Background M3 muscarinic acetylcholine receptor (M3R) is evaluated to be one of the auto-antigens of Sjögren's syndrome (SS). Our previous studies have shown that Rag1–/– mice transferred with splenocytes of M3R–/– mice immunized with M3R peptides mixture (three N-terminal regions; N1, N2, N3, and three extracellular loops; 1st, 2nd, 3rd) (M3R–/–<Mix>→Rag1–/–) developed sialoadenitis like SS (M3R induced autoimmune sialoadenitis; MIS) . Besides, altered peptide ligands (APLs), the peptides with substitutions in amino acid residues at T-cell receptor contact sites, are reported to regulate the activation of antigen-specific T cells.
Objectives To develop antigen-specific therapy, we tried to clarify the T cell epitopes of M3R reactive T cells and also to evaluate the suppressive ability of APLs in MIS both in vitro and in vivo.
Methods 1) The splenocytes of M3R–/– mice immunized with M3R peptides mixture were cultured with each M3R peptide. The cytokines (IFN-g and IL-17) production was measured by ELISA. In a similar manner, the splenocytes of M3R–/– mice immunized with each N1 and 1st peptide, which was the candidate for the T cell epitopes, were cultured and evaluated.
2) The splenocytes of M3R–/– mice, immunized with each N1 and 1st peptide, were transferred into Rag1–/– mice (each M3R–/–<N1>→Rag1–/–, M3R–/-<1st>→Rag1–/–). On day 45 after transfer, the salivary glands of Rag1–/– mice were pathologically examined.
3) APLs of each N1 and 1st peptide were designed.
4) CD4+ and CD11c+ cells were isolated from M3R–/– mice immunized with each N1 and 1st peptide. Each APL was loaded to CD11c+ cells pre-cultured with each suboptimal concentration of N1 and 1st peptide (N1: 1.5 μM, 1st: 4.5 μM). Afterward, CD4+ T cells were added and cytokines production was measured by ELISA.
5) 100 μg of antagonistic APLs were administered to MIS (M3R–/–<Mix>→Rag1–/–) intravenously on day 7 and 10 after the cell transfer.
Results 1) Splenocytes immunized with M3R peptide mixture produced IL-17 and IFN-g against N1 and 1st peptide. Splenocytes immunized with N1 and 1st peptide produced IL-17 and IFN-g against each corresponding peptide.
2) Both M3R–/–<N1>→Rag1–/– and M3R–/–<1st>→Rag1–/– developed sialoadenitis. The majority of infiltrating cells in salivary glands were CD4+ T cells, with only few CD8+ T cells and B220+ B cells.
3) Seven APLs of N1 peptide (N1-APL1-7) and eight APLs of 1st peptide (1st-APL1-8) were designed.
4) N1-APL5 (AA15 N→T), N1-APL6 (AA15 N→C) and N1-APL7 (AA15 N→S) significantly suppressed the production of IFN-g (Inhibition ratio: N1-APL5 89.3%, N1-APL6 84.9%, N1-APL7 89.5%) (p<0.05). As well, 1st-APL8 (AA140 A→M) significantly suppressed IL-17 (Inhibition ratio: 1st-APL8 86.5%) (p<0.05).
5) All the antagonistic APLs in vitro also suppressed MIS in vivo, especially N1-APL7 (Focus score: N1-APL5: 0.78±0.51, N1-APL6: 0.89±0.77, N1-APL7: 0.11±0.12, 1st-APL7: 0.67±0.58, control: 1.44±0.51) (N1-APL7; p<0.05).
Conclusions The major T cell epitopes in MIS might be both N1 terminal region and 1st extracellular loop of M3R. Antagonistic APLs in vitro suppressed the induction of MIS in vivo.
Iizuka M, et al: Pathogenic role of immune response to M3 muscarinic acetylcholine receptor in Sjögren's syndrome-like sialoadenitis. J Autoimmun 2010; 35: 383-389.
Disclosure of Interest : None declared
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