Leveraging Human iPSC-derived beta-cells to Probe Antigen Specificity of Anti-islet Memory T Cells in T1D

项目摘要/摘要 1型糖尿病(T1D)是一种自身免疫性疾病，但破坏β细胞的相关自身抗原很少 有定义的，尤其是在人类中。有证据表明，胰岛素是早期抗胰岛自身免疫的驱动抗原， 然而，随着疾病的发展，针对其他抗原的抗体出现，表明胰岛损伤是 伴随着抗原的传播。因此，与疾病过程中β细胞破坏相关的抗原 入门阶段可能不同于导致晚期疾病的入门阶段。关于哪些抗原介导β细胞的知识 已建立的T1D患者的自身免疫对新出现的细胞替代策略具有重要的临床意义 使用自体诱导的多能干细胞(IPSCs)。IPSC-β-CELS可能会触发内存扩展 抗β细胞T细胞导致移植物破坏。自体移植物优势抗原和表位的鉴定 排斥反应可以直接用于治疗，如ipsc-β--人体免疫系统看不见的细胞。 T1D可以通过基因编辑产生。抗β细胞记忆CD_4~+T细胞抗原特异性的探讨 并评估是否发生功能相关的抗原扩散，我们已经开发了相关工具。这些 包括MHC-II四聚体、用于表征CD4+T细胞和配对TCR激活状态的单细胞分析 测序，携带编辑的自身抗原的NOD小鼠，以及IPSC-β-细胞和 携带T1D相关人类白细胞抗原的人外周血单核细胞 第二类DQ8变种。拟议的项目将建立在这些工具的基础上来研究MHC-II介导的抗原 长期存在T1D患者的表现和记忆中的CD4+T细胞反应。要实现这一点，请在 目的1比较幼年非糖尿病NOD小鼠和糖尿病NOD小鼠移植过程中CD4+T细胞的变化 并评估抗原扩散的程度。以指导人类发现新抗原的努力 记忆曲目，我们将在β细胞中进行免疫肽组研究，并从功能上测试候选抗原 在陈述分析中。这些实验将评估小鼠的记忆CD4+T细胞库，并提供 深入了解临床前模型中与功能相关的抗原传播。在目标2中，我们将使用类似的方法 建立共培养与人源化小鼠模型研究人类记忆CD_4~+T细胞 IPSC-β-细胞和PBMC。然后我们将编辑IPSC中的候选自身抗原和表位，并测试它们的 在记忆、CD4+T细胞刺激和移植物排斥反应中的作用。这个高风险、高回报的项目，由一个 T1D免疫学家和β细胞生物学和IPSCs方面的专家，具有实质性的翻译影响和构建 依靠我们团队开发的技术进步。

PROJECT SUMMARY/ABSTRACT Type 1 diabetes (T1D) is an autoimmune disease, yet the relevant autoantigens for β-cell destruction are poorly defined, especially in humans. There is evidence that insulin is the driving antigen in early anti-islet autoimmunity, however, with disease progression antibodies against other antigens arise, indicating that islet damage is accompanied by antigen spreading. Therefore, the antigens relevant for β-cell destruction during disease initiation might differ from those leading to advanced disease. Knowledge of which antigens mediate β-cell autoimmunity in established T1D has significant clinical importance for emerging cell replacement strategies using autologous induced pluripotent stem cells (iPSCs). IPSC-β-cells will likely trigger expansion of memory anti-β-cell T cells leading to graft destruction. The identification of dominant antigens and epitopes for autograft rejection could find immediate therapeutic applications as iPSC-β-cells invisible to the immune system of people with T1D could be produced by gene editing. To probe antigen specificity of anti-β-cell memory CD4+ T cells and assess whether functionally relevant antigen spreading occurs, we have developed relevant tools. These include MHC-II tetramers, single-cell assays to characterize activation states of CD4+ T cells and paired TCR sequencing, NOD mice carrying edited autoantigens, as well as an autologous system of iPSC-β-cells and peripheral blood monocytes (PBMCs) derived from people carrying the T1D-associated human leukocyte antigen class II DQ8 variant. The proposed project will build on these tools to study MHC-II-mediated antigen presentation and the memory CD4+ T cell response in people with long-standing T1D. To accomplish this, in Aim 1 we will compare the CD4+ T cell repertoire in young non-diabetic and diabetic NOD mice during graft rejection and evaluate the extent of antigen spreading. To guide the effort of discovering new antigens in the memory repertoire, we will conduct immunopeptidome studies in β-cells and functionally test candidate antigens in presentation assays. These experiments will evaluate the memory CD4+ T cell repertoire in mice and provide insight into functionally relevant antigen spreading in a preclinical model. In Aim 2, we will use a similar approach to study the human memory CD4+ T cell repertoire by establishing co-cultures and a humanized mouse model of iPSC-β-cells and PBMCs. We will then edit candidate autoantigens and epitopes in iPSCs and test their function in memory CD4+ T cell stimulation and graft rejection. This high-risk, high-reward project, co-led by a T1D immunologist and an expert in β-cell biology and iPSCs, has substantial translational impact and builds upon technological advances that our team has developed.