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.