Temporal control of differentiation and epigenetics of Exhausted CD8 T cells by Tox

摘要 在慢性感染和癌症期间，T细胞耗尽是常见的，并限制了疾病的控制。以Tex为目标 阻断PD-1：PD-L等通路可以激活这些细胞，从而在癌症中产生显着的临床效果。 然而，大多数患者并没有获得持久的临床益处。尽管PD-1通路被阻断后重新焕发活力 Tex功能，并导致转录变化，染色质景观和 功能变化是不可持续的。因此，我们针对TeX的能力可在癌症和慢性疾病中获得治疗益处 感染受到这些细胞表观遗传顽固性的限制。更好地理解启动、稳定 而TeX表观遗传学特性的可逆性应该揭示新的治疗可能性。 我们和其他人最近确定Tox是Tex的表观遗传谱系编程者。没有Tox，Tex就不能 形式。毒素是启动Tex染色质重塑所必需的，但它抑制末端被盖的分化。然而， Tox计划表观遗传学的机制尚不清楚。一个主要的问题是染色质会发生什么 如果在已建立的纹理中去除了Tox，则景观和纹理差异。解决这个问题是一个主要目标。 特克斯的异质性现在也指向了一种发育生物学层次，具有谨慎的、功能相关的 分化阶段--或Tex亚群--由转录因子回路控制。这些子集也不同 表观遗传学表明，Tox的关键角色尚未经过测试，同时也存在机会。 这些观察表明Tox在Tex的表观遗传学身份中发挥了关键作用，但也提出了关于 一旦Tex成立，Tox的持续角色就会出现。我们的总体假设是可诱导的Tox缺失 已建立的TeX将揭示TeX的表观遗传稳定性的机制和治疗机会 在慢性感染和癌症期间有所改善。我们将在以下目标中检验这一假设： 目的1：测试在已建立的TeX中TOX的缺失是否改变了TeX的分化、转录 程序，开放的染色质景观和/或纹理子集的动力学。我们假设毒物的清除 在已建立的德州将恢复特克斯表观遗传计划，并将与功能，分化 和转录变化，这些变化将被PD-1阻断和/或去除抗原所增强。为了测试这一点 我们将使用新的诱导性毒物删除策略，并结合对 细胞发育生物学、转录和表观遗传程序以及对PD-1通路阻断的反应。 目标2：测试互补或下游表观遗传或转录回路如何配合 德克萨斯州的毒物。我们假设，体内CRISPR筛查和候选测试的组合将揭示 毒素在TEX中的表观遗传和转录机制。我们将结合使用此CRISPR方法和 强制表达策略和一种新的Tox驱动的可诱导Cre报告程序来定义分子和 Tox介导的Tex谱系启动和维持的基因组机制。

SUMMARY T cell exhaustion is common during chronic infections and cancer and limits control of disease. Targeting TEX by blocking pathways such as PD-1:PD-L can reinvigorate these cells leading to dramatic clinical effects in cancer. However, most patients do not receive durable clinical benefit. Although PD-1 pathway blockade re-invigorates TEX function, and results in transcriptional changes, there is little change in the chromatin landscape and functional changes are not sustained. Thus, our ability to target TEX for therapeutic benefit in cancer and chronic infections is limited by the epigenetic inflexibility of these cells. A better understanding of the initiation, stability and reversibility of TEX epigenetic identity should reveal new therapeutic possibilities. We and others have recently identified Tox as the epigenetic lineage programmer of TEX. Without Tox, TEX cannot form. Tox is required to initiate chromatin remodeling for TEX but represses terminal TEFF differentiation. However, the mechanisms of how Tox programs epigenetics are unclear. A major question is what happens to chromatin landscape and TEX differentiation if Tox is removed in established TEX. Addressing this question is a major goal. TEX heterogeneity is also now pointing to a developmental biology hierarchy with discreet, functionally relevant stages of differentiation – or TEX subsets - controlled by transcription factor circuits. These subsets also differ epigenetically suggesting key roles for Tox that are as yet untested as well as opportunities. These observations suggest a key role for Tox in the epigenetic identity of TEX but raise key questions about the ongoing role of Tox once TEX are established. Our overall hypothesis is that inducible deletion of Tox in established TEX will reveal mechanisms of epigenetic stability of TEX and opportunities for therapeutic improvement during chronic infections and cancer. We will test this hypothesis in the following Aims: AIM 1: TEST WHETHER DELETION OF TOX IN ESTABLISHED TEX ALTERS TEX DIFFERENTIATION, TRANSCRIPTIONAL PROGRAM, OPEN CHROMATIN LANDSCAPE AND/OR DYNAMICS OF TEX SUBSETS. We hypothesize that removal of Tox in established TEX will revert the TEX epigenetic program and will be associated with functional, differentiation and transcriptional changes that will be augmented by PD-1 blockade and/or removal of antigen. To test this idea we will use new inducible Tox deletion strategies combined with deep mechanistic interrogation of the cellular developmental biology, transcriptional and epigenetic program and response to PD-1 pathway blockade. AIM 2: TEST HOW COMPLEMENTARY OR DOWNSTREAM EPIGENETIC OR TRANSCRIPTIONAL CIRCUITS COOPERATE WITH TOX IN TEX. We hypothesize that a combination of in vivo CRISPR screening and candidate testing will reveal epigenetic and transcriptional mechanisms of Tox in TEX. We will use this CRISPR approach together with enforced expression strategies and a novel Tox-driven inducible Cre reporter to define the molecular and genomic mechanisms of Tox-mediated initiation and maintenance of the TEX lineage.