Figure 3.

Impact of effective anti-PD-1 treatment on the kinetics and plasticity of CTL immune cells

(A) Tracking strategy. ToC videos of lung cancer cells (IGR-Heu) and autologous CTLs (H5B) were acquired with high temporal resolution (every 30 s) for 6 h. A cancer cell and an immune were considered to interact when their distance was closer than the interaction radius (here defined as 34 μm, twice the sum of the average radius of detected CTLs and cancer cells). See also Video S2.

(B) Representative output of the Cell Hunter tracking algorithm.

(C) Quantification of the time of interaction between cancer cells and CTLs. 669 time events with a duration longer than 10 min were counted in total. Statistical significance was assessed using Mann-Whitney test.

(D) Quantification of the number of interactions between cancer cells and CTLs. The number of interactions counted for each cancer cell was normalized by the total number of cancer cell trajectories detected along the video. 2,880 interaction events were counted in total. Mann-Whitney statistical test was used.

(E) Number of interactions per each immune CTL. 625 interactions in total were counted.

(F) Number of interactions per each cancer cell. 382 interactions in total were counted.

(G) Quantification of the speeds of immune CTLs. 1,542 speed values were counted in total. Mann-Whitney test was used.

(H) Quantification of the track curvatures of immune CTLs. 1,542 curvature values were counted in total. Mann-Whitney test was used.

(I) Experimental design for analysis of T cell plasticity in ToC co-cultures. CTLs were co-cultured in microfluidics devices with or without autologous cancer cells and treated with the isotype control or with anti-PD-1 (nivolumab). After 3 days, the cells were retrieved from the gel by collagenase digestion, stained, and analyzed by flow cytometry. The following markers were measured: CD25 and CD69 (activation markers); PD-1, TIM-3, TIGIT, LAG-3, CD244, and CTL-4 (inhibitory immune checkpoints); and OX-40, CD137, and GITR (activatory immune checkpoints). It was not possible to measure the PD-1 marker in the presence of anti-PD-1 treatment because of antibody competition. Three conditions were assessed: CTLs only, with cancer cells without anti-PD-1, and with cancer cells with anti-PD-1. Heatmaps report averages from 2–4 independent experiments depending on the condition.

(J) Fold change of specific MFI for CTL markers of H5B cells. The specific MFI for the condition CTLs only is set as 1.

(K) Percentage of positive H5B cells for CTL markers.

See Figure S1 for full datasets.