Parameters that influence CD4+ T cell diabetogenic potential

描述（由申请人提供）：1型糖尿病（T1D）是一种复杂的疾病，由T细胞进入胰岛并随后破坏产生胰岛素的B细胞介导。控制这些事件的机制知之甚少，更深入的了解可能会导致新的治疗策略的发展。解决这些复杂问题的一个重大限制是缺乏适当的遗传工具。我们已经开发了一种新的方法用于快速生成TCR Tg小鼠，我们称之为逆转录（Rg）小鼠。我们将使用一组TCR特异性的两个主要自身抗原的相当大的兴趣，胰岛素（Ins）和嗜铬粒蛋白A（ChgA），具有非常广泛的胰岛素和糖尿病的潜力，新的小鼠模型和创新，尖端技术，以确定生物物理和时间参数，调节CD4 + T细胞胰岛素和糖尿病。这是该项目的主要重点和长期目标。最简单的假设是"高度致糖尿病的CD4 + T细胞在胰腺淋巴结（PLN）中最佳地产生和活化，并且具有决定其致病性的独特生物物理、动力学和迁移特征"。将在两个高度整合的目的中检验这一假设并定义参数：目的1：影响CD4 + T细胞致糖尿病性的生物物理参数。(A)TCR的生物物理特性决定了CD4 + T细胞的胰岛素生成和糖尿病生成潜力吗？与Brian Evavold（埃默里大学）合作，我们将测定Ins-和ChgA-特异性TCR对其同源肽-MHC复合物（pMHC）的2D亲和力、结合（开）速率和解离（关）速率。结合TCR Rg T细胞在功能测定中的敏感性，我们将确定这些生物物理参数是否决定其致糖尿病潜力。(B)抗原可用性是否影响CD4 + T细胞胰岛进入和糖尿病发生？我们将产生NOD小鼠，其中模型自身抗原GAD 65的B细胞限制性表达可以被外部控制。我们将评估抗原表达水平和TCR功能亲和力如何合作影响T细胞致病性。目的2：影响CD4 + T细胞致糖尿病性的时间参数。(A)CD4 + T细胞进入胰岛的动力学是否决定了糖尿病发生的可能性？我们将比较通过流式细胞术和组织学分析评估的T细胞胰岛浸润的动力学，以及使用活小鼠的非侵入性生物发光成像确定的B细胞破坏率。(B)胰岛内CD4 + T细胞的迁移特性是否决定了糖尿病发生的可能性？与亚历山大Chervonsky（大学芝加哥）合作，多光子活体显微镜将用于跟踪胰岛内的CD 4 + T细胞迁移，以确定其迁移特征与其胰岛素和糖尿病发生潜力之间是否存在相关性。 公共卫生相关性：1型糖尿病（T1D）是一种复杂的疾病，其由T细胞进入胰岛和随后破坏产生胰岛素的B细胞介导。控制这些事件的分子和机制知之甚少。它们的鉴定是至关重要的，并可能导致新的治疗策略的发展，以治愈或限制T1D和改善人类健康。

DESCRIPTION (provided by applicant): Type 1 diabetes (T1D) is a complex disease that is mediated by T cell entry into pancreatic islets and the subsequent destruction of insulin-producing b cells. The mechanisms that control these events are poorly understood, a greater understanding of which could lead to the development of novel therapeutic strategies. A significant limitation in addressing these complex issues has been the availability of appropriate genetic tools. We have developed a new approach for the rapid generation of TCR Tg mice, which we refer to as retrogenic (Rg) mice. We will use a panel of TCRs specific for two primary autoantigens of considerable interest, insulin (Ins) and chromogranin A (ChgA), that possess a very broad range of insulitogenic and diabetogenic potentials, novel mouse models and innovative, cutting-edge techniques to determine the biophysical and temporal parameters that regulate CD4+ T cell insulitogenicity and diabetogenicity. This is the primary focus and long-term objective of this project. The simplest hypothesis is that 'highly diabetogenic CD4+ T cells are optimally generated and activated in the pancreatic lymph node (PLN), and have a unique biophysical, kinetic and migratory signature which dictates their pathogenicity'. This hypothesis will be tested and parameters defined in two highly integrated Aims: Aim 1: Biophysical parameters that influence CD4+ T cell diabetogenicity. (A) Do the biophysical properties of the TCR dictate the insulitogenic and diabetogenic potential of CD4+ T cells? In collaboration with Brian Evavold (Emory Univ.), we will determine the 2D affinity, association (on) rate, and dissociation (off) rate of the Ins- and ChgA-specific TCRs for their cognate peptide-MHC complex (pMHC). Together with the sensitivity of TCR Rg T cells in functional assays, we will determine if these biophysical parameters dictate their diabetogenic potential. (B) Does antigen availability affect CD4+ T cell islet entry and diabetogenicity? We will generate NOD mice in which the b cell-restricted expression of a model autoantigen, GAD65, can be externally controlled. We will assess how antigen expression levels and TCR functional avidity cooperate to influence T cell pathogenicity. Aim 2: Temporal parameters that influence CD4+ T cell diabetogenicity. (A) Does the kinetics of CD4+ T cell islet entry determine diabetogenic potential? We will compare the kinetics of T cell islet infiltration, assessed by flow cytometric and histological analysis, and the rate of b cell destruction, determined using non- invasive, bioluminescent imaging of live mice. (B) Do the intra-islet migratory characteristics of CD4+ T cells dictate diabetogenic potential? In collaboration with Alexander Chervonsky (Univ. Chicago), multiphoton intravital microscopy will be used to track CD4+ T cell migration within the islets to determine whether there is a correlation between their migratory characteristics and their insulitogenic and diabetogenic potential. PUBLIC HEALTH RELEVANCE: Type 1 diabetes (T1D) is a complex disease that is mediated by T cell entry into pancreatic islets and the subsequent destruction of insulin-producing b cells. The molecules and mechanisms that control these events are poorly understood. Their identification is of critical importance and could lead to the development of novel therapeutic strategies to cure or limit T1D and improve human health.