Chapter 13 Immunogenetic mechanisms in myasthenia gravis

S.-M. Aquilonius and P.-G. Gillberg (Eds.) Progress in Brain Research Vol. 84 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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CHAPTER 13

Immunogenetic mechanisms in myasthenia gravis Lawrence Steinman Departments of Neurologv, Pediatrics and Genetics, Stanford University, Stanford CA 94305, U.S.A.

Introduction T cells can recognize fragments of self-constituents, such as acetylcholine receptor, and provide specific stimulatory signals for the activation of B cells which differentiate into autoantibodysecreting plasma cells. Myasthenia gravis (MG) results from an antibody-mediated autoimmune response to the nicotinic acetylcholine receptor (AChR). Individuals with MG have a higher frequency of certain major histocompatibility complex (MHC) antigens, particularly HLA-B8, DR3 and DQw2 (Sawenberg et al., 1978). Since the target antigen is known, MG will be one of the first human autoimmune diseases to be fully characterized at the molecular level. HLA-DR, DQ and DP antigens have already been sequenced in MG patients (Todd et al., 1988; Mantegazza et al., 1989), some T and B cell epitopes have been characterized (Brocke et al., 1988; Harcourt et al., 1988; Hohlfeld et al., 1989), and T cell receptor , (TcR) gene polymorphisms associated with the disease have been described (Oksenberg et al., 1989). The ternary interaction of class II major histocompatibility molecules, the T cell receptor, and AChR peptides

This subject has been recently reviewed by us (Wraith et al., 1989). The minimal requirement for activation of helper T (TH) cells is the occupation of their TcR by a complex formed between frag-

ments of antigen and class I1 MHC molecules. Class I1 molecules are encoded by I-A and I-E genes of the H-2 complex in mice and DP, DQ and DR genes of the HLA complex in man. In addition to this molecular triad, accessory (cell differentiation) molecules on the surface of the T cell (CD4 for T, and CD8 for cytotoxic T (T,) cells) interact with MHC molecules on the surface of the antigen-presenting cell (APC), increase the overall affinity of cell-cell contact, and permit recognition of the antigen-MHC complex by low affinity TcR. The majority of antigens for T cells are recognized in a denatured or fragmented form (Moller et al., 1987). This requirement presumably allows for association of fragments with the antigen-binding “cleft” of class I (Bjorkman et al., 1987) and the predicted “cleft” of class I1 MHC molecules (Brown et al., 1988). The majority of polymorphic MHC residues are clustered around this “cleft’, and this indicates how these residues directly influence antigen binding and interaction with TcR (Bjorkman et al., 1987). The immune system can respond to a wide variety of different protein antigens, and this implies that any one MHC molecule can associate with a vast assortment of antigenic structures. MHC molecules are highly polymorphic, and products of separate alleles can associate with different sets of peptide antigenic structures. In spite of the promiscuous nature of MHC-antigen associations, it has proved difficult to measure peptide binding. This can be explained by the

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relatively low-affinity constant for association between MHC and peptide (Babbit et d., 1985; Buus et al., 1986) or by the premise that MHC proteins are normally occupied by endogenous self-peptides, as suggested by recent studies (Buus et al., 1988). This information, combined with the demonstration that APC from normal tissues constitutively present self-antigens, such as hemoglobin (Lorenz and Allen, 1988), is evidence that the recognition of self-antigen-MHC complexes may be involved in immunoregulation (Kourilsky and Claverie, 1986). However, the overall significance of endogenous peptides to immune responsiveness and tolerance to self-antigens awaits further experimentation. It is now quite clear that there is a single antigen binding site on class I1 molecules, since cross-linking of class I1 MHC with radiolabeled peptide probes produces complexes of the appropriate size for a 1:l ratio of binding (Luescher et al., 1988; Wraith et al., unpublished data). Furthermore, peptides can compete with one another for MHC binding and T cell activation (Guillet et al., 1987). The furthest extension of this observation has been the demonstration that a mouse lysozyme peptide can competitively inhibit priming with a foreign lysozyme peptide on co-injection into a mouse strain normally responsive to the foreign peptide (Adorini et al., 1988). The mouse lysozyme peptide binds to one of the two responder MHC molecules (I-A’), thus specifically blocking binding of and preventing an immune response to the foreign peptide. This experiment was successful since, even though the peptide bound to the I-Ak molecule, the responder mouse was tolerant to the self-lysozyme peptide. Ideally, an effective immune system would be able to respond to any foreign antigen and yet would remain unresponsive to, or tolerant of, its own self-antigens throughout life. To a large extent the immune system of higher animals has evolved to achieve this end. Immunological tolerance is believed to be induced during a perinatal period when immature lymphocytes are exposed to self-antigens. In humans this period terminates

