Fig. 4: NKG2D mediates CRC targeting by DOT cells.
a, Flow cytometry histograms of NKR ligand expression in different CRC cell lines. b, Correlation of geometric mean of the mean fluorescence intensity of NKR ligand(s) on different CRC lines and targeting by DOT cells, assessed by the increase in the percentage of annexin V+ tumor cells upon incubation with DOT cells. Spearman’s correlation coefficients (r) and P values are shown. Data are representative of three independent experiments. c, Analysis of NKG2D ligand expression, assessed by FPKM, in normal colon (n = 41 donors), primary colon cancer (n = 453 donors) and AML (n = 151 donors) obtained from TGCA repository. Data are presented as the means ± s.e.m. and were analyzed by a Kruskal–Wallis test with Dunn’s multiple-comparisons test. d, Quantification by flow cytometry of CD69, CD107a and TNF expression on DOT cells after 48-h incubation with plate-bound MICA–Fc chimera (n = 3 DOT donors). Data were analyzed by a two-tailed unpaired t-test. e, SW620 tumor cell death assessed by flow cytometry over a 3-h killing assay, performed with freshly thawed DOT cells and DOT cells prestimulated with plate-bound MICA–Fc chimera for 48 h (n = 2 DOT donors). Data were analyzed by a one-way ANOVA with Tukey’s post hoc test. In d,e, data are one representative of three experiments. f, SW620 tumor cell death assessed by flow cytometry over a 3-h killing assay performed with DOT cells in the presence of anti-NKG2D and anti-DNAM1 blocking antibodies or their isotype controls. Lines connect individual DOT donors (n = 7 DOT donors, pool of seven assays). Data were analyzed by a repeated-measures one-way ANOVA with Tukey’s post hoc test. g, SW620 tumor cell death assessed by flow cytometry over a 3-h killing assay performed with control or KLRK1-knockout DOT cells. Data are presented as the means of the technical replicates of n = 2 independent experiments and were analyzed by a one-way ANOVA with Tukey’s post hoc test. Data in c, d and g are presented as the means ± s.e.m. Data in e are presented as the means.
