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

总结 T细胞耗竭在慢性感染和癌症期间很常见，并限制了疾病的控制。通过以下方式瞄准TEX 阻断通路如PD-1：PD-L可以重振这些细胞，从而在癌症中产生显著的临床效果。 然而，大多数患者并未获得持久的临床益处。虽然PD-1通路阻断剂可以使 TEX功能，并导致转录变化，染色质景观几乎没有变化， 功能性变化不能持续。因此，我们靶向TEX以在癌症和慢性疾病中获得治疗益处的能力， 感染受到这些细胞的表观遗传可移植性的限制。更好地理解启动、稳定性 TEX表观遗传特性的可逆性应该揭示新的治疗可能性。 我们和其他人最近已经确定Tox是TEX的表观遗传谱系程序员。如果没有Tox，TEX就不能 form. Tox需要启动TEX的染色质重塑，但抑制终末TEFF分化。然而，在这方面， Tox如何编程表观遗传学的机制尚不清楚。一个主要的问题是染色质 景观和特克斯差异，如果在建立特克斯去除毒性。解决这个问题是一个主要目标。 TEX异质性现在也指向一个发育生物学等级， 分化阶段-或TEX亚群-由转录因子电路控制。这些子集也不同 表观遗传学表明毒性的关键作用，尚未测试以及机会。 这些观察结果表明，Tox在TEX的表观遗传特性中起着关键作用，但也提出了关于TEX的表观遗传特性的关键问题。 一旦TEX建立，Tox将继续发挥作用。我们的总体假设是，诱导性缺失Tox基因， 建立TEX将揭示TEX的表观遗传稳定性机制和治疗的机会， 改善慢性感染和癌症。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中删除TOX是否会改变TEX分化，过渡性 特克斯亚群的开放染色质景观和/或结构。我们假设去除毒素 在建立TEX将恢复TEX表观遗传程序，并将与功能，分化， 以及将通过PD-1阻断和/或去除抗原而增强的转录变化。为了验证这一 我们将使用新的可诱导的Tox缺失策略，结合对Tox基因的深层机械询问， 细胞发育生物学、转录和表观遗传程序以及对PD-1通路阻断的应答。 目标2：测试互补或下游的表生或过渡型微生物如何与 毒性单位：特克斯。我们假设体内CRISPR筛选和候选人测试的结合将揭示 TEX中Tox的表观遗传和转录机制。我们将使用这种CRISPR方法， 强制表达策略和一种新的Tox驱动的诱导型Cre报告基因，以定义分子和 Tox介导的TEX谱系起始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.