1016 Enforcement of exhausted T cell epigenetic fate by HMG-box transcription factor TOX

Background

T cell exhaustion is one of the major barriers limiting efficacious anti-tumor therapy. Exhausted CD8 T cells (T_EX) 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 T_EX function, the underlying epigenetic landscape of T_EX remains largely unchanged and these ‘reinvigorated’ T_EX revert to less effective antitumor T cells. This fate inflexibility is likely attributed to epigenetic events during T_EX differentiation that cement commitment to the T_EX fate. Our lab and others recently identified the HMG-box transcription factor TOX as an essential transcriptional and epigenetic initiator of T_EX differentiation. However, it was unclear whether TOX maintained T_EX fate commitment and, thus, whether TOX removal would reprogram T_EX 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 T_EX differentiation. To decouple the role of TOX in T_EX maintenance from its previously reported role in T_EX initiation, we established an inducible-Cre system to temporally delete TOX from LCMV-specific T_EX only after exhaustion was established.

Results

Induced TOX ablation in committed T_EX reduced T_EX numbers and impaired expression of PD-1 and other canonical T_EX inhibitory receptors. This effect of TOX loss appeared to be driven by altered T_EX proliferation and survival. Accordingly, blocking apoptosis numerically rescued TOX-deficient T_EX; yet, PD-1 loss and other key T_EX phenotypic differences were retained. This incomplete rescue alluded to a global role for TOX in enforcing T_EX differentiation, beyond directly regulating T_EX survival. Indeed, single-cell RNAseq and ATACseq revealed that TOX was required to maintain expression and chromatin accessibility of key T_EX modules. Furthermore, when exposed to signals that drive effector differentiation, TOX-deficient T_EX 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 T_EX.

Conclusions

Together, these findings suggest that TOX transcriptionally and epigenetically enforces critical components of the T_EXprogram, and that TOX manipulation provides an avenue for rewiring T_EX identity. By improving molecular understanding of the role of TOX in enforcing T_EX identity and in constraining T_EX fate reprogramming, this study will inform future immunotherapies that seek to re-engineer T_EX into customized differentiation states with amplified potential for tumor control.