Selective p110α inhibitors have lesser effect on IFN-γ production in anti-NKG2D- or anti-NK1.1-stimulated NK cells.

<p>IL-2-expanded mouse NK cells were stimulated with plate-bound anti-NKG2D mAb (<b>A–B</b>) or anti-NK1.1 mAb (C) in the presence of 1 µM indicated inhibitors (TGX-221 was 0.5 µM) (A) or 250 nM, 500 nM, and 1 µM indicated inhibitors (B–C). After 18 h stimulation, the supernatants were collected and IFN-γ secretion was determined by ELISA (A, left). After 6 h stimulation, total RNA was isolated and IFN-γ mRNA expression was determined by real-time RT-PCR using SYBR Green (A, right). The relative transcript quantities were calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099486#s4" target="_blank">Materials and Methods</a> section and normalized using the unstimulated group defined as 1. After 18 h stimulation, the NK cells were harvested and the intracellular IFN-γ level was determined by flow cytometry (B–C). Brefeldin A was added for the last 4 h before cell harvest and IFN-γ production was measured in CD3⁻NK1.1⁺ NK cells by intracellular staining. The data are expressed as the means ± SEM of three independent experiments. Statistical analysis was performed with one-way ANOVA using Prism 6 (GraphPad Software, Inc.) to compare the differences between vehicle and each inhibitor-treated group. *, <i>p</i><0.05, **, <i>p</i><0.01, and ***, <i>p</i><0.001. (<b>D</b>) NK cells were pre-treated with vehicle (0.1% DMSO) or 1 µM GDC-0941 for 15 min, followed by stimulation with plate-bound anti-NKG2D mAb in the absence or presence of IL-12 and IL-18 for 18 h. The intracellular IFN-γ level was determined as in B–C.