Cryptic epitopes on the nicotinic acetylcholine receptor are recognized by autoreactive CD4⁺ cells

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 α, γ, and δ subunits. Only three epitopes on the α subunit were recognized consistently. Mice immunized with large doses (nanomoles) of TAChR clearly recognized only the immunodominant sequence Tα150-169. Anti-TAChR CD4⁺ cells did not cross- react with murine α subunit sequences, or with any synthetic sequence of human γ and δ 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 α, γ, and δ subunits. In addition to the three immunodominant α subunit epitopes, other epitopes were recognized by CD4⁺ cells within the sequences Tα304-322, Tγ105-124, Tγ120-139, Tγ401-420, Tγ357-376, Tδ16-35, Tδ61- 80, Tδ121-140, and Tδ301-320. CD4⁺ cells thus sensitized cross-reacted with the mammalian sequences α304-322, γ105-124, γ120-139, and δ301-320. Mice were immunized with large doses (~40 nmol) of individual TAChR synthetic cryptic epitopes. CD4⁺ cells sensitized to five cryptic epitopes (the ones listed above plus δ121-140) cross-reacted with autologous sequences. We determined the dose dependence of the sensitization of CD4⁺ cells in vivo to the strongly immunodominant epitope peptide Tα150-169 and to the cryptic epitope peptides Tγ120-139 and Tδ301-320 by immunizing mice with increasing doses of peptide (~1.2 to ~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 μg 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.