Foxp3 drives context-dependent epigenetic programs that define regulatory T cell molecular identity and function

Regulatory T cells (T_reg cells) express the master regulator, Foxp3, and display distinctive epigenetic landscapes ensuring T_reg cell-specific gene expression and stable suppressive functions, yet Foxp3's contribution to this epigenetic identity remains unclear. Leveraging Foxp3-transduced conventional T cells as a gain-of-function probe in mice, we identified a previously unrecognized subset that acquires endogenous Foxp3 expression, T_reg cell-like transcriptomic and chromatin features, and suppressive functions exclusively in vivo. These Foxp3-driven features were conserved in T_reg cells but impaired in Foxp3-mutant T_reg-like cells, demonstrating a Foxp3 requirement. Induction of endogenous Foxp3 expression in vivo required reduced AKT-mTOR signaling and Foxp3-dependent engagement of STAT5 and nuclear factor κB (NF-κB). Temporal chromatin profiling revealed stepwise Foxp3-driven regulatory programs, including a core program shared across T_reg cell subsets and effector-specific programs, both associated with NF-κB activity and Foxp3 binding. Thus, Foxp3 integrates cell-intrinsic and environmental contexts to drive epigenetic programs defining T_reg cell identities and functions, with implications for Foxp3-based therapies.