Apoptosis induction after PI3K pathway inhibition in T-LGL. (A) Healthy-donor PBMCs and T-LGL PBMCs were incubated overnight in RPMI with 10% FCS at 37°C either untreated or treated with CH11 (anti-Fas; 1 μg/mL), LY294002 (50 μM), or CH11 (1 μg/mL) + LY294002 (50 μM), as indicated. After 20 hours the cells were stained for FITC–annexin V and PC5-CD8 and analyzed via FACS. For analysis, cells were gated on the lymphocyte population based on forward and scatter profile. The percentage of CD8⁺ cells that are annexin V positive are indicated by the numbers in the top right quadrants. (B) PBMCs from a T-LGL patient with a large Vβ2 clonal expansion (90% of all PBMCs) were incubated overnight in RPMI with 10% FCS at 37°C either untreated, or with 50 μM LY294002. After 20 hours, the cells were stained for FITC–annexin V and PE-Vβ2 and analyzed via FACS. For analysis, cells were gated on the Vβ2 population. The percentage of Vβ2⁺ cells that are annexin V positive are indicated by the numbers in the top right quadrants. (C) T-LGL PBMCs were incubated overnight in RPMI with 10% FCS at 37°C either untreated (red) or with 6.25 μM LY294002 (gray), 12.5 μM LY294002 (orange), 25 μM LY294002 (green), or 50 μM LY294002 (blue). After 16 hours the cells were stained for FITC–annexin V and PC5-CD8 and analyzed via FACS. For analysis, cells were gated on the lymphocyte population based on forward and scatter profile.