Continuous expression of TOX safeguards exhausted CD8 T cell epigenetic fate

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Doctor of Philosophy (PhD)
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Cell and Molecular Biology
Immunology and Infectious Disease
T cell differentiation
T cell exhaustion
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Huang, Yinghui Jane, E

CD8 T cell exhaustion is a major barrier limiting anti-tumor therapy. Though checkpoint blockade temporarily improves exhausted CD8 T cell (Tex) function, the underlying epigenetic landscape of Tex remains largely unchanged, preventing their durable “reinvigoration.” Whereas the transcription factor (TF) TOX has been identified as a critical initiator of Tex epigenetic programming, it remains unclear whether TOX plays an ongoing role in preserving Tex biology after cells commit to exhaustion. Here, we decoupled the role of TOX in the initiation versus maintenance of CD8 T cell exhaustion by temporally deleting TOX in established Tex. Induced TOX ablation in committed Tex resulted in apoptotic-driven loss of Tex, reduced expression of inhibitory receptors including PD-1, and a pronounced decrease in terminally differentiated subsets of Tex cells. Simultaneous gene expression and epigenetic profiling revealed a critical role for TOX in ensuring ongoing chromatin accessibility and transcriptional patterns for key Tex gene modules in committed Tex cells. Moreover, when exposed to effector-driving conditions, inducibly TOX-deleted established Tex acquired an altered chromatin landscape with increased accessibility at cytotoxic genes typically accessible in Teff cells, thus undergoing partial reprogramming into a more functional state. Together, these findings suggest that continuous TOX expression in established Tex acts as a durable epigenetic barrier to reinforce the Tex developmental fate by simultaneously maintaining Tex epigenetic commitment while restraining differentiation into Teff. Manipulation of TOX even after Tex establishment could therefore provide a therapeutic opportunity to rewire Tex biology in settings of chronic infection or cancer. The secondary goal of this dissertation was to develop a novel Tex fate-mapping mouse model driven to track the fate of developing Tex and manipulate Tex in a lineage-restricted fashion. Given the selectively high expression of TOX in Tex, compared to other peripheral non-Tex CD8 lineages, we used the Tox locus to drive this model (termed ToxTREx), which was engineered with a T2A-hmKO2-P2A-CreERT2 cassette knocked into the Tox locus after the last exon. We confirmed intact TOX function, hmKO2 reporter detection and TOX-driven Cre recombinase activity in this model and identified further optimization that will be necessary to improve Cre efficiency and specificity of this model for the Tex lineage. Nevertheless, the ToxTREx model could enable insightful studies that address existing and emerging questions in Tex ontogeny, differentiation and function.

Wherry, E John
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