Foxp1 and Foxp3 coordinate the regulation of CTLA-4 expression in Treg cells.

(A) Heatmap of the representative common target genes of Foxp1 and Foxp3 in rTreg cells. (B) The relative mRNA levels of CTLA-4 in purified rTreg and aTreg cells from Foxp3Cre and Foxp1f/fFoxp3Cre mice, n = 4. (C) Flow cytometry analysis of CTLA-4 expression in splenic rTreg and aTreg cells from Foxp3Cre and Foxp1f/fFoxp3Cre mice. (D) Predicted conserved forkhead-binding site in the Ctla4 promoter (left panel); Foxp1 ChIP analysis was performed in Foxp3Cre and Foxp1f/fFoxp3Cre iTreg cells (right panel) (n = 3); −2.4 kb upstream of TSS at the Ctla4 locus was used as a negative control. (E) Foxp3 ChIP was performed in Foxp3Cre and Foxp1f/fFoxp3Cre iTreg cells to detect Foxp3 binding to the Ctla4 promoter region in the presence or absence of Foxp1, n = 2. A region in the Gmpr locus was used as a negative control. (F) The relative mRNA levels of CTLA-4 in Foxp1f/fFoxp3Cre iTreg cells infected with control retroviruses (RV-ctrl), retroviruses expressing Foxp1A (RV-Foxp1A), retroviruses expressing Foxp3 (RV-Foxp3), or retroviruses expressing both RV-Foxp1A and RV-Foxp3 (RV-Foxp1A+Foxp3), n = 3. WT iTreg cells infected with control retroviruses were used as a control. Data in (A, D-F) are representative of at least two independent experiments. Data in (B, C) are representative of at least three independent experiments. Data in (B, D, E, F) 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; ChIP, chromatin immunoprecipitation; CTLA-4, cytotoxic T-lymphocyte-associated protein 4; Foxp1, forkhead box P1; Foxp3, forkhead box P3; Gmpr, guanosine monophosphate reductase; iTreg, induced Treg cells; rTreg, resting Treg; Treg, regulatory T; TSS, transcription start site; WT, wild-type.