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. 2020 Oct 13;9:e56554. doi: 10.7554/eLife.56554

Figure 2. CTLs migrate rapidly and directionally towards sites of cognate target engagement due to a diffusing homotypic signal.

(A) Schematic of assay enabling 3D tracking of CTL movements relative to a tumouroid exposing a straight interface (scale bar: 50 µm). (B) Average speed and forward migration index (FMI) of CTLs adjacent to tumouroids containing cognate (SIINFEKL-pulsed) EL4 tumour cells or non-cognate (unpulsed) EL4, and without (-) or with pre-embedded tumour-reactive CTLs at indicated CTL:tumour cell ratios. (C) Left: Schematic showing H-2Kb/SIINFEKL cognate antigen-coated beads co-embedded with CTLs (OT1) in a dense mass, with the migration of adjacent fluorescent CTLs (OT1GFP) tracked in 3D over time. Right: FMI of CTLs migrating towards tumouroids containing OT1 CTLs and non-cognate or cognate tumour cells or beads. (D) Transmigration of CTLs towards supernatant obtained from CTLs conjugated with non-cognate or cognate cells and beads. Bars: mean from three independent experiments (data points). Error bars: SEM. p-values for comparisons of cognate and non-cognate conditions by t tests. (E) FMI of OT1 or gBT1 CTLs migrating towards tumouroids pre-embedded at a 1:1 ratio with OT1 or gBT1 CTLs respectively, showing ‘self-recruitment’ of CTLs. (F) FMI of OT1 or gBT1 CTLs migrating towards cognate tumoroids (EL4 pulsed with SIINFEKL or SSIEFARL as indicated) pre-embedded at a 1:1 ratio with gBT1 or OT1 CTLs, showing ‘cross-recruitment’ of CTLs of different antigen specificity towards tumour-reactive CTLs. Tumouroid densities are kept constant, and when pre-embedded with CTLs contain only half the number of tumour cells compared to masses in 2B constituted exclusively of tumour cells. (G) FMI of polyclonal CD8+CD44+ T cells migrating towards tumouroids containing OT1 CTLs with cognate or non-cognate tumour cells. Box-whiskers indicate medians and the interquartile range (IQR) with outliers outside whiskers. Red bars: mean of pooled data from three independent experiments. Data points: mean of each individual experiment. n: number of tracks. ns: p>0.05, where FMI in (B) to (G) were compared to a theoretical median of 0 using the two-tailed Wilcoxon signed rank test and average speeds in (B) were compared using the Kruskal-Wallis test followed by Dunn’s multiple comparisons tests.

Figure 2—source data 1. Source data file for Figure 2.

Figure 2.

Figure 2—figure supplement 1. CTLs transmigrate towards cognate supernatants.

Figure 2—figure supplement 1.

(A) Schematic illustration of the transmigration assay. CTLs were co-incubated with SIINFEKL-pulsed EL4 tumour cells or unpulsed non-cognate EL4 cells for 3 hr. The supernatant was filtered and used in the lower compartment of transwell chambers to quantify its chemotactic effect on CTLs. (B) Transmigration of CTLs towards cognate or non-cognate supernatant obtained from different CTL: tumour cell ratios. Data points from three independent experiments. (C) Transmigration of CTLs embedded in 3D collagen matrices towards cognate or non-cognate supernatant using transwells. Data points from three independent experiments. (D) Quantification of the killing efficiency (cytotoxicity index) of OT1 and gBT1 CTLs in the presence or absence of their respective cognate antigen-presenting cells (+SFKL for OT1; SFARL for gBT1). Data points from four independent experiments. (B–D) Bars: mean; error bars: SEM. ns: p>0.05, p-values by ANOVA and Tukey’s multiple comparison test of transmigration indices and cytotoxic indices in (B) to (C).
Figure 2—figure supplement 1—source data 1. Source data file for Figure 2—figure supplement 1.