Experimental autoimmune myasthenia gravis is induced in C57BL/6 mice by injection of Torpedo
nicotinic acetylcholine receptor (TAChR). We investigated here the presence of cryptic CD4+
epitopes on the TAChR molecule, and their relationship with potentially autoreactive CD4+ cells, which survived clonal deletion. CD4+ cells from C57BL/6 mice immunized with native or denatured TAChR were challenged in vitro with overlapping synthetic
peptides, 20-residue long, screening the sequences of TAChR alpha, gamma, and delta subunits. Only three
epitopes on the alpha subunit were recognized consistently. Mice immunized with large doses (nanomoles) of TAChR clearly recognized only the immunodominant sequence T alpha 150-169. Anti-TAChR CD4+ cells did not cross-react with murine alpha subunit sequences, or with any synthetic sequence of human gamma and delta subunits, which are very similar to the corresponding murine subunits. To facilitate recognition of cryptic
epitopes, we injected mice with pools of synthetic
peptides corresponding to the sequences of TAChR alpha, gamma, and delta subunits. In addition to the three immunodominant alpha subunit
epitopes, other
epitopes were recognized by CD4+ cells within the sequences T alpha 304-322, T gamma 105-124, T gamma 120-139, T gamma 401-420, T gamma 357-376, T delta 16-35, T delta 61-80, T delta 121-140, and T delta 301-320. CD4+ cells thus sensitized cross-reacted with the mammalian sequences alpha 304-322, gamma 105-124, gamma 120-139, and delta 301-320. Mice were immunized with large doses (approximately 40 nmol) of individual TAChR synthetic cryptic
epitopes. CD4+ cells sensitized to five cryptic
epitopes (the ones listed above plus delta 121-140) cross-reacted with autologous sequences. We determined the dose dependence of the sensitization of CD4+ cells in vivo to the strongly
immunodominant epitope peptide T alpha 150-169 and to the cryptic
epitope peptides T gamma 120-139 and T delta 301-320 by immunizing mice with increasing doses of
peptide (approximately 1.2 to approximately 20 nmol), and testing the in vitro anti-
peptide response of the CD4+ cells. No difference was found for the
epitopes tested. Doses of 3 to 10 micrograms induced a strong CD4+ sensitization, and the dose dependence of the in vitro response of the sensitized cells to the relevant
peptide was comparable. Production of cryptic
epitopes upon in vitro TAChR processing was investigated by testing
peptide-sensitized CD4+ cells with native TAChR: only two cryptic
epitopes were produced.