Fig. 5.
Amplified γc cytokine-dependent S6 phosphorylation and increased sensitivity to PI3K/mTOR inhibition in obinutuzumab-experienced NK cells. Primary cultured NK cells were stimulated (2:1) for 18 h with biotinylated rituximab (RTX-exp)-, obinutuzumab (GA101-exp)-opsonized (a–c) or not opsonized Raji (Ctrl-exp) (b) and immunomagnetically purified by negative selection. Experienced NK cells were treated with 10 μM idelalisib (a), 20 nM rapamycin (c) (white histograms) or with the same volume of DMSO as vehicle (grey histograms) for 2 h and then stimulated for additional 18 h with IL-2 (500 U/ml) or IL-15 (100 ng/ml), as indicated, in the presence of the inhibitors. Supernatants were then collected and assessed for IFN-γ levels. Data (mean ± SEM) from n = 8 donors from three independent experiments are depicted in bar graphs. ***p < 0.0008, **p < 0.0006, *p < 0.03. b Experienced NK cells were stimulated with IL-2 (500 U/ml) (top panels) or IL-15 (100 ng/ml) (bottom panels) for the indicated times. The phosphorylation levels of ribosomal protein S6 in S235/236 residues were evaluated by FACS analysis in fixed and permeabilized samples. Graphs depict for each time point the MFI value calculated as follow: (MFI of cytokine activated sample-MFI of untreated sample). Three representative donors and mean ± SEM from n = 6 donors are shown. *p = 0.031