MOLECULAR EVENTS IN ISLET ANTIGEN PRESENTATION

最近，一些胰岛抗原被认为与IDDM有关，但 控制MHC II类分子呈现的因素是 未知。这项提案的主要目标是确定各组成部分 参与产生循环中识别的表位 自身反应性T细胞，以及不同谱系的细胞在其中发挥的作用 疾病进展，使用IDDM的NOD模型。 在特定目标1中，不变链和DM在生成中所起的作用 第II类：将检查来自胰岛抗原的多肽复合体。 我们将创建仅在这些辅因子的表达上不同的APC 然后评估胰岛自身抗原的呈递情况。此外, 将进行生化分析以评估NOD II级 分子表现出独特的细胞内转运模式或非典型 与II和/或DM的交互来评估一些独一无二的 与这个II类等位基因相关的疾病与全身性 II类限制性的细胞内事件的特征 演示文稿。 在特定目标2中，不同谱系的细胞产生 将检查循环中自身免疫T细胞识别的表位 将专注于巨噬细胞、树突状细胞、内皮细胞和 胰岛β细胞。我们将评估这些细胞是否产生 激活T细胞表位，或者如果它们产生拮抗剂 多肽；MHC II类复合体。在这个目标中，我们还将评估 外周循环T细胞识别胰岛抗原的潜能 IDDM 9GAD，羧肽酶H，HSP 60)将由内源性 第二类呈现途径。这些都没有在可察觉到的情况下表达 在细胞表面，每个水平都有一个独特的亚细胞 可能导致产生独特多肽的定位。 最后，在目标3中，将测试MHC II类分子的表达 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 构成地表达他们自己的同源基因II类的 它们的胰岛β细胞上的分子。除了监控之外 针对转基因小鼠的发病情况，我们还将采用几种收养方式 转移策略以加速自身免疫依恋。如果胰腺 构成表达II类分子的β细胞能够 显示II类胰岛抗原特异性表位，如果它们的附属 函数仍然不足以区分T的TjhI子集 细胞，它们可能能够拮抗肿瘤的启动或进展 这种疾病。自身免疫的CD4T细胞的表型特征将 在体内和体外都要进行评估。最后，为了直接演示 体质引起的疾病拮抗的机制 Nod-II类在胰腺β细胞上的表达与 具体到他们缺乏辅助功能，我们将越过RIP-I- Ag7小鼠移植到RIP-B7-1小鼠，这将赋予胰岛β细胞 具有很强的激活T细胞的辅助活性。

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.