Combined stimulation of TLRs enhances production of IL-6 and RANTES and upregulation of DC activation markers. BMDCs from C57BL/6 mice were generated with GM-CSF for 6 days. On day 6, the immature DCs were activated with single-TLR ligands or various combinations thereof: Poly (I:C) (IC) 50 μg/mL, R-848 1 μg/mL, LPS 100 ng/mL, CpG ODN1668 (CpG) 1 μM, Pam₃CysSKKK (P3C) 1 μg/mL. After 18 hours of stimulation, culture supernatants were collected for ELISA or Luminex analysis. (A) IL-6 (▪) and RANTES (□). (B) Two days after TLR ligand induced maturation expression of activation markers, CD40 (▪), CD70 (##), Ox40 ligand (##), and CD86 (□) were analyzed by FACS. The depicted results show mean and standard deviation from 4 independent experiments where mean fluorescence intensity as measure of surface expression of the respective activation marker was analyzed on viable CD11c⁺ cells. (C-D) Supernatants from BMDCs stimulated for the indicated period of time (4 to 72 hours) with medium (▪), I:C (##), R-848 (##), or I:C + R-848 (□) in combination were analyzed for IL-6 (C) and RANTES (D). (E-F) BMDCs were stimulated as described in “Generation and activation of mouse bone marrow–derived dendritic cells.” After 20 hours, supernatants were collected and the TLR ligand–containing medium was exchanged for medium without stimulus. Supernatants were collected again 20 hours later (after stimulus removal) and analyzed for the production of IL-6 (E) and RANTES (F) by ELISA or Luminex, respectively. All data are presented as mean and standard deviation of triplicate wells from 1 representative of at least 3 independent experiments for IL-6 and RANTES (A,C-F). *P < .001 by ANOVA among the respective single- and combined TLR ligand–stimulated cells.