Mar1 5a3

For the differentiation of T_H1 cells35 (link), CD45.1⁺OT-II CD4⁺ T cells (2×10⁵) were cultured with pDCs (2×10⁴) in the presence or absence of IMQ (5 μg/ml) in combination with OVA_323-339 peptide (1 μM), anti-IL-4 mAb (10 μg/ml; 11B11, BD Biosciences) and recombinant mouse IL-2 (0.2 ng/ml; Wako Pure Chemicals) for 3 days in 96-well flat-bottomed plates. For the differentiation of T_H17 cells35 (link), CD45.1⁺OT-II CD4⁺ T cells (2×10⁵) were cultured with pDCs (2×10⁴) in the presence or absence of IMQ (5 μg/ml) in combination with OVA_323–339 peptide (1 μM), anti-IFN-γ mAb (10 μg/ml; R4-6A2, BD Biosciences), anti-IL-4 mAb (10 μg/ml; 11B11), recombinant mouse IL-2 (0.2 ng/ml) and recombinant human transforming growth factor (TGF)-β1 (10 ng/ml; Wako Pure Chemicals) for 3 days in 96-well flat-bottomed plates. In some experiments, anti-IL-6 mAb (10 μg/ml; MP5-20F3, eBioscience), anti-IL-12 mAb (10 μg/ml; C17.8, eBioscience) or anti-IFNAR1 mAb (10 μg/ml; MAR1-5A3, eBioscience) was added to the culture. Analysis of the expression of IFN-γβ and/or IL-17 among gated CD4⁺ T cells was performed by flow cytometry as described above.

All knockdown experiments were performed using the Lipofectamine RNAiMAX transfection reagent (Invitrogen) with siRNA delivered at a concentration of either 20 or 50 nM in serum-depleted medium (OptiMEM; Invitrogen) as per the manufacturer’s instructions. Reverse transfections were employed with cells added to preformed siRNA:lipofectamine complexes pre-incubated for 20 minutes. Subsequent cell stimulations or viral infections were performed 48 hours after initial siRNA knockdown. Reporter constructs were transfected using the Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s instructions.
MEFs were stimulated by transfection of 2 µg/ml poly(I:C) into the cells using Lipofectamine 2000 transfection reagent (Invitrogen) following the manufacturer’s instructions. Alternatively, MEFs were stimulated with 500 U/ml recombinant IFN-β (R&D Systems) for the indicated times. To block IFN-β-induced signaling, MEFs were pre-incubated with 10 µg/ml IFNαR1 antibody (MAR1-5A3, eBioscience, catalogue number 16-5945).