Roles of Spi-B in in vitro pDCs responses to TLR7/9 stimuli. (A) MACS-purified BM pDCs were stimulated with 8-mercaptoguanosine (8-MG, a TLR7 agonist), polyuridylic acid (polyU, a TLR7 agonist), VSV (a TLR7 agonist), CpG DNA (a TLR9 agonist), or R848 (a TLR7 agonist). 8-MG was used at 1mM. PolyU was added at 3μg/mL as a complex with lipofectamine 2000. As CpG DNA, D19 was used at 3μM for measurement of IFN-α and IFN-β and ODN1668 was used at 1μM for measurement of IL-12p40 and TNF-α. R848 was used at 100nM. VSV was used at a multiplicity of infection of 0.5. After 20-24 hours, cytokine production was measured by ELISA. Data are representative of 3 independent experiments (means ± SD). **P < .01. (B) CD86 expression in TLR9-stimulated pDCs. MACS-purified splenic or BM pDCs were stimulated with (shaded histograms) or without (open histograms) 1μM ODN1668 for 12 hours. Surface expression of CD86 in CD19⁻CD11c⁺BST2⁺ cells was analyzed with a FACSCalibur. Data are representative of 2 independent experiments. (C) Gene induction in TLR7/9-stimulated pDCs and cDCs. FACS-sorted BM or splenic pDCs were stimulated with 3μM D19 or 3 μg/mL of polyU. FACS-sorted splenic cDCs were stimulated with 1μM ODN1668 or 100nM R848. Cells were harvested at the indicated time points and subjected to quantitative real-time PCR. Data are representative of 2 independent experiments (means ± SD). (D) 293T cells were transiently transfected with an Ifna4, Ifnb1, Il12b, or Tnf promoter-driven luciferase reporter plasmid alone or together with a combination of expression vectors for Spi-B (2.1 or 8.4 ng/well), NF-κB p65 (8.4 ng/well), and IRF-7 (8.4 ng/well). After 18 hours, cell lysates were prepared and subjected to the luciferase assay. Data are representative of 2 independent experiments (means ± SD).