Background/Purpose: Previous studies have shown that Rag1^-/- mice transferred with splenocytes of M3 muscarinic acetylcoline receptor (M3R)^-/- mice immunized with M3R peptides mixture (three N-terminal regions; N1, N2, N3, and three extracellular loops; 1^st, 2^nd, 3^rd) (M3R^-/-→Rag1^-/-) developed sialadenitis like Sjögren’s syndrome (M3R induced autoimmune sialadenitis; 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 T cells. To develop antigen-specific therapy, we tried to clarify the T cell epitopes of M3R reactive T cells in MIS and also to evaluate the antagonistic APLs.

Methods:

1) Splenocytes of M3R^-/- mice immunized with M3R peptides mixture were cultured with each M3R peptide. The cytokines (IFN-g, IL-17, and Il-4) production was measured by ELISA. Similarly, the splenocytes of M3R^-/- mice immunized with each N1 and 1^st peptide, which was the candidate for the T cell epitopes, and the combination of N1 and 1^st peptides were cultured and evaluated.

2) The splenocytes of M3R^-/- mice, immunized with N1 alone, 1^st alone, the combination of N1 and 1^st peptides, or PBS as control were transferred into Rag1^-/- mice (each M3R^-/-→Rag1^-/-, M3R^-/-<1^st>→Rag1^-/-, M3R^-/-st>→Rag1^-/-, and M3R^-/-→Rag1^-/-). On day 45 after transfer, the salivary glands of Rag1^-/- mice were pathologically examined.

3) APLs of each N1 and 1^s peptide were designed.

4) CD4+ and CD11c+ cells were isolated from M3R^-/- mice immunized with each N1 and 1^st peptide. Each APL was loaded to CD11c+ cells pre-cultured with each suboptimal concentration (N1: 1.5 µM, 1^st: 4.5 µM) of N1 and 1^st peptide. Afterward, CD4+ T cells were added and cytokines (IFN-g and IL-17) production was measured by ELISA.

Results:

1) Splenocytes immunized with M3R peptide mixture produced IL-17 and IFN-g against N1 and 1^st peptide. IL-4 production could not be detected. Splenocytes immunized with N1 and 1^st peptide produced IL-17 and IFN-g, but  not IL-4, against each corresponding peptide.

2) M3R^-/-→Rag1^-/-, M3R^-/-<1^st>→Rag1^-/-, and M3R^-/-st>→Rag1^-/- developed sialoadenitis. The majority of infiltrating cells in salivary glands were CD4⁺ T cells, only few with CD8⁺ T cells and B220⁺ B cells. The focus scores of these mice (1.0±0.3 in M3R^-/-st>→Rag1^-/-, 1.3±0.3 in M3R^-/-→Rag1^-/-, and 0.7±0.3 in M3R^-/-<1^st>→Rag1^-/-) were significantly higher than that of control mice (0.1±0.2 in M3R^-/-→Rag1^-/-) (p<0.05).

3) Seven APLs of N1 peptide (N1-APL 1-7) and eight APLs of 1^st peptide (1^st-APL 1-8) were designed.

4) N1-APL5 (AA15 N→T), N1-APL6 (AA15 N→C) and N1-APL7 (AA15 N→S) suppressed the production of IFN-g (N1-APL5 38.8±11.6 pg/mL, N1-APL6  54.9±16.5 pg/mL, N1-APL7 38.0±15.7 pg/mL, control 363.6±65.1 pg/mL). 1^st-APL8 (AA140 A→M) suppressed both IL-17 and IFN-g production (IL-17: 1^st-APL8 403.4±14.1 pg/mL, control 720.3±49.0 pg/mL. IFN-g: 1^st-APL8 185.4±1.6 pg/mL, control 268.8±94.8 pg/mL).

Conclusion: The major T cell epitopes in MIS might be both N1 terminal region and 1^st extracellular loops of M3R. N1-APL5, 6, 7 and 1^st-APL8 were the candidate for antagonistic APLs, which might have a possibility to suppress MIS in vivo.

---

« Back to 2013 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-suppressive-ability-of-altered-peptide-ligands-to-m3r-reactive-t-cells-in-m3r-induced-autoimmune-sialadenitis/