Akt promotes NK cell degranulation. (A) WT and ItpkB^−/− splenic NK cells were pretreated with DMSO, Akt inhibitor VIII, or LY294002 for 1 hour before coculture with RMA/S targets for 2 hours. Degranulation was assessed by cell-surface staining of CD107a. Results shown are representative of 3 independent experiments. (B) Degranulation (mean ± SEM, n = 3) by WT and ItpkB^−/− splenic NK cells stimulated with RMA/S or RMA/S-Rae1ϵ cells or PMA/ionomycin after pretreatment with DMSO, Akt inhibitor VIII, or LY294002. Asterisks indicate statistical significance of P < .05 for the indicated comparisons (paired Student t test, n = 3). (C-D) Akt activation was assessed in WT and ItpkB^−/− splenic NK cells stimulated without (control) or with Abs against NK1.1 without (anti-NK1.1) or together with 25μM PI3K-inhibitor (anti-NK1.1 + LY294002) or 50μM cell-permeable IP₄-ester (anti-NK1.1 + IP₄) by intracellular staining with phospho-Akt-S₄₇₃ (pAKT)–specific Abs. Results shown are representative of 3 independent experiments. (E) WT and ItpkB^−/− NK cells contain similar total Akt protein amounts. Total Akt protein levels in WT (black open histogram) or ItpkB^−/− (hatched open histogram) splenic CD3⁻NK1.1⁺ NK cells were determined via intracellular staining and flow cytometry. Shaded gray histogram indicates the negative control stain. Results shown are representative of 3 independent experiments. (F) ItpkB^−/− and WT splenic NK cells were stimulated without (control, shaded gray histograms) or with anti-NK1.1 Abs (solid open histograms) or PMA (hatched open histograms) for 10 minutes, followed by flow cytometric analysis for phospho-Erk (pThr₂₀₂/pTyr₂₀₄) content (pERK). Numbers indicate the percentage of pERK⁺ cells after stimulation with plate-bound anti-NK1.1 Abs (5 μg/mL).