 s

Not determined

89-101

+

l-ActSAI3s

VI~ 17 predominantly

96-109

+

1-A~tSAI3s

Not determined

92-103

+

1.ActSAI3s

Not determined

substitution in the Acl-11 sequence, alanine for lysine at position 4, yields a peptide that binds with greater affinity than does A c l - l l to I-Au. This peptide is also able to powerfully stimulate in vitro encephalitogenic T cell clones responsive to Ac 1-11. This peptide was predicted to be a strong inducer of disease. Surprisingly, mice coimmunized with Acl-11 [4a] were protected from EAE (Wraith et al. 1989) (Table 2). From these results, it is clear that a variety of synthetic peptides can have regulatory effects on the autoimmune phenomenon. This raises the possibility of therapy using "designer" peptides, competing for the antigen binding site. Attempts to reverse established disease have been only partially successful (Smilek et al. 1991). At present, we are not

yet able to accurately predict which peptide will be an effective therapeutic agent, and which epitope should be blocked. The information about immunodominant antigen epitopes in multiple sclerosis (MS) reveals a complicated picture (Table 3). Several groups have now identified immunogenic epitopes of MBP in MS patients and in healthy controls. Hailer and colleagues showed that both patients and controls who were DR2, DQwl proliferate to MBP:84102. A second epitope related to DRwl l was MBP:I 43168 (Ota et al. 1990). Martin et al. (1990) have reported results in studies with cytotoxic T cell lines that recognize MBP. Both MS patients and healthy controls responded frequently to MBP:87-106, but several HLA DR molecules could serve as restriction elements. Finally, Chou et al. (1991) demonstrated that T cell clones from HLA DR2 homozygous donors responded to a variety of epitopes, indicating that this molecule is permissive in its ability to restrict T cell responses. Some epitopes, like MBP: 149--170 were restricted by different MHC molecules. Overall, these results suggest that therapies aiming to block the MHC-peptide interaction may be more complicated to implement in human systems than anticipated from experimental systems. An alternative possibility is the use of synthetic peptides to induce tolerance to MBP. Characterization of natural peptides bound by class I molecules shows that they are smaller than expected, about 7-9 amino acids (peptides of 12-15 amino acid are frequently used in in vitro experiments), matching more precisely the crystallographic data currently available (Schumacher et al. 1991). From sequencing both peptide mixtures and individual peptides bound by particular MHC molecules, conserved motifs of "anchoring residues", one at the C terminus and others close to the N terminus, distinguish sets of binding peptides for different class I molecules (Sette et al. 1992). The extension of these findings to class II molecules (Hunt et al. 1992) may lead to a more rational design of therapeutic blocking peptides. In addition, since it

TABLE 2 PEPTIDE THERAPY ON H-2 u MICE

Peptide

Sequence

Encephalitogenic capabilities

Stimulates encephalitogenic T cells

Binding to I-A u

Acl-9

Ac-ASQKRPSQR

induces disease

+++

+

Ac 1- 11

A¢-ASQKRPSQRHG

induces disease

+++

+

Ac 1-11 [4A]

Ac-ASQARPSQRHG

inhibits disease

+++

+++

ASQKRPSQRHG SKYLATAST

inhibits disease

+

inhibits disease

4-

1-20

Ac9-20

Ac-RHGSKYLATAST

1-11

Ac-ASQKRPSQRHG

?

Ac2-11

Ac-SQKRPSQRHG

?

At1-11 13A,4A]

Ac-ASAARPSQRHG

+4-+

$31 TABLE 3 DISCRETE T-CELL EP1TOPES OF MBP IN MULTIPLE SCLEROSIS Epitope 84-102 142-168

87-106

Several

Class II restriction

TCR usage

Comments

DR2,DQw 1

V]317 and V]312

DRwl 1

V]317 infrequent

Over 15 000 short-term T-cell lines were established. The epitope 142168 was recognized equally in MS patients and controls

DR2, 4

V]36.6-J132.3

DR2(*1501)

V[35.2_JI32.4

DR4,13

V[38-J[31.6/1.4

HLA-DR2

V135.2 (predominant)

144 45-89

HLA-DR

Heterogeneous

One epitope is recognized in the context of four different HLA molecules and different TCRs

HLA-DR2 as a permissive restriction element for a variety of epitopes MBP specific lines could be isolated with comparable efficiency from MS patients and controls

90-170 90-170 149 162

H LA-DR

149 171

is possible that a given MHC molecule has a single functional antigen-binding site, peptides from unrelated antigens can compete with self-peptides for T cell activation. For example, it was possible to block the proliferative response of acetylcholine receptor a-subunit-T cell clones and lines by the I-A b restricted synthetic peptide (T,G)-A L, but not by the I-A k restricted polypeptide (H,G)-A-L (Brocke et al. 1990). Results with other substances like copolymer I (Cop 1) appear promising. Cop I decreased the exacerbation rate and slowed disease progression, particularly in relapsing-remitting MS patients (Bornstein et al. 1987). Cop I may act by blocking the presentation of MBP by HLA class II molecules or generation of antigen specific suppressor cells.

