Stabilizing CD8+ Treg plasticity in inflammatory environments through FOXP3 regulation

Cell therapy using regulatory T cells (Tregs) has demonstrated therapeutic potential in treating several immune diseases in both preclinical and clinical studies. However, their stability and persistence following adoptive transfer into inflammatory environments remain critical challenges impacting both safety and therapeutic efficacy. CD8+ Tregs express several cytokine receptors, making them susceptible to the environment, and their plasticity is unknown. In this study, we explored the susceptibility of CD8+ Tregs to pro-inflammatory conditions, with a particular focus on the role of FOXP3 in maintaining Treg stability. While short-term culture with IL-6 and TNFa or IFN? had no impact on Treg stability, exposure to TGFß combined with IL-6 and IL-1ß or IL-21 and IL-23 resulted in substantial transcriptomic changed, with 348 and 267 differentially expressed genes, respectively, including a significant decrease in FOXP3 expression. Forced expression of FOXP3 in CD8+ Tregs using a lentiviral vector enhanced their suppressive activity in vitro and stabilized their phenotype in an inflammatory environment. Interestingly, FOXP3 knockout CD8+ Tregs maintained their suppressive activity for up to two weeks. Additionally, FOXP3 expression could be increased by transduction of mTOR pathway regulators SESN2 and FLCN, or the SAGA complex component TAF5L, with SESN2 and TAF5L being individually sufficient to enhance CD8+ Treg suppressive activity. Overall, our findings provide new insights to enhance the stability of Treg-based cell therapies, offering promising avenues for the treatment of inflammatory diseases.