PI3Kδ^−/− NK cells fail to efficiently lyse leukemic target cells. (A) The cytolytic function of IL-2–expanded PI3Kδ^+/− and PI3Kδ^−/− NK cells was assessed by [⁵¹Cr]-release assay using various cell lines as target cells as indicated. PI3Kδ^−/− NK cells showed consistently impaired specific lysis when compared with PI3Kδ^+/− NK cells, regardless of the target cell line used. The complete table of all target lines including statistics is provided in Table S3. (B) Top panels: typical confocal images of CD107a⁺ granules of PI3Kδ^+/− and PI3Kδ^−/− NK cells are given. Bottom panel: quantification of granule numbers. Confocal images of CD107a⁺ granules were acquired in the z-stack mode on a Zeiss LSM510 confocal microscope with a 100× oil-immersion objective (NA 1.3) with the 488-nm line of the Ar laser and a LP 505-nm emission filter. About 24 slices were acquired with a distance of 0.3 μm between the slices. Images were imported into ImageJ software, and maximum projection images were calculated. Stained vesicles were selected by auto-thresholding, followed by quantification and analysis of all selected regions. Horizontal lines indicate data mean. (C) A FACS-based degranulation assay measuring surface expression of the late endosomal marker CD107a was performed. FACS histograms illustrate the percentage of PI3Kδ^+/− (top row) and PI3Kδ^−/− (bottom row) NK cells expressing CD107a under basal conditions (left panels) and after stimulation by coincubation with v-abl–transformed target cells. Significantly more PI3Kδ^+/− NK cells were stimulated to express CD107a on their surface compared with PI3Kδ^−/− NK cells (data mean ± SEM). (D) Bar graphs depicting different stimuli used to induce degranulation in PI3Kδ^+/− and PI3Kδ^−/− NK cells: none of the target cells or specific antibodies induced degranulation of PI3Kδ^−/− NK cells. (E) Electrophysiologic capacitance measurements were performed and confirmed the defect of PI3Kδ^−/− NK cells in Ca²⁺-dependent exocytosis/degranulation. Electrophysiologic capacitance measurement relies on recording the passive current, which has to be injected into the cell to keep a constant voltage and this corresponds to the size (surface area) of a cell: the bigger the cell, the more current has to be injected. In vitro–expanded murine NK cells are round cells (means of absolute capacitance of unstimulated PI3Kδ^+/− NK cells, 12.4 ± 5.4 pF; of PI3Kδ^−/− NK cells, 13.73 ± 5.9 pF). Two typical examples of the exocytotic response under the superfusion with calcimycin are illustrated (left panel). The mean increase in relative capacitance after superfusion of PI3Kδ^+/− and PI3Kδ^−/− NK cells was compared (mean increase in relative capacitance for PI3Kδ^+/− NK cells: 21% ± 2%; for PI3Kδ^−/−NK cells: 8.5% ± 1%; P < .001; n = 10 for each genotype; the experiment was repeated with 3 independent NK-cell preparations). The increase in relative capacitance was obtained by comparing the peak increase in relative capacitance after superfusion with calcimycin with resting cell capacitance under superfusion with control solution (right panel). Error bars represent SEM. n.s. indicates P > .05; *P < .05; and **P < .01.