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

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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.