Major histocompatibility complex genes and disease susceptibility In 1981 it was demonstrated by Steinman, McDevitt and colleagues that EAE could be prevented by injection of anti I-A antibodies prior to immunization with spinal cord homogenate (Steinman et al. 1981). In rhesus monkeys, treatment of paralytic disease was successful with polymorphic anti HLA-DQ or HLA-DR antibodies that react with RHLAD (Jonkers et al. 1988). Therapy with monoclonal anticlass II antibodies is a very efficient way to control autoimmunity in experimental systems. It is partially specific, blocking only responses restricted by a given class II isotype. For example, although anti I-A blocks EAE, experimental myasthenia gravis and thyroiditis, in each of these

Heterogeneous

Predominant V[3 gene usage among each individual. No correlation with the epitope or LHA restriction molecule

diseases responses to PPD were left intact (Waldor et al. 1983; Vladutui and Steinman 1987). At present however, no specific HLA allele or sequence has yet been identified which is essential for the development of MS and could be a potential target of therapeutical monoclonal antibodies (mAbs) (Cowan et al. 1991). The association between a particular HLA determinant and multiple sclerosis was first described in 1972 (Bertrams et al. 1972; Maito et al. 1972). Since then a large number of studies have been done to correlate HLA antigens with MS. The results consistently showed an association with HLA-DRwl5 (formerly DR2/Dw2) in Northern European and North American Caucasoid populations, with relative risks ranging from 2 to 4 (Jersild et al. 1973). The association with HLA-DR2 was also evident in Black American patients (Dupont et al. 1977). In addition, a distinction was made in Australian patients between a relatively benign exacerbating-remitting MS of early onset, associated with the haplotype HLA-A3, B7, DR2; and more severe, progressive MS of later onset, associated with the haplotype HLA-A1, B8, DR3 (Hammond et al. 1988). In Italian patients, the association with DR2 was confirmed, and was found to be related to the severity and age of onset of disease (Veneroni et al. 1986). In other populations, other haplotypes have been associated with the disease. For example, HLA DR4 shows a moderate increase in Jordanian Arab and Sardinian MS populations (Kurdi et al. 1977; Marrosu et al. 1988), while HLA-DRw6 in Japanese (Naito et al. 1978) and in Mexican MS populations (Gorodezky et al. 1986). All these associations are however, relatively weak and need to be confirmed in larger

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studies. In fact, a recent study by Spurkland et al. (1991) shows no significant deviation in frequency of any HLA class II specificity in Japanese MS patients. No HLA-D region antigen is significantly associated with MS in lsraeli Jews (Brautbar et al. 1982). With more detailed molecular information about the HLA-DQ loci, it was possible to evaluate its role in MS susceptibility. Indeed, some studies suggest that the association with DRwl5 (DR2), was merely a marker for the stronger association with DQw6 (consisting of DQA 1"0102/DQB 1"0602 heterodimers encoded either in cis or trans configuration) (Olerlup et al. 1989; Vartdal et al. 1989; Spurkland et al. 1991). This association was not confirmed in a large group (n = 179) of Swedish MS patients (Olerlup et al. 1991). Recently, an association between HLA DQB 1 "0201 and "0203 and MS susceptibility in Sardinian patients was reported (Marrosu et al. 1992). The almost exclusive use of HLA-DR gene products as restriction elements by T cell clones responding to MBP, or to fragments of MBP also complicates the potential role of HLA DQ in MS pathogenesis. As was hypothesized by Attman et al. (1991), the role of HLA-DQ may be manifested in thymic repertoire selection. From these results it is clear that HLA-DR/DQ alleles alone can not determine susceptibility to MS. If other HLA loci contribute to susceptibility, they cannot be within or tightly linked to the DR/DQ loci. HLA-DP genes have little or no linkage disequilibrium with DQ and DR genes. In one study, 42 of 45 MS patients carried DPw4 as determined by primed lymphocyte typing (Odum et al. 1988). On the other hand, using the polymerase chain reaction (PCR) and nonisotopic sequence specific oligonucleotide (550) probes for the HLA-DP13 locus, no DP[33 allele was found to be increased in Californian (n = 24) or Japanese (n = 23) patients by Begovich et al. (1990). These results were recently confirmed for by Japanese, French Canadian and Danish study groups (Fugger et al. 1990; Howell et al. 1991; Spurkland et al. 1991). In summary, considerable contradictory data have been reported by different groups. Geographical and ethnic variation may account for some of the differences. It is likely that different genetic backgrounds may provide different susceptibility patterns for various environmental factors. Most probably, in addition to environmental influences, other loci may also be involved. Allelic variants of the human peptide transporter genes involved in antigen processing have been described (Colonna et al. 1992; Monaco 1992; Powis et al. 1992), and their possible involvement in susceptibility to MHC-associated diseases, including MS, is under investigation in a number of laboratories. Since the antigen specific T cell receptor plays a central role in the immune recognition, variations in the genes encoding this receptor could potentially contribute to the development and inheritance of autoimmune diseases.

