Figure 5. Characterization of synergistic dual GLA-AF+BGP activation of newborn MoDCs.

Adult and newborn MoDCs were stimulated for 18 hours with GLA-AF (1000 ng/ml), BGP (100 μg/ml) or the combination. Secreted cytokines were measured by multiplexing bead array (A,B) (n=3–6, Paired Student’s t-test, *p<0.05, **p<0.01 and ***p<0.001). IFN-γ- and IL-4-producing cells were quantified by flow cytometry following treatment of autologous naïve CD4⁺ T cells with MoDC culture supernatant for 6 days and subsequent addition of Brefeldin A. Mean relative percentage of IFN-γ producing cells (C), mean relative percentage of IL-4 producing cells (D), and ratio of IFN-γ producing cells over IL-4 producing cells (E) show an increase in Th1 polarization after treatment with culture supernatants from R848+TDB-activated MoDCs (n=4, mean+SEM, paired Student’s t-test, *p<0.05, **p<0.01 and ***p<0.001). The expression of a panel of 80 innate immune pathway-related genes was measured by qRT-PCR and the mean (n=3) depicted as a volcano plot (F). Inflammasome activation was confirmed by incubation of cells that were treated as indicated with FITC-labeled Caspase-1 substrate FAM-YVAD-FMK (representative experiment shown, n=3) (G) Activation of NF-κB was confirmed by detection of IκBa degradation in lysates from cells treated as indicated (representative experiment shown, n=3) (H).