before birth, while in rodents the process is complete shortly after birth. Recent studies have clarified the role of the thymus in preparing T lymphocytes for life in an effective immune system. Studies in normal and transgenic mice have revealed how interaction of TcRS with an appropriate MHC molecule can lead to increased cell numbers in the periphery (positive selection) (reviewed by Von Boehmer et al., 1989). Conversely, analysis of the distribution of either MLS (mouse lymphocyte stimulating) locus reactive or MHC class I1 antigen (I-E) specific T cells in normal mice has shown how developing T cells can be deleted from the repertoire following interaction of TcR with the self-antigen-MHC complex in the thymus (negative selection) (reviewed by Marrack and Kappler, 1988). By subjecting T cells to such education, the system selects for cells that are “obsessed” with self-MHC and yet are unable to react with self-MHC combined with any self-antigen that the T cell encountered during the later stages of ontogeny. How the thymus manages to turn MHC recognition from providing a positive signal at one stage to a negative signal at another is not clear at this time. One explanation relies on the differential recognition of, or different affinities for, MHC molecules on epithelial as opposed to bone marrow-derived macrophages and dendritic cells by the maturing T lymphocytes (Sprent et al., 1988). T cell-dependent autoimmune diseases arise from a breakdown in self-tolerance. There are essentially four ways in which tolerance to self-antigens can be viewed. First, an individual can be unresponsive to self if the antigen, or fragment thereof, is unable to bind to self-MHC. Secondly, potentially self-antigen-reactiveT cells can be deleted from the T cell repertoire by clonal deletion in the thymus. Unfortunately, neither of the first two possibilities would explain why autoreactive T cells, specific for myelin basic protein (MBP) for example, can readily be isolated from the mature T cell repertoire and yet do not normally cause disease (Schluesner and Wekerle, 1984). A third possibility, which would account for this paradox,

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is that self-antigens, such as MBP, are sequestered from the immune system either physically, by the blood-brain barrier, or by being presented in a nonimmunogenic fashion. According to this theory, self-antigens associated with class I1 negative cells may never be immunogenic unless they are reprocessed by class I1 positive cells such as B lymphocytes, macrophages, dendritic or interdigitating cells. However, it should be noted that some cells can be induced to express class I1 molecules when treated with lymphokines (e.g., y-interferon). Ectopic MHC expression is believed to contribute to the presentation of “sequestered” antigens in autoimmune disease (Bottazzo et al., 1986). Finally, in cases where clonal deletion does not eliminate potentially autoreactive TH cells, autoimmune attack may be guarded against by the action of T suppressor (T,) cells. In certain mouse strains, TH cells specific for autoantigens are fupctional and remain quiescent because of the action of such antigen-specificT, cells (Jensen and Kapp, 1985). If homeostasis in the system is seen as a “balance” between T cell help and suppression, and self-antigen-specificT cells are present in the normal individual, one can imagine how the “balance” could be tipped by stimulation with crossreactive antigen contained in an infectious bacterium or virus.

