TLR8 agonists efficiently induce CD40 expression on neonatal and adult mDCs. Whole blood was stimulated with TLR agonists for 19 hours prior to measurement of CD40 expression on mDCs (lin1^–/HLA-DR⁺/CD11c⁺ cells) by flow cytometry using a PE-conjugated anti-CD40 mAb as described in “Materials and methods.” Representative examples are provided of imiquimod (TLR7)–induced (A) and of 3M-002 (TLR8)–induced (B) CD40 expression on neonatal mDCs with isotype control (white), buffer control (gray), and TLR-stimulated (black) conditions indicated. The “R14” gate defines CD40⁺ events in the examples provided, 1.2% for the TLR7 (A) and 41% for the TLR8 agonist (B). (C) Composite analysis of such data demonstrating the percentage of mDCs positive for CD40 in relation to the indicated concentrations of TLR agonists. Data represent means ± SEM. Statistical comparison is made in relation to the TLR7 agonist imiquimod at 500 μM (open arrow, newborns; filled arrow, adults). n = 3-4, *P < .05. Table S2 presents comparisons of CD40 expression induced by TLR8 and TLR7/8 agonists with that induced by each TLR7 agonist at every dose.