Generation of Allergen-Reactive Th17 Cells

Allergen-reactive Th17 cells were developed as previously described [4 (link),8 (link)]. In brief, ovalbumin (OVA)-reactive CD4⁺ T cells were prepared from spleen cells of BALB/c background transgenic mice, DO11.10/RAG2^−/−, by magnetic cell sorting with an EasySep Mouse CD4⁺ T Cell Isolation Kit (Veritas, Santa Clara, CA, USA). The cells were co-cultured in the presence of X-ray-irradiated spleen cells in AIM-V medium (Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal calf serum. At the beginning of culture, we added 0.3 μM OVA323-339 synthetic peptide (Scrum Inc., Tokyo, Japan), 10 ng/mL human IL-1β (PeproTech, Rocky Hill, NJ, USA), 20 U/mL IL-2 (PeproTech), 20 ng/mL IL-6 (PeproTech), 10 ng/mL IL-23 (R & D Systems, Minneapolis, MN, USA), 1 ng/mL human TGF-β (BioLegend, San Diego, CA, USA), 10 ng/mL TNF-α (PeproTech), 10 μg/mL anti-IL-4 (Abcam, Cambridge, UK), and 10 μg/mL anti-IFN-γ (R4-6A2, eBioscience, San Diego, CA, USA). Cells were collected following seven-day culture and used for the transfer. Successful polarization of Th17 cells has been reported elsewhere [4 (link)], and was confirmed by comparing Il4 and Il17a-expressing activity with that of Th2 cells (Supplementary Figure S1).