Foxp1 helps maintain Treg cell suppressive function through controlling stable Foxp3 expression.

(A) Relative Foxp3 mRNA levels in highly purified rTreg (n = 4) and aTreg cells (n = 6) from Foxp3Cre and Foxp1f/fFoxp3Cre mice. (B) Immunoblot analysis of Foxp3 in rTreg and aTreg cells from Foxp3Cre and Foxp1f/fFoxp3Cre mice. (C) Flow cytometry analysis of the expression of Foxp3 in rTreg and aTreg cells from the spleens of Foxp3Cre and Foxp1f/fFoxp3Cre mice (left panel), and the corresponding mean fluorescence intensity (MFI) of Foxp3 (right panel) (n = 4). (D) Foxp1 ChIP was performed in Foxp3Cre and Foxp1f/fFoxp3Cre iTreg cells to detect the Foxp1 binding to Foxp3 CNS0, CNS2, and promoter regions; a region in the Gmpr locus was used as a negative control, n = 3–4. (E, F) Sorted rTreg and aTreg cells from Foxp3Cre and Foxp1f/fFoxp3Cre mice were labeled with CellTrace and cultured in the plate coated with 1 μg/mL anti-CD3 and -CD28 antibodies in the presence of 200 U/mL IL-2 for 3 days. (E) Foxp3 expression during Treg cell division in rTreg and aTreg cells. The numbers below the red line represent the percentages of Foxp3low Treg cells. (F) Foxp3 expression in rTreg and aTreg cells during Treg cell division; numbers adjacent to the histograms represent Foxp3+ Treg frequencies. (G) Foxp3 expression in Foxp3Cre and Foxp1f/fFoxp3Cre rTreg and aTreg cells cultured with plate-bound anti-CD3/CD28 antibodies in the presence or absence of IL-4 for 3 days. (H-I) EAE was induced in Foxp1f/fFoxp3Cre mice by immunization with MOG peptide and pertussis toxin (n = 3–8), followed by transfer of sorted Foxp3Cre and Foxp1f/fFoxp3Cre Treg cells infected with control retroviruses (RV-ctrl) or retroviruses expressing Foxp3 (RV-Foxp3) at d4. The severity of EAE was evaluated by the clinical score (H) and intracellular staining of cytokines in the T cells from the spinal cords and the brains (I); the Foxp1f/fFoxp3Cre mice without Treg cell transfer were controls. The numbers adjacent to the outlined area indicate the percentage of gated cells. Data in (A-I) represent at least three independent experiments. Data in (A, C: right panel, D, H) are mean ± SEM, *P P P t test). Data associated with this figure can be found in the supplemental data file (S1 Data). aTreg, activated Treg; CD, cluster of differentiation; ChIP, chromatin immunoprecipitation; CNS0, conserved noncoding sequence 0; CNS2, conserved noncoding sequence 2; d, day; EAE, experimental autoimmune encephalomyelitis; Foxp1, forkhead box P1; Foxp3, forkhead box P3; Gmpr, guanosine monophosphate reductase; IL, interleukin; iTreg, induced Treg cells; max, maximum; MFI, mean fluorescence intensity; MOG, myelin oligodendrocyte glycoprotein; rTreg, resting Treg; RV-ctrl, control retrovirus; RV-Foxp3, retrovirus expressing Foxp3; TCR, T-cell receptor; Treg, regulatory T.