MOLECULAR EVENTS IN ISLET ANTIGEN PRESENTATION

最近，几种胰岛抗原与IDDM有关，但是， 控制其通过MHC II类分子呈递的因子是 未知 本提案的主要目标是确定 参与产生由循环免疫球蛋白识别的表位， 自身反应性T细胞，以及不同谱系的细胞在 疾病进展，使用IDDM的NOD模型。 在具体目标1中，不变链和DM在生成中的作用 II类：将检查来自胰岛抗原的肽复合物。 我们将创建APC，其差异仅在于这些辅因子的表达 然后评估胰岛自身抗原的呈递。 此外，本发明还提供了一种方法， 将进行生化分析，以评估是否NOD II类 分子显示独特的细胞内运输模式或非典型的 与Ii和/或DM的相互作用，以评估一些独特的 与该II类等位基因相关的疾病涉及全身性 参与II类限制性细胞内事件的特征 演示文稿. 在具体的目标2中，不同谱系的细胞产生 将检测循环自身免疫T细胞识别的表位 并将重点放在巨噬细胞，树突细胞，内皮细胞和 胰腺β细胞 我们将评估这些细胞是否产生 活化T细胞表位，或者如果它们产生拮抗剂， 肽;MHC II类复合物。 为此，我们还将评估 胰岛抗原被循环T细胞识别的潜力 IDDM 9 GAD，羧肽酶H，HSP 60）由内源性 II类介绍途径。 这些都没有表现出明显的 在细胞表面的水平，每一个都有一个独特的亚细胞 这可能导致产生独特的肽。 最后，在目标3中，将测试MHC II类分子的表达是否 缺乏辅助功能的细胞上的分子，特别是胰腺 β细胞对抗自身免疫过程 转基因NOD小鼠将 衍生的组成型表达其自身的同源II类 胰腺β细胞上的分子。 除了监测 转基因小鼠的发病率，我们也将使用几个过继 转移策略来促进自身免疫性附着。 如果胰腺 组成型表达II类分子的β细胞能够 显示II类胰岛抗原特异性表位，并且如果它们的辅助表位 功能仍然不足以区分T细胞的TjhI亚群， 细胞，它们可能能够拮抗 这种疾病 自身免疫性CD 4 T细胞的表型特征将 在体内和体外进行评估。 终于，直接证明 组成性疾病引起的疾病拮抗机制 胰腺β细胞上的NOD II类表达与 特别是他们缺乏辅助功能，我们将越过RIP-I- Ag 7小鼠至RIP-B7-1小鼠，其将赋予胰腺β细胞 对T细胞活化有很强的辅助活性。

Recently, several islet antigens have been implicated in IDDM, but the factors that control their presentation by MHC class II molecules is unknown. The main objective of this proposal is to identify the components involved in generation of the epitopes recognized by the circulating autoreactive T cells, and the role that cells of distinct lineages play in disease progression, using the NOD model of IDDM. In specific aim 1, the roles that invariant chain and DM play in generation of class II: peptide complexes from the islet antigens will be examined. We will create APC that differ only in the expression of these cofactors and then evaluate presentation of the islet autoantigens. In addition, biochemical analyses will be performed to evaluate whether the NOD class II molecule displays unique intracellular trafficking patterns or atypical interactions with Ii and/or DM to evaluate whether some of the unique disease association with this class II allele relate to generalized characteristics of intracellular events involved in class II restricted presentation. In specific aim 2, the capacity of cells of different lineages to generate the epitopes recognized by circulating autoimmune T cells will be examined and will focus on macrophages, dendritic cells, endothelial cells and pancreatic beta cells. We will evaluate whether these cells generate the activating T cell epitope, or alternatively if they generate antagonist peptide;MHC class II complexes. In this aim we will also evaluate the potential of the islet antigens recognized by circulating T cells during IDDM 9GAD, carboxypeptidase H, HSP 60) to be presented by an endogenous class II presentation pathway. None of these are expressed at appreciable levels at the cell surface, and each has a distinctive subcellular localization that might lead to generation of unique peptides. Finally, in aim 3, it will be tested whether expression of MHC class II molecules on cells that lack accessory function, specifically pancreatic beta cells, antagonizes the autoimmune process. Transgenic NOD mice will be derived that constitutively express their own syngeneic class II molecules on their pancreatic beta cells. In addition to monitoring transgenic mice for disease incidence, we will also use several adoptive transfer strategies to precipitate autoimmune attach. If the pancreatic beta cells that constitutively express class II molecules are able to display class II islet antigen specific epitopes, and if their accessory function remains insufficient for differentiation of the TjhI subset of T cells, they may be capable of antagonizing the initiation or progression of the disease. The phenotypic characteristics of autoimmune CD4 T cells will be evaluated both in vivo and in vitro. Finally., to directly demonstrate that the mechanism involved in disease antagonism caused by constitutive expression NOD class II on the pancreatic beta cells is related specifically to their lack of accessory function, we will cross the RIP-I- Ag7 mouse to the RIP-B7-1 mouse, which will endow the pancreatic beta cell with potent accessory activity for T cell activation.