The anergy induction of M3 muscarinic acetylcholine receptor-reactive CD4⁺ T cells suppresses experimental Sialadenitis-like Sjögren's syndrome

Objective Autoreactive CD4⁺ T cells are involved in the pathogenesis of Sjögren's syndrome (SS). The aim of the present study was to clarify the dominant T cell epitopes of M3 muscarinic acetylcholine receptor (M3R) and to establish a new antigen-specific therapy for SS using an experimental mouse model. Methods Production of cytokines from M3R-reactive CD4+ T cells, after culture with various M3R peptides, was analyzed by enzyme-linked immunosorbent assay. Adoptive cell transfer was performed using splenocytes from M3R^-/- mice that were immunized with M3R peptides or phosphate buffered saline plus H37Ra as a control. Rag1^-/- mice were inoculated with the splenocytes and examined for the development of sialadenitis. Altered peptide ligands (APLs) of the T cell epitopes, with substitutions in amino acid residues at T cell receptor contact sites, were synthesized, and the ability of the APLs to suppress sialadenitis was evaluated. The mechanisms underlying such effects were assessed. Results CD4+ M3R-reactive T cells produced interleukin-17 (IL-17) and interferon-γ (IFNγ) in response to the N-terminal 1 (N1) and 1st extracellular loop peptides of M3R, and Rag1^-/- mice that received N1- and/or 1st peptide-immunized splenocytes developed sialadenitis. Among the designed APLs, N1-APL7 (N→S at amino acid 15) significantly suppressed IFNγ production in vitro, and also suppressed sialadenitis in vivo. Levels of early growth response 2 in CD4+ T cells from the cervical lymph nodes of N1-APL7-treated mice were significantly higher than those of control mice, and cell proliferation was reversed by administration of exogenous IL-2. Levels of the anergy-related molecules itchy homolog E3 ubiquitin-protein ligase, Casitas B-lineage lymphoma b, gene related to anergy in lymphocytes, and Deltex-1 were significantly higher in CD4+ T cells cultured with N1-APL7. Conclusion The major T cell epitopes were from the N1 and 1st peptide regions. Moreover, N1-APL7, selected as the antagonistic APL in vitro, also suppressed sialadenitis through the induction of anergy. This is a potentially useful strategy for regulating pathogenic T cell infiltration in SS.