THU0044 The Suppressive Ability of Altered Peptide Ligands to M3R Induced Autoimmune Sialoadenitis in Vivo
Authors: H. Asashima, H. Tsuboi, M. Iizuka, C. Hagiya, T. Hirota, Y. Kondo, I. Matsumoto, T. Sumida
Journal: Annals of the Rheumatic Diseases (2014)

<h3>Background</h3> M3 muscarinic acetylcholine receptor (M3R) is evaluated to be one of the auto-antigens of Sjögren9s syndrome (SS). Our previous studies have shown that Rag1<sup>–/–</sup> mice transferred with splenocytes of M3R<sup>–/–</sup> mice immunized with M3R peptides mixture (three N-terminal regions; N1, N2, N3, and three extracellular loops; 1st, 2nd, 3rd) (M3R<sup>–/–</sup>&lt;Mix&gt;→Rag1<sup>–/–</sup>) developed sialoadenitis like SS (M3R induced autoimmune sialoadenitis; MIS) [1]. 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. <h3>Objectives</h3> 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 <i>in vitro</i> and <i>in vivo</i>. <h3>Methods</h3> 1) The splenocytes of M3R<sup>–/–</sup> 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<sup>–/–</sup> 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<sup>–/–</sup> mice, immunized with each N1 and 1st peptide, were transferred into Rag1<sup>–/–</sup> mice (each M3R<sup>–/–</sup>&lt;N1&gt;→Rag1<sup>–/–</sup>, M3R<sup>–/-</sup>&lt;1st&gt;→Rag1<sup>–/–</sup>). On day 45 after transfer, the salivary glands of Rag1<sup>–/–</sup> mice were pathologically examined. 3) APLs of each N1 and 1st peptide were designed. 4) CD4+ and CD11c+ cells were isolated from M3R<sup>–/–</sup> 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<sup>–/–</sup>&lt;Mix&gt;→Rag1<sup>–/–</sup>) intravenously on day 7 and 10 after the cell transfer. <h3>Results</h3> 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<sup>–/–</sup>&lt;N1&gt;→Rag1<sup>–/–</sup> and M3R<sup>–/–</sup>&lt;1st&gt;→Rag1<sup>–/–</sup> developed sialoadenitis. The majority of infiltrating cells in salivary glands were CD4<sup>+</sup> T cells, with only few CD8<sup>+</sup> 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&lt;0.05). As well, 1st-APL8 (AA140 A→M) significantly suppressed IL-17 (Inhibition ratio: 1st-APL8 86.5%) (p&lt;0.05). 5) All the antagonistic APLs <i>in vitro</i> also suppressed MIS <i>in vivo</i>, 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&lt;0.05). <h3>Conclusions</h3> The major T cell epitopes in MIS might be both N1 terminal region and 1st extracellular loop of M3R. Antagonistic APLs <i>in vitro</i> suppressed the induction of MIS <i>in vivo</i>. <h3>References</h3> Iizuka M, et al: Pathogenic role of immune response to M3 muscarinic acetylcholine receptor in Sjögren9s syndrome-like sialoadenitis. J Autoimmun 2010; 35: 383-389. <h3>Disclosure of Interest</h3> : None declared <h3>DOI</h3> 10.1136/annrheumdis-2014-eular.1933