Background T cell exhaustion is one of the major barriers limiting efficacious anti-tumor therapy. Exhausted CD8 T cells (TEX) develop following persistent antigen stimulation and are characterized by a unique epigenetic state, expression of PD-1 and other inhibitory receptors, dampened effector function, and limited capacity to control disease. Though checkpoint blockade temporarily improves TEX function, the underlying epigenetic landscape of TEX remains largely unchanged and these ‘reinvigorated’ TEX revert to less effective antitumor T cells. This fate inflexibility is likely attributed to epigenetic events during TEX differentiation that cement commitment to the TEX fate. Our lab and others recently identified the HMG-box transcription factor TOX as an essential transcriptional and epigenetic initiator of TEX differentiation. However, it was unclear whether TOX maintained TEX fate commitment and, thus, whether TOX removal would reprogram TEX into more functional CD8 lineages with enhanced capacity for tumor control.
Methods Here, we used an adoptive T cell transfer approach in mice, which we infected with lymphocytic choriomeningitis virus (LCMV), to model TEX differentiation. To decouple the role of TOX in TEX maintenance from its previously reported role in TEX initiation, we established an inducible-Cre system to temporally delete TOX from LCMV-specific TEX only after exhaustion was established.
Results Induced TOX ablation in committed TEX reduced TEX numbers and impaired expression of PD-1 and other canonical TEX inhibitory receptors. This effect of TOX loss appeared to be driven by altered TEX proliferation and survival. Accordingly, blocking apoptosis numerically rescued TOX-deficient TEX; yet, PD-1 loss and other key TEX phenotypic differences were retained. This incomplete rescue alluded to a global role for TOX in enforcing TEX differentiation, beyond directly regulating TEX survival. Indeed, single-cell RNAseq and ATACseq revealed that TOX was required to maintain expression and chromatin accessibility of key TEX modules. Furthermore, when exposed to signals that drive effector differentiation, TOX-deficient TEX acquired cytotoxic-specific chromatin accessibility and exhibited greater reprogramming into highly functional and cytotoxic effector-like cells—thus identifying TOX as one of the epigenetic barriers that constrain the fate flexibility of TEX.
Conclusions Together, these findings suggest that TOX transcriptionally and epigenetically enforces critical components of the TEXprogram, and that TOX manipulation provides an avenue for rewiring TEX identity. By improving molecular understanding of the role of TOX in enforcing TEX identity and in constraining TEX fate reprogramming, this study will inform future immunotherapies that seek to re-engineer TEX into customized differentiation states with amplified potential for tumor control.
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