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 T1D患者和健康对照(HC)的哪种免疫系统是从造血干细胞产生的 个体化免疫(PI)小鼠的细胞(HSCs)和转基因(TG)自身反应性TCRs导入的模型 他们的造血干细胞和T细胞。我们还开发了生成胸腺上皮细胞(TEC)祖细胞的方法 以及来自人类多能干细胞的β细胞，这将增加我们测试这些细胞的影响的能力 T1D风险的关键细胞群。我们将使用这些工具来解决HSC固有的和 T1D患者胸腺固有遗传变异导致胸腺对β细胞抗原的异常选择 自动反应TCR。我们将：目标1：确定T1D倾向基因对选择的影响 人胸腺中的自身反应性T细胞。我们已经证明了Tg-HLADQ8-的阴性选择 限制胰岛素B9-23多肽在HU小鼠胸腺中的表达。我们将评估额外的β单元- 自身反应性I类和II类限制性TCR，决定HSC和AIRmTEC抗原的影响 T1D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： TECs T1D易感基因对胸腺β细胞反应性T细胞选择的影响 人性化的老鼠。我们将利用一种新的模型，在这种模型中，hPSC-TECs在活的支架上创造一个“杂交胸腺”。 支持人类T细胞发育的胎猪胸腺组织在HU小鼠身上。我们将使用此模型与 基因工程hPSCs对人TEC表达TID相关基因的影响 选择自身反应性TCR的变体；目标3：评估hPSC来源的自身免疫相互作用 人源化小鼠β细胞和自身反应性T细胞。我们已经开发了拒绝hPSC来源的模型 HU小鼠体内的β细胞和通过将自身反应性TCR转导到其T细胞来破坏自身免疫的β细胞。我们 已经产生了一种hPSC细胞株，除了HL A-A2外，没有其他所有的HL A，从而避免了HL A-A2免疫中的排斥反应 系统。这些将在体内和体外使用，有和没有表达人类白细胞抗原II类分子DQ8， 模拟TCR TG T1D患者和HC来源的T细胞的自身免疫破坏，评估对 CD8细胞介导的自身免疫性β细胞的破坏。我们的合作努力将 生成新的和强大的系统来模拟人类T1D，增强对其发病机制和 为检测免疫疗法提供了一个平台。

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.