Acetylcholine Receptor Epitopes Research Articles

The susceptibility to experimental myasthenia gravis of STAT6-/- and STAT4-/- BALB/c mice suggests a pathogenic role of Th1 cells.

Autoantibodies to the muscle acetylcholine receptor (AChR) cause the symptoms of human and experimental myasthenia gravis (EMG). AChR-specific CD4+ T cells permit development of these diseases, but the role(s) of the Th1 and Th2 subsets is unclear. The STAT4 and STAT6 proteins, which mediate intracellular cytokine signaling, are important for differentiation of Th1 and Th2 cells, respectively. Wild-type (WT) BALB/c mice, which are prone to develop Th2 rather than Th1 responses to Ag, are resistant to EMG. We have examined the role of Th1 and Th2 cells in EMG using STAT4 (STAT4-/-)- or STAT6 (STAT6-/-)-deficient BALB/c mice. After AChR immunization, STAT6-/- mice were susceptible to EMG: they developed more serum anti-AChR Ab, and had more complement-fixing anti-AChR IgG2a and 2b and less IgG1 than WT or STAT4-/- mice. The susceptibility to EMG of STAT6-/- mice is most likely related to the Th1 cell-induced synthesis of anti-AChR Ab, which trigger complement-mediated destruction of the neuromuscular junction. CD4+ T cells of the STAT6-/- mice had proliferative responses to the AChR comparable to those of WT and STAT4-/- mice, and recognized similar AChR epitopes. STAT6-/- mice had abundant AChR-specific Th1 cells, which were nearly absent in WT and STAT4-/- mice. Spleen and lymph nodes from STAT6-/- mice contained cells that secreted IL-4 when cultured with AChR: these are most likely STAT6-independent cells, stimulated in a non-Ag-specific manner by the cytokines secreted by AChR-specific Th1 cells.

Evidence of a diverse T cell receptor repertoire for acetylcholine receptor, the autoantigen of myasthenia gravis

We utilized two methods to look for T cell clonal expansions in myasthenia gravis (MG). We analyzed TCRBV CDR3 length polymorphism (spectratyping) to look for evidence of clonal expansion of CD4 or CD8 T cells directly from peripheral blood of MG patients. No statistically significant differences were found between the diversity of TCR repertoires in MG patients compared to normal control individuals when analyzed as groups. Rare oligoclonal expansions were detected in some individual MG patients but the significance of these findings is unclear. Next, we analyzed a panel of T cell hybridomas from acetylcholine receptor (AChR) immunized, MG-susceptible HLA-DR3 transgenic mice. The epitope specificity, TCRBV gene usage and CDR3 sequences of these hybridomas were highly diverse. We conclude there is only limited evidence for restricted TCR repertoire usage in human MG and suggest this may be due to the inability of HLA-DR molecules to select for restricted TCR recognition of AChR epitopes.

Breakage of tolerance to hidden cytoplasmic epitopes of the acetylcholine receptor in experimental autoimmune myasthenia gravis

The acetylcholine receptor (AChR) is the major autoantigen in the antibody-mediated disease myasthenia gravis (MG) and its animal model experimental autoimmune myasthenia gravis (EAMG). This study demonstrates that rats immunized with a recombinant fragment corresponding to the normally exposed extracellular region of the rat AChR α-subunit first develop antibodies to the injected extracellular portion only, but later develop antibodies to intracellular cytoplasmic epitopes of AChR. The presence of autoantibodies to intracellular epitopes seems to be correlated with development of clinical signs of disease. We propose that a similar process of epitope spreading may take place in the natural course of myasthenia.

