IL-12 production and polarization of Th1 cells, synergistically induced by combined TLR agonist combination, are inhibited by exogenous CCL2. (A) MD-DCs were seeded and cultured, as described in “Cell separation and culture.” Cells were pretreated for 30 minutes with exogenous CCL2 (50 ng/mL) or left untreated before the addition of LPS (100 ng/mL), R848 (2 μg/mL), or their combination. After 18 hours, supernatants were harvested and frozen for IL-12p70 determination. Data are expressed as mean ± SD of duplicate wells and are representative of 6 independent experiments. The P values were calculated by Student t test, and statistical significance is indicated comparing results from TLR ligand-stimulated cells in the presence versus in the absence of exogenous CCL2. (B-D) MD-DCs were stimulated for 18 hours with LPS, R848, or LPS plus R848 with or without exogenous CCL2 (50 ng/mL), and then were washed and cultured with allogeneic naive CD4⁺ T cells. (B) Intracellular cytokine staining for IFN-γ and IL-4 in naive CD4⁺ T cells. After 5 days, cells were incubated for additional 7 days in the presence of IL-2 (10 IU/mL), and then proliferating T cells were tested for their capacity to produce IFN-γ and IL-4 after stimulation with PMA and ionomycin. One representative experiment of 3 is shown. (C) ELISA determination of IFN-γ and (D) IL-10 in culture supernatants of cell populations primed and expanded, as described in (B). Data in (C-D) are expressed as mean ± SD of culture duplicates and are representative of 4 independent experiments. The P values were calculated by ANOVA, and statistical significance is indicated comparing results from TLR ligand-stimulated cells in the presence versus in the absence of exogenous CCL2.