ItpkB and IP₄ inhibit NK cell degranulation. (A) WT or ItpkB^−/− splenic NK cells were pretreated with DMSO, cell-permeable IP₄, or the PI3K inhibitor LY294002 for 1 hour and then cocultured with RMA/S targets for 2 hours. Degranulation was assessed by cell-surface staining of CD107a. (B) Degranulation (mean ± SEM, n = 3) by WT and ItpkB^−/− splenic NK cells in response to media, RMA/S, or RMA/S-Rae1ϵ target cells after pretreatment with DMSO, cell-permeable IP₄, or LY294002. Asterisks indicate statistical significance of P < .05 for the indicated comparisons (paired Student t test, n = 3). (C) WT and ItpkB^−/− splenic NK cells were stimulated without (gray shaded, hatched histograms) or with (open solid histograms) the indicated concentrations of plate-bound Abs against NKp46, NK1.1, or 2B4 for 2 hours. Degranulation was assessed as in panel A. Numbers indicate the percentage of cells in the CD107a⁺ gate after stimulation. (D) WT and ItpkB^−/− NK cells express similar levels of granzyme B. Splenocytes from WT (solid open histogram) and ItpkB^−/− (hatched open histogram) mice were stained for surface CD3 and NK1.1, followed by intracellular staining with anti–granzyme B (open histograms) or isotype control (shaded gray histogram, isotype control, WT only) Abs and analyzed by flow cytometry. Numbers denote the percentage of cells in the indicated granzyme B (GrzB)⁺ gate for ItpkB^+/+ and ItpkB^−/− mice.