Figure 7.

MutuDC lines efficiently produce IL-12 p70 and show synergism in response to different combinations of TLR-Ls, IFNγ and aCD40. (A) DC lines (n = 4) and purified splenic cDC subsets (n = 2 per subset) were stimulated with different combinations of TLR-Ls (CpG, PolyIC), IFNγ, and cross-linking anti-CD40, as indicated. The supernatants were collected after 15 h and analyzed for IL-12 p40 and p70 secretion by ELISA. Data are presented as mean ± SD, with the corresponding Spearman’s rank correlation (and its p-value) coefficient comparing all three types of DC. (B) MutuDC lines (n = 3) were stimulated for 20 h with different combinations of TLR-Ls (CpG or PolyIC), IFNγ, and cross-linking anti-CD40, as indicated, and mRNA expression was quantified by Nanostring technology. Genes shown in the heat map displayed a clear synergistic effect upon stimulation with multiple agents (green: low expression, to red: high expression). For each gene, a value for the synergistic increase is given (right side of the gene row) based on the fold increase in the stimulation with the full combination of reagents (CpG, PolyIC, IFNγ, and aCD40) versus the sum of stimulations with each reagent individually (Equation = value for “CpG, PolyIC, IFNγ, aCD40” divided by the sum of values for “CpG” + “PolyIC” + IFNγ + “aCD40,” with background subtraction for each value). (C) Impact of the lack of 1IFNR on mRNA expression of several genes upon stimulation. DC lines genetically deficient for the 1IFNR (n = 2 per treatment; derived from DC tumors in 1IFNR^−/− mice) were stimulated and analyzed as in, (B) compared to WT MutuDC lines. The gene expression data for eight representative genes upon stimulation in WT versus 1IFNR^−/− DC lines are shown in more detail in Figure A5 in Appendix.