PI5KI isoform silencing impairs NK92 cell cytotoxic function but not IFN-γ release. (A) Uninfected (NI), shRNA-ctr, shRNA-PI5KIα, or shRNA-PI5KIγ populations were assessed in a 51Cr release assay against P815 target cells in the presence of anti-MHC class I (ctr) or anti-2B4 mAb. One representative experiment of 5 performed is shown. Differences between shRNA-PI5KIα or shRNA-PI5KIγ populations and NI or shRNA-ctr populations were significant in 5 independent experiments at all E:T ratios. (B) shRNA-ctr, shRNA-PI5KIα, or shRNA-PI5KIγ NK92 cells were stimulated by 2B4-induced redirected killing as in Figure 2 (top panels). Cell conjugates were fixed, stained with anti-perforin mAb, and analyzed by fluorescence microscopy. The percentage of NK92 cells conjugated with target cells containing polarized granules was calculated on randomly acquired fields of 3 independent experiments (mean ± SD, n = 100 conjugates). The difference obtained between the groups is not significant. (C) The same cell populations were stimulated with plastic-immobilized mAbs or PMA plus ionomycin as indicated. After 4 hours, cell supernatants were collected and assessed for BLT esterase release. Data represent the percentage (mean ± SD) of specific release (sample/total release) from 3 independent experiments. Differences between shRNA-PI5KIα or shRNA-PI5KIγ populations and NI or shRNA-ctr populations were significant (*P < .002). (D) The same cell populations were stimulated with plastic-immobilized anti-2B4 mAb or with rIL-2 (200 U/mL). After 18 hours, supernatants were collected and assessed for IFN-γ levels. Data are expressed as mean plus or minus SD from 3 independent experiments. The difference obtained between the groups is not significant.
