Treatment with PI3K inhibitors differentially impairs Tregs in Eμ-TCL1 mice. (A) In vivo activity of PI3K inhibitors was assessed by monitoring levels of pS473-AKT in splenic T cells after 3 days of treatment (100 mg/kg, once daily, oral gavage) in wild-type mice (n = 4 per group). (B) Eμ-TCL1 CLL-bearing recipient mice were randomized and treated for 21 days with the indicated inhibitors (100 mg/kg, once daily, n = 5 mice per group). The plasma concentration of each PI3K inhibitor was measured by high-performance liquid chromatography after collection of peripheral blood at 1 hour and 16 hours after oral gavage. (C) Antitumor efficacy of inhibitors was determined by quantifying CLL B cells in peripheral blood following 21 days of treatment (100 mg/kg, n = 5 mice per group). (D) Total CD3⁺ T-cell count in peripheral blood in the animals treated in (C). CD4/CD8 ratio (E) and Treg count (F) in peripheral blood in the animals treated in (C). (G-H) Total CD3⁺ splenic T cells from the mice treated in (C) were stimulated ex vivo for 24 hours with anti-CD3/CD28. Expression of functional markers on the surface of Tregs was determined by flow cytometry, and IL-10 secretion was measured in the supernatant by cytometric bead array. MFIs of functional markers were normalized to fluorescence minus 1 controls. Graphs show mean + standard deviation. Data are representative of 3 independent in vivo experiments. *P < .05, **P < .005, ***P < .0005, ordinary 1-way ANOVA.