Enhancing the Effectiveness of Immunotherapies by T Cell Epigenetic Reprogramming

项目总结 癌症和慢性病毒感染是发病率和死亡率的重要原因。而细胞毒CD8 T细胞 细胞是肿瘤或病毒感染细胞的主要杀手，在慢性疾病期间持续刺激CD8T细胞 感染或癌症导致其细胞毒功能逐渐丧失，因为T细胞向完全... 精疲力竭状态。而免疫检查点阻断(ICB)疗法允许部分耗尽的CD8T细胞 通过阻断抑制信号来恢复功能，终末耗尽的T细胞对此仍然没有反应 心理治疗。ICB后终末耗尽的T细胞无法恢复可能解释了为什么许多癌症患者 无法安装对ICB的持久响应。我们最近发现从头开始的DNA甲基化编程是 与T细胞进入终末期衰竭和对ICB反应差有关。重要的是 我们发现，靶向T细胞内在表观遗传程序可以协同ICB在慢性疾病中的疗效 感染或癌症。然而，以下问题代表了我们目前对T细胞理解的主要差距 衰竭：(1)这些表观遗传变化是如何在衰竭的T细胞中获得的？(2)上游是什么 调节耗竭T细胞和功能T细胞中从头DNA甲基化程序的特异性的信号？ (3)我们能否将耗尽的T细胞的表观遗传编程逆转到功能状态，同时避免 转型？弥合这些差距将使我们能够识别和定位应用“表观遗传刹车”的因素 CD8 T细胞功能。为了解决这些问题，我们开发了一种新的稳定的人T细胞体外模型 细胞功能障碍作为一种易于处理的工具，可以通过提供第一线的机制来指导我们的体内实验 学习。首先，我们将采用尖端方法，如CRISPR-Cas9基因编辑和逆转录病毒 转导，以检验特定的肿瘤微环境信号调节表观遗传的假设 持续刺激的CD8T细胞中的编程，这增加了它们对ICB治疗的抵抗力。我们的目标是 通过以此为目标的组件来阻止和/或恢复到最终耗尽状态的进程 信号途径，同时促进抵消途径。第二，我们的目标是重新平衡特定的 微环境信号恢复终末期耗尽的T细胞的功能和反应。使用新颖的In- T细胞功能障碍的体外模型系统和慢性病毒感染和癌症的体内互补模型， 以及分析DNA甲基化、开放染色质图谱和转录组的尖端技术 在CD8T细胞中，我们提议的研究可以确定靶向特定因素是否可以将染色质重塑回 在效应器和/或干细胞相关基因上的可接近状态，导致功能恢复的T细胞。 这些拟议的研究将提供关于表观遗传编程如何在 临床上T细胞耗竭的进展可转化为对终末耗尽的T细胞进行重新编程 环境，最终提高T细胞免疫疗法的疗效。

PROJECT SUMMARY Cancer and chronic virus infections are significant causes of morbidity and mortality. While cytotoxic CD8 T cells are the main killers of tumors or virus-infected cells, persistent stimulation of CD8 T cells during chronic infections or cancer results in a gradual loss of their cytotoxic function as T cells progress towards a fully- exhausted state. While immune checkpoint blockade (ICB) therapy allows partially-exhausted CD8 T cells to functionally recover by blocking inhibitory signals, terminally-exhausted T cells remain nonresponsive to this therapy. The inability of terminally-exhausted T cells to recover after ICB may explain why many cancer patients fail to mount durable responses to ICB. We recently showed that de novo DNA methylation programming is causally linked to the progression of T cells toward terminal exhaustion and poor response to ICB. Importantly, we discovered that targeting T cell-intrinsic epigenetic programs synergized the efficacy of ICB during chronic infection or cancer. Yet, the following questions represent major gaps in our current understanding of T cell exhaustion: (1) How are these epigenetic changes acquired in exhausted T cells? (2) What are the upstream signals that regulate the specificity of de novo DNA methylation programs in exhausted versus functional T cells? (3) Can we reverse the epigenetic programming in exhausted T cells to the functional state while avoiding transformation? Bridging these gaps will allow us to identify and target factors that apply “epigenetic brakes” to CD8 T cell function. To address these questions, we have developed a novel in-vitro model of stable human T cell dysfunction as a tractable tool that can guide our in-vivo experiments by providing first-line mechanistic studies. First, we will employ cutting-edge approaches, such as CRISPR-Cas9 gene editing and retroviral transduction, to test the hypothesis that specific tumor microenvironmental signals regulate epigenetic programming in persistently stimulated CD8 T cells, which promotes their resistance to ICB therapy. We aim to block and/or revert the progression toward the terminally-exhausted state by targeting components of this signaling pathway while promoting counteracting pathways. Second, we aim to rebalance specific microenvironmental signals to restore functionality and response in terminally-exhausted T cells. Using novel in- vitro model systems of T cell dysfunction and complementary in-vivo models of chronic viral infection and cancer, as well as cutting-edge technologies to profile DNA methylation, open chromatin landscape, and transcriptome in CD8 T cells, our proposed studies can determine if targeting specific factors can remodel chromatin back into an accessible state at effector and/or stemness-associated genes, leading to functionally-rejuvenated T cells. These proposed studies will provide insights into how epigenetic programming can be reversed during progression to T cell exhaustion that can be translated to reprogram terminally-exhausted T cells in clinical settings, ultimately enhancing the efficacy of T cell immunotherapies.