Stabilization of Human CD8 ⁺ Treg in Inflammatory Environments Through FOXP3 Expression

ABSTRACT Cell therapy using regulatory T cells (Tregs) holds therapeutic promise in immune‐mediated diseases, but their stability and persistence after transfer into inflammatory environments remain major challenges. CD8 + Tregs, in particular, express multiple cytokine receptors, raising questions about their plasticity. Here, we investigated their susceptibility to pro‐inflammatory signals and the role of FOXP3 in maintaining stability. Short‐term culture with IL‐6 and TNFα or IFNγ had no impact, but exposure to TGFβ combined with IL‐6 and IL‐1β or IL‐21 and IL‐23 induced substantial transcriptomic changes, including marked FOXP3 downregulation. Enforced FOXP3 expression via lentiviral transduction enhanced suppressive activity in vitro and stabilized phenotype under inflammation. Notably, FOXP3 knockout CD8 + Tregs retained suppressive function for up to 2 weeks, suggesting FOXP3 is not strictly required for short‐term activity. Moreover, FOXP3 expression was boosted by transduction of mTOR regulators SESN2 or FLCN, or the SAGA complex component TAF5L, with SESN2 and TAF5L alone sufficient to enhance suppressive function. Together, these findings reveal mechanisms controlling CD8 + Treg stability and identify molecular targets to optimize next‐generation Treg‐based cell therapies for immune‐mediated diseases.