Expression of a noncleavable form of CD16 prevents reduction of perforin in sequential stimulation. (A) Schematic representation of CD16-WT and CD16-S197P indicating a single point mutation, S197P, which renders CD16 insensitive to ADAM17. (B) The NK92 cell line was transduced to express CD16-WT or CD16-S197P. Representative histograms of surface CD16 on parental cell line (gray) and NK92/CD16-WT or NK92/CD16-S197P before (black) and after activation with Daudi-rituximab (Rtx; red) or PMA/ionomycin (orange). The same histogram for a parental cell line is shown in both panels. (C) CD16 expression levels normalized to unstimulated (US) CD16⁺ cells (n = 5 independent experiments; mean ± SD). (D and E) Transfected NK92 cells were sequentially activated through CD16 on slides coated with rituximab and ICAM-1. Secreted perforin was captured with anti-perforin mAb and visualized by a noncompeting Alexa Fluor 488–labeled anti-perforin mAb. Median fluorescence of perforin for NK92/CD16-WT (D) or NK92/CD16-S197P (E). n = 7; mean ± SEM; symbols represent different experiments. **, P < 0.01 calculated by two-way ANOVA (C) or Friedman test (D and E).