Cloning of molecular targets of Cell Mediated Autoimmunity in Type 1 Diabetes

描述(由申请人提供)：这项修订的竞争性更新的主要目标是开发可转化为人类的1型糖尿病抗原特异性预防疗法。到目前为止，我们的研究旨在从机制上理解NOD小鼠模型的致病过程，重点是8类蛋白酪氨酸磷酸酶家族，这些酶是人类体液自身免疫的主要靶标。我们已经确定了人类和小鼠CD4+T细胞克隆的共同靶标的保守抗原肽序列，以及糖尿病自身抗体识别的构象表位，这些表位在3亿多年的进化过程中一直被保守。我们已经实施了体外评估T细胞细胞因子反应(ELISPOT)的新技术，以及使用逆转录小鼠快速筛选致病T细胞受体的方法，以进一步评估这些抗原的致病重要性。这笔赠款还支持对人类锌转运蛋白8的体液和细胞免疫反应的初步表征，锌转运蛋白8是一种主要的SS细胞特异性人类糖尿病自身抗原；这是十多年来首次发现的。目前的提案建立在这些初步数据的基础上，体现了重点的转移，使用NOD小鼠作为临床前模型，开发以机械为基础的预防人类1型糖尿病的新疗法。具体目标1：NOD小鼠自发产生的识别ZnT8的自身反应T细胞的特征。我们的方法在很大程度上是经验性的，目的是识别多个候选的抗原特异性TCR，并通过在NOD小鼠模型中的回生来评估它们的疾病潜力。其基本目标是从锌离子中筛选出5-10个在疾病发病机制中起关键作用并可能具有治疗潜力的多肽。我们还将测试在上一个资助期确定的Phogrin TCR的糖尿病发生。具体目标2：对NOD小鼠进行以锌为导向的干预措施的临床前研究这个目的是探索以锌为基础的治疗方法的实际发展，同时注意在疾病的早期和晚期可能发生这种抑制的机制。在实验上，这包括分析缺乏ZnT8的NOD小鼠的T1D的发生率和进展率，以及基于主要的ZnT8或phogrin T细胞表位多肽或编码cDNA的多肽和DNA疫苗策略的发展。我们预计，我们的结果将证实，这两种自身抗原在NOD小鼠糖尿病发生的早期和晚期都可以成为有效的治疗靶点，并将为最终可移植到人类的药物和方案的设计提供重要的新见解。

DESCRIPTION (provided by applicant): The major goal of this revised competing renewal is the development of antigen-specific preventative therapies of type 1 diabetes that can be translated to man. Our studies to date have been aimed at a mechanistic understanding of the pathogenic processes in the NODmouse model focused on a family of class 8 protein tyrosine phosphatases that are major targets of humoral autoimmunity in humans. We have identified conserved antigenic peptide sequences that are common targets of both human and mouse CD4+ T cell clones, and conformational epitopes recognized by diabetes autoantibodies that have been conserved through over 300 million years of evolution. We have implemented new technologies for the in vitro evaluation of T cell cytokine responses (ELISPOT), and methodologies for rapidly screening pathogenic T cell receptors using retrogenic mice to further evaluate the pathogenic importance of these antigens. The grant also supported the initial characterization of humoral and cell-mediated immune responses to human Zn transporter 8, a major new ss-cell specific human diabetes autoantigen; the first to be discovered in over a decade. The current proposal while building upon these preliminary data embodies a shift in emphasis by using the NOD mouse as a pre-clinical model for the development of mechanistically-based new therapies for prevention of type 1 diabetes in humans. Specific Aim 1: Characterization of spontaneously-arising autoreactive T cells recognizing ZnT8 in NOD mice. Our approach here is largely empirical aimed at identifying multiple candidate antigen-specific TCRs and evaluating their disease potential by retrogenesis in a NOD mouse model. The fundamental objective is to identify 5-10 peptides from ZnT8 that are key to the pathogenesis of disease and which may have therapeutic potential. We will also test the diabetogenesis of the phogrin TCRs identified in the previous funding period. Specific aim 2: Pre-clinical studies of ZnT8-directed interventions in NOD mice This aim explores the practical development of ZnT8-and phogrin-based therapies, while at the same time giving heed to the mechanisms by which such suppression might occur in early and late stages of the disease. Experimentally this involves analysis of the incidence and rate of progression of T1D in NOD mice lacking ZnT8, and development of peptide and DNA based vaccination strategies based on dominant ZnT8 or phogrin T cell epitope peptides, or encoding cDNA. We anticipate that our results will confirm that both autoantigens can be effective therapeutic targets in NOD mice, both early and late during diabetogenesis, and will provide important new insight into the design of drugs and protocols that can ultimately be translated to humans.