TOX enforces the immunosuppressive program of tumor-infiltrating regulatory T cells

Regulatory T (Treg) cells accumulate in the tumor microenvironment (TME) to suppress anti-tumor immunity, but the transcriptional regulators stabilizing their immunosuppressive state remain poorly defined. Here we show that transcriptional factor TOX is selectively upregulated in tumor-infiltrating (TI) Treg cells across human cancers and mouse tumor models, while remaining low in peripheral and naive Treg populations. Treg-specific deletion of TOX reduced tumor burden, impaired TI Treg-mediated immune suppression, and enhanced effector functions of CD8⁺ and CD4⁺ T cells. In mosaic mice, TOX-deficient Tregs were selectively depleted from tumors, accompanied by increased apoptosis. Single-cell RNA sequencing and TCR clonotype analysis linked TOX expression to an effector-like TI Treg state with clonal expansion, whereas TOX loss shifted cells toward a TCF7-associated progenitor-like phenotype. ATAC-seq revealed enrichment of AP-1 motifs in TOX-sufficient TI Tregs. In contrast, TCF7, LEF1, and FOXO1 motifs in TOX-deficient counterparts, uncovering the opposing transcriptional networks downstream of TOX. Furthermore, TOX deficiency augmented CD8⁺ T cell responses to PD-1 blockade. Together, these findings establish TOX as a key regulator of TI Treg fitness and stability, and identify it as a potential therapeutic target to enhance the efficacy of PD-1-based immunotherapy.