University of Tsukuba, Tsukuba, Japan.
Fureai Higashitotsuka Hospital, Yokohama, Japan.
Arthritis Rheumatol. 2015 May;67(8):2213-25. doi: 10.1002/art.39163.
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.
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.
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.
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.
自身反应性 CD4+T 细胞参与干燥综合征（SS）的发病机制。本研究旨在阐明 M3 毒蕈碱乙酰胆碱受体（M3R）的主要 T 细胞表位，并通过实验性小鼠模型建立新的针对 SS 的抗原特异性治疗方法。
通过酶联免疫吸附试验分析与各种 M3R 肽培养后的 M3R 反应性 CD4+T 细胞产生的细胞因子。使用 M3R 肽免疫的 M3R(-/-) 小鼠的脾细胞进行过继细胞转移，或用磷酸盐缓冲盐水加 H37Ra 作为对照。将 Rag1(-/-) 小鼠接种脾细胞，并检查涎腺炎的发展情况。合成 T 细胞表位的改变肽配体（APL），在 T 细胞受体结合部位的氨基酸残基进行取代，并评估 APL 抑制涎腺炎的能力。评估了这种效应的机制。
CD4+M3R 反应性 T 细胞对 M3R 的 N 端 1（N1）和 1 号细胞外环肽产生白细胞介素-17（IL-17）和干扰素-γ（IFNγ），接受 N1 和/或 1 号肽免疫脾细胞的 Rag1(-/-) 小鼠发生涎腺炎。在设计的 APL 中，N1-APL7（N→S 在 15 号氨基酸处）在体外显著抑制 IFNγ 的产生，并在体内也抑制涎腺炎。接受 N1-APL7 治疗的小鼠颈淋巴结 CD4+T 细胞中的早期生长反应因子 2 水平明显高于对照组，并且通过给予外源性 IL-2 逆转了细胞增殖。在与 N1-APL7 培养的 CD4+T 细胞中，瘙痒同源物 E3 泛素蛋白连接酶、Casitas B 谱系淋巴瘤 b、淋巴细胞无反应性相关基因和 Deltex-1 的无反应性相关分子水平明显升高。
主要 T 细胞表位来自 N1 和 1 号肽区。此外，体外选择的 N1-APL7 作为拮抗 APL，也通过诱导无反应性来抑制涎腺炎。这是一种调节 SS 中致病性 T 细胞浸润的潜在有用策略。