Adoptive protection from experimental myasthenia gravis with T cells from mice treated nasally with acetylcholine receptor epitopes

Nasal administration of synthetic CD4 + epitopes of the acetylcholine receptor (AChR) prevents experimental myasthenia gravis (EMG) in C57Bl/6 mice, but not in IL4-deficient C57Bl/6 (IL4 −/−) mice. Here we verify that nasal tolerance requires IL4, by showing that CD4 + cells from C57Bl/6 mice treated nasally with a pool of AChR CD4 + epitopes protected IL4 −/− mice from EMG and caused a reduced production of anti-AChR antibody. CD4 + cells from C57Bl/6 mice treated with unrelated peptides or sham-treated did not induce protection. CD4 + cells from C57Bl/6 mice treated with just one AChR peptide protected IL4 −/− mice from EMG without affecting antibody synthesis.

Scanning a DRB3*0101 (DR52a)-Restricted Epitope Cross-Presented by DR3: Overlapping Natural and Artificial Determinants in the Human Acetylcholine Receptor

A recurring epitope in the human acetylcholine receptor (AChR) alpha subunit (alpha146-160) is presented to specific T cells from myasthenia gravis patients by HLA-DRB3*0101-"DR52a"-or by DR4. Here we first map residues critical for DR52a in this epitope by serial Ala substitution. For two somewhat similar T cells, this confirms the recently deduced importance of hydrophobic "anchor" residues at peptide p1 and p9; also of Asp at p4, which complements this allele's distinctive Arg74 in DRbeta. Surprisingly, despite the 9 sequence differences in DRbeta between DR52a and DR3, merely reducing the bulk of the peptide's p1 anchor residue (Trp149-->Phe) allowed maximal cross-presentation to both T cells by DR3 (which has Val86 instead of Gly). The shared K71G73R74N77 motif in the alpha helices of DR52a and DR3 thus outweighs the five differences in the floor of the peptide-binding groove. A second issue is that T cells selected in vitro with synthetic AChR peptides rarely respond to longer Ag preparations, whereas those raised with recombinant subunits consistently recognize epitopes processed naturally even from whole AChR. Here we compared one T cell of each kind, which both respond to many overlapping alpha140-160 region peptides (in proliferation assays). Even though both use Vbeta2 to recognize peptides bound to the same HLA-DR52a in the same register, the peptide-selected line nevertheless proved to depend on a recurring synthetic artifact-a widely underestimated problem. Unlike these contaminant-responsive T cells, those that are truly specific for natural AChR epitopes appear less heterogeneous and therefore more suitable targets for selective immunotherapy.

Interleukin-4 deficiency facilitates development of experimental myasthenia gravis and precludes its prevention by nasal administration of CD4 + epitope sequences of the acetylcholine receptor

Immunization with acetylcholine receptor (AChR) causes experimental myasthenia gravis (EMG). We investigated EMG in interleukin (IL)-4 knock out B6 (KO) mice, that lack Th2 cells. EMG was more frequent in KO than in wild type B6 mice. KO and B6 mice developed similar amounts of anti-AChR antibodies. They were IgG2a and IgG2b in KO mice, IgG1 and IgG2b in B6 mice. CD4 + cells from KO and B6 mice recognized the same AChR epitopes. Nasal administration of synthetic AChR CD4 + epitopes reduced antibody synthesis and prevented EMG in B6, not in KO mice. Thus, Th2 cells may have protective functions in EMG.

Myasthenia in SCID mice grafted with myasthenic patient lymphocytes: role of CD4+ and CD8+ cells.

