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

描述（由申请人提供）：本次修订竞争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。迄今为止，我们的研究旨在了解NODmouse模型中致病过程的机制，重点关注8类蛋白酪氨酸磷酸酶家族，这是人类体液自身免疫的主要靶点。我们已经确定了保守的抗原肽序列，这些序列是人类和小鼠CD4+ T细胞克隆的共同靶点，以及糖尿病自身抗体识别的构象表位，这些构象表位已经保守了超过3亿年的进化。我们已经实施了用于体外评估T细胞细胞因子反应的新技术（ELISPOT），以及使用逆转录小鼠快速筛选致病性T细胞受体的方法，以进一步评估这些抗原的致病重要性。该基金还支持了对人类Zn转运蛋白8（一种主要的新的ss细胞特异性人类糖尿病自身抗原）的体液和细胞介导的免疫反应的初步表征；这是十多年来首次被发现。目前的建议是建立在这些初步数据的基础上，通过使用NOD小鼠作为开发基于机械的预防人类1型糖尿病新疗法的临床前模型，体现了重点的转变。特异性目的1：表征NOD小鼠中自发产生的识别ZnT8的自身反应性T细胞。我们的方法在很大程度上是经验性的，旨在鉴定多种候选抗原特异性tcr，并通过NOD小鼠模型中的逆行性评估其疾病潜力。基本目标是从ZnT8中鉴定出5-10个在疾病发病机制中起关键作用的肽，这些肽可能具有治疗潜力。我们还将测试在之前的资助期内确定的phogrin tcr的糖尿病发生。本研究旨在探索以znt8和phogrin为基础的治疗方法的实际发展，同时关注这种抑制在疾病早期和晚期可能发生的机制。实验研究包括分析缺乏ZnT8的NOD小鼠中T1D的发病率和进展速度，以及基于显性ZnT8或phogrin T细胞表位肽或编码cDNA的肽和DNA疫苗策略的开发。我们预计，我们的研究结果将证实，这两种自身抗原都可以成为NOD小鼠糖尿病发生早期和晚期的有效治疗靶点，并将为最终可转化为人类的药物设计和方案提供重要的新见解。

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.