Continuous expression of TOX safeguards exhausted CD8 T cell epigenetic fate [coassay_TOXKO]

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 HMG-box transcription factor 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 cells. 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 strong effector-differentiation signals, established Tex in which TOX had been deleted acquired an altered chromatin landscape with increased accessibility at cytotoxic genes typically accessible in Teff cells, undergoing partial reprogramming into a more functional state. Together, these findings suggest that continuous TOX expression in established Tex functions as a durable epigenetic barrier to reinforce the Tex developmental fate by simultaneously maintaining Tex epigenetic commitment while restraining effector reprogramming. Manipulation of TOX even after Tex establishment could therefore provide a therapeutic opportunity to rewire Tex biology in settings of chronic infection or cancer