Epitope sepcificity relates to GAD65-specific regulation of pathogenesis

描述(由申请人提供)： 谷氨酸脱羧酶(GAD65)是最早诱导糖尿病相关T、B细胞自身免疫反应的胰岛β细胞抗原之一。然而，最近的证据支持，GAD65多肽的特异性T细胞也可以作为调节剂来抑制非肥胖糖尿病(NOD)小鼠的1型糖尿病(T1D)。由于抗原处理和提呈细胞(APC)是第一个遇到外来和自身抗原的细胞参与者，它们的相互作用对于启动适应性免疫反应和建立自我耐受是必不可少的，我们假设T1、D易感和耐药、MHC匹配的个体之间抗原处理机制中存在的差异对于致病效应物的产生而不是调节因子的产生是必不可少的，反之亦然。我将研究GAD65决定簇的处理和呈现，以及它们在敏感的NOD株和同种耐药的NOR株中的特异性CD4+T细胞的激活。将考察两种可能但相互非排他性的处理机制，它们可能影响MHC II分子上呈现的GAD65中肽决定因素的选择：1)在遥远的决定因素中与MHC II结合的可及性--可能与MHC有高亲和力的外源性免疫诱导决定子与可能隐蔽的内源性自发产生的决定簇；2)在糖尿病相关决定簇区域内重叠表位的侧翼氨基酸残基的差异酶切或修剪。针对GAD65多肽的T细胞系和杂交瘤克隆将被用来检测它们的特定多肽在敏感和抗性动物中处理后或在不同APC群体的培养中的呈现情况。将进行过继转移实验，以检测GAD65特异性T细胞的胰岛渗透和激活，这些T细胞被染料和可区分的表面标志标记。最重要的是，我将使用一种强大的谱系分析方法，CDR3长度光谱分型(免疫显微镜)，来识别和跟踪细胞系中的主要T细胞克隆，以及在它们转移后的胰岛中。最后，我将研究与糖尿病相关的候选基因Idd5.2及其候选基因Nramp1(自然抵抗相关巨噬细胞蛋白1)，它们在NOD和NOR品系中不同，以确定它们在NOD小鼠明显异常的处理和呈现机制中的作用。

DESCRIPTION (provided by applicant): Glutamic acid decarboxylase (GAD65) is one of the earliest pancreatic beta-cell antigens that induces a diabetes-related T and B cell autoimmune response. However, recent evidence supports that T cells specific for GAD65 peptides could also function as regulatory agents to inhibit type 1 diabetes (T1D) in non-obese diabetic (NOD) mice. Since antigen processing and presentation cells (APC) are the first cellular players to encounter foreign and self antigens, whose interactions are essential for initiating adaptive immune responses and establishing self tolerance, we hypothesize that the existing differences within the antigen processing machinery between T1 D-susceptible and -resistant, MHC-matched individuals are essential for the generation of the pathogenic effectors rather than the regulators, or vice versa. I will study processing and presentation of GAD65 determinants and activation of their specific CD4+ T cells in the susceptible NOD strain and a congenic resistant NOR strain. Two possible, but mutually non-exclusive, processing mechanisms that could affect the choice of peptide determinants from GAD65 to be presented on MHC II molecules will be examined: 1) accessibility to MHC II binding among distant determinants -the exogenous, immunization-induced determinants, likely of high affinity for MHC, vs. the endogenous, spontaneous-arising determinants, that are possibly cryptic; 2) differential enzymatic cutting or trimming of flanking amino acid residues of overlapping epitopes within the diabetes-associated determinant regions. T cell lines and hybridoma clones specific for GAD65 peptides will be used to examine the presentation of their specific peptides following processing in the susceptible and resistant animals or in culture by various APC populations. Adoptive transfer experiments will be performed to examine islet infiltration and activation of GAD65-specific T cells that are labeled with dyes and distinguishable surface markers. Most importantly, I will use a powerful repertoire analysis method, CDR3-length spectratyping (Immunoscope), to identify and track the dominant T cell clones in the cell lines, as well as in the islets after their transfer. Finally, I will examine one candidate diabetes-associated locus, Idd5.2, and its candidate gene, Nramp1 (Natural Resistance-Associated Macrophage Protein 1), which are different between the NOD and NOR strains, for their contribution to the apparent abnormal processing and presentation machinery in NOD mice.