Project 1 - Immunologic and Cellular Mechanisms of T1D Modifier Mutations

项目摘要/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参与改变NOD/Nck H小鼠的T1D表型，并确定细胞 这些突变影响T1D发育的机制。就像我们为Dusp10所做的那样，一个 在我们实验室通过表型选择培育出纯合的自发突变，我们将分析每一个 选择突变以确定哪些显著的免疫病理步骤-被识别为T1D的特征 --开始和/或进展--受到影响。为此，我们将使用诸如胰岛分析等工具 浸润性(即：胰岛炎症)：不同淋巴的数量和体内功能能力的测量 促糖尿病或调节性CD4+FoxP3+T淋巴细胞器官；胰岛抗原提呈分析 过继转移表达转基因BDC2.5 T细胞受体的糖尿病细胞 含有胰岛素和嗜铬粒蛋白A序列的自体抗原融合肽)注入4周大的受体。 我们将特别关注胰岛素抵抗的机制，以及增加的标志物的定义。 β-细胞对特定基因突变的敏感性。在这样做的情况下，这部小说是有道理的 免疫病理步骤将被发现。其次，我们将调查特定组织或 携带每种突变的细胞类型对T1D发育的影响 在新生儿中，胰岛移植，组织特异性条件基因敲除小鼠，以及胰岛器官培养 免疫细胞的存在或缺失。结果将为项目2提供重要的指导，重点是分子 发病机制。我们将进行细胞机制研究，以了解Paqr8基因突变的影响 (保护性)，以及Dusp10、Rapgef1和XpnPep1(加重)，在我们最初的合作中确定。我们预计 积极进行一次四到六种蛋白质的机理研究，每年通过相互补充一种新的蛋白质 与项目2达成一致。我们预计项目之间的强大协同将加快我们的进度。一个 我们将解决的重要问题是我们的发现与人类健康的相关性。要了解如何 在NOD小鼠中发现的突变与人类T1D相关，我们将研究NOD中选定的突变的影响 携带T1D敏感人源化免疫系统的小鼠被修改为包含或不包含同源基因 人类造血干细胞和/或人类胰岛细胞的突变。我们还将识别人类T1D患者 基因突变与我们在NOD小鼠筛查中发现的基因同源。我们的发现将揭开 治疗T1D的新治疗靶点，并有助于提高人类T1D发病的预测。

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.