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

TGF- (cid:1) induces Foxp3 expression in stimulated T cells. These Foxp3 (cid:2) cells (induced regulatory T cells (iTreg)) share functional and therapeutic properties with thymic-derived Foxp3 (cid:2) 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- (cid:1) up-regulated Foxp3 in CD4 (cid:2) Foxp3 (cid:3) 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- (cid:1) led to a loss of Foxp3 expression after an (cid:1) 4-day lag. Most iTreg transferred into wild-type mice down-regulated Foxp3 within 2 days, and these Foxp3 (cid:3) cells were concentrated in the blood, spleen, lung, and liver. Few of the Foxp3 (cid:3) cells were detected by 28 days after transfer. However, some Foxp3 (cid:2) 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 (cid:2) 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 (cid:3) cells, Foxp3 (cid:2) iTreg retained CD25 and glucocorticoid-induced TNFR family-related gene. Thus, Foxp3 expression in naı¨ve-stimulated T cells is transient in vitro, dependent on TGF- (cid:1) activity within a TGF- (cid:1) induces Foxp3 expression in stimulated T cells. These Foxp3 (cid:2) cells (induced regulatory T cells (iTreg)) share functional and therapeutic properties with thymic-derived Foxp3 (cid:2) 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- (cid:1) up-regulated Foxp3 in CD4 (cid:2) Foxp3 (cid:3) 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- (cid:1) led to a loss of Foxp3 expression after an (cid:2) 4-day lag. Most iTreg transferred into wild-type mice down-regulated Foxp3 within 2 days, and these Foxp3 (cid:3) cells were concentrated in the blood, spleen, lung, and liver. Few of the Foxp3 (cid:3) cells were detected by 28 days after transfer. However, some Foxp3 (cid:2) 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 (cid:2) 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 (cid:3) cells, Foxp3 (cid:2) iTreg retained CD25 and glucocorticoid-induced TNFR family-related gene. Thus, Foxp3 expression in naı¨ve-stimulated T cells is transient in vitro, dependent on TGF- (cid:1) activity within