Figure 5.

Heterozygous RhoA deletion in Treg cells induces Treg cell plasticity and increases CD4⁺ effector T cells but does not result in autoimmunity. (A) Body weight of RhoA^+/+Foxp3^YFP-Cre and RhoA^Flox/+Foxp3^YFP-Cre mice. (B) Images of H&E staining of the indicated organs. (C) Representative flow cytogram of Foxp3 staining in CD4⁺ cells from the spleen of RhoA^+/+Foxp3^YFP-Cre and RhoA^Flox/+Foxp3^YFP-Cre mice. The numbers indicate percentages of CD4⁺Foxp3⁺ Treg cells. (D) Left, average percentages of CD4⁺Foxp3⁺ Treg cells. Right, cell numbers of CD4⁺Foxp3⁺ Treg cells. (E) Proliferation of Foxp3⁺YFP⁺ Treg cells from RhoA^+/+Foxp3^YFP-Cre/+ and RhoA^Flox/+Foxp3^YFP-Cre/+ female mice. Percentages of Foxp3⁺YFP⁺ Treg cells incorporated with BrdU are shown. (F) Apoptosis of Foxp3⁺YFP⁺ Treg cells from RhoA^+/+Foxp3^YFP-Cre/+ and RhoA^Flox/+Foxp3^YFP-Cre/+ female mice. The expression levels (MFI: mean fluorescence intensity) of active caspase 3 in Foxp3⁺YFP⁺ Treg cells are shown. (G) The expression levels of Foxp3 in Treg cells from RhoA^+/+Foxp3^YFP-Cre and RhoA^Flox/+Foxp3^YFP-Cre mice. (H) Representative flow cytogram of IFN-γ, IL-17 and IL-4 staining in CD4⁺Foxp3⁺ Treg cells. The numbers indicate percentages of IFN-γ⁺, IL-17⁺ and IL-4⁺ Treg cells. (I) Average percentages of IFN-γ⁺, IL-17⁺ and IL-4⁺ Treg cells. (J) Representative flow cytogram of RORγT, T-bet and GATA3 staining in CD4⁺Foxp3⁺ Treg cells. The numbers indicate percentages of RORγT ⁺, T-bet⁺, and GATA3⁺ Treg cells. (K) Average percentages of RORγT ⁺, T-bet⁺, and GATA3⁺ Treg cells. (L) Representative flow cytogram of IFN-γ, IL-17 and IL-4 staining in CD4⁺Foxp3^- cells. The numbers indicate percentages of IFN-γ⁺CD4⁺, IL-17⁺CD4⁺ and IL-4⁺CD4⁺ effector T cells. (M) Average percentages of IFN-γ⁺CD4⁺, IL-17⁺CD4⁺ and IL-4⁺CD4⁺ effector T cells. (N, O) In vitro Treg suppressive activity. (N), 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. (O), 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/+Foxp3^YFP-Cre/+ female mice. (A, C–O) Error bars indicate SD of 5-6 mice (A, C–M) or triplicates (N, O). Data are representative of two independent experiments. Error bars indicate SD. **p < 0.01. (B) Data are representative of 3 mice.