In a 3D environment, CD16 shedding allows more interactions with target cells. (A–G) NK cells and Daudi-rituximab were resuspended in Matrigel at E:T = 1:3 ± TAPI-0. Cell interactions were recorded every 3 min for 8 h. (A and B) Representative time-lapse–enlarged regions showing a composite of fluorescence and brightfield images. NK cells (blue), Daudi-rituximab (green), and dead cells (red) are shown at times indicated. Bars, 20 µm. (A) An NK cell (blue) formed a conjugate with a target (green; 0 min; yellow arrow). The NK cell formed a contact with another target (9 min; white arrow). The second target cell was killed (99 min), whereas the first one remained intact. The NK cell detached from both targets and moved to new target cells (132 min). (B) In the presence of TAPI-0, an NK cell established contact with a target cell (51 min; yellow arrow) and killed it (165 min). The NK cell remained attached to dead target until the end of acquisition (480 min). (C) Percentage of interactions that resulted in target cell lysis. (D) Percentage of NK cells that detached from target cells whether or not target cell was lysed. (E) Duration of all cytolytic NK cell–target cell contacts. Each point represents an individual contact. Yellow points indicate contacts still intact at the end of the acquisition (480 min). These points underestimate true contact times (median ± IQR). (F) Percentage of NK cells interacting with one, two, three, or more target cells. (G) Percentage of NK cells killing one or more target cells. Mean ± SD; symbols indicate different donors. n = 392 interactions from three independent experiments. *, P < 0.05; ***, P < 0.001 calculated by Student’s t test (C), one-way ANOVA (D), Mann-Whitney test (E), or two-way ANOVA (F and G).