MHC and MG Recent molecular studies of several autoimmune conditions reveal that MHC class I1 genes play a major role. In patients with rheumatoid arthritis, 80-90% are either HLA-DR1 or HLA-DR4 (Dw4, Dw14 or Dw15). The HLA-DR4 DwlO variant is associated with resistance to rheumatoid arthritis. HLA-DR1 and HLA-DR4 (Dw4, Dw14 or Dw15) have a very similar sequence in the third hypervariable region of HLA-DR, while HLA-DR4 DwlO is strikingly different (Todd et al., 1988). A similar analysis of HLA class I1 genes in insulindependent diabetes mellitus localizes susceptibility to residue 57 in the DQ beta chain (Todd et al., 1988). In pemphigus vulgaris (PV), a severe autoim-

mune disease of the skin mediated by autoantibodies to an epidermal cell surface protein, there is a striking association of disease with MHC class I1 genes. Thus, nearly 100%of individuals with PV are HLA-DR4, DRw6, or DR4/DRw6 heterozygotes. The DR4 susceptibility is highly associated with the DwlO, DRPl allele, implicating polymorphic residues in the third hypervariable region, while the DRw6 susceptibility is strongly associated with a rare DQj? allele (DQP1.9). This allele differs from a common DQPallele (DQP1.l) only by a valine to aspartic acid substitution at position 57 (Scharf et al., 1988; Sinha et al., 1988). Susce‘ptibilityto MG correlates with HLA-DR3, DQw2. Serological techniques have established a relative risk of 3.5 for MG if HLA-DR3 is present (SZfwenberg et al., 1978). RFLP studies on German and Northern California Caucasoid MG patients revealed a closer association of HLA-DQ and MG. An HLA-DQ beta polymorphism in MG patients revealed a relative risk of 36 (Bell et al., 1986). This polymorphism may be closely linked to a genetic locus encoding a binding site for an AChR epitope. Susceptibility to experimental allergic myasthenia gravis (FAMG), like susceptibility to MG, is linked to the MHC (Berman and Patrick, 1980a,b). The immune response to AChR has been mapped to the I-A genes of H-2 (the murine homologue of the human DQ region)(Christadoss et al., 1979). Studies with B6bm12and B6 mice have shown that particular I-A alleles play an essential role in the development of an antibody response to AChR and clinical FAMG. The bm12 mouse differs from the B6 mouse only in the I-A beta chain at amino acid residues 68, 71 and 72 (Waldor et al., 1986). These three amino acid changes result in the conversion of a strain which is susceptible to the induction of EAMG, B6, to one that is resistant to EAMG, bm12. Since I-A molecules exert an essential role in the development of an immune response to AChR, we investigated whether in vivo administration of monoclonal anti-I-A antibodies could suppress the immune response to AChR.

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Reduction of anti-AChR antibody titers C57BL/6 (H-2b) and SJL/J (H-29 are both high responders to AChR and susceptible to EAMG (Berman and Patrick, 1980a). Monoclonal anti-I-A antibody was administered in vivo to these high-responder mice before immunization with soluble AChR or AChR in complete Freund's adjuvant. Four mg of monoclonal antibody (0.5 ml ascites fluid) were injected intraperitoneally, 1 day before and 1 day after immunization with AChR. This injection regimen was repeated at the time of secondary immunization. In vivo treatment with anti-I-A antibody prevented the secondary antibody response to soluble AChR (Table I). Anti-I-A antibody treatment only prevented the anti-AChR antibody response in the appropriate strain of mice. Thus, anti-I-Ab antibody had no effect on the anti-AChR antibody response in SJL/J (H-2') mice, and likewise, anti-I-As antibody did not alter the anti-AChR antibody response in C57BL/6 (H-2b) mice (Table I). Anti-IA injection also reduced anti-AChR antibody titers in mice immunized with AChR in complete Freund's adjuvant (Table 11). Prevention of clinical EAMG The effect of in vivo anti-I-A treatment on clinical EAMG in SJL/J mice was assessed. Myasthenia symptoms included a characteristic hunched posture with drooping of the head and TABLE I Anti-I-A antibody treatment prevents the secondary antibody response to soluble AChR Treatment * *

Anti-AChRantibodyle~els(plXlO-~) * SJL/J(H-2*)

C57BL/6(H-2b)

None Anti-I-A' Anti-I-Ab

22.3f 5.9 0.2f 0.4 18.6 f 17.7

7.5 f 7.6 6.2f4.8 1.6 f 3.9

* Mean f standard deviatibn of the anti-AChR antibody levels determined by ELISA 1week after secondary immunization with soluble AChR. * * 4 mg of the monoclonal antibody in ascites fluid were injected intraperitoneally on the day before and the day after primary and secondary immunization with 50 pg of soluble AChR.

