Modeling autoimmune pathogenesis and beta cell destruction by T1D immune systems

项目摘要 1型糖尿病（T1 D）发病机制的研究一直受到动物模型不足的限制， 患者人群的异质性，他们从HLA和大约100个基因的不同解释中获得遗传风险， 60种非HLA遗传变异。考虑到这种复杂性，需要改进的人类T1 D模型 发病机制涉及不同的遗传背景。我们已经开发了人源化（HU）小鼠模型， 哪种T1 D患者和健康对照（HC）免疫系统由造血干细胞从头产生 在个体化免疫（PI）小鼠和将转基因（Tg）自身反应性TCR引入个体化免疫（PI）小鼠的模型中， 他们的造血干细胞和T细胞我们还开发了产生胸腺上皮细胞（TEC）祖细胞的方法 以及来自人类多能干细胞（hPSC）的β细胞，这将提高我们测试这些细胞影响的能力。 关键细胞群对T1 D风险的影响。我们将使用这些工具来解决HSC内在和 T1 D个体的胸腺内在遗传变异导致β细胞抗原的异常胸腺选择- 自身反应性TCR我们将：目标1：确定T1 D易感基因型对选择的影响。 人类胸腺中的自身反应性T细胞。我们已经证明了Tg HLA-DQ 8的负选择， HU小鼠的HLA-DQ 8+胸腺中的限制性胰岛素B 9 -23肽特异性TCR。我们将评估更多的β细胞- 自身反应性I类和II类限制性TCR，确定HSC和AIRE+ mTEC抗原的影响 T1 D患者与携带β细胞反应性TCR的HC胸腺细胞的表达和比较选择;目的2： 确定TEC的T1 D倾向基因型对胸腺选择β细胞反应性T细胞的影响， 人源化小鼠。我们将利用一种新的模型，其中hPSC-TEC在活支架上产生“杂交胸腺 胎猪胸腺组织，其支持HU小鼠中的人T细胞发育。我们将使用这个模型， 基因工程化的hPSC以确定人TEC表达对TID相关基因表达的影响。 目的3：评估hPSC衍生的TCR与自身免疫性TCR之间的自身免疫相互作用; 人源化小鼠中的β细胞和自身反应性T细胞。我们已经开发了hPSC衍生的排斥模型， 用于HU小鼠中的自身免疫性β细胞和通过将自身反应性TCR转导到其T细胞中的自身免疫性β细胞破坏。我们 已经产生了一种hPSC细胞系，该细胞系缺乏除HLA-A2之外的所有HLA，从而避免了HLA-A2+免疫排斥反应。 系统.这些将在体内和体外使用，表达和不表达HLA II类分子，DQ 8， 为了模拟TCRTg T1 D患者和HC衍生的T细胞的自身免疫破坏，评估对 II类HLA和CD 4有助于CD 8细胞介导的自身免疫β细胞破坏。我们的合作将 产生新的和强大的系统来模拟人类T1 D，增强对其发病机制的理解， 提供了一个测试免疫疗法的平台。

Project Summary The study of Type 1 diabetes (T1D) pathogenesis has been limited by the insufficiency of animal models and the heterogeneity of patient populations, who derive genetic risk from HLA and different assortments of about 60 non-HLA genetic variants. Given this complexity, there is a need for improved models of human T1D pathogenesis involving diverse genetic backgrounds. We have developed humanized (HU) mouse models in which T1D patient and healthy control (HC) immune systems are generated de novo from hematopoietic stem cells (HSCs) in Personalized Immune (PI) mice and models introducing transgenic (Tg) autoreactive TCRs into their HSCs and T cells. We have also developed methods of generating thymic epithelial cell (TEC) progenitors and β cells from human pluripotent stem cells (hPSCs) that will increase our ability to test the influence of these key cell populations on T1D risk. We will use these tools to address the hypothesis that HSC-intrinsic and thymus-intrinsic genetic variants in T1D individuals lead to abnormal thymic selection of β cell antigen- autoreactive TCRs. We will: Aim 1: Determine the impact of T1D-prone genotypes on selection of autoreactive T cells in the human thymus. We have demonstrated negative selection of a Tg HLA-DQ8- restricted insulin B9-23 peptide-specific TCR in HLA-DQ8+ thymi of HU mice. We will assess additional β cell- autoreactive class I- and class II-restricted TCRs, determine the impact of both HSC and AIRE+ mTEC antigen expression and compare selection of T1D patient vs HC thymocytes bearing β cell-reactive TCRs; Aim 2: Determine the impact of T1D-prone genotypes of TECs on thymic selection of β cell-reactive T cells in humanized mice. We will utilize a novel model in which hPSC-TECs create a “hybrid thymus” on a living scaffold of fetal pig thymic tissue that supports human T cell development in HU mice. We will use this model with genetically engineered hPSCs to determine the impact of human TEC expression of TID-associated genetic variants on selection of autoreactive TCRs; Aim 3: Assess autoimmune interactions between hPSC-derived β cells and autoreactive T cells in humanized mice. We have developed models for rejection of hPSC-derived β cells in HU mice and for autoimmune β cell destruction by transducing autoreactive TCRs into their T cells. We have generated a hPSC cell line that lacks all HLA except HLA-A2, evading rejection in HLA-A2+ immune systems. These will be used in vivo and in vitro, with and without expression of an HLA Class II molecule, DQ8, to model autoimmune destruction by TCR Tg T1D patient and HC-derived T cells, assessing the requirement for Class II HLA and CD4 help for CD8 cell-mediated autoimmune β cell destruction. Our collaborative effort will generate novel and robust systems to model human T1D, enhancing understanding of its pathogenesis and providing a platform for testing of immunotherapies.