Humanized Mouse Avatars for T1D

摘要 我们对调节人类T1D效应阶段的关键分子和细胞的忽视可能是很好的基础 缺乏有希望的新治疗干预措施。本补充资金申请日期为2023年6月1日 到2024年5月31日，我们的HIRN项目5U01DK104218，“T1D的人性化鼠标头像”。这 建议的时间段是基于在体内执行许多操作中的每一项所需的2-4个月 实验。预算申请将仅支持我们未能执行的关键实验 与COVID相关的延误。这些问题已经解决，我们准备完成实验 这将为体内研究人SC-胰岛/自身反应性T细胞相互作用建立模型。 这笔资金将允许我们：1)只保留执行这些实验所需的必要人员，2)保持 并用所需的免疫缺陷小鼠的新种群进行实验，3)保存独特的IPSC 我们为该项目开发的线路，4)生成SC-Islet，这些SC-Islet对于所有提议的 研究，以及5)进行建立人体自身反应活体模型所需的基本实验 T细胞介导的SC-胰岛破坏。我们的团队目前由来自4个机构的教员组成，梅尔顿博士 在哈佛，波士顿儿童医院的戴利博士、杰克逊实验室的舒尔茨博士和格雷纳博士， 布雷姆、梅尔、肯特和哈兰，来自马萨诸塞州大学医学院。梅尔顿博士的实验室将于4月30日关闭， 2023年，该项目所需的从IPSC生产人SC-胰岛的过程已经无缝进行 由哈伦博士在马萨诸塞州大学承担。虽然最初的提议有两个目的，但我们将重点 仅基于完成目标1所需的关键实验，即人体T1D的效应器阶段模型。至 为了实现这一目标，我们将使用来自T1D患者(Brehm博士，Greiner博士)血液中的自身反应性T细胞，T 将细胞系(由Kent博士产生)和IPSC来源的CAR T细胞(由Daley博士产生)嫁接在一起 将自体SC-胰岛细胞(由哈兰博士产生)转化为OPTI小鼠(由舒尔茨博士产生)。我们会 确定浸润性效应T细胞的TCR特异性和体内β细胞对免疫攻击的反应 使用10倍基因组测序(Maehr博士)。我们只请求支持必要的试剂，鼠标 进行这些关键研究所需的菌株和人员。对这些模型的验证将提供 非常需要体内模型系统来研究介导β细胞破坏的T细胞和 它们与自体干细胞-胰岛的体内相互作用。我们预计这些模型将被广泛应用于 科学界确定许多已识别的T1D自身反应性T细胞特异性中的哪些是“因果”的 它们是非致病的，允许询问自身反应性T细胞的效应器反应和 β细胞对自身免疫攻击的反应，并提供检测T细胞特异性的平台 科学界的免疫疗法。

ABSTRACT Our ignorance of the key molecules and cells mediating the effector phase of human T1D may well underlie the paucity of promising new therapeutic interventions. This supplemental funding request is for June 1, 2023 through May 31, 2024 for our HIRN project 5U01DK104218, “Humanized Mouse Avatars for T1D”. This proposed time period is predicated on the 2-4 months needed to perform each of many in vivo experiments.The budget request will support only the critical experiments that we were not able to perform due to COVID-related delays. These issues have been resolved and we are poised to complete the experiments that will establish models for investigation of human SC-islet/autoreactive T cell interaction in vivo. This funding will permit us: 1) to retain only essential staff needed to perform these experiments, 2) to maintain and carry out experiments with the novel stocks of immunodeficient mice required, 3) to preserve unique iPSC lines we developed for this project, 4) to generate the SC-islets that are essential for all of the proposed studies, and 5) to perform the essential experiments needed to establish in vivo models for human autoreactive T cell-mediated destruction of SC-islets. Our group currently consists of faculty from 4 institutions, Dr. Melton at Harvard, Dr. Daley at Boston Children's Hospital, Dr. Shultz at The Jackson Laboratory, and Drs. Greiner, Brehm, Maehr, Kent and Harlan at UMass Chan Medical School. Dr. Melton' laboratory is closing on April 30, 2023 and the production of human SC-islets from iPSCs required for this project has been seamlessly assumed by Dr. Harlan at UMass Chan. Although there are two aims in the original proposal, we will focus solely on the critical experiments needed to accomplish Aim 1, the effector phase model of human T1D. To accomplish this aim we will use autoreactive T cells from the blood of T1D individuals (Drs. Brehm, Greiner), T cell lines (generated by Dr. Kent) and iPSC-derived CAR T cells (generated by Dr. Daley) engrafted together with autologous SC-islet cells (generated by Dr. Harlan) into OPTI-MICE (generated by Dr. Shultz). We will identify the TCR specificity of the infiltrating effector T cells and the in vivo beta cell response to immune attack using 10X Genomic sequencing (Dr. Maehr). We request support only for the essential reagents, mouse strains, and personnel that are required to perform these critical studies. Validation of these models will provide much needed in vivo model systems for the investigation of the T cells that mediate beta cell destruction and their in vivo interaction with autologous SC-islets. We anticipate that these models will be widely used by the scientific community to identify which of the many identified T1D autoreactive T cell specificities are “causal” and which are “non-pathogenic”, allow interrogation of the effector response of autoreactive T cells and the response of beta cells to autoimmune attack, and to provide a platform for testing T cell specific immunotherapeutics by the scientific community.