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.

The critical contribution of TGF‐β to the induction of Foxp3 expression and regulatory T cell function

“…It is fair to point out that some other groups [6,7] have failed to demonstrate T reg function, stability of Foxp3 expression, or survival in vivo of TGF-binduced T reg even though they express high levels of Foxp3 immediately after induction. From our own studies, it is clear that Foxp3 cannot be induced in T cells following an initial period of activation in vitro.…”

“…Under our culture conditions, we observed induction of Foxp3 expression after 24 h, prior to any cell divisions. Others [6] have only seen significant Foxp3 expression after 3 days and changes may have been induced in the responder cells downstream from Foxp3 that preclude T reg function. Lastly, trivial differences in cell culture protocols may be important.…”

STAT6 Inhibits TGF-β1-mediated Foxp3 Induction through Direct Binding to the Foxp3 Promoter, Which Is Reverted by Retinoic Acid Receptor

“…Ϫ naïve T cells in vitro (8). Recently, an enhancer in the Foxp3 gene in which NFAT and Smad3 bind and cooperatively induce Foxp3 expression was identified (9).…”

“…Recently, retinoic acid (RA) has been discovered as a potent inducer and preserver of Foxp3 in iTregs (14 -18). Unlike that in nTregs, Foxp3 expression in iTregs has been shown to be transient both in vitro and in vivo (8,19). However, the molecular mechanisms for the suppression of Foxp3 induction by IL-6 and IL-4 as well as those for transient induction of Foxp3 have not been clarified.…”

Induction of regulatory T-cells from memory T-cells is perturbed during acute exacerbation of multiple sclerosis

“…However, subsequent observations of transient FOXP3 induction in activated CD4 + CD25 − T-cells complicated the perception of FOXP3 as a distinctive identifier of Treg populations. Many studies have since described additional markers towards discriminating between thymic-derived nTregs and peripherally-induced CD4 + FOXP3 + T-cells, however, all markers to date are found to be expressed by both regulatory populations, including CTLA-4 [8], GITR [9], CD127 [10], and more recently, HELIOS [11, 12]. Despite the lack of unique identifiers, iTregs remain a highly-studied population.…”