Acetylcholine receptor (AChR)-specific CD4+ cells are present in MG patients, and synthesis of the high-affinity immunoglobulin G (IgG) autoantibodies (autoAb) against the muscle AChR that causes MG symptoms requires intervention of CD4+ cells. The role of CD4+ cells in MG pathogenesis has been postulated but never proven. MG patients do not have anti-AChR cytotoxic phenomena, and it has been assumed that CD8+ cells do not have a pathogenic role in MG. However, CD8+ cells may facilitate rodent experimental MG, raising the possibility that CD8+ cells might be necessary also in MG. In this study we examined whether CD4+ and CD8+ cells play a role in the pathogenesis of MG and whether CD4+ cells specific for AChR epitope sequences recognized by most MG patients ("universal" epitopes) drive the synthesis of pathogenic antibodies. First we characterized a chimeric human-mouse model of MG in severe combined immunodeficiency (SCID) mice engrafted with blood lymphocytes (BL) from MG patients. We used that model to determine whether CD4+ and CD8+ cells are necessary for transfer of MG symptoms. We engrafted SCID mice intraperitoneum with BL from 19 MG patients and 5 healthy controls. We engrafted some mice with either BL, BL depleted in CD4+ or CD8+ cells from the same patient, or CD4+ depleted BL reconstituted with CD4+ T cells from the same patient, specific for "universal" AChR epitopes or for two unrelated antigens, tetanus and diphtheria toxoids. We tested the mice for myasthenic symptoms for 7 to 18 weeks. Mice transplanted with BL, or CD8+ depleted BL, or CD4+-depleted BL reconstituted with anti-AChR CD4+ cells from MG patients frequently developed myasthenic weakness. The mice had human anti-AChR Ab in the serum and bound to muscle AChR. Mice transplanted with BL from controls, or CD4+-depleted BL from MG patients, or CD4+-depleted BL from an MG patient reconstituted with CD4+ cells specific for tetanus or diphtheria toxoids did not develop myasthenic weakness or anti-AChR Ab. CD4+ cells are necessary for MG pathogenesis; CD8+ cells may not be. CD4+ cells specific for "universal" AChR epitopes help the synthesis of pathogenic Ab.

TCR-Vβ Usage in the Thymus and Blood of Myasthenia Gravis Patients

In myasthenia gravis (MG) the muscle acetylcholine receptor (AChR) is the target of an autoimmune response. The anti-AChR response may originate in the thymus, which is abnormal in most MG patients and contains anti-AChR T and B cells. Microbial superantigens (sAg) may trigger autoimmune responses and in this study we sought clues as to whether sAg play a role in the pathogenesis of MG. We investigated the frequency of use of the different TCR Vβ families by the thymus and blood T cells in MG patients and in control subjects, using a multi-primer PCR assay. Identical TCR-Vβ usage was found in the thymi of MG patients and controls, except Vβ2, which showed a small increase in MG patients’ thymi. Blood T cells of MG patients used Vβ4, Vβ6, Vβ15, Vβ16 and Vβ24 significantly more than those of the controls. Vβ4 and Vβ6 are the gene families most frequently used by anti-AChR CD4+cells in MG patients. Blood T cells from MG patients used Vβ12, Vβ14, Vβ17 and Vβ18 significantly less than controls. MG patients used Vβ4 and Vβ6 significantly more in the blood than in the thymus, while the opposite occurred for Vβ7, Vβ12 and Vβ14. Controls used Vβ17 more and Vβ24 less in the blood than in the thymus. The preferential expansion of Vβ4 and Vβ6 in MG patients might reflect the immunodominance of certain AChR epitopes, or the action of a sAg outside the thymus. The minimal differences in the TCR-Vβ usage in the blood and thymus of control subjects might be due to expansion of T cell clones specific for common antigens. Identical Vβ usage in the thymi of MG patients and controls does not support an important role of the thymus as the location of anti-AChR sensitization when MG is clinically evident. The differences observed in the Vβ usage in blood and thymi of MG patients are likely to be due to preferential Vβ usage by the anti-AChR T cells in the blood.

A pathogenetic role for the thymoma in myasthenia gravis. Autosensitization of IL-4- producing T cell clones recognizing extracellular acetylcholine receptor epitopes presented by minority class II isotypes.

