Tregs display high PTEN expression levels during stimulation and PTEN deficiency partially antagonizes resistance of Tregs toward RAPA. (A) Levels of PTEN protein expression are analyzed by FACS for naive (left histogram) or activated (right histogram) Tconv (dark gray-filled histogram) and Tregs (light gray-filled histogram) cells. Activation was by allo-Ag (CD11c⁺ H-2k^d, 30 Gy γ-irradiation) and IL-2 (100 IU/mL) for 48 hours. The black open histogram represents the isotype control. MFI for PTEN is higher in Tregs compared with Tconv after activation (241 vs 82, P < .001). (B) Western blot analysis demonstrates that Treg cells display slightly higher amounts of PTEN protein compared with Tconv cells while both were in the naive state. (C) Specific recombination at the PTEN locus in the presence of Cre is shown by PCR amplification of genomic DNA isolated from CD4 + T cells from Cre^negative (+/+), Pten^flox/+Cre⁺ (+/−), and Pten^flox/floxCre⁺ (^−/−) littermates. Western blot analysis demonstrated absence of PTEN in Pten^flox/floxCre⁺ (^−/−) mice. (D) Percentage of proliferating wild-type () or PTEN-deficient () Tconv or Treg after 48-hour stimulation with (CD11c⁺ H-2k^d, 30 Gy γ-irradiation) and IL-2 (100 IU/mL), in the presence or absence of RAPA (R, 10 ng/mL) as indicated (*P < .05; **P < .01). Proliferation was assessed by serial CFSE dilution.