S1P-mediated signaling plays an important role in survival of leukemic LGLs. (A) PBMCs from healthy donors (N PBMCs 1,2), normal activated PBMCs (AC PBMCs), or LGL leukemia patients (TLGLs 1,2) were treated with DMSO (□) or 5 μM FTY720 (■) as described. Flow cytometric analysis for induction of apoptosis was done. Cells were gated for CD3⁺CD57⁺ double-positive cells (LGL cells) and further analyzed for apoptosis. Leukemic LGLs showed 20-fold higher apoptosis compared with naive normal LGLs. (B) Treatment with FTY720 sensitizes LGL leukemia PBMCs to Fas-mediated apoptosis. PBMCs from LGL leukemia patients were incubated with vehicle or 5 μM FTY720 for 1 hour. Each treatment group was further divided into 2 and left untreated or treated with 1 μg/mL CH11 and further incubated for 6 hours. In addition to inducing spontaneous apoptosis, treatment with 5 μM FTY720 further sensitizes leukemic LGLs to Fas-mediated apoptosis (*P = .001). (Results shown are representative of 3 independent experiments.) (C) Activated PBMCs from healthy donors were cultured in RPMI-1640 supplemented with 1% FBS for 18 hours. The cells were treated with either vehicle or indicated concentrations of S1P for 1 hour. CH11 (1 μg/mL) was added to the wells and cells were further incubated for an additional 3.5 hours. The graph shows that increasing amounts of S1P in culture protects cells from Fas-mediated apoptosis in a dose-dependent manner. At 0.5-μM and 0.05-μM concentrations, S1P inhibits Fas-mediated apoptosis by more than 55% and 30% (*P < .03), respectively. The results shown are a representative of 1 of the 3 individual experiments performed.