TABLE I1 Anti-I-A treatement reduces the secondary antibody response to AChR in complete Freund's adjuvant Treatment *

Anti-AChR antibody levels in C57BL/6 mice (H-2b) * * ( p l x 10-2)

None Anti-I-Ab Anti-I-A"

5.4i-0.5 1.0 f 0.4 4.6 f 0.6

* Mice were immunized with 15 p g of AChR in CFA in the hind footpads and at the base of the tail. One week later a booster injection of 50 pg of soluble AChR was given intraperitoneally. Antibody treatments were as in Table I. * * Mean f standard deviation of anti-AChR antibody levels determined by ELISA 1week after secondary immunization.

neck, exaggerated arching of the back, splayed limbs, abnormal walking, and difficulty in righting. Weakness was alleviated with 5-10 min of administration of neostigmine bromide and atropine sulfate intraperitoneally. Clinical disease was apparent in 11of 19 control animals and only 2 of 10 anti-I-As treated mice.

TcR and MG To investigate which parts of the AChR are involved in the initiation and development of MG, peptides representing different sequences of the human AChR a-subunit were synthesized. These peptides were tested for their ability to stimulate T cells of myasthenic patients and healthy control patients in proliferation assays and to bind to sera antibodies. Three of eight peptides discriminated significantly between the two groups in the proliferation assay, as well as in their ability to bind to serum antibodies. HLA-DR3 and DR5 were associated with proliferative responses to specific AChR peptides in the group of myasthenics. Acetylcholine receptor epitopes which might play a specific role in myasthenia gravis were thus demonstrated. Proliferative responses of PBL of MG patients to peptides representing sequences of the human AChR PBL of MG patients and of healthy controls were tested for their ability to proliferate in the

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TABLE 111 Synthetic peptides of the AChR a-subunit Peptides

Species

Sequence

p195-212 p257-269 p310-327 p169-181 p183-196 p185-196 p351-368 p394-409

Human Human Human Human Human Human Human Human

Asp Leu Asn Asn Gly Lys Ile Asn

Thr Leu T rp Phe T rp His Ser Ala

Pro Val Va t Met Lys Ser Gly ALa

Tyr ILe Arg Glu His Val Lys GLu

Leu Val Lys Ser Ser Thr Pro GLu

ASP Glu Ite Gly Val T yr Gly Trp

ILe Leu Phe Glu Thr Ser Pro Lys

presence of an in vitro stimulus with the various peptides representing regions of the AChR a-subunit listed in Table 111. Table IV summarizes the results of the proliferative assays specific to eight sequences of the AChR performed with PBL of MG patients and healthy controls. As shown in the table, the myasthenic patients responded better to all the peptides. However, only three peptides (p195-212, p257-269 and p310-327) could discriminate significantly between MG patients and controls on the basis of the proliferative responses of PBL. The differences between MG patients and controls were the most significant when the responses to p195-212 were measured (Table IV). Antibody levels specific to synthetic peptides representing AChR in sera of MG patients

It was of interest whether sera of patients with myasthenia gravis possess antibodies that react TABLE IV

Percentages of positive proliferative PBL responses of MG patients and healthy controls to peptides representing different sequences of the human AChR Peptides

MG patients

Healthy controls

p195-212 p257-269 p310-327 p169-181 p183-196 p185-196 p351-368 p394-409

72.5% (20/28) 56 % (14/25) 44.4% (12/27) 26.9% (7/26) 26.9%(7/26) 5.4% (4/26) 35.7%(10/28) 26.9%(7/26)

21.2% (7/33) p < 0.001 16 S (4/25) p < 0.01 15.6%(5/32) p < 0.01 9.5%(2/21) 19.2%(5/26) 3.8% (1/26) 26.9% (7/26) 19.2%(5/26)

ILe Ile Ile Trp Tyr Cys Pro Tyr

Tyr Pro Asp Val Ser Cys Pro Val

H i s Phe V a l Met GLn Arg Leu Pr o Leu Ser Thr Ile Cys Pro Met ALa

Thr Ile Lys Cys Asp GLy Met

Ser Pro GLu Pro Thr Phe Val

Ser Asn I l e Met Phe Phe Ser Ser Asp Thr

H i s Ser Pr o Leu I L e Lys I l e Asn H i s I L e

with the different peptides representing the AChR a-subunit. Table V sumarizes the percentages of positive antibody titers specific to the seven peptides in sera of MG patients and controls. It can be seen in the table that the percentages of sera with antibody titers specific to p195-212, p257-269, and p310-327 are significantly higher than those determined in sera of controls.

