Project 1 - Immunologic and Cellular Mechanisms of T1D Modifier Mutations

项目总结/摘要 项目1的目标是详细了解ENU诱导突变的病理后果 核心B和核心C涉及修饰NOD/NckH小鼠的T1 D表型，并确定细胞凋亡。 这些突变影响T1 D发展的机制。正如我们对Dup 10所做的那样， 在我们实验室基于表型选择培育纯合性的自发突变中，我们将分析每个 选择突变，以确定哪些显著的免疫病理学步骤被认为是T1 D的标志 启动和/或进展受到影响。为此，我们将使用诸如胰岛分析等工具， 浸润（即胰岛炎）;测量各种淋巴细胞中的数量和体内功能能力。 致糖尿病器官或调节性CD 4 + FoxP 3 + T淋巴细胞;以及胰岛抗原呈递分析 使用表达转基因BDC2.5 T细胞受体（对糖尿病特异性）的致糖尿病细胞的过继转移， 掺入胰岛素和嗜铬粒蛋白A序列的自身抗原融合肽）植入4周龄的接受者。 我们将特别关注胰岛素抵抗的机制，以及胰岛素抵抗增加的标志物的定义。 β细胞敏感性与特定的基因突变有关。在这样做的时候，小说似乎是合理的， 将发现免疫病理学步骤。第二，我们将研究特定组织的贡献， 使用骨髓移植、过继转移， 在新生儿中，胰岛移植、组织特异性条件性基因敲除小鼠和胰岛类器官培养物在新生儿中的应用， 免疫细胞的存在或不存在。结果将为项目2提供重要指导，重点是分子生物学。 发病机制我们将进行细胞机制研究，以了解Paqr 8突变的影响， （保护性）和Dusp 10，Rapgef 1和Xpnpep 1（加重），在我们最初的合作中确定。我们预计 同时积极进行4到6种蛋白质的机制研究，每年增加一种新的蛋白质， 与项目2达成协议。我们希望项目之间的强大协同作用将加速我们的进展。一个 我们要解决的一个重要问题是我们的发现与人类健康的相关性。了解如何 在NOD小鼠中鉴定的突变与人类T1 D相关，我们将研究选择的NOD突变的影响， 携带T1 D易感性人源化免疫系统的小鼠，所述人源化免疫系统经修饰以含有或缺乏所述正向免疫球蛋白 人造血干细胞和/或人胰岛细胞中的突变。我们还将识别人类T1 D患者， 与我们在NOD小鼠筛选中所涉及的那些基因正交的基因突变。我们的发现将揭示 T1 D治疗的新治疗靶点，并有助于改善对人类T1 D发作的预测。

PROJECT SUMMARY/ABSTRACT The goal of Project 1 is to understand in detail the pathological consequences of ENU-induced mutations implicated by Core B and Core C to modify the T1D phenotype of NOD/NckH mice, and to determine the cellular mechanisms by which those mutations impact the development of T1D. As we have done for Dusp10, a spontaneous mutation bred to homozygosity in our lab based on phenotypic selection, we will analyze each selected mutation to determine which salient immunopathological steps—recognized as hallmarks of T1D initiation and/or progression—are affected. To that end, we will use tools such as analysis of pancreatic islet infiltration (i.e. insulitis); measurement of the numbers and the in vivo functional capacity in various lymphoid organs of diabetogenic or regulatory CD4+FoxP3+ T lymphocytes; and analysis of islet antigen presentation using adoptive transfer of diabetogenic cells expressing the transgenic BDC2.5 T cell receptor (specific for an autoantigenic fusion peptide incorporating insulin and chromogranin A sequences) into 4-week-old recipients. We will pay particular attention to mechanisms of insulin resistance, and to the definition of markers of increased β-cell sensitivity witnessed with particular genetic mutations. In doing so it is plausible that novel immunopathological steps will be discovered. Second, we will investigate the contribution of particular tissues or cell types bearing each mutation to the development of T1D using bone marrow transplantation, adoptive transfer in neonates, islet transplantation, tissue-specific conditional knockout mice, and islet organoid cultures in the presence or absence of immune cells. Results will provide important guidance to Project 2, focused on molecular pathogenesis. We will perform cellular mechanistic studies to understand of the effects of mutations in Paqr8 (protective), and Dusp10, Rapgef1, and Xpnpep1 (exacerbating), identified in our initial collaboration. We expect to actively pursue mechanistic studies of four to six proteins at a time, adding one new protein per year by mutual agreement with Project 2. We expect that strong synergy between Projects will accelerate our progress. An important question we will address is the relevance of our findings to human health. To understand how mutations identified in NOD mice relate to human T1D, we will study the effects of selected mutations in NOD mice bearing a T1D-susceptible humanized immune system modified to contain or lack the orthologous mutations in human hematopoietic stem cells and/or human islet cells. We will also identify human T1D patients with mutations in genes orthologous to those we implicated in our screen of NOD mice. Our findings will uncover new therapeutic targets for treatment of T1D and help to improve prediction of T1D onset in humans.