The T cell receptor and the development of selective therapy The ideal therapy for autoimmune diseases would be to selectively eliminate or tolerize the autoagressive cells. Investigations in EAE shows that in PL/J and PLSJ Fl mice the majority o f T cell clones reactive to MBP share a similar specificity for the encephalitogenic peptide fragment of the protein, associated with particular MHC molecules and TCR proteins. These clones are specific for the N-terminal nonapeptide ofMBP Ac 1-9, and are restricted to I-Au. They use mainly the same TCR V138.2 segment and Va2 segment with restricted heterogeneity in the junctional region (AchaOrbea et al. 1988; Wraith et al. 1989; Steimnan 1991). Consequently, a possible approach would be to generate mAbs that target unique determinants expressed on these immunopathogenic T cells. Successful prevention and even reversal of EAE was achieved using an anti TCR V138 antibody (Acha-Orbea et al. 1988). Other studies noted that cocktails of mAbs directed against different TCR V regions might be necessary to optimize antibody treatment (Sakai et al. 1989; Zaller et al. 1990). The situation in SJL/J mice is complicated by the presence of nested epitopes among the major immunostimulatory regions of MBP. Sakai and coworkers found that only half of the encephalitogenic clones recogmzing p89-101 utilized V[317, and anti-V1317 alone does not ameliorate disease induced with MBP (Sakai et al. 1989). This result suggests that the identification of immunopathogenic T cells in human disease could be a formidable challenge. An early approach to identify variations in TCR genes that could affect the development of disease was based on the detection of polymorphic markers using restriction fragment length polymorphism (RFLP) analysis. Several polymorphisms in a and 13 human TCR genes have been described (Robinson et al. 1985; Concannon et al. 1987) and, in certain cases were found to correlate with incidence of MS (Martell et al. 1987; Beall et al. 1989; Oksenberg et al. 1989; Seboun et al. 1989; Charmley et al. 1991; Sherrit et al. 1992;). In contrast, some studies have revealed no associations between germ-line polymorphisms of the TCR and susceptibility to MS (Fugger et al. 1990; Hillert et al. 1991; Hashimoto et al. 1992). The RFLP-defined TCR haplotype V138, V1311, C13 was linked to DR2-positive MS patients, with a relative risk of 3.3 by Beall et al. (1989). This study was followed by the description of a new DNA polymorphism within the TCR variable region gene segment which appears to localize the MS susceptibility gene 5' (centromeric) of V138 (Charmley et al. 1989). These results suggest that the susceptibility gene does not map to the D, J or C region genes of the TCR, and help to explain some negative studies showing a lack of association of TCR 13 genes and MS. In a recent "Report Card on the Genome

