Modeling autoimmune pathogenesis and beta cell destruction by T1D immune systems

项目摘要 1型糖尿病(T1D)发病机制的研究一直受到动物模型和 患者群体的异质性，他们从人类白细胞抗原和不同种类的约 60个非人类白细胞抗原基因变异。鉴于这种复杂性，需要改进的人类T1D模型 发病机制涉及不同的遗传背景。我们已经开发了人性化(HU)小鼠模型， 包括T1D患者和健康对照(HC)免疫系统从头产生 个体化免疫(PI)小鼠的造血干细胞(HSCs)和转基因(TG)模型 自身反应性TCR进入他们的HSCs和T细胞。我们已经开发出产生胸腺上皮细胞的方法 (Tec)人多能干细胞(HPSC)的祖细胞和β细胞。我们将使用这些工具来解决 T1D个体的HSC固有和胸腺固有遗传变异导致 β细胞抗原自身反应性TCR的胸腺选择异常。我们将：目标1：确定 T1D倾向基因对人胸腺自身反应性T细胞选择的影响。我们已经证明了 人胸腺HLA-DQ8-Tg限制性胰岛素B9-23多肽特异性TCR的阴性选择 老鼠。我们将评估额外的β细胞--自身反应I类和II类限制性TCR，确定其影响 HSC和AIRE+mTEC抗原的表达及T1D患者与HC胸腺细胞的比较选择 携带β细胞反应性TCR；目的2：确定TECs T1D易感基因对胸腺的影响 人源化小鼠β细胞反应性T细胞的筛选。我们将利用一种新的模型，其中hPSC-TEC 在支持人类T细胞发育的胎猪胸腺组织的活支架上创造出“杂交胸腺”。 胡鼠。我们将使用这个模型和基因工程的hPSCs来确定人类TEC的影响 TID相关基因变异在自身反应性TCR选择中的表达；目的3：评估自身免疫 人源化小鼠β细胞与自身反应性T细胞的相互作用我们有 HU小鼠hPSC来源的β细胞的排斥反应和自身免疫β细胞的破坏模型的建立 将自身反应性TCR转化为T细胞。我们已经产生了一个hPSC细胞株，它除了 人类白细胞抗原A2，在人类白细胞抗原A2+免疫系统中逃避排斥反应。这些药物将在体内和体外使用，无论有没有 人类白细胞抗原II类分子DQ8的表达以模拟TCR TG T1D患者和 HC来源的T细胞，评估CD8细胞介导的自身免疫对II类人类白细胞抗原和CD4帮助的需求 β细胞破坏。我们的合作努力将产生新的和强大的系统来模拟人类的T1D， 加强对其发病机制的了解，为免疫疗法的检测提供平台。

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, including one 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 developed methods of generating thymic epithelial cell (TEC) progenitors and β cells from human pluripotent stem cells (hPSCs). 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.