Delineating mechanisms underlying the enhanced stability and functionality of CD2-KO Tregs and chimeric antigen receptor (CAR) Tregs and their application in xenotransplantation

项目总结 调节性T细胞(Treg)疗法已被广泛研究用于控制不同模型的免疫反应 自身免疫和移植。尽管使用Tregs和嵌合抗原受体的结果很有希望 (CAR)Tregs用于耐受诱导，关于Tregs的可塑性和效果仍存在重大担忧 在临床应用之前，需要解决这些细胞上T细胞耗尽的调节方案。 我们已经发现，CD2-KO树和CAR树不仅对 包括抗CD2抗体(Siplizumab)，但也增加了FOXP3的表达和Treg功能 体外和体内。CD2是一种共刺激分子，存在于包括Treg在内的所有T细胞中。潜在的直接 或者CD2信号和FOXP3之间的间接联系尚不清楚。我们的初步数据显示CD2-KO树 增加mTORC1活性、糖酵解和线粒体呼吸。我们假设新陈代谢- 活性CD2-KO Tregs通过组蛋白修饰或翻译后修饰产生代谢物 提高FOXP3水平。在目标1中，我们将解决这些假设。我们将在以下位置研究CD2+和CD2-KO树 基因组、表观基因组、转录组和蛋白质水平以及它们的代谢和信号 小路。在目标2中，我们将探索这一发现在异种移植环境中的翻译方面。我们 已经开发并验证了一种针对通用猪抗原(猪I类白细胞抗原)的CAR 并表明SLA Car Tregs具有较强的抑制抗猪免疫的能力 与多克隆Tregs的体外反应进行比较。我们假设CD2-KO SLA汽车树抵抗 转化为效应性T细胞，并以抗原特异性的方式保护移植的猪器官。我们将评估 CD2-KO-SLA CAR树在炎症条件下对转化的抵抗力及CD2- Ko SLA Car Tregs在人源化异种胰岛小鼠模型中预防猪胰岛排斥反应的实验研究 移植。综上所述，这项工作可能会导致更安全和更高效的开发 基于Treg的免疫疗法。机械学研究可能揭示相关的分子和/或信号通路 在决定Tregs的命运/功能时，靶向Tregs可能会改变免疫平衡，使其倾向于耐受。

PROJECT SUMMARY Regulatory T cell (Treg) therapy has been widely investigated for control of immune responses in different models of autoimmunity and transplantation. Despite promising results in using Tregs and chimeric antigen receptor (CAR) Tregs for tolerance induction, there are still major concerns regarding the plasticity of Tregs and the effect of T cell-depleting conditioning regimens on these cells that need to be addressed before their clinical application. We have discovered that CD2-KO Tregs and CAR Tregs are not only resistant to a conditioning regimen that includes anti-CD2 antibody (siplizumab), but also have increased FOXP3 expression and Treg functionality in vitro and in vivo. CD2 is a costimulatory molecule that is present in all T cells, including Tregs. Potential direct or indirect link between CD2 signaling and FOXP3 is not known. Our preliminary data show that CD2-KO Tregs have increased mTORc1 activity, glycolysis and mitochondrial respiration. We hypothesize that metabolically- active CD2-KO Tregs generate metabolites that through histone modification or post-translational modifications increase FOXP3 levels. In Aim 1, we will address these hypotheses. We will study CD2+ and CD2-KO Tregs at the genome, epigenome, transcriptome and protein levels and evaluate their metabolism and signaling pathways. In Aim 2, we will explore the translational aspects of this discovery in xenotransplantation setting. We have developed and validated a CAR against a universal pig antigen (class-I swine leukocyte antigen (SLA)) and have shown that SLA CAR Tregs harbor higher suppressive capacity in suppressing anti-pig immune responses in vitro as compared to polyclonal Tregs. We hypothesize that CD2-KO SLA CAR Tregs resist conversion to effector T cells and protect transplanted pig organs in an antigen-specific manner. We will evaluate the resistance of CD2-KO SLA CAR Tregs to conversion in inflammatory conditions and also the efficacy of CD2- KO SLA CAR Tregs in preventing the rejection of pig islets in a humanized mouse model of xeno-islet transplantation. Taken together, this work can potentially lead to the development of safer and more efficient Treg-based immunotherapies. Mechanistic studies could uncover molecules and/or signaling pathways involved in determining the fate/function of Tregs, targeting of which could alter the immune balance toward tolerance.