Fig 2: Morphological and migratory features of NK cells imaged, segmented, and tracked on ICAM-1 or VCAM-1 in the presence of IL-15Freshly isolated human NK cells were pre-incubated briefly (10 min) with IL-15 then imaged on B) ICAM-1 or C) VCAM-1 surfaces in the presence of IL-15. Timelapse images of NK cells derived from PBMCs plated onto B) ICAM-1 or C) VCAM-1 coated glass slides. Dashed box (left) shows a zoomed region (middle) and example cell segmentation and tracking (right). Red arrows denote different morphologies of cells identified qualitatively as described in text. D) Number of cells detected per condition. E) Number of timepoints each cell was tracked. Blue = cells from donor 1, yellow: cells from donor 2. Large dots: mean per donor. n = 14,825 and 6,584 cells tracked respectively from two donors.

Fig 3: Morphological and migratory features of NK cells imaged, segmented and tracked on ICAM-1 or VCAM-1 in the presence of IL-15. (A) Freshly isolated human NK cells from Donor 1 labelled with membrane and DNA dye were imaged on ICAM-1 or VCAM-1 surfaces in the presence of IL-15. Timelapse images of NK cells on (B) ICAM-1 or (C) VCAM-1-coated glass. The dashed box (left) highlights the location of the magnified region (middle) and example cell segmentation and tracking (right). Red arrows denote different morphologies of cells identified qualitatively as described in the text. Representative images from 14,825 cells tracked from Donor 1. See also Movies 1 and 2.

Fig 4: Distinct behaviours de-abstractified from NK cells migrating on ICAM-1 or VCAM-1 in the presence of IL-15. (A) Eight clusters of distinct cell behaviours based on morphological and motility-based metrics identified by UMAP followed by HDBSCAN cluster analysis. (B) Percentage cluster ID by condition. (C–J) Exemplar cells representing each cluster shown as cell contours and tracks overlaid onto raw data accompanied by the metrics that best define membership in that cluster (scaled values in parentheses). n=14,825 cells from Donor 1.

Fig 5: Features of NK cell speed and cell area on VCAM-1 and ICAM-1 in the presence of IL-15. Freshly isolated human NK cells from Donor 1 were imaged on ICAM-1 or VCAM-1 surfaces in the presence of IL-15. Cells were segmented and tracked as in Fig. 2 and features of cell shape and cell migration were calculated using cellPLATO. Plots of differences for (A) cell speed and (B) cell area show data distribution (left) and effect size (right). (C) Timeplot of mean cell speed on ICAM-1 or VCAM-1 in the presence of IL-15. (D) Timeplot of effect size for cell speed comparing VCAM-1 (orange line; VCAM-1+IL-15) relative to ICAM-1 control (green line; ICAM-1+IL-15). (E) Timeplot of mean cell area of cells on ICAM-1 or VCAM-1 in the presence of IL-15. (F) Timeplot of effect size for cell area comparing VCAM-1 (orange line; VCAM-1+IL-15) to ICAM-1 (green line; ICAM-1+IL-15). n=14,825 cells from Donor 1. A Kruskal–Wallis test was used to compare conditions and produce P-values. Plots in A and B display the distribution of the data as points (left) or a violin plot (right) showing the median (dot), interquartile range (IQR; thick line) and range (thin whiskers). Plots in C–F display the mean value per timepoint (line) and 95% c.i. (edge denoted by pale shades).