Galectin-9 restores chemokine-driven cDC2 migration. (A) Schematic representation of the experimental setup. Primary human cDC2s were matured overnight with a MC for 24 h prior to being harvested and replated in the presence of melanoma-derived CM for 24 h. Exogenous galectin-9 was supplemented for the last 2 h of cDC2 incubation with melanoma-derived CM. Tumor-primed cDC2s were collected and analyzed for the surface expression levels of galectin-9 and CCR7 or for their migratory capacity. (B) Schematic representation of the transwell migration assay. cDC2s were seeded in the top chamber of a transwell chamber containing a 5-µm porous membrane and subjected to a chemokine gradient of the CCR7 ligands CCL19 and CCL21. Migratory cDC2s were collected after 3 h and quantified. (C) Histograms showing the surface expression of galectin-9 and CCR7 of a representative cDC2 donor analyzed by flow cytometry. (D) Percentage of positive cDC2s for galectin-9 and CCR7 for each of the indicated treatments. (E) Relative cDC2 migration under each treatment was determined by normalizing each treatment to the migration given by mature cDC2s unexposed to the melanoma-derived CM for every donor. Violin plots in D and E show mean of five independent donors. One-way ANOVA followed by Dunnett’s test for multiple comparisons was performed. *P < 0.05; **P < 0.01; ***P < 0.001. MC, maturation cocktail.