HLA typing of MG patients The HLA-A, B, C, DR and DQ phenotypes of 45 MG patients were tested. Fifteen patients (33%) possessed HLA-B8. Fourteen of the 45 patients (31%) possessed HLA-DR3. In 12 cases of the latter, DR3 occurred jointly with HLA-BS. Note that both HLA-B8 and DR3 are present in relatively low frequencies of 9.4 and 10.4%, respectively, in the Israeli population. Moreover, the joint occurrence of B8 and DR3 in the healthy

TABLE V Percentages of positive serum antibody levels of MG patients and healthy controls to peptides representing different sequences of the human AChR Peptides

MG patients

Healthy controls

p195-212 p257-269 p310-327 p169-181 p185-196 p351-368 p394-409

79.5% (31/39) 47.5% (19/40) 49% (21/43) 14%(6/43) 5% (2/40) 5% ( 2 / W 16%(7/43)

6.5% (2/31) p < O.ooO1 3.3% (1/30) p < 0.001 6.5% (2/31) p < 0.001 6.5% (2/31) 3.3% (1/30) 3.3% (1/30) 6.5% (2/31)

122 TABLE VI Correlation between HLA-DR3 and proliferative responses to ~257-269 Responder HLA-DR3 myasthenic Not HLA-DR3 myasthenic HLA-DR healthy Not HLA-DR3 healthy a

Nonresponder

7/7 a/b (100%) 0/7 a (0%) 6/16 (38%) 10/16 (62%) 1/1(100%) o/o (0%) 3/17 (18%) 14/17 (82%)

p < 0.0003, Fisher’s exact test. p < 0.006, Fisher’s exact test.

Israeli population is rather low (5.7%) but sigmficantly higher ( x 2 = 11.3, p c 0.001) in the Israeli MG patients. The relative risk for B8, DR3 carriers is 6.1. A possible correlation was sought between the proliferative capacity of PBL of MG patients and control individuals to the various peptides of the AChR and HLA. Table VI demonstrates that all seven HLA-DR3-type patients responded to p257-269 ( p K 0.0003). The one HLA-DR3 healthy individual also responded to p257-269. Non-HLA-DR3 patients also responded to p257269, but less frequently (7 of 7 vs. 6 of 16; p ~0.006).Table VII demonstrates that 10 of 12 (83%) of the HLA-DR5 myasthenic patients responded to p195-212 by proliferation, compared with 2 of 12 (17%) of myasthenic DR5 carriers who did not respond. In the group of HLA-DR5 healthy controls, the distribution was different, with 3 of 14 (21%) responding and 11 of 14 not responding to p195-212 ( p < 0.007). Non-HLADR5 myasthenics also responded frequently to ~195-212 (9 Of 15 VS. 10 Of 12, P=NS). NO TABLE VII Correlation between HLA-DR5 and proliferative responses to ~195-212 Responder

Nonresponder

HLA-DR5 myasthenic 10/12 (83%) 2/12 (17%) Not HLA-DR5 myasthenic 9/15 (60%) 6/15 (40%) HLA-DR5 healthy 3/14 a (21%) 11/14 (79%) 3/15 (20%) 12/15 (80%) Not HLA-DR5 healthy

