Purified experienced NK cells (5 × 105/ml) were resuspended in complete medium and left untreated or stimulated for 18 h at 37 °C with 500 U/ml of IL-2 (#200-02), or 10 ng/ml of IL-12 (#200-12), or 100 ng/ml of IL-15 (#200-15) (all from PeproTech). Where required, 10 μM idelalisib (CAL-101, GS1101; #S2226, Selleck Chemicals) or 20 nM rapamycin (#R0395, Merck) was added 2 h before cytokine treatment, and maintained until the end of stimulation. An equivalent volume of DMSO (#D5879, Merck), as vehicle, was added to control samples.
Stimulation of activating receptors was performed by plastic-immobilized goat F(abʹ)2 anti-mouse IgG (H + L) (1 μg/106) followed by anti-NKp46 (0.5 μg/106), anti-NKG2D (1 μg/106) or anti-2B4 (0.24 μg/106) mAbs, used alone or in combination. IFN-γ was quantified in the supernatants by commercial ELISA kit (#EHIFNG2, Thermo Fisher Scientific), according to the manufacturer’s instructions.
Stimulation of activating receptors was performed by plastic-immobilized goat F(abʹ)2 anti-mouse IgG (H + L) (1 μg/106) followed by anti-NKp46 (0.5 μg/106), anti-NKG2D (1 μg/106) or anti-2B4 (0.24 μg/106) mAbs, used alone or in combination. IFN-γ was quantified in the supernatants by commercial ELISA kit (#EHIFNG2, Thermo Fisher Scientific), according to the manufacturer’s instructions.