PTEN deficiency reversed low mTOR pathway usage and renders Tregs sensitive to RAPA. (A) Treg cells were isolated after 48 hours of culture. Where indicated, IL-2 (100 IU/mL) and/or RAPA (10 ng/mL) was present in the culture. The amount of phosphorylated p70S6 and 4E-BP1 was quantified by phospho-flow analysis within wt or PTEN^−/− Tregs. The amount of phosphorylated protein as measured in MFI was significantly higher for phospho-p70S6 (left panel) phospho-4E-BP1 (right panel) in PTEN^−/− compared with wt Treg cells (* P < .01). Addition of RAPA (10 ng/mL) to the culture reduced the amount of phospho-p70S6 and phospho-4E-BP1 in PTEN^−/− Treg cells. (B) BALB/c mice were given 5 × 10⁶ TCD-BM together with 5 × 10⁵ wt or PTEN^−/− Treg (C57BL/6) after lethal irradiation with 800 cGy and were injected with PBS or RAPA (1.5 mg/kg) daily. The amount of phospho-p70S6, phospho-STAT5 in wt, or PTEN^−/− donor type Treg cells (H-2k^b) is displayed (*P < .01). Percentage of dividing donor type (H-2k^b) CFSE labeled Tregs was higher in PTEN^−/− donors compared with wt donors (44% vs 19.6%, P < .05). Increased expansion of PTEN^−/− Tregs was antagonized when the recipients were treated with RAPA (44% vs 26.5%, P < .05).