Figure 5. PTEN deficiency impairs T_reg stability.

(a,b) Expression of CD44, CD69, CD62L (a) and ICOS, PD-1, GITR and CTLA4 (b) in T_reg cells from the spleen of WT and Pten^fl/flFoxp3-Cre mice. Mean fluorescent intensity (MUFI) is presented in the plots. (c) Expression of CD25 and Foxp3 (gated on CD4⁺TCRβ⁺ cells) in the spleen of WT and Pten^fl/flFoxp3-Cremice; the numbers above the graphs indicate the mean fluorescent intensity (MUFI) of Foxp3 in CD25⁻ and CD25⁺ subsets. Right, quantification of Foxp3⁺CD25⁻ cells. (d) Expression of Blimp1 in the splenic T_reg cells of WT and Pten^fl/flFoxp3-Cre mice. (e) Foxp3-YFP and GFP expression in CD4⁺ T cells from Pten^+/+Foxp3-Cre Rosa26^GFPand Pten^fl/flFoxp3-Cre Rosa26^GFPmice. (f) Intracellular staining of IFN-γ and IL-17 (right, quantification of IFN-γ⁺ and IL-17⁺ producing cells in T_reg cells), and RNA analysis of IFN-γ in T_reg cells of WT and Pten^fl/flFoxp3-Cre mice (IL-17 RNA was undetectable). (g) WT and Pten^fl/flFoxp3-Cre T_reg (CD45.2⁺) were transferred into CD45.1⁺ recipients, followed by analysis of donor cell percentages (upper) and Foxp3 and CD25 expression (lower). Right, direct overlays of donor-derived WT and Pten^fl/flFoxp3-Cre T_reg cells for Foxp3 (upper right) and CD25 levels (lower right). (h) Flow cytometry analysis of Foxp3-YFP⁺CD25⁻ cells in WT, Pten^fl/flFoxp3-Cre, Ifng^−/− and Pten^fl/flFoxp3-Cre Ifng^−/− splenic T_reg cells. Data are representative of at least three independent experiments (a-f, h) and two independent experiments (g). NS, not significant; *P < 0.05 and **P < 0.001. Data are mean ± s.e.m.