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

Abstract

TGF-β 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-β 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-β led to a loss of Foxp3 expression after an ∼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-β activity within a highly restricted window after activation and continuous TGF-β presence. In vivo, a subset of transferred iTreg persist long term, potentially providing a lasting source for regulatory activity after therapeutic administration.