$33 Project", ttood describes the localization of this MS susceptibility gene to a region on chromosome 7, just 175 000 bases long. He and his colleagues will sequence this entire region in the search for the MS susceptibility gene(s). We have shown that polymorphic markers in genes encoding the Vetl2.1 and Cot chains of the TCR are related to MS in three geographically distinct MS populations: California, Australia and Japan (Oksenberg et al. 1989). A synergistic effect of TCR Cet region gene and HLA-DPB 0401 on the relative risk to develop MS, was observed by Bernard and colleagues in the Australian population (Sherrit et al. 1992). However, thus far there has been no demonstration that these germline genes which are associated with disease susceptibility actually play a functional role in disease pathogenesis. Another approach to verify the TCR involvement in MS is to test families with multiple-affected siblings sharing TCR haplotypes. Seboun and colleagues studied the inheritance of TCR [3 chain genes in the germ line of families with 40 sibling pairs with relapsing/remitting MS using both human and murine probes (Seboun et al. 1989). The mean proportion of TCR beta haplotypes identical by descent inherited by MS sibling pairs was significantly increased compared with expected values, whereas the distribution of haplotype sharing was random when MS patients were compared to their unaffected siblings. The association between MS and the sharing of TCR [3 haplotypes in families was not absolute. Nevertheless, these data support the concept that TCR genes contribute to MS susceptibility (Steinman et al. 1992). Several groups have analyzed the distribution of rearranged TCRs in T lymphocytes reactive to MBE Some of these studies indicate that these T cells may rearrange a restricted number of TCR Vet and V[3 genes (Table 3). Wucherpfennig studied 83 T cell lines in man that react to the peptide MBP:84 102 (Wucherpfennig et al. 1990). In one patient who was HLA-DR2/DR7, 24 of 31 clones used V[3 17. Other clones from other DR2 patients used V[31, 3, 4, 5, 6, 7, 8, 12, 14 and 17. In a second study by Martin et al. (1991) four cytotoxic T cell clones from MS patients with different immunogenetic backgrounds, specific for the p89 99 core sequence of the 87 106 epitope of MBP, expressed different V[3-D[3-J[3 gene rearrangements. Kotzin, Vandenbark, Hashim and colleagues suggested that in the context of HLA-DR2 the TCR family V[35.2 is preferentially used in response to different BP epitopes (Chou et al. 1991; Kotzin et al. 1991). It is possible that differences in cloning techniques, antigen used in stimulation and patient populations accounted for the contrasting results among these groups. The experimental observation that MBP reactive cells can be cloned from peripheral blood from both MS patients and controls further complicates the evaluation of their relevance in disease (Pette et al. 1990). To overcame this problem, Allegretta et al. (1990) used a somatic muta-

tion marker, hypoxantine guanine phosphoribosyltransferase (hrpt) to select for activated PBLs. The approach is based on the fact that dividing cells acquire random mutations during DNA synthesis. Mutations in the hprt gene result in inactivation of the hrpt enzyme. Thus, mutant cells do not metaboilze thioguanine to a cytotoxlc metaboilte, which allows selection of these mutants in culture. Although interpatient variability was seen, mutant clones were more frequent in patients and 4% (11 of 258) of these reacted strongly to MBP, where no such clones (none of 114) were observed in controls (Allegretta et al. 1990). These data support the notion that an anti-MBP reaction is activated in MS patients. Recently, we have used the PCR method to specificaliy amplify TCR Vet sequences from transcripts derived from MS brain lesions in 3 patients (Oksenberg et al. 1990). In each of the 3 MS post-mortem brain samples, a limited number of rearranged TCR Vet transcripts was detected. No Vet sequences could be found in 3 control brains without inflammation. Sequence analysis of 25 cDNA clones from MS white matter plaques with Vet 12.1-JetCet rearrangements demonstrated only two Jet sequences, reinforcing further the notion of a limited heterogeneity of TCR transcripts in MS brain. The extension of this study by us and others, may result in the elucidation of genetic susceptibility patterns where particular TCR V genes are expressed in regions of demyelination. Particular V genes might be expressed in the brains of individuals who possess certain HLA class II genes. Will be of interest, to correlate the histology of demyelination with the diversity of TCR expression (Wucherpfennig et al. 1992). As sequences become available from T cell clones reactive to other myelin proteins like PLP, MAG and MOG, correlations may emerge between T cell clones with defined specificities and TCR rearrangements in affected areas of the MS central nervous system. These findings may lead to an understanding of how the trimolecular complex (MHC-antigenTCR) triggers the autoimmune response in MS, and might have therapeutic implications, given the success of reversing or preventing EAE with reagents targeting TCR V genes. Vaccination with TCR peptides Cohen and associates have shown that it is possible to use autoimmune T cell clones or lines as vaccines to prevent or reverse autoimmune disease (Lider et al. 1981). In the EAE system for example, animals inoculated with MBP reactive T cells remained free of disease for prolonged times, and disease could not be induced with MBP in adjuvant. The detailed knowledge of the TCR repertoire involved in EAE (Steinman 1991) allowed the development of even more selective vaccination therapies based on TCR-

