mInsA2-10改造肽诱生抗原特异性CD8+CD25+ Tregs防治I型糖尿病的机制研究

Autoreactive CD8+ T cells play a fundamental pathogenic role in type 1 diabetes (T1D). Immune tolerance of CD8+ T cells induced autoantigen and altered peptide ligand (APL) became a research hotspot of prevention of T1D. Insulin was a key target of pathogenic T cells and the first β-cell protein to which an autoimmune response was documented in T1D. Therefore, immune tolerance of insulin-specific CD8+ T cells might prevent the development of T1D. We already targeted the InsA2-10 which is murine and human immunodominant CD8+ T cell epitope. Many APLs were designed by molecular simulation, and the APLs of significant antagonist was screened in vitro. We found multiple intraperitoneal injection of the APL DQ4 could prevent diatetes of HLA-transgenic NOD mice. Immune tolerance of InsA2-10-specific CD8+ T cells was confirmed, and CD8+CD25+ regulatory T cells (Tregs) were induced. .Specific CD8+CD25+Tregs induced by the DQ4 shall be further studied on immunosuppression function, phenotype, cytokine secretion, immunosuppression mechanism, and creation mechanism, which shall provide new strategy and experiment data for prevention of T1D, and new candidate drug for clinic transformation.

自身反应性CD8+T细胞是I型糖尿病（T1D）发生的关键病因，用自身抗原肽或改造肽（APL）诱导自身反应性CD8+T细胞免疫耐受，成为T1D防治研究热点。胰岛素是最早启动T1D的关键靶抗原，靶向诱导其特异性CD8+T细胞耐受，可能防止T1D发生。前期我们以人鼠同源优势CD8+T细胞表位mInsA2-10为靶标设计了若干APL，通过体外实验筛选出显著拮抗性APL，在人源化T1D小鼠中证实了多次腹腔注射APL DQ4能致自身反应性CD8+T细胞耐受，且CD8+CD25+Tregs显著上调表达，而具有T1D保护作用。本研究拟结合细胞和分子生物学实验方法，通过体内外实验研究DQ4诱生的抗原特异性CD8+CD25+Tregs的免疫抑制功能、表型特征和免疫抑制机制，同时探讨DQ4特异性的CD8+CD25+Tregs的产生机制，这将为T1D防治提供新策略和实验依据，也为临床转化提供新的候选药物。

自反应性CD8+ T细胞在胰岛β细胞的破坏和1型糖尿病(T1D)的发生中起着直接的关键作用。胰岛素是T1D中一种必需的β细胞自身抗原。胰岛素A链mInsA2-10是HLA-A*0201转基因人源化T1D小鼠中CD8+ T细胞的免疫优势表位。T细胞受体（TCR）作用位点的氨基酸替换可产生改造肽 (APL)，APL可能通过改变自身反应T细胞的TCR信号来调节其效应。本研究采用计算机分子模拟策略，通过在mInsA2-10的潜在TCR作用位点(位置4和6)上用D -氨基酸取代原L -氨基酸从而产生两条APL mInsA2-10DQ4和mInsA2-10DC6。通过体内实验，我们发现，腹腔多次注射DQ4，而不是DC6，可显著抑制人源化T1D小鼠的T1D发展。进一步发现，mInsA2-10DQ4治疗不仅显著抑制了mInsA2-10的自身反应性CD8+ T细胞反应，而且也阻止了胰腺组织中CD4+ T和CD8+ T细胞的浸润，以及胰腺炎症反应。APL DQ4处理显著增加人源化NOD雌鼠脾脏和胰腺淋巴结中CD8+CD25+ T和CD8+CD25+Foxp3+ T细胞的频率；mInsA2-10DQ4系统处理人源化NOD雌鼠诱导产生的CD8+CD25+ T 细胞具有典型的调节性T细胞的表型特征，且CD8+CD25+ T 细胞具有肽特异性的免疫抑制功能。mIns A2-10 DQ4在体外诱导的CD8+CD25+ T细胞不是来源于CD8+CD25- T细胞；脾淋巴细胞体外诱导的CD8+CD25+ T细胞具有肽特异性免疫抑制功能。因此，mInsA2-10DQ4在T1D防治中可能具有潜在的临床应用价值。