Epitope sepcificity relates to GAD65-specific regulation of pathogenesis

描述（由申请人提供）： 谷氨酸脱羧酶（GAD 65）是诱导糖尿病相关的T和B细胞自身免疫应答的最早的胰腺β细胞抗原之一。然而，最近的证据支持GAD 65肽特异性T细胞也可以作为调节剂来抑制非肥胖糖尿病（NOD）小鼠中的1型糖尿病（T1 D）。由于抗原加工和呈递细胞（APC）是遇到外来和自身抗原的第一个细胞参与者，它们的相互作用对于启动适应性免疫应答和建立自身耐受性是必不可少的，因此我们假设T1 D易感和抗性、MHC匹配个体之间抗原加工机制内的现有差异对于致病效应物而不是调节物的产生是必不可少的，或者反之亦然。我将研究GAD 65决定簇的加工和呈递及其特异性CD 4 + T细胞在易感NOD株和同源耐药NOR株中的活化。两种可能的，但相互不排斥的，加工机制，可能会影响选择的肽决定簇从GAD 65上提出的MHC II分子将被检查：1）可及性MHC II结合之间的遥远的决定簇-外源性，免疫诱导的决定簇，可能高亲和力的MHC，与内源性，自发产生的决定簇，可能是隐蔽的; 2）糖尿病相关决定簇区内重叠表位的侧翼氨基酸残基的差异酶切或修整。将使用对GAD 65肽具有特异性的T细胞系和杂交瘤克隆来检查在易感和抗性动物中或在各种APC群体的培养物中处理后其特异性肽的呈递。将进行连续转移实验以检查用染料和可区分的表面标记物标记的GAD 65特异性T细胞的胰岛浸润和活化。最重要的是，我将使用一种强大的库分析方法，CDR 3长度谱分析（Immunoscope），来识别和跟踪细胞系中以及转移后胰岛中的优势T细胞克隆。最后，我将研究一个候选糖尿病相关基因座，Idd5.2，和它的候选基因，Nramp 1（天然耐药相关巨噬细胞蛋白1），这是不同的NOD和NOR菌株之间，他们的贡献明显异常的加工和呈现机制在NOD小鼠。

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.