Mice with autologous human T1D-derived immune systems and iPSC-derived beta cells

 描述（由申请人提供）：直到最近，对自身免疫性疾病（如1型糖尿病（T1 D））的研究一直受到动物模型不足和人类患者样本仅限于外周血的限制，这可能无法反映终末器官的免疫力。此外，患者人群在疾病持续时间、治疗、合并症、遗传背景和环境暴露方面具有异质性，因此难以确定病理生理机制。我们已经开发了一种“个性化免疫”（PI）人源化小鼠模型，该模型通过允许在免疫缺陷小鼠中从1型糖尿病（T1 D）患者和健康对照（HC）的造血干细胞（HSC）同步从头开发功能性人免疫系统来克服这些限制。T细胞在人胸腺移植物中从小鼠队列中的CD 34 + HSC发育，所述小鼠队列由小的床边骨髓穿刺液产生。我们现在建议将该模型进一步开发为人类T1 D生物学的个体化模型，将来自相同T1 D患者和健康对照供体的完整免疫系统和干细胞衍生的β细胞结合起来。我们已经证明了从来自用于构建PI小鼠的相同TlD患者和HC志愿者的皮肤成纤维细胞的iPSC产生的β细胞的体内功能。我们建议进一步开发该模型以诱导攻击iPSC衍生的β细胞的β细胞自身免疫。我们将：1）通过改善人APC再增殖和淋巴结构并从源自成人供体的iPSC产生自体胸腺上皮细胞（TEC）来优化由成人HSC产生的人免疫系统的功能。从iPSC产生TEC将提供自体HLA/肽复合物，用于阳性选择与外周中的自体APC最佳相互作用的T细胞。所有研究中免疫功能的读数将包括对用于免疫的抗原的抗体、增殖和细胞因子应答以及控制EBV感染的能力; 2）优化iPSC衍生的辟田胞作为PI小鼠中自身免疫的靶标的使用，并比较TlD衍生的iPSC衍生的辟田胞与HC衍生的iPSC衍生的辟田胞以及“天然”辟田胞的免疫原性。我们将开发提高iPSC衍生的内分泌细胞的纯度的方法，优化植入方法和部位，并表征与来自TlD和HC受试者的成人胰岛β细胞相比在体内成熟的iPSC衍生的内分泌细胞的免疫原性; 3）在PI小鼠中开发胰岛炎/糖尿病模型。我们将尝试使用各种操作在PI小鼠中诱导针对天然胰腺和自体iPSC衍生的辟田胞的胰岛炎。这种优化的PI小鼠将用作“金标准”基线，其中应用产生iPSC衍生的辟田胞和HSC的能力的改进，可视化引发和驱动TlD的免疫相互作用，并评估疾病的环境沉淀物。通过该联盟，该模型将允许分析T1 D发病机制中涉及的早期和晚期事件，并作为一个模型，以建立从iPSC和其他技术进步中扩增或生成HSC的方法。

 DESCRIPTION (provided by applicant): The study of autoimmune diseases such as Type 1 diabetes (T1D) has, until recently, been limited by the insufficiency of animal models and by the restriction of human patient samples to peripheral blood, which may not reflect immunity in the end organs. Moreover, patient populations are heterogeneous with respect to the duration of disease, treatments, comorbidities, genetic background and environmental exposures, making it difficult to identify pathophysiologic mechanisms. We have developed a "Personalized Immune" (PI) humanized mouse model that overcomes these limitations by allowing synchronized de novo development, in immunodeficient mice, of functional human immune systems from hematopoietic stem cells (HSCs) of Type 1 diabetic (T1D) patients and healthy controls (HCs). T cells develop in human thymus grafts from CD34+ HSCs in cohorts of mice generated from a small bedside bone marrow aspirate. We now propose to develop this model further as an individualized model of human T1D biology, incorporating complete immune systems and stem cell-derived β cells from the same T1D patient and healthy control donors. We have demonstrated in vivo function of β cells generated from iPSCs from skin fibroblasts of the same T1D patient and HC volunteers used to construct PI mice. We propose to further develop this model to induce β cell autoimmunity that attacks iPSC-derived β cells. We will: 1) Optimize the functionality of human immune systems generated from adult HSCs by improving human APC repopulation and lymphoid structure and generating autologous thymic epithelial cells (TECs) from iPSCs derived from adult donors. Generation of TECs from iPSCs will provide autologous HLA/peptide complexes for positive selection of T cells that optimally interact with autologous APCs in the periphery. Readouts of immune function in all studies will include antibody, proliferative and cytokine responses to antigens used for immunization and ability to control EBV infections; 2) Optimize the use of iPSC-derived β cells as a target for autoimmunity in PI mice and compare the immunogenicity of T1D- vs HC-derived iPSC-derived β cells and of "natural" β cells. We will develop methods of enhancing the purity of iPSC-derived endocrine cells, optimize implantation methods and sites and characterize the immunogenicity of iPSC-derived endocrine cells that mature in vivo in comparison to adult human pancreatic islet β cells from T1D and HC subjects; 3) Develop an insulitis/diabetes model in PI mice. We will attempt to induce insulitis against the native pancreas and autologous iPSC-derived β cells in PI mice using a variety of manipulations. This optimized PI mouse will serve as a "gold standard" baseline in which to apply improvements in the ability to generate iPSC-derived β cells and HSCs, visualize immune interactions that initiate and drive T1D and assess environmental precipitants of disease. Through the Consortium, the model will allow analyses of both early and late events involved in T1D pathogenesis, and serve as a model to build upon as methods of expanding or generating HSCs from iPSCs and other technologies advance.