Myasthenia gravis (MG) is caused by helper T cell-dependent autoantibodies against the muscle acetylcholine receptor (AChR). Thymic epithelial tumors (thymomas) occur in 10% of MG patients, but their autoimmunizing potential is unclear. They express mRNAs encoding AChR alpha and epsilon subunits, and might aberrantly select or sensitize developing thymocytes or recirculating peripheral T cells against AChR epitopes. Alternatively, there could be defective self-tolerance induction in the abundant maturing thymocytes that they usually generate. For the first time, we have isolated and characterized AChR-specific T cell clones from two MG thymomas. They recognize extracellular epitopes (alpha75-90 and alpha149-158) which are processed very efficiently from muscle AChR. Both clones express CD4 and CD8alpha, and have a Th-0 cytokine profile, producing IL-4 as well as IFN-gamma. They are restricted to HLA-DP14 and DR52a; expression of these minority isotypes was strong on professional antigen-presenting cells in the donors' tumors, although it is generally weak in the periphery. The two clones' T cell receptor beta chains are different, but their alpha chain sequences are very similar. These resemblances, and the striking contrasts with T cells previously cloned from non-thymoma patients, show that thymomas generate and actively induce specific T cells rather than merely failing to tolerize them against self antigens.

Synthetic peptides fail to induce nasal tolerance to experimental autoimmune myasthenia gravis

Nasal administration of Torpedo acetylcholine receptor (AChR) to Lewis rats prior to induction of experimental autoimmune myasthenia gravis (EAMG) is highly efficient in prevention of clinical weakness, and suppression of AChR-specific T and B cell responses. To identify possible antigenic determinants within the receptor which can modulate EAMG and anti-AChR response, we evaluated the effects of nasal administration of α 61–76, α 100–116, α 146–162, δ 354–367, and α 261–277 of Torpedo AChR at different doses on the tolerance induction against EAMG irrespective if given at lower, the same or higher doses than whole Torpedo AChR protein, that was confirmed to be highly efficient as tolerogen to EAMG. None of these peptides, neither administrated alone nor in combination, induced tolerance to EAMG. Peptide administration did not affect the levels or affinities of anti-AChR antibodies when compared with non-tolerized control EAMG rats, while administration of whole AChR protein affected both variables. The results may indicate that the T and B cell heterogeneity of AChR epitopes makes it difficult to induce tolerance using synthetic peptide.

Acetylcholine receptor epitopes in ocular myasthenia.

Annals of the New York Academy of SciencesVolume 841, Issue 1 p. 309-319 Acetylcholine Receptor Epitopes in Ocular Myastheniaa HENRY J. KAMINSKI, Corresponding Author HENRY J. KAMINSKI Department of Neurology; Case Western Reserve University School of Medicine; Department of Veterans Affairs Medical Center in Cleveland; University Hospitals of Cleveland; Cleveland, OhioAddress for correspondence: Department of Neurology. University Hospitals of Cleveland, Ohio 44106.Search for more papers by this author HENRY J. KAMINSKI, Corresponding Author HENRY J. KAMINSKI Department of Neurology; Case Western Reserve University School of Medicine; Department of Veterans Affairs Medical Center in Cleveland; University Hospitals of Cleveland; Cleveland, OhioAddress for correspondence: Department of Neurology. University Hospitals of Cleveland, Ohio 44106.Search for more papers by this author First published: 07 February 2006 https://doi.org/10.1111/j.1749-6632.1998.tb10937.xCitations: 11 a This work was supported by NIH Grant No. EY-00332 and the Office of Research and Development, Medical Research Service of the Department of Veterans Affairs. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume841, Issue1MYASTHENIA GRAVIS AND RELATED DISEASES: DISORDERS OF THE NEUROMUSCULAR JUNCTIONMay 1998Pages 309-319 RelatedInformation

Reactivity of T cells from seronegative patients with myasthenia gravis to T cell epitopes of the human acetylcholine receptor.

Seronegative (SN) patients with myasthenia gravis (MG) have clinical and electrophysiologic features similar to those of seropositive (SP) patients, and they respond to the same therapeutic measures. However, because SN patients lack detectable (by standard radioimmunoassays) serum antibodies to acetylcholine receptor (AChR), which are considered to have a crucial role in MG, the pathophysiologic basis for the disease is not clear. We therefore compared the ability of peripheral blood lymphocytes (PBL) of SN patients (11) and SP patients (39) to respond to myasthenogenic T cell epitopes of human AChR. We tested two aspects that relate to T-cell immunity: 1) T cell responses to myasthenogenic peptides by proliferation and IL-2 production, and 2) the ability of antigen-presenting cells to bind these T-cell epitopes. T cells of SN patients did not differ from those of SP patients in their ability to respond and to bind the two human AChR-derived myasthenogenic peptides. This supports the belief that most SN patients indeed suffer from an autoimmune disease directed against the AChR. The presence of T-cell immunity in the absence of antibodies may emphasize the importance of AChR-specific T cells in MG.

Th1 epitope repertoire on the alpha subunit of human muscle acetylcholine receptor in myasthenia gravis.

In myasthenia gravis (MG), CD4+ T helper cells recognize the muscle acetylcholine receptor (AChR) alpha subunit. We investigated the epitope repertoire of anti-AChR blood CD4+ Th1 cells from 13 myasthenic patients and three healthy controls, using overlapping synthetic peptides screening the alpha subunit sequence and an enzyme-linked immunospot (ELISPOT) assay that detects antigen-induced interferon-gamma secretion of individual Th1 cells. All patients recognized a pool of the alpha subunit peptides. All but one patient recognized numerous peptides. Each patient had an individual pattern of peptide recognition, but most or all patients recognized four sequences (residues 48-67, 101-137, 304-322, and 403-437) that stimulated relatively large numbers of Th1 cells. They include previously identified "immunodominant" sequences recognized by AChR-specific CD4+ T cell lines from myasthenic patients. Peptide 1-14 was also recognized frequently. The controls recognized, with a low precursor frequency, the peptide pool and a few peptides that frequently included the immunodominant sequences described above. The present results demonstrate that Th1 cells are involved in the anti-AChR response in MG and that their epitope repertoire is very complex. This indicates that when MG is clinically evident, the AChR itself is the sensitizing antigen and the target of the autoimmune Th1 cells, although it does not exclude that molecular mimicry between one AChR epitope and a microbial structure may have triggered this autoimmune response. Although the complexity of the Th1 repertoire suggests that development of specific immunosuppressive treatments targeted on epitopes recognized by autoimmune T cells will be difficult, the existence of immunodominant T epitope sequences might facilitate that task.

Paraneoplastic autoimmunity in thymus tumors.

Autoimmune phenomena are more frequent in thymic epithelial tumors (TET) than in any other human tumor. Mysthenia gravis (MG) is by far the most common autoimmune disease in thymoma patients. MG is characterized by muscle weakness due to autoantibodies against the acetylcholine receptor (AChR), and CD4 +AChR-specific T cells play a pivotal role for the production of these autoantibodies. About 10% of MG patients have a thymoma and, interestingly, only such thymomas exhibit an MG association that maintains thymuslike morphological and functional features with respect to the homing and differentiation of immature T cells. Since AChR protein is not expressed in thymomas, the specificity of the autoimmunity in thymoma-associated MG is thought to be determined by nonreceptor proteins with AChR epitopes. Such proteins are overexpressed in cortical-type MG-associated thymomas, and medullary thymomas express these proteins at barely detectable levels. Aside from this quantitative difference, the pathogenesis of anti-AChR autoimmunity might be qualitatively different in these thymoma subtypes. Our findings suggest that an antigen-specific abnormal Tcell selection by cortical-type TET may contribute to the pathogenesis of paraneoplastic MG. In contrast, an abnormal (intratumorous) activation of autoreactive T cells may be operative in medullary thymomas.

