Proteomics Analysis of T Cell Autoantigens in TID2

描述（由申请人提供）： 了解T1 D疾病过程的一个至关重要的部分是识别刺激糖尿病致T细胞的蛋白质。该项目的总体假设是，任何有助于发病机制的自身抗原都可能是治疗和诊断靶点。因此，从定义T细胞的抗原肽中实现的一个益处是提供可能有用地应用于诱导耐受性的试剂。我们的目标是利用生物化学分离和鉴定蛋白质（称为蛋白质组学）的方法学的最新进展来定义致病性T细胞的自身抗原，这些T细胞在成熟的小鼠模型中自发地出现在T1 D中，并且也可能参与人类疾病。具体而言，我们在该试验性应用中提出鉴定一组6-8个胰岛反应性、致糖尿病性CD 4 + T细胞克隆的抗原。我们的目标是：（1）通过对从刺激性和非刺激性胰岛素瘤获得的总细胞和所选细胞器蛋白质组的比较分析来鉴定自身反应性T细胞的候选抗原;（2）获得能够定量测量复杂混合物中溶解的抗原的稳健的生物测定，并在制备色谱和/或电泳分离后使用它们来鉴定来自胰腺b细胞的候选抗原;和（3）克隆和表达目的1和/或2中鉴定的候选蛋白的全长或片段形式，以确认其特异性免疫原性。

DESCRIPTION (provided by applicant): A critically important part of understanding the disease process in T1D is the identification of proteins that stimulate diabetogenic T cells. The overall hypothesis underlying this project is that any autoantigen that contributes to pathogenesis is potentially both a therapeutic and diagnostic target. Thus one benefit to be realized from defining antigenic peptides for T cells is the provision of reagents that might usefully be applied to the induction of tolerance. Our objective is to take advantage of recent advances in methodology for biochemical isolation and identification of proteins (termed proteomics) to define autoantigens for pathogenic T cells that spontaneously arise in T1D in a well-established mouse model, and which might also be involved in the human disease. Specifically, we propose in this pilot application to identify the antigens for a panel of 6-8 islet-reactive, diabetogenic CD4+ T cell clones. Our aims are: (1) to identify candidate antigens for autoreactive T cells through a comparative analysis of total cellular, and selected organellar, proteomes obtained from stimulatory and non-stimulatory insulinomas; (2) to derive robust bioassays capable of the quantitative measurement of solubilized antigens in complex mixtures and use them to identify candidate antigens from pancreatic b-cells following preparative chromatographic and/or electrophoretic separations; and (3) to clone and express full-length or fragmented forms of the candidate proteins identified in aims 1 and/or 2 for confirmation of their specific immunogenicity.