Mouse bone marrow-derived DCs were generated as previously described [10 (link)]. Briefly, bone marrow precursors were cultured in complete IMDM (10% FBS, antibiotics and 2-mercaptoethanol) containing 3.3 ng/ml GM-CSF (BD Biosciences, San Jose, USA) or 1% conditioned media from GM-CSF-secreting B7H1 cells. At day 6–8, cDCs were stimulated with LPS (100 ng/ml), R848 (1 μg/ml), or CpG 1826 (10 μg/ml) and harvested after 1.5 hours for Western Blot and 6 hours for RT-PCR, while supernatants and cells were collected after 24 hours for ELISA and Flow analysis. Anti-IFNAR (10 μg/ml, clone MAR1–5A3, Leinco Technologies I-400 or Gentex GTX14637) or isotype control antibody (10 μg/ml, IgG1, Leinco Technologies I-102 or Gentex GTX35014) were added 24 hours and again 30 minutes before or 1 hour before TLR stimulation. ERK inhibitor (10 μM, PD98059, Cell Signaling #9900) was used 30 minutes before TLR stimulation. 1μg/ml anti-IL-27 (Polyclonal goat IgG, R&D AF1834) or 1μg/ml IgG control antibodies (R&D AB-108-C) were added one hour prior to TLR stimulation.