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Myasthenia gravis (MG) is a T-cell regulated autoimmune disease. A peptide representing a sequence of the human acetylcholine receptor alpha-subunit (p195-212) was previously shown to stimulate proliferative responses of peripheral blood lymphocytes from MG patients and to be an immunodominant and myasthenogenic T-cell epitope in SJL mice. The authors generated a panel of analogues of p195-212 that contain single amino acid substitutions. Three of the analogues (203PHE, 204GLY and 207ALA) triggered low to no proliferative responses of a p195-212-specific T-cell line designated TCSJL195-212. Two of these analogues were able to stimulate the line to produce interleukin-2 (IL-2) and IL-4 (203PHE and 204GLY), whereas one analogue, 207ALA, did not stimulate the line to produce these cytokines. Binding assays revealed that the binding affinity of an altered peptide for a given major histocompatibility complex (MHC) molecule is not sufficient to determine whether it will be stimulatory or inhibitory to a native peptide-specific T-cell line. Two of the analogues, 204GLY and 207ALA, inhibited proliferative responses of cells of the TCSJL195-212 line when co-cultured with p195-212 and syngeneic antigen presenting cells (APC). The two inhibitory analogues were also able to inhibit proliferative responses of the TCSJL195-212 line when APC were pre-pulsed with p195-212, indicating that MHC blockade is not the only mechanism of action of these peptides. Moreover, both analogues inhibited the ability of p195-212 to prime lymph node cells for proliferative responses even when the analogues were administered in a soluble form. Thus the altered peptide ligands 207ALA and 204GLY can modulate T-cell responses both in vitro and in vivo and may have therapeutic potential for the treatment of MG.

Myasthenia gravis is an autoimmune disease in which T cells specific to epitopes of the autoantigen, the human acetylcholine receptor, play a role. We identified two peptides, p195-212 and p259-271, from the alpha subunit of the receptor, which bound to major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (APCs) from peripheral blood lymphocytes of myasthenia gravis patients and stimulated lymphocytes of >80% of the patients. We have prepared analogs of these myasthenogenic peptides and tested their ability to bind to MHC class II determinants and to interfere specifically with T-cell stimulation. We first determined relative binding efficiency of the myasthenogenic peptides and their analogs to APCs of patients. We found that single substituted analogs of p195-212 (Ala-207) and p259-271 (Lys-262) could bind to human MHC molecules on APCs as efficiently as the original peptides. Moreover, dual analogs containing the two single substituted analogs in one stretch (either sequentially, Ala-207/Lys-262, or reciprocally, Lys-262/Ala-207) could also bind to APCs of patients, including those that failed to bind one of the single substituted analogs. The single substituted analogs significantly inhibited T-cell stimulation induced by their respective myasthenogenic peptides in >95% of the patients. The dual analogs were capable of inhibiting stimulation induced by either of the peptides: They inhibited the response to p195-212 and p259-271 in >95% and >90% of the patients, respectively. Thus, the dual analogs are good candidates for inhibition of T-cell responses of myasthenia gravis patients and might have therapeutic potential.

MG is an autoimmune disease in which T cells specific to T-cell epitopes of the human acetylcholine receptor play a role. We have identified two peptides, p195–212 and p259–271, of the human acetylcholine receptor α-subunit, to which PBLs of MG patients responded by proliferation. Nevertheless, proliferation assays are relatively complicated to perform and might be affected by medications taken by the patients. Therefore, we tested the possibility of using a different assay to determine recognition of these peptides by MG patients. Thus, we performed a direct binding assay using biotinylated peptides and APCs from peripheral blood of MG patients and healthy controls. With this assay we detected the binding of the two peptides to the surface of intact APCs of both MG patients and control donors. Moreover, the presentation of peptide p259–271 by individuals with MG was significantly higher than that observed in healthy subjects. The peptides were specifically bound to HLA class II determinants on the APCs, as shown by inhibition with antibodies to the HLA. class II haplotypes of the individuals investigated. Moreover, the binding of these peptides was in correlation with their ability to induce specific proliferative responses of peripheral blood T cells of these patients. The ability to screen for potentially pathogenic epitopes in each patient is of importance for the future design of specific inhibitory analogues that might be used to treat MG.

Myasthenia gravis is a T-cell-regulated, antibody-mediated autoimmune disease. The synthetic peptides p195-212 and p259-271, which represent sequences of the human acetylcholine receptor α-subunit, preferentially stimulated T cells of patients with myasthenia gravis and were found to be immunodominant T cell epitopes in SJL and BALB/c mice, respectively. Therefore, we established a p195-212-specific T cell line from SJL mice and a p259-271-specific T cell line from BALB/c mice. N- and C-terminal truncated and/or extended peptides differed in their ability to stimulate proliferative responses of the lines and of their derived clones. Activated cells of the lines were inoculated into naive syngeneic mice. In both strains of mice, peptide-specific antibodies and antibodies to the murine acetylcholine receptor were detected. In addition, decremental compound muscle action potentials consistent with impairment of neuromuscular transmission were recorded from the line-inoculated mice. Thus, these T cell lines, clones, and epitopes constitute a useful model for investigating T cell pathogenicity in autoimmune manifestations related to myasthenia gravis.

