胰岛β细胞ERS诱导的MHC-Ⅰ类分子相关新生抗原肽鉴定及其在NOD小鼠胰岛早期启动CD8+T细胞活化中的作用

The autoreactive CD8+T cells against islet β cells are the key pathogenic factors for the early initiation of insulitis and T1D. Adaptive CD8+ T cells specifically recognize MHC I-presented peptides on the surface of target cells, then activate and exert effects. These MHC I-presented peptides are collectively referred to as the MHC-I associated immunopeptidome(MIP). However the islet β autoantigens, which mediate the activation and destruction of islet cells of autoreactive CD8+T cells in the early stage, are not fully understood. Endoplasmic reticulum stress (ERS) is one of the earliest pathological characteristic of islet in T1D. Recent studies have demonstrated that pathological ERS can induce abnormal protein post-translational modifications, fusion peptide and defect ribosome transcription product, etc., and subsequently produce "neo-antigens", which can be recognized as foreign antigens by autoreactive T cells. Over the past years, the ability of mass spectrometry to analyze complex peptide mixtures has provided unprecedented insights into the repertoire of peptides displayed by MHC molecules and recognized by T cells. Here we will applicate mass spectrometry to analysis of MIP from islet β cell under steady state and ERS condition respectively, and to identify ERS related neo-antigenic peptides , which are recognized by CD8+ T cells, through in vitro and in vivo immunological experiments using NOD mice model. We question whether pathological ERS of the islet β cell induce series MHC -Ⅰrelated neo-antigenic peptide, which can drive the autoreactive CD8 + T cell activation directly, mediated insulitis in early stage. This project is helpful to further elucidate the pathogenesis of T1D, and to provide new autoantigen targets for the prevention and control of T1D.

胰岛β细胞自身反应性CD8+T细胞是胰岛炎早期启动和T1D发生的关键致病因素。CD8+T细胞识别靶细胞表面MHC-Ⅰ类分子结合肽是其活化及发挥效应的前提。但驱动自身反应性CD8+T细胞早期活化并破坏胰岛β细胞的自身抗原谱尚不清楚。内质网应激（ERS）是T1D胰岛早期的病理特征之一，最新研究显示病理性ERS可通过诱导异常的蛋白质翻译后修饰、肽融合以及缺陷核糖体转录产物等，产生“新生抗原Neo-antigens”，进而活化T细胞并介导疾病。本课题拟利用高分辨率质谱技术，全貌鉴定稳态和ERS状态下胰岛β细胞表面MHC-I类分子结合肽组，并筛选出ERS相关的新生抗原肽，结合 NOD小鼠离体、在体免疫学功能实验明确胰岛β细胞病理性ERS是否诱生系列MHC-Ⅰ类分子相关新生抗原肽，从而驱动胰腺早期CD8+T 细胞直接激活，介导胰腺炎发生，进一步阐明T1D的发病机制，并为T1D防治提供新的自身抗原靶点。

Ⅰ型糖尿病（T1D）是由自身反应性T细胞介导的器官特异性自身免疫病，其中自身反应性CD8+ CTL细胞通过其TCR特异性识别胰岛β细胞表面MHC-Ⅰ类分子-自身抗原肽复合物而直接破坏β细胞。有核细胞产生的MHC-I类分子相关肽的总和称为MHC-Ⅰ相关免疫肽组（MIP），其组成受到细胞内在或外在刺激因素的调控，而该过程中新出现的自身抗原肽，可能成为特定状态下CD8+T细胞免疫耐受被打破的重要机制。内质网应激（ERS）作为T1D早期胰岛重要病理特征之一，与T1D发生发展密切关系。研究提示病理性ERS可增强胰岛β细胞免疫原性，该过程是否通过重塑β细胞MIP而新产生某些免疫原性自身肽尚不清楚。用20mM 葡萄糖（HG）和经典ERS诱导剂Thapsigargin（TG）处理，可诱导NOD小鼠来源的胰岛β细胞系NIT-1细胞发生显著增强的ERS，并显著增强NIT-1细胞刺激NOD小鼠脾脏CD8+ T细胞分泌IFN-γ的能力，该效应可被抗MHC-I类分子抗体阻断。提示：HG和TG诱导NIT-1细胞产生的ERS可能伴随着MHC-Ⅰ类分子相关新生抗原肽的出现。利用LC-MS/MS深度鉴定稳态和ERS态胰岛β细胞系NIT-1细胞MⅠPs，差异分析显示HG和TG诱导的ERS重塑了NIT-1细胞MⅠPs，发现了若干ERS态NIT-1细胞MⅠPs特有肽和丰度上调肽。进一步 利用NOD小鼠鉴定出若干具有免疫原性的ERS态NIT-1特有的新生抗原肽，其中SYT12291-299和OTUB258-66在NOD小鼠中具有免疫优势性。 NOD小鼠体内存在SYT12291-299和OTUB258-66特异性CD8+ T细胞，且能在相应肽刺激下发生特异性增殖和杀伤功能，被动回输NOD/scid小鼠有致糖尿病作用。高糖饮水可显著促进NOD小鼠肽特异性CD8+ T细胞反应。提示SYT12291-299和OTUB258-66可能是参与NOD小鼠T1D致病的重要自身抗原。人工多次免疫肽SYT12291-299和OTUB258-66可显著降低NOD小鼠的T1D发病率，机制研究提示：特异性免疫干预诱导了肽特异性CD8+ T细胞及其他致病性T细胞数量减少及功能的降低，可能与诱导凋亡和功能耗竭有关。SYT12291-299和OTUB258-66可能成为潜在的特异性免疫干预防治T1D的新靶点。

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