TOX Phase Separation Locks T Cells in a Terminal Exhausted State, and Disrupting It Can Revive Anti-Tumor Immunity

One major obstacle limiting the efficacy of tumour immunotherapy is terminal exhaustion of T cells. Although the molecular mechanisms sustaining the tired state are still unclear, the transcription factor TOX is thot to be a crucial driver of this process. The purpose of this study was to determine whether TOX can “lock in” terminal fatigue by forming condensates thru liquid-liquid phase separation and to evaluate the possibility of targeting phase separation to restore anti-tumor immunity. We discovered that the N-terminal intrinsically disordered region (IDR) of TOX can spontaneously form droplet-like condensates thru sequence analysis and in vitro recombinant assays. This property is markedly amplified in the nucleus of tired T cells after persistent antigen stimulation.We verified that TOX condensates are dynamic and capable of enriching transcriptional co-repressor complexes using methods such as optogenetic clustering and fluorescence recovery following photobleaching. By creating a phase-separation-deficient mutant without the IDR (TOX-IDRm) and adoptively introducing it into tumor-bearing mice, the percentage of terminally fatigued T cells was considerably decreased while effector cytokine production and proliferation were restored. TOX condensates directly occupy and maintain chromatin in an open state at important exhaustion genes like Pdcd1 and Havcr2, according to single-cell transcriptomic and epigenomic investigations.In melanoma models, interfering with TOX phase separation not only inhibited tumour growth but also shown a synergistic impact with anti-PD-1 antibodies, greatly increasing animal survival. In conclusion, TOX serves as the foundation for sustaining terminal exhaustion by physically solidifying the exhaustion transcriptional program by phase separation. The functional limitations on T cells can be effectively lifted by targeting TOX phase separation, offering a new potential target for the development of immunotherapy techniques akin to “phase separation intervention”.