PI3K inhibitors differentially impair normal and CLL Tregs in a similar manner. (A) CD3⁺ T cells were isolated from normal donor PBMCs and cultured with the indicated concentrations of PI3K inhibitors in dose titration in the presence of anti-CD3/anti-CD28 for 72 hours. Graphs display mean of technical triplicates + standard deviation (SD) from 1 representative donor. (B) PD-1 and CTLA-4 expression on Tregs in (A). Heat map depicts average MFI of technical triplicates from 1 representative donor. (C) CD3⁺ T cells were isolated from normal donor PBMCs (n = 6) and cultured with PI3K inhibitors (1 μM, 72 hours) in the presence of anti-CD3/anti-CD28. (D) CD3⁺ T cells were isolated from previously frozen CLL patient PBMCs (n = 6) and cultured with PI3K inhibitors (1 μM, 72 hours) in the presence of anti-CD3/anti-CD28. Immunophenotyping was performed via flow cytometry. PD-1 and CTLA-4 MFIs were normalized to DMSO to stabilize variance. Graphs display mean + SD. *P < .05, **P < .005, ***P < .0005, ordinary 1-way ANOVA.