Figure 3.

Treg cells bearing homozygous RhoA deletion are plastic and outcompeted by Treg cells bearing intact RhoA in RhoA^Flox/FloxFoxp3^YFP-Cre/+ female mice. (A) Left, representative flow cytogram of Foxp3⁺YFP⁺ and Foxp3⁺YFP^- cells in CD4⁺Foxp3⁺ Treg cells from the spleen of RhoA^+/+Foxp3^YFP-Cre/+ and RhoA^Flox/FloxFoxp3^YFP-Cre/+ female mice. The numbers indicate percentages of Foxp3⁺YFP⁺ and Foxp3⁺YFP^- cells. Middle, cell numbers of Foxp3⁺YFP⁺ and Foxp3⁺YFP^- cells. Right, the ratio of percentages of Foxp3⁺YFP⁺ versus percentages of Foxp3⁺YFP^- cells. (B) Foxp3⁺YFP⁺ Treg cell proliferation. (C) Foxp3⁺YFP⁺ Treg cell apoptosis. (D) The expression levels (MFI: Mean fluorescence intensity) of Foxp3 in Foxp3⁺YFP⁺ and Foxp3⁺YFP^- cells from the spleen of RhoA^Flox/FloxFoxp3^YFP-Cre/+ female mice. (E) Left, representative flow cytogram of IL-17, IFN-γ, and IL-4 staining in CD4⁺Foxp3⁺YFP⁺ and CD4⁺Foxp3⁺YFP^- cells from the spleen of RhoA^Flox/FloxFoxp3^YFP-Cre/+ female mice. The numbers indicate percentages of IL-17⁺, IFN-γ⁺, and IL-4⁺ cells. Right, average percentages of IL-17⁺, IFN-γ⁺, and IL-4⁺ cells. (F) In vitro Treg suppressive activity. Left, representative flow cytogram of CFSE dilution in T cells (responder). The numbers above the gating are percentages of cells low for CFSE, representing proliferating T cells. Right, average percentages of cells low for CFSE. Foxp3⁺YFP⁺ Treg cells were obtained by flow cytometry sorting of CD4⁺YFP⁺ cells from the spleen of RhoA^+/+Foxp3^YFP-Cre/+ and RhoA^Flox/FloxFoxp3^YFP-Cre/+ female mice. Error bars indicate SD of 3 mice (A–E) or triplicates (F). Data are representative of two independent experiments. *p < 0.05, **p < 0.01.