Expression of a noncleavable form of CD16 leads to prolonged contacts with opsonized target cells. (A and B) NK92/CD16-WT or NK92/CD16-S197P cells were incubated with Daudi-rituximab (Rtx) in 450 × 450 µm2 microwells. Representative time lapse–enlarged regions show composite fluorescence and brightfield images. NK cells (blue), Daudi-rituximab (green), and dead cells (red) at times indicated. Bars, 20 µm. (A) An NK92/CD16-WT cell (blue) formed a contact with Daudi-rituximab (green; 45 min; yellow arrow). The NK92 cell detached without killing and established a contact with a new target (102 min; white arrow). The second target cell was killed (123 min), and the NK92 cell was detached (156 min). (B) An NK92/CD16-S197P cell formed a contact with a target cell (15 min; yellow arrow) and killed it (36 min). The NK92/CD16-S197P cell died while still attached (387 min). (C) Percentage of NK cell–target interactions that resulted in target cell lysis. (D) Percentage of NK cells that detached by the end of the acquisition. n = 195 interactions from three independent experiments (mean ± SD). (E and F) NK92 cells and transfectants were incubated on surfaces coated with ICAM-1 (−) or ICAM-1 with rituximab for 5 min. Cells were fixed and stained with Alexa Fluor 488–labeled phalloidin marking F-actin. (E) Representative images of spreading on rituximab with ICAM-1. Bars, 20 µm. (F) Percentage of NK92 cells forming dense peripheral rings of F-actin. n = 3 independent experiments (mean ± SD). (G) Representative dot plot assessing the degranulation marker, CD107a, by flow cytometry. NK92 cells and transfectants were activated on ICAM-1 (−) or rituximab with ICAM-1 for 4 h. **, P < 0.01; ***, P < 0.001 calculated by Student’s t test (C) or one-way ANOVA (D and F). SSC-W, side-scatter–width.
