Fig 2. Foxp1 enforces the quiescent state of Treg cells and inhibits aTreg cell differentiation.

(A) Flow cytometry analysis of YFP⁺ Treg cells in CD4⁺ T cells in indicated organs from 6–8-week-old sex- and age-matched Foxp3^Cre and Foxp1^f/fFoxp3^Cre mice. Numbers adjacent to the outlined areas indicate the percentages of YFP⁺ Treg cells. (B) The frequencies of YFP⁺ Treg cells in CD4⁺ T cells in indicated organs from mice as in (A) (n = 3–7). (C) Quantification of total Treg cells in the thymuses, the spleens, and the lymph nodes from the mice as in (A) (n = 3–6). (D) Flow cytometry analysis of CD44 and CD62L expression in Treg cells from 6–8-week-old sex- and age-matched Foxp3^Cre and Foxp1^f/fFoxp3^Cre mice; numbers adjacent to the outlined areas indicate the percentages of rTreg cells (CD44^lowCD62L^high) or aTreg cells (CD44^highCD62L^low). (E) Kinetics of frequencies and numbers of rTreg and aTreg cells in Foxp3^Cre and Foxp1^f/fFoxp3^Cre mice at different ages (n = 3–6). (F) Flow cytometry analysis of Ki-67 expression in rTreg and aTreg cells in the spleens from 8-week-old Foxp3^Cre and Foxp1^f/fFoxp3^Cre mice; numbers above bracketed lines indicate percentages of Ki-67⁺ Treg cells. (G) Proliferation of rTreg cells stimulated by different concentrations of plate-bound anti-CD3/CD28 antibodies in the presence of 200 U/mL IL-2 for 3 days, as shown by the dilution of CellTrace; numbers above bracketed lines indicate percentages of Treg cells with two or more divisions. Data in (A-E) are representative of at least three independent experiments. Data in (F-G) are representative of two independent experiments. Data in (B, C, E) are mean ± SEM, *P < 0.05, ** P < 0.01, *** P < 0.001 (two-tailed Student t test). Data associated with this figure can be found in the supplemental data file (S1 Data). aTreg, activated Treg; CD, cluster of differentiation; Foxp1, forkhead box P1; FSC-H, forward scatter-height; IL, interleukin; Ki-67, antigen identified by monoclonal antibody Ki 67; rTreg, resting Treg; SI, small intestine; Treg, regulatory T; YFP, yellow fluorescent protein.