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

利用人 iPSC 衍生的 β 细胞探测 T1D 中抗胰岛记忆 T 细胞的抗原特异性

• 批准号: 10589556
• 负责人: Luc Teyton
• 金额: $ 76.25万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10589556
• 关键词: Antibodies; Antigen Presentation; Antigenic Specificity; Antigens; Autoantigens; Autoimmune; Autoimmune Diseases; Autoimmunity; Autologous; Autologous Transplantation; Automobile Driving; B-Lymphocytes; Beta Cell; Biological Assay; C-Peptide; CD4 Positive T Lymphocytes; Cell Compartmentation; Cell Line; Cell Therapy; Cells; Cellular Assay; Cellular biology; Clinical; Coculture Techniques; Development; Diabetes Mellitus; Diabetic mouse; Disease; Disease Progression; Engineering; Engraftment; Epitope spreading; Epitopes; Genes; Genotype; Goals; Graft Rejection; HLA Antigens; HLA-DQ8 antigen; Haplotypes; Histocompatibility Antigens Class II; Human; Immune; Immune system; Immunologic Memory; Immunologist; In Vitro; Inbred NOD Mice; Individual; Insulin; Insulin-Dependent Diabetes Mellitus; Knowledge; Mediating; Memory; Minor; Modeling; Mus; Nature; Persons; Play; Pluripotent Stem Cells; Pre-Clinical Model; Role; Source; Specificity; Study models; Subgroup; System; T cell receptor repertoire sequencing; T cell response; T memory cell; T-Lymphocyte; T-cell diversity; Technology; Testing; Therapeutic; Time; Transplantation; Variant; advanced disease; diabetic; experimental study; high reward; high risk; humanized mouse; induced pluripotent stem cell; innovation; insight; insulin dependent diabetes mellitus onset; islet; islet autoimmunity; memory CD4 T lymphocyte; monocyte; mouse model; non-diabetic; peripheral blood; prevent; response; single cell analysis; tool; translational impact; type I diabetic

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.

项目概要/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却很少 定义，特别是在人类中，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，随着疾病进展，针对其他抗原的抗体出现，表明胰岛损伤正在发生 因此，与疾病期间β细胞破坏相关的抗原。 启动可能不同于导致晚期疾病的抗原介导 β 细胞的知识。 既定 T1D 中的自身免疫对于新兴细胞替代策略具有重要的临床意义 使用自体诱导多能干细胞（iPSC）可能会引发记忆扩展。 抗 β 细胞 T 细胞导致移植物破坏，识别自体移植物的显性抗原和表位。 由于 iPSC-β 细胞对人类免疫系统来说是不可见的，排斥反应可能会立即得到治疗应用 可以通过基因编辑产生具有 T1D 的抗体，以探测抗 β 细胞记忆 CD4+ T 细胞的抗原特异性。 并评估是否发生功能相关的抗原扩散，我们开发了相关工具。 包括 MHC-II 四聚体、单细胞测定，以表征 CD4+ T 细胞和配对 TCR 的激活状态 测序，携带编辑过的自身抗原的 NOD 小鼠，以及 iPSC-β 细胞的自体系统和 来自携带 T1D 相关人类白细胞抗原的人的外周血单核细胞 (PBMC) II 类 DQ8 变体将基于这些工具来研究 MHC-II 介导的抗原。 长期 T1D 患者的表现和记忆 CD4+ T 细胞反应 目标 1 我们将比较年轻的非糖尿病和糖尿病 NOD 小鼠在移植过程中的 CD4+ T 细胞库 排斥反应并评估抗原传播的程度，以指导发现新抗原的努力。 记忆库，我们将在β细胞中进行免疫肽组研究并对候选抗原进行功能测试 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功能相关的抗原扩散。在目标 2 中，我们将使用类似的方法。 通过建立共培养物和人源化小鼠模型来研究人类记忆 CD4+ T 细胞库 然后，我们将编辑 iPSC 中的候选自身抗原和表位并测试它们。 记忆 CD4+ T 细胞刺激和移植物排斥中的功能 这个高风险、高回报的项目由一位科学家共同领导。 T1D 免疫学家和 β 细胞生物学和 iPSC 专家，具有重大转化影响并构建 基于我们团队开发的技术进步。