Fig. 4

Activation of dendritic cells co-cultured with LOAd-infected melanoma cells. Monocytes were isolated from peripheral blood mononuclear cells and cultured with GM-CSF/IL-4 to induce immature dendritic cells (DCs). DCs were co-cultured with Mel526 cells that were either infected with LOAd(-), LOAd703 or LOAd732, or left untreated (DC:Mel526 ratio = 2:1). As control condition, DCs were infected directly with the respective LOAd virus or left untreated and cultured alone. Cells were cultured for 48 h before cell culture supernatants and cells were harvested and analyzed for DC maturation with flow cytometry and multiplex analysis. For flow cytometry analysis, DCs were gated based on CD1a expression and the mean fluorescence intensity of the respective marker was determined for CD1a + cells based on matched isotype control antibodies. The expression of activation markers in A is displayed as percentage fold change over control condition (DCs cultured alone) (A). In B, cytokine and chemokine levels in the culture supernatants of the co-cultures are shown in pg/mL (B). Bar graphs display the mean ± SD of three biological repeats. Statistical differences between LOAd-infected and untreated cells were determined with Kruskal–Wallis test followed by Dunn’s multiple comparison test (*p < 0.05, **p < 0.01)