Foxp1 regulates iTreg cell differentiation and maintenance.

(A)Foxp1f/fRosa26YFP and Foxp1f/fCre-ERT2+Rosa26YFP mice were treated with tamoxifen for 6 days, and CD4+ T cells were isolated and cultured in the plate coated with 1 μg/mL anti-CD3/CD28 antibodies in the presence of 0.3 μM 4-hydroxytamoxifen, 5 ng/mL TGF-β, and various concentrations of recombinant human IL-2 for 2 days. Cells were detached and cultured for an additional day. Foxp3 and CD25 expression were analyzed by flow cytometry; numbers adjacent to the outlined areas represent the percentages of gated cells. (B-D) Sorted YFP+ Foxp1f/fFoxp3Cre or Foxp3Cre iTreg cells differentiated in vitro were cotransferred with sorted CD44lowCD62LhighCD25− WT naive CD4+ T cells into Rag1−/− mice (n = 3). Mice were analyzed at 6 weeks post T-cell transfer. (B) Flow cytometry analysis of Foxp3+YFP+ Treg cells in the spleens and the colons of the recipient mice (left panel) and quantification of YFP+ Treg cell frequencies (right panel); the numbers adjacent to the outlined area indicate percentage of YFP+ Treg cells. (C) The severity of colitis was evaluated by loss of body weight of the recipient mice and (D) intracellular staining of cytokines in T cells from the colons; the numbers adjacent to the outlined area indicates percentage of gated cells. Data represent at least two independent experiments. Data in (B: right panel, C) are mean ± SEM, *P t test). Data associated with this figure can be found in the supplemental data file (S1 Data). CD, cluster of differentiation; Foxp1, forkhead box P1; Foxp3, forkhead box P3; IFNγ, interferon gamma; IL, interleukin; iTreg, induced Treg cells; Rag1, recombination activating 1; TGF-β, transforming growth factor, beta; Treg, regulatory T; WT, wild-type; YFP, yellow fluorescent protein.