Modeling autoimmune pathogenesis and beta cell destruction by T1D immune systems

项目概要 1型糖尿病（T1D）发病机制的研究一直受到动物模型和研究不足的限制。 患者群体的异质性，他们从 HLA 和不同种类的基因中获得遗传风险 60 个非 HLA 遗传变异。鉴于这种复杂性，需要改进人类 T1D 模型 发病机制涉及不同的遗传背景。我们开发了人源化（HU）小鼠模型， 其中包括一种 T1D 患者和健康对照 (HC) 免疫系统从头产生的方法 个性化免疫 (PI) 小鼠中的造血干细胞 (HSC) 和引入转基因 (Tg) 的模型 自身反应性 TCR 进入 HSC 和 T 细胞。我们开发了产生胸腺上皮细胞的方法 (TEC) 祖细胞和来自人类多能干细胞 (hPSC) 的 β 细胞。我们将使用这些工具来解决 T1D 个体中 HSC 内在和胸腺内在遗传变异导致的假设 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 易感基因型对人胸腺中自身反应性 T 细胞选择的影响。我们已经证明了 HU HLA-DQ8 胸腺中 Tg HLA-DQ8 限制性胰岛素 B9-23 肽特异性 TCR 的阴性选择 老鼠。我们将评估额外的 β 细胞自身反应性 I 类和 II 类限制性 TCR，确定影响 HSC 和 AIRE mTEC 抗原表达并比较 T1D 患者与 HC 胸腺细胞的选择 携带 β 细胞反应性 TCR；目标 2：确定易患 T1D 的 TEC 基因型对胸腺的影响 在人源化小鼠中选择β细胞反应性T细胞。我们将利用一种新模型，其中 hPSC-TEC 在胎猪胸腺组织的活体支架上创建“混合胸腺”，支持人类 T 细胞的发育 胡老鼠。我们将使用该模型与基因工程 hPSC 来确定人类 TEC 的影响 TID 相关遗传变异在自身反应性 TCR 选择中的表达；目标 3：评估自身免疫 人源化小鼠中 hPSC 衍生的 β 细胞与自身反应性 T 细胞之间的相互作用。我们有 开发了 HU 小鼠中 hPSC 衍生 β 细胞排斥和自身免疫 β 细胞破坏的模型 将自身反应性 TCR 转导至 T 细胞中。我们已经生成了缺乏所有 HLA 的 hPSC 细胞系，除了 HLA-A2，逃避 HLA-A2 免疫系统的排斥反应。这些将在体内和体外使用，有或没有 HLA II 类分子 DQ8 的表达，以模拟 TCR Tg T1D 患者的自身免疫破坏和 HC 衍生的 T 细胞，评估 II 类 HLA 和 CD4 的需求，以帮助 CD8 细胞介导的自身免疫 β细胞破坏。我们的合作将产生新颖且强大的系统来模拟人类 T1D， 增强对其发病机制的了解并为免疫疗法测试提供平台。

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.