correlation could be observed between proliferative responses to p310-327 and any HLA-DR antigen. Antibody reactivity to p195-212, p257269 and p310-327 in the sera of MG patients was not associated with any particular HLA type. This study suggests that peptides of AChR are particularly immunogenic for certain HLA-DR types. HLA-DRS individuals responded better than healthy controls to p195-212. Moreover, all HLA-DR3 respond to p257-269. It has been demonstrated that immunogenic peptides bind directly to class I1 MHC products (Babbitt et al., 1985). In this context, it is possible that p195-212 binds more readily with an MG-associated class I1 HLA product, whereas p257-269 binds preferentially to HLA-DR3. The fact that HLA-DR3 class I1 molecules have identical sequences in MG patients and controls may underlie the observation that some healthy individuals respond to some AChR peptides (Todd et al., 1988). This may account for the fact that individuals with different HLA-class I1 genotypes can respond to a given AChR peptide. These studies represent one of the first attempts to analyse the determinants of the AChR molecule involved in the aberrant immune response in MG in humans. Hohlfeld and others have reported responses to three NH,-terminal AChR peptides in T cell lines from two MG patients that react with Torpedo AChR (Hohlfeld et al., 1988). Atassi and colleagues have studied the response in inbred mouse strains to various AChR peptides from the a subunit of Torpedo AChR (Atassi et al., 1987). Our studies demonstrate significant differences between MG patients and healthy controls in the immune response potential to various sequences of the human AChR and its association with HLA. Other epitopes of AChR, when tested, may also be shown to play a critical role in the pathogenesis of MG. Polymorphic markers in genes encoding the a chain of the human TcR have been detected by Southern blot analysis in PssI digests. Polymorphic bands were observed at 6.3 and 2.0 kilobases (kb) with frequencies of 0.30 and 0.44, respectively, in the general population. Using the poly-

123 TABLE VIII Distribution of TcR V" and C" in MG patients 6.3 kb MG Stanford Control Stanford

~~

2.0 kb

+

-

+

-

14 21

3 49

17 31

0 39

merase chain reaction (PCR) method, we amplified selected sequences derived from the full-length TcR a cDNA probe. These PCR products were used as specific probes to demonstrate &at the 6.3-kb polymorphic fragment hybridizes to the variable (V)region probe and the 2.0-kb fragment hybridizes to the constant (C) region probe. Segregation of the polymorphic bands was analysed in family studies. To look for associations between these markers and autoimmune diseases, we have studied the restriction fragment length polymorphism (RFLP) distribution of the PssI markers in patients with multiple sclerosis, myasthenia gravis and Graves disease. Significant differences in the frequency of the polymorphic V, and C, markers were identified between MG patients and healthy controls. For MG patients, the 6.3-kb positive phenotype is significantly more common than in controls and confers a relative risk of 11 (x2= 15.6), while the 2.0-kb positive phenotype is also significantly more common than in controls (17 of 17 MG patients versus 31 of 70 controls; x2 = 17.2) (Table VIII). Conclusion

Autoimmune disease results from a breakdown in self-tolerance. The root cause of many serious human diseases is the aberrant activation of selfreactive T cells. Two striking features are emerging. First, individuals expressing certain class I1 MHC genes are more likely to be affected. Furthermore, there appears to be a common thread between the susceptible MHC genes in that, as shown for RA and IDDM, susceptibility alleles share regions of allelic hypervariability. We be-

lieve that the similarity between susceptible alleles reflects the role of MHC molecules in the maintenance of T cell tolerance. However, it is not yet clear whether this lies at the level of self-antigen binding to MHC, thymic T cell education, or immunoregulation via T suppressor cells. We need to know much more about the molecular nature of T cell recognition in human diseases to identify the antigens recognized and to correlate MHC restriction of disease-associated T cells with known susceptibility alleles. Even so, the striking association between disease and MHC class I1 alleles holds promise for immunotherapy. Second, a common finding in many experimental models is the oligoclonality in some human autoimmune conditions, though it is not certain that the T cell populations studied were the primary effectors of disease. When this can be established, specific TcR-targeted immunotherapy may also be feasible for human autoimmune diseases (Acha-Orbea et al., 1988). References Acha-Orbea, H., Mitchell, D.J., Timmerman, L., Wraith, D.C., Waldor, M.K, Tausch, G.S., Zamvil, S.S.,McDevitt, H.O. and Steinman, L. (1988) Limited heterogeneity of T cell receptors from lymphocytes mediating autoimmune encephalomyelitis allows specific immune intervention. Cell, 54: 263-273. Adorini, L., Muller, S., Cardinaux, F., Lehmann, P.V., Falcioni, F. and Nagy, Z.A. (1988) In vivo competition between self peptides and foreign antigens in T-cell activation. Nature, 334: 623-625. Atassi, M.I., Mulac-Jericevic, B., Yokoi,T. and Manshour, T. (1987) Localization of the functional sites on the a chain of acetylcholine receptor. Fed. Proc., 46:2538-2547. Babbit, D.P., Allen, P.M., Matsueda, G., Heber, E. and Unanue, E.R. (1985) Binding of immunogenic peptides to la histocompatibility molecules. Nature, 317: 359-361. Bell, J., Smoot, S., Newby, C., Toyka, K., Rassenti, L., Smith, K., Hohlfeld, R., McDevitt, H.O. and Steinman, L. (1986) HLA-DQ beta chain polymorphism linked to myasthenia gravis. Lancet, ii: 1058-1060. Berman, P. and Patrick, J. (1980a) Linkage between the frequency of muscular weakness and loci that regulate responsiveness in EAMG. J. Exp. Med, 152: 507-520. Berman, P. and Patrick, J. (1980b) Experimental myasthenia gravis. J. Exp. Med., 151: 204-223. Bjorkman, P.J., Saper, M.A., Samraoui, B., Bennett, W.S., Strominger, J.L. and Wiley, D.C. (1987a) Structure of the

