Recombinant mouse il 2

For the differentiation of T_H1 cells44 (link), CD45.1⁺OT-II CD4⁺ T cells (2 × 10⁵) were cultured with CD8α⁻ cDCs (2 × 10⁴) in the presence or absence of Pam3CSK4 (1.25–1 μg ml⁻¹), poly(I:C) (12.5–50 μg ml⁻¹), LPS (0.25–1 μg ml⁻¹) or CpG-B (0.025–0.1 μM) in combination with OVA_323–339 peptide (1 μM), anti-IL-4 mAb (10 μg ml⁻¹; 11B11, BD Biosciences) and recombinant mouse IL-2 (0.2 ng ml⁻¹; Wako Pure Chemicals) for 3 days in 96-well flat-bottomed plates. For the differentiation of T_H17 cells44 (link), CD45.1⁺OT-II CD4⁺ T cells (2 × 10⁵) were cultured with CD8α⁻ cDCs (2 × 10⁴) in the presence or absence of TLR ligands as described above in combination with OVA_323–339 peptide (1 μM), anti-IFN-γ mAb (10 μg ml⁻¹; R4-6A2, BD Biosciences), anti-IL-4 mAb (10 μg ml⁻¹; 11B11), recombinant mouse IL-2 (0.2 ng ml⁻¹) and recombinant human transforming growth factor-β1 (10 ng ml⁻¹; Wako Pure Chemicals) for 3 days in 96-well flat-bottomed plates. Analysis of the expression of IFN-γ or IL-17 among gated CD4⁺ T cells was performed by flow cytometry as described above.

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.