Protection of M07Sb cells from apoptosis and STAT6 activation in PB progenitors committed to the pro-NK lineage by r-IL-15/s-IL-15Rα complex. (A) Protection of M07Sb cells from apoptosis by r-IL-15/s-IL-15Rα complex. M07Sb cells that had been starved of GM-CSF for 24 hours were treated for 24 hours with r-IL-15 (10 and 0.1 ng/mL) and with 0.1 ng/mL r-IL-15 bound to decreasing concentrations of the soluble IL-15Rα chain (1000-1 ng/mL). The percentage of apoptotic cells was determined by flow cytometry as described in the legend to Figure 6. As a control of apoptosis, cells were also treated with a membrane uncoupler, carbonyl cyanide m-chlorophenylhydrazone (ClCCP; Sigma Chemical), at a final concentration of 50 μM, for 15 minutes at 37°C under 5% CO₂. The data presented are representative of 3 independent experiments. R-IL-15 at 10 ng/mL but not at 0.1 ng/mL induced significant protection from apoptosis (^*P < .05 compared with the group without cytokine). Association of decreasing concentrations of soluble IL-15Rα with 0.1 ng/mL r-IL-15 induced powerful antiapoptotic effects (^*P < .05 compared with the group treated with 0.1 ng/mL r-IL-15). Values are means ± SD (n = 3). Horizontal bars identify the sample on which statistic significance has been observed. (B) STAT6 activation analysis by confocal microscopy of PB progenitors committed to the pro-NK lineage using r-SCF/r-Flt3-L medium. Overlay pictures (original magnification, ×63). (Bi) Negative control. (Bii) basal conditions. (Biii) 30 minutes of incubation at 37°C with 0.1 ng/mL human r-IL-15. (Biv) 30 minutes of incubation at 37°C with 0.1 ng/mL human r-IL-15 bound to 1 μg/mL of the soluble IL-15Rα chain. (Bv) 30 minutes of incubation at 37°C with 100 ng/mL human r-IL-15. As negative controls, cells were incubated with rabbit IgG, the second reagent, and propidium iodide. The data presented are representative of 3 independent experiments. Images were acquired with a 63 ×/1.30 objective lens.