124 human class I histocompatibility antigen, HLA-A2. Nature, 329: 506-512. Bjorkman, P.J., Saper, M.A., Samraoui, B., Bennett, W.S., Strominger, J.L. and Wiley, D.C. (1987b)The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens. Nature, 329: 512-518. Bottazzo, G.F., Todd, I., Mirakian, R., Belfiore, A. and h j o l Borrell, R. (1986) Organ-specific autoimmunity. Immunol. Rev., 94: 137-169. Brocke, S., Brautbar, L., Steinman, L., Abromsky, O., Rothbard, J. and Mozs, E. (1988) In vitro proliferative responses and antibody titres specific to human acetylcholine receptor synthetic peptides in patients with myasthenia gravis and relation to MHC class I1 genes. J. Clin. Inuest., 82: 1894-1900. Brown, J.H., Jardetzky, T., Saper, M.A., Samraoui, B., Bjorkman, P.J. and Wiley, D.C. (1988)A hypothetical model of the foreign antigen binding site of class I1 histocompatibility molecules. Nature, 332: 845-850. Buus, S., Colon, S., Smith, C., Freed, J.H., Miles, C. and Grey, H.M. (1986)Interaction between a ''processed" ovalbumin peptide and Ia molecules. Pfoc. NatL Acad Sci, USA, 83: 3968-3971. Buus, S., Sette, A., Colon, S.M. and Grey, H.M. (1988)Autologous peptides constitutively occupy the antigen binding site on Ia. Science, 242: 1045-1047. Christadoss, P., Lennon, V. and David, C. (1979) Genetic control of experimental allergic myasthenia gravis. J. Immunol., 123: 2540-2545. Guillet, 3.-G., Lai, M.Z., Briner, T.J., Buus, S., Sette, A., Grey, H.M., Smith, J.A. and Gefter, M.L. (1987) Immunological self non-self discrimination. Science, 235: 865-870. Harcourt, G.C., Sommer, N., Rothbard, J., Willcox, H.N.A. and Newsom-Davis, J. (1988) A juxta-membrane epitope on the human acetylcholine receptor recognized by T cells in myasthenia gravis. J. Clin. Invest., 82: 1295-1300. Hohlfeld, R., Toyka, K., Miner, L., Walgrave, S. and ContiTronconi, B. (1988) Amphipathic segment of the nicotinic AChR a-subunit contains epitopes recognized by Tlymphocytes in myasthenia gravis. J. Clin. Znuest., 81: 657-660. Jensen, P.E. and Kapp, J.A. (1985)Genetics of insulin-specific helper and suppressor T cells in non-responder mice. J. Immunol., 135: 2990-2995. Kourilsky, P. and Claverie, J.M. (1986)The peptidic self model: a hypothesis on the molecular nature of the immunological self. Ann. Znst. Pmtew; 137D: 3-21. Lorenz, R.G. and Allen, P.M. (1988) Direct evidence for

functional self protein/La-molecule complexes in vivo.

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