Fig. 6

NK cell adhesion and lysis of PAECs under the flow. (A) Live-cell wide-field microscopy of NK cells on control (Φ, left) and pTNF-stimulated (right) PAECs at given time points. The channels showing NK cells (green) and dead cells (Draq7⁺, red) were overlaid on the bright field image to correlate with the PAECs monolayer. For easier visualization and quantification, only the fluorescent channels are shown on the right. Arrows show the three spatial coordinates (magenta, yellow and green), with magenta indicating the flow direction. Scale bar = 100 μm. (B) Analysis throughout the entire 3D channels seeded with control (Φ) or pTNF-stimulated PAECs, at the endpoint (120 min), E:T ratio 1:1. Bottom left plot shows the number of attached NK and dead PAECs at an E:T ratio of 1:1; middle plot shows the number of attached NK and dead PAECs at an E:T ratio of 1:20; right plot shows the number of attached NK cells and dead EA.hy926 cells at an E:T ratio of 1:20. Data were analyzed from three independent experiments with three different healthy donors, multiple paired t-test. Scale bar = 200 μm. (C) The bar-plot shows number of NK cells attached to one region of the channel (0.1 mm) under flow, as well as and the number of cells that migrated on top of control (Φ) and pTNF-stimulated (right) PAECs. Data were analyzed from three different experiments consisting of different healthy donors, one specific region per channel. (D) The left panel displays NK cell trajectories in control (Φ) and pTNF-treated PAECs in one field of view (0.1 mm) of a 3D channel during a 20 min time-lapse, with x- and y-axis coordinates given in pixels (px). The right panel displays the percentage of different NK cell movements, categorized by small and intermediate diameter trajectories, in control (Φ) and pTNF-activated PAECs. The magenta arrow indicates the flow direction. (E) Average speed of NK cell movements on control (Φ) and pTNF-treated target cells. Data were analyzed from a heterogeneous population of NK cells in one region of a 3D channel, from one healthy donor, using an in-house developed Python program. Statistics were obtained using a multiple paired t-test.