TCR Vβ Usage by Acetylcholine Receptor-Specific CD4+T Cells in Myasthenia Gravis

In myasthenia gravis the muscle acetylcholine receptor (AChR) is the target of an autoimmune response. AChR epitopes recognized by CD4+T cells in myasthenic patients have been identified. AChR-specific CD4+cell lines can be propagated by stimulation of blood lymphocytes with synthetic or biosynthetic AChR sequences. We analysed, using a semi-quantitative PCR assay, the T cell receptor (TCR) Vβ usage of 16 anti-AChR polyclonal CD4+T cell lines of known epitope specificity, propagated from myasthenic patients using pools of overlapping peptides corresponding to the sequence of an AChR subunit, or individual synthetic AChR sequences. Twelve lines had been propagated for less than 2 months, four lines for 3.5–5 months. Most lines had limited Vβ usage, but in most cases different Vβ regions were used for different epitopes in the same patient, and for the same epitope in different patients. In a few patients, the same Vβ regions were used for recognition of different epitopes. The Vβ4 and Vβ6 regions were used most frequently. These findings suggest that the potentially autoimmune T cells that survive clonal deletion have a limited TCR repertoire. Although the present data do allow conclusions on the role of a superantigen in triggering the anti-AChR autoimmune response, the finding that different Vβ regions were used in different patients does not support an important role of a superantigen in the maintenance of the CD4+response in myasthenia gravis.

TCR gene usage in experimental autoimmune myasthenia gravis pathogenesis. Usage of multiple TCRBV genes in the H-2b strains.

Experimental autoimmune myasthenia gravis (EAMG) is an Ab-mediated autoimmune disease. The pathogenic auto-antibody production depends on the activation of CD4+ cells after their TCR interact with dominant T cell epitopes within acetylcholine receptor (AChR) in the context of the MHC class II molecule. In vitro analysis suggested that the TCRBV6 was the predominant TCR that recognized AChR and one of the dominant epitopes, alpha 146-162, in C57BL6 (B6, H-2b) mice. However, in vivo depletion of TCRBV6 cells in H-2b mice by anti-TCRBV6 mAb neither suppressed the in vitro immune response to AChR nor prevented development of EAMG. Moreover, B10.TCRc (H-2b) strain with a genomic deletion of TCRBV genes including TCRBV6, and B10.V beta 8.2 transgenic mice with a restricted TCRBV8S2 T cell repertoire, responded to AChR, alpha 146-162, and developed EAMG after immunizations with AChR/CFA. These data suggest that more than one TCRBV-bearing cell having the affinity for AChR-dominant peptides is involved in pathogenesis. Therefore, depletion of a single TCRBV (e.g., TCRBV6) with mAb may not be sufficient to completely suppress the response to AChR and development of EAMG. However, if a similar amino acid sequence in the TCR-VDJ (e.g., CDR3) region among different TCRBV gene(s) could be involved in recognizing the dominant AChR epitope(s), then motif-specific mAb reactive to the common motif within the VDJ region of different TCR could be used to eliminate the T cell clones involved in EAMG.

Myasthenia gravis. Residues of the alpha and gamma subunits of muscle acetylcholine receptor involved in formation of immunodominant CD4+ epitopes.

We propagated from myasthenia gravis (MG) patients by stimulation in vitro with synthetic sequences (alpha 48-67, alpha 304-322, gamma 75-94 and gamma 321-340) of the human muscle acetylcholine receptor (AChR), CD4+ lines against four 20-residue sequence regions of the AChR alpha and gamma subunits that are recognized by Th cells of most MG patients. Most lines secreted IL-2 and not IL-4, suggesting that they comprise Th1 cells. For three lines we verified that, as reported previously, AChR epitopes are presented by DR molecules: their response to the relevant peptide was abolished by anti-DR Abs. The DR molecules presenting AChR epitopes were identified by testing the response of the lines to the relevant peptide, using APC from donors homozygous for the different DR alleles of the line. We tested the lines with single residue-substituted analogues of the epitope sequence. The results of these experiments indicated that the lines were polyclonal and recognized overlapping epitopes. Their response was abolished by some substitutions, identifying residues common to all epitopes within a given region, whereas other substitutions reduced but did not obliterate the response, indicating residues included in some but not all epitopes recognized by the line. Comparison of the residues involved in epitope formation for different lines supported the conclusion that within the 20-residue immunodominant regions investigated here, the same sequence segment is involved in formation of epitopes, even in DR-discordant patients.

