Figure 7.
Figure 7. Inhibition of CD16 shedding impairs detachment of NK cells from opsonized target cells. (A–M) NK cells were incubated in microwells (50 × 50 µm2 [A–F] or 450 × 450 µm2 [G–M]) with Daudi-rituximab ± TAPI-0 and imaged every 3 min for 8 h. Images show fluorescence and brightfield images overlaid. (A) Representative image of 81 wells (50 × 50 µm2) showing NK cells (blue), Daudi-rituximab (green), and dead cells (red). Yellow indicates cells beginning to die. (B and C) Representative time-lapse images of individual wells at times indicated. Bars, 20 µm. (B) NK cell (blue) formed a conjugate with a target cell (green; 0 min; yellow arrow). The NK cell formed a contact with another target (white arrow, 126 min). First target cell was killed (234 min), the NK cell detached from killed cell (249 min) and detached from the second target without killing (315 min). (C) In the presence of TAPI-0, an NK cell contacted two targets (15 min; yellow and white arrows), and both were killed (42 and 114 min), but the NK cell was unable to detach (480 min). (D) Percentage of interactions that resulted in target cell lysis (mean ± SD). (E) Percentage of NK cells that detached from target cells whether or not the target cell was lysed (mean ± SD). (F) The duration of all cytolytic NK cell–target cell contacts. Each point represents an individual contact. Contacts still intact at the end of the acquisition (480 min) are plotted in yellow. These points underestimate the true contact time (median ± IQR). n = 886 interactions from five independent experiments; symbols indicate different donors. (G and H) Representative time-lapse zoomed-in regions from 450 × 450 µm2 microwells. Bars, 20 µm. (G) An NK cell (blue) formed a contact with Daudi-rituximab (green; 21 min; yellow arrow). After target cell lysis (24 min), the NK cell detached and moved onto a second target (51 min; white arrow). The second target was not killed, and the NK cell detached (213 min). (H) With TAPI-0, an NK cell contacted a target cell (3 min; yellow arrow). After target cell lysis (105 min), the NK cell did not detach and itself died (351 min). (I) Percentage of all NK cell–target cell interactions that resulted in target cell lysis. (J) Percentage of NK cells that detached from Daudi-rituximab cells whether or not the target cell was lysed. (K) Average time that NK cells were in contact with a target cell in which the target cell was lysed. Conjugates that remained attached at the end of the acquisition are plotted in yellow (median ± IQR; each point represents an individual contact). (L) Percentage of NK cells that died after target cell contact. (M) Percentage of NK cells that died after failing to detach. Mean ± SD; symbols indicate different donors. n = 705 interactions from six independent experiments. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 calculated by Student’s t test (D, I, L, and M), one-way ANOVA (E and J), or Mann-Whitney test (F and K). See also Videos 3 and 4.

Inhibition of CD16 shedding impairs detachment of NK cells from opsonized target cells. (A–M) NK cells were incubated in microwells (50 × 50 µm2 [A–F] or 450 × 450 µm2 [G–M]) with Daudi-rituximab ± TAPI-0 and imaged every 3 min for 8 h. Images show fluorescence and brightfield images overlaid. (A) Representative image of 81 wells (50 × 50 µm2) showing NK cells (blue), Daudi-rituximab (green), and dead cells (red). Yellow indicates cells beginning to die. (B and C) Representative time-lapse images of individual wells at times indicated. Bars, 20 µm. (B) NK cell (blue) formed a conjugate with a target cell (green; 0 min; yellow arrow). The NK cell formed a contact with another target (white arrow, 126 min). First target cell was killed (234 min), the NK cell detached from killed cell (249 min) and detached from the second target without killing (315 min). (C) In the presence of TAPI-0, an NK cell contacted two targets (15 min; yellow and white arrows), and both were killed (42 and 114 min), but the NK cell was unable to detach (480 min). (D) Percentage of interactions that resulted in target cell lysis (mean ± SD). (E) Percentage of NK cells that detached from target cells whether or not the target cell was lysed (mean ± SD). (F) The duration of all cytolytic NK cell–target cell contacts. Each point represents an individual contact. Contacts still intact at the end of the acquisition (480 min) are plotted in yellow. These points underestimate the true contact time (median ± IQR). n = 886 interactions from five independent experiments; symbols indicate different donors. (G and H) Representative time-lapse zoomed-in regions from 450 × 450 µm2 microwells. Bars, 20 µm. (G) An NK cell (blue) formed a contact with Daudi-rituximab (green; 21 min; yellow arrow). After target cell lysis (24 min), the NK cell detached and moved onto a second target (51 min; white arrow). The second target was not killed, and the NK cell detached (213 min). (H) With TAPI-0, an NK cell contacted a target cell (3 min; yellow arrow). After target cell lysis (105 min), the NK cell did not detach and itself died (351 min). (I) Percentage of all NK cell–target cell interactions that resulted in target cell lysis. (J) Percentage of NK cells that detached from Daudi-rituximab cells whether or not the target cell was lysed. (K) Average time that NK cells were in contact with a target cell in which the target cell was lysed. Conjugates that remained attached at the end of the acquisition are plotted in yellow (median ± IQR; each point represents an individual contact). (L) Percentage of NK cells that died after target cell contact. (M) Percentage of NK cells that died after failing to detach. Mean ± SD; symbols indicate different donors. n = 705 interactions from six independent experiments. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 calculated by Student’s t test (D, I, L, and M), one-way ANOVA (E and J), or Mann-Whitney test (F and K). See also Videos 3 and 4.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal