A Kinetic and Dynamic Analysis of Foxp3 Induced in T Cells by TGF-β

Abstract

TGF-beta induces Foxp3 expression in stimulated T cells. These Foxp3 cells (induced regulatory T cells (iTreg)) share functional and therapeutic properties with thymic-derived Foxp3 regulatory T cells (natural regulatory T cells (nTreg)). We performed a single-cell analysis to better characterize the regulation of Foxp3 in iTreg in vitro and assess their dynamics after transfer in vivo. TGF-beta up-regulated Foxp3 in CD4(+)Foxp3 T cells only when added within a 2- to 3-day window of CD3/CD28 stimulation. Up to 90% conversion occurred, beginning after 1-2 days of treatment. Foxp3 expression strictly required TCR stimulation but not costimulation and was independent of cell cycling. Removal of TGF-beta led to a loss of Foxp3 expression after an approximately 4-day lag. Most iTreg transferred into wild-type mice down-regulated Foxp3 within 2 days, and these Foxp3 cells were concentrated in the blood, spleen, lung, and liver. Few of the Foxp3 cells were detected by 28 days after transfer. However, some Foxp3 cells persisted even to this late time point, and these preferentially localized to the lymph nodes and bone marrow. CXCR4 was preferentially expressed on Foxp3 iTreg within the bone marrow, and CD62L was preferentially expressed on those in the lymph nodes. Like transferred nTreg and in contrast with revertant Foxp3 cells, Foxp3 iTreg retained CD25 and glucocorticoid-induced TNFR family-related gene. Thus, Foxp3 expression in naïve-stimulated T cells is transient in vitro, dependent on TGF-beta activity within a highly restricted window after activation and continuous TGF-beta presence. In vivo, a subset of transferred iTreg persist long term, potentially providing a lasting source for regulatory activity after therapeutic administration.