Fig. 6: EVs secreted by Cal27 induce apoptosis in NK cells.

a Cal27-WT and KO derived EVs were analyzed by flow cytometry for known NKG2D ligands MICA/B, ULBP2/5/6, ULPB3, and for TRAIL and FasL, proteins involved in intrinsic or extrinsic apoptosis pathway (green histograms). In blue is shown the isotype control. Representative of n = 2 independent experiments. b Flow cytometry CASP3/7 viability assay showing increasing NK cell death once exposed to EVs from Cal27 in a time course of 4-hours. The error bars represent ±SEM across n = 3 replicates. Two-way ANOVA and Bonferroni’s post hoc test was performed to determine statistical significance. Representative of n = 3 independent experiments. c 24 hour time course viability assay quantified using the IncuCyte real-time imaging system. N = 5 images/well taken for each of three replicates. The error bars represent ±SEM across n = 3 replicates. Statistical analysis was performed by two-way ANOVA and Bonferroni’s post hoc multiple comparison test. Representative of n = 2 independent experiments. d 4 hour cytotoxicity assay using NK cells as effectors against Cal27-WT or KO as target cells. Here we show how Cal27-derived EVs reduce the killing in Cal27-KO. Error bars represent ±SEM across n = 3 replicates. Statistical analysis was performed by one-way ANOVA and Bonferroni’s post hoc test (ns, not significant). e antibodies blocking MICA/B, TRAIL, or a combination of the two (aMICA/B and aTRAIL) limit the inhibitory effect of EV on NK cell-mediated killing. 4-hour cytotoxicity assay using NK cells as effectors against Cal27-WT or KO as target cells (5:1) incubated with Cal27-WT-derived EV and with MICA/B and TRAIL blocking antibodies or respective isotypes controls. Error bars represent ±SEM across n = 3 replicates. Statistical analysis was performed by two-way ANOVA and Bonferroni’s post hoc multiple comparison test. d–e Representative of n = 2 independent experiments.