Residues within the α subunit sequence 304-322 of muscle acetylcholine receptor forming autoimmune CD4⁺ epitopes in BALB/c mice

BALB/c mice develop myasthenic symptoms after immunization with rodent acetylcholine receptor (AChR). After immunization with Torpedo AChR (TAChR), their CD4⁺ cells become strongly sensitized against a conserved region of the TAChR α subunit sequence (residues α304-322), and cross-react vigorously with the homologous sequences of mouse and human AChR, which are almost identical. Therefore AChR-specific potentially autoreactive CD4⁺ cells exist in this strain. We immunized BALB/c mice with the synthetic TAChR sequence α304-322. The CD4⁺ cells thus sensitized responded to TAChR, indicating that they recognize an epitope(s) produced upon TAChR processing. They recognized peptide α304-322 in association with the I-A(d) molecule. Anti-α304-322 CD4⁺ cells cross-reacted well with the corresponding murine and human synthetic sequences. To identify residues involved in formation of an autoimmune epitope(s). CD4⁺ cells from mice immunized with peptide α304- 322 were challenged in vitro with single residue glycine-substituted analogues of this sequence. Substitution of residue W₃₁₁, and of any residue within the sequence α313-319 (RKVFIDT), consistently and, in some cases, strongly affected the CD4⁺ cells response. Substitution of residues in the region α311-319 had variable effects in different experiments, and in general affected moderately the CD4⁺ response. These results suggest that anti-α304-322 CD4⁺ cells comprise several clones, recognizing overlapping epitopes which share residues α311-319. The importance of the sequence region α311-319 for formation of CD4⁺ cell epitope(s) was verified by testing CD4⁺ cells sensitized to Tα304-322 with analogues of this sequence, carrying non-conservative substitutions at positions Q₃₁₀, K₃₁₄ and D₃₁₈. Substitution of Q₃₁₀ had minimal or no effects, while those of K₃₁₄ or D₃₁₈ strongly affected the CD4⁺ cell response.