Fig 2: 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 were pre-incubated briefly with IL-15 then imaged on ICAM-1 or VCAM-1 surfaces in the presence of IL-15. Cells were segmented and tracked as in Fig. 1, and parameters of cell shape and cell migration were calculated using cellPLATO. A) Plot of differences (left) and effect size (right) for cell speed. B) Timeplot of mean cell speed over time, C) Timeplot of difference of effect size of cell speed of NK cells on VCAM-1 (orange line; VCAM-1 + IL-15) relative to control (blue line; ICAM-1 + IL-15). D) Plot of differences (left) and effect size (right) for cell area. E) Timeplot of mean cell area over time. F) Timeplot of difference of effect size of cell area of VCAM-1 (orange line; VCAM-1 + IL-15) relative to control (blue line; ICAM-1 + IL-15). n = 14,985 cells from 1 donor.

Fig 3: Behavioural trajectory cluster analysis reveals differential trajectories of NK cells migrating on ICAM-1 or VCAM-1. Trajectory analysis of NK cells from Donor 1 generated by pairwise similarity comparison of cluster ID time sequences. (A) UMAP plot coloured by trajectory ID. (B) Fingerprint plot showing the percentage trajectory ID per condition. (C–F) Graphical representations (top) and time plots of behavioural cluster ID (bottom) for each trajectory ID. See also Movies 3–6. (G) Fingerprint plot showing the frequency of single timepoint behavioural cluster IDs for each trajectory ID. (H) Graphical depictions of single timepoint behaviours represented in G and reproduced from Fig. 4C–J. (I) Plasticity of cells per behavioural trajectory ID measured by the median number of cluster switches. (J) Median number of cluster switches per condition. n=187 cells filtered to include only cells with 200 to 220 timepoints randomly selected from 14,825 cells from Donor 1. Kruskal–Wallis followed by Wilcoxon rank-sum testing with Bonferroni correction was used to compare between trajectories (I) or conditions (J); P<0.00001 for all pairwise comparisons. The data distribution is shown as a violin plot with median (dot, printed value), interquartile range (IQR; thick line) and range (thin whiskers).

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: 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.