2.1 Mice and cells
C57BL/6 and Balb/c mice were purchased from Japan SLC (Hamamatsu, Japan), and OT-II mice were obtained from The Jackson Laboratory (USA).Nrf2 -/- mice were previously generated7. Mice were maintained under specific pathogen-free conditions. All animal experiments were performed in accordance with the guidelines of the Institutional Review Board of Tokyo University of Science. The current study was specifically approved by the Animal Care and Use Committees of Tokyo University of Science: K21004, K20005, K19006, K18006, K17009, and K17012. Bone marrow-derived DCs (BMDCs) were generated from whole BM cells by cultivation in RPMI-1640-based media supplemented with 20 ng/mL mGM-CSF (BioLegend, San Diego, CA, USA) as previously described 8. Naïve CD4+ T cells were isolated from the spleen by using the MojoSort Mouse Naïve CD4+ T Cell Isolation Kit (#480040, BioLegend). CD4+ T cells isolated from the OT-II spleen were cocultured with BMDCs, which were generated from C57BL/6 mice and were preincubated with OVA peptide 323-339 (POV-3636-PI, Peptide Institute, Inc., Osaka, Japan). To stimulate CD4+ T cells with plate-coated antibodies (Abs), anti-CD3ε Ab (clone 145-2111C, BioLegend) and anti-CD28 Ab (clone 37.51, TONBO Bioscience) were used. For Th1 polarization, 10 ng/mL mIL-12 (PeproTech Inc., Rocky Hill, NJ, USA) and 10 µg/mL anti-IL-4 Ab (clone 11B11, BioLegend) were added to the culture media. Th2 polarization was induced with 20 ng/mL IL-4 (PeproTech) and 10 µg/mL anti-IL-12 Ab (clone C17.8, BioLegend).
Damasone-β (#34059, Vigon International, East Stroudsburg, PA, USA) was diluted with DMSO. LPS (#L3024, Wako), poly-I:C (#P0913, Sigma), R848 (AG-CR1-3582-M005, AdipoGen, Liestal, Switzerland), and CpG (ODN1826, InvivoGen, San Diego, CA, USA) were used to stimulate BMDCs. DAPI (#11034-56, Nacalai Tesque Inc., Kyoto, Japan) was used to determine cell viability.