Specific tolerance to an acetylcholine receptor epitope induced in vitro in myasthenia gravis CD4+ lymphocytes by soluble major histocompatibility complex class II-peptide complexes.

In autoimmune disorders, inactivation of pathogenic antigen-specific T cells, rather than global immunosuppression, would be highly desirable. One way to achieve this would be to deliver the first antigen-specific signal to the T cell in the absence of the second costimulatory signal. Myasthenia gravis (MG) is a well-characterized autoimmune disease in which T cell-dependent autoantibodies are directed against the acetylcholine receptor (A ChR) at the neuromuscular junction. AChR-specific T cells have been cloned from MG patients, and in this study, we have induced long-lasting tolerance in vitro in one particular clone (PM-A1) with a known peptide epitope (alpha 144-163) and MHC class II restriction (DR4 Dw14.2 or 4.2) by using soluble MHC-class II peptide complexes. Preincubation of PM-A1 T cells with such complexes induced death by apoptosis in < or = 40-50% of the AChR-specific cells. Surviving cells remained refractory to stimulation with AChR-derived synthetic peptides or recombinant polypeptides for < or = 38 d after complex treatment. These effects were highly specific, dose-dependent and required > 2 h preincubation. The T cells could be protected from the tolerizing effects of complex by coincubation with DR-matched or -mismatched antigen-presenting cells. This work shows that antigen-specific T cells can be selectively killed or anergized using soluble MHC class II: peptide complexes. Such an antigen-specific therapy offers a rational approach to the immunotherapy of autoimmune or allergic disease in vivo.

Processing requirements of two acetylcholine receptor derived peptides for binding to antigen presenting cells and stimulation of murine T cell lines.

We have previously identified two myasthenogenic T cell epitopes of the human acetylcholine receptor (AChR) alpha subunit, peptides p195-212 and p259-271. These peptides were the immunodominant T cell epitopes of AChR in SJL and BALB/c mice respectively, and only cryptic in C3H.SW strain. In order to find out whether these mouse strains differ in their requirements for processing of the same T cell epitopes, we used p195-212 specific T cell lines from SJL, TCSJL195-212, and C3H.SW, TCSW195-212, mice, and p259-271 specific T cell lines from BALB/c, TCBALB/c259-271, and C3H.SW, TCSW259-271, mice. The peptide-specific proliferative responses of the lines TCSW195-212 and TCSW259-271, originated from strains in which these peptides are cryptic epitopes, were inhibited significantly in the presence of several inhibitors of proteases or glutaraldehyde-fixed antigen presenting cells (APC). In contrast, the proliferative responses of the lines TCSJL195-212 and TCBALB/c259-271, established from strains in which these peptides are immunodominant, were only slightly affected by the above inhibitors or by fixation of the APC. Using a direct binding assay of biotinylated peptides to live intact APC, we showed that peptides p195-212 and p259-271 preserved their binding capacity to APC of SJL and BALB/c mice respectively when processing was inhibited. Thus, the AChR peptides that represent cryptic T cell epitopes have to be processed before they can be recognized by T cells, whereas no further processing is necessary for APC presentation and T cell stimulation when these peptides are immunodominant epitopes.

V beta-specific immunotoxin selectively kills acetylcholine receptor-reactive T lymphocytes from mice with experimental autoimmune myasthenia gravis.

Immunization of C57BL/6 mice with purified acetylcholine receptor (AChR) is known to induce a T cell-dependent antibody response that results in experimental autoimmune myasthenia gravis (EAMG). Since past observations link V beta 6+ T cells with a prominent AChR epitope specificity, a V beta 6-specific immunotoxin (VIT6) was tested in vitro for its ability to selectively kill monoclonal and polyclonal T cells that demonstrate reactivity against AChR. Results described below clearly demonstrate the ability to selectively kill AChR-reactive T cells based on their expression of a particular V beta-associated antigen receptor.