Effect of TLR inhibitors and ligands and addition of RNase. (A, B) pDCs were stimulated with HL5B either alone or together with IRS661, IRS869 (each 0.5μM), or R848 (1 μg/mL) as indicated. Relative expression of IFNα and TNFα mRNA normalized to β-actin. All values represent mean ± SD of 5 independent experiments. *P < .05; **P < .005 for HL5B versus HL5B plus inhibitor or activator, respectively. (C) Human pDCs were stimulated with HL5B (100 ng/mL) or a combination of HL5B and R848 (1 μg/mL) either alone or in the presence of RNase (10 μg/mL). RNAse was added 30 minutes before the other stimuli. Quantitative RT-PCR was performed after different periods of time, for up to 6 hours. Relative expression of TLR7 (left) or IFNα (right) was normalized to β-actin. Values represent mean ± SD of at least 5 independent experiments. *P < .01, **P < .005 for HL5B versus HL5B plus RNase.