The synthetic peptides p195-212 and p259-271, representing amino acids 195-212 and 259-271 of the alpha subunit of the human acetylcholine receptor, preferentially stimulate T cells of patients with myasthenia gravis and are immunodominant T-cell epitopes in SJL and BALB/c mice, respectively. We designed and synthesized analogs of these peptides that contain single amino acid substitutions. An analog of peptide p195-212, no. 455 (Met-207-->Ala), was capable of inhibiting up to 100% of the proliferative responses of a p195-212-specific T-cell line originating from the high-responder strain SJL. Similarly, an analog of p259-271, no. 306 (Glu-262-->Lys), was capable of inhibiting up to 93% of the proliferative responses of the p259-271-specific T-cell line originating from high-responder BALB/c mice. Analog 306 also inhibited up to 43% of the proliferative responses of p259-271-primed lymph node cells in an in vitro proliferation assay. To test the in vivo inhibitory activity of the analogs, mice were primed with the myasthenogenic peptides in complete Freund's adjuvant concomitant with administration of the analogs in aqueous solution. Administration of analogs 455 and 306 led to decreased proliferative responses of up to 70% by peptide p199-212-primed lymph node cells and up to 85% by peptide p259-271-primed lymph node cells. Similar results were obtained whether the analogs were administered i.v. or i.p. Thus, these analogs are good candidates for specific immunomodulatory therapy for patients with myasthenia gravis.

Myasthenia gravis (MG) is a T-cell-regulated autoimmune disease in which a pathological autoantibody response is mounted against the nicotinic acetylcholine receptor of the neuromuscular junction. Our laboratory previously identified a T cell epitope, p195-212, derived from the human acetylcholine receptor alpha subunit, which triggered PBL to proliferate from about 70% of MG patients tested. p195-212 was also found to be an immunodominant T cell epitope in SJL mice and a cryptic epitope in C3H.SW mice. Inoculation of naive SJL mice with cells from a p195-212-specific syngeneic T cell line caused MG-related autoimmune manifestations in those mice. In these studies we analyzed TCR alpha and beta chain sequences used by T cell lines and clones from both high- and low-responder mouse strains in response to p195-212. T cell lines generated from either strain expressed single TCR V beta gene segments (V beta 17 in SJL mice and V beta 8 in C3H.SW mice). By deleting V beta 17-expressing T cells in p195-212-immunized SJL mice we established a T cell line that expressed the V beta 6 gene product, suggesting that SJL mice are not limited to using a single V beta gene segment in response to p195-212. In addition, we found that N- and/or C-terminal-truncated peptides of p195-212, presented under the same culture conditions to different clones bearing the same TCR alpha beta chain, could elicit very different proliferative responses from the clones. Thus, even within a constrained system, factors other than TCR sequence contribute to the differential stimulation of T cell responses.

Myasthenia gravis (MG) is a T cell-regulated, antibody-mediated autoimmune disease. Two peptides representing sequences of the human acetylcholine receptor α-subunit, p195-212 and p259-271, were previously shown to stimulate peripheral blood lymphocytes of patients with MG and were found to be immunodominant T cell epitopes in SJL and BALB/c mice, respectively. Single amino acid substituted analogs of p195-212 (analog Ala-207) and p259-271 (analog Lys-262) were synthesized. We showed that analogs Ala-207 and Lys-262 inhibited,in vitroandin vivo, the proliferative responses of T cell lines specific to the relevant peptide and lymph node cells of mice immunized to p195-212 and p259-271, respectively. To inhibit T cell responses to both peptides (p195-212 and p259-271), we synthesized dual analogs composed of the tandemly arranged two single (Ala-207 and Lys-262) analogs (dual analog) either sequentially (Ala-207–Lys-262) or reciprocally (Lys-262–Ala-207). In the present study, we report that both dual analogs could bind to major histocompatibility complex class II molecules on antigen-presenting cells of SJL and BALB/c mice. Analog Lys-262–Ala-207, which bound more efficiently to major histocompatibility complex class II molecules, was found to inhibit the proliferative responses of both p195-212- and p259-271-specific T cell lines. Furthermore, the analog inhibited thein vivopriming of lymph node cells of both SJL and BALB/c mice when administered i.v., i.p., orper os. The dual analog Lys-262–Ala-207 could also immunomodulate myasthenogenic manifestations in mice with experimental autoimmune MG induced by inoculation of a pathogenic T cell line. Thus, a single peptide that is composed of analogs to two epitope specificities can be used to regulate T cell responses and disease associated with each epitope.