Parameters that influence CD4+ T cell diabetogenic potential

描述(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尚不清楚，对这些机制的更多了解可能会导致新的治疗策略的开发。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快速产生TCRTG小鼠的新方法，我们称之为逆转录(RG)小鼠。我们将使用一组针对两种主要自身抗原的TCR，胰岛素(INS)和嗜铬粒蛋白A(ChgA)，这两种抗原具有非常广泛的促胰岛素和促糖尿病潜能，新的小鼠模型和创新的尖端技术来确定调节CD4+T细胞的促胰岛素和促糖尿病的生物物理和时间参数。这是该项目的主要重点和长期目标。最简单的假设是“高度糖尿病的CD4+T细胞是在胰腺淋巴结(PLN)产生和激活的，并且具有独特的生物物理、动力学和迁移特征，这决定了它们的致病性。”这一假说将在两个高度整合的目标中进行检验和参数定义：目标1：影响CD4+T细胞糖尿病原性的生物物理参数。(A)TCR的生物物理特性是否决定了CD4+T细胞的绝缘性和致糖尿病潜能？与Emory大学的Brian Evavold合作，我们将确定它们的同源肽-MHC复合体(PMHC)的INS和CHgA特异性TCR的2D亲和力、缔合(ON)率和解离(OFF)率。结合TCR Rg T细胞在功能分析中的敏感性，我们将确定这些生物物理参数是否决定了它们的致糖尿病潜力。(B)抗原的可获得性是否影响CD4+T细胞进入胰岛和糖尿病的发生？我们将产生NOD小鼠，在其中，一种模型自身抗原GAD65的b细胞限制性表达可以受到外部控制。我们将评估抗原表达水平和TCR功能亲和力如何协同影响T细胞的致病性。目的2：影响CD4+T细胞致糖尿病作用的时间参数。(A)CD4+T细胞进入胰岛的动力学是否决定了糖尿病的发生潜力？我们将比较通过流式细胞术和组织学分析评估的T细胞胰岛渗透的动力学，以及通过非侵入性的活体小鼠生物发光成像确定的b细胞破坏率。(B)CD4+T细胞在胰岛内的迁移特性是否决定了糖尿病的发生？与Alexander Chervonsky(大学)合作在芝加哥)，多光子活体显微镜将被用来跟踪CD4+T细胞在胰岛内的迁移，以确定它们的迁移特征与它们的胰岛素和糖尿病形成潜力之间是否存在关联。

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.