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sequences peptides. Immunization with synthetic peptides corresponding to variable regions of the TCR now appears to be feasible as a treatment modality for autoimmune conditions. Recently, EAE in the Lewis rat was prevented by immunization with a nonapeptide spanning the V-D-J region of VI38:p72-86 (Howell et al. 1989) and a 21-amino acid sequence that included the second complementarity determining region (CDR2), Vfl8:39-59, predicted to be immunogenic for T cells (Vanderbark et al. 1989). Moreover, TCR peptides were used to treat established disease in rats. Injection of TCR VI38:p39-59 (DMGHGKRLIHYSYDVN-STEKG) without adjuvant, to animals with clinical signs of EAE reduced disease severity and speeded recovery (Ofner et al. 1991). The apparent mechanisms may be active by stimulating anti VI38+ T cells and by inducing antibodies raised naturally in response to encephalitogenic VI38 cells. A pilot trial of treatment of MS with TCR-135.2 synthetic peptides is underway in Portland, Oregon. The wide application of these therapies in man will depend upon increased knowledge of the TCR repertoire utilized by pathogenic T cells. Anti-CD4 mAb

The CD4 surface protein is associated with "helper/inducer" T cell functions. Anti-CD4 therapy is the most extensively studied antibody used in the treatment of experimental autoimmunity. Anti-CD4 mAbs have been employed successfully by our group (Waldor et al. 1983) and by Brostoff et al. (1984) for reversal of EAE. One drawback of this approach is that administration of anti-CD4 antibody might generate general immunosuppression and might induce tolerance to dangerous pathogens. Though restoration of tolerance to self antigens is advantageous in autoimmunity, it could have deleterious consequences when given to individuals with chronic or persistent infection, such as toxoplasmosis (Vollmer et al. 1987). Despite these considerations, phase I clinical trials with chimeric anti-CD4 are now underway in Stanford Medical Center with patients with the chronic progressive form of MS. Opportunistic infections have not occurred in the first 19 patients treated with the anti-CD4 antibody. Construction of hybrid chimeric antibodies, humanizing murine mAbs may avoid the stimulation of human anti mouse responses, while retaining and even increasing the potency of the antibody (Alpers et al. 1990). Oral tolerance

A pilot trial of treatment of MS with oral feeding of bovine myelin is underway. Oral tolerance to myelin antigens was accomplished in treating experimental allergic encephalomyelitis (EAE) (Higgins et al. 1988), collagen

arthritis and uveitis (Marx 1991). Superantigens

Intrathymic elimination of autoreactive T lymphocytes expressing specific T-cell receptor VI3 chains in mice, depends on the expression of certain autosomal gehes like those encoding the so-called minor lymphocyte stimulatory (Mls) antigens (Blackman et al. 1988) or integrated retroviral sequences (Marrack et al. 1991). Some bacterial toxins exert similar effects as Mls antigens (Marrack et al. 1990). The observation by Marrack and colleagues on the high frequency of VI314 in the T cells associated with inflammation in the synovial fluid of patients with rheumatoid arthritis and the paucity of Vl314 T cells in the blood, suggest that the biased usage is due to superantigen stimulation of selfreactive cells. A microbial superantigen specific for VI314 has led to the activation of this cells, a few of which, because of their cross-reaction with self-antigens, have homed to the joints, where they initiate disease. This study has important implications for the understanding of fundamental mechanisms of autoimmunity (Paliard et al. 1991). The correlation between infection and demyetinating diseases has been the subject of continuous interest and controversy. A link between superantigens and infectious agents like retroviruses in MS has proven elusive. However, HTLV-1 has been definitively excluded as a potential retroviral trigger for MS (Hauser et al. 1986; Bangham et al. 1989; Richardson et al. 1989; Oksenberg et al. 1990). Conclusion

The application of advances in molecular and cellular immunology has increased our understanding of how tolerance to self antigens is broken and autoimmunity develops. The precise and detailed knowledge of the complexity of autoantigens, and the structural analysis as well as diversity of MHC antigens and T cell receptors linked to disease susceptibility will allow the formulation of appropriate protocols for trials in human disease. Many lessons have been learned in the EAE system, and are now in the process of been verified in human. Indeed, EAE has served as a model for testing novel forms of specific immunotherapy, and some of these approaches are being attempted in MS at the present time. In established disease, the pathological immune response may involve multiple epitopes or even multiple antigens, and it is likely that an individual HLA background shapes the TCR repertoire. Consequently, therapy will be customized to a certain extent. On the other hand, a valid interpretation of the currently available experimental data points to the complexity of the autoimmune reaction, forecasting the likely success of less selective modes of immunotherapy.

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