Il 23

Murine naïve CD4⁺T cells were purified from the spleen of male C57BL/6 mice by magnetic cell negative selection using a mouse naïve CD4⁺T cell isolation kit (Miltenyi Biotec, 130-104-453), according to manufacturer’s instructions. Cells were stimulated in vitro with plate-bound anti-CD3 (BD Pharmingen, 553057) and anti-CD28 antibodies (BD Pharmingen, 553294) (both for 2 μg/mL) in RPMI 1640, supplemented with 10% heat-inactivated fetal bovine serum and 100 U/mL penicillin/streptomycin. To induce the differentiation of Th17 cells, cells were activated in the presence of 25 ng/mL IL-6 (Novoprotein, CG39), 20 ng/mL IL-23 (Novoprotein, CI18), 2.5 ng/mL TGF-β (Novoprotein, CA59). PNS, Tepp-46 (Apexbio, C4181), and SAICAR (Apexbio, C3413) were dissolved at a concentration of 10 mM in dimethyl sulfoxide (DMSO) and stored at −20 °C until use. For PNS treatment, IL-6/IL-23/TGF-β were added to each well, followed by treatment with PNS (5, 10, 20 μg/mL) and incubation for 72 h. For Tepp-46 and SAICAR treatment, cells were pre-incubated at 37 °C for 24 h with Tepp-46 (50, 100, 150 μM) or SAICAR (2, 4, 8 μM) before IL-6/IL-23/TGF-β activation. For co-treatment, cells were pretreated with SAICAR (4 μM) or Tepp-46 (100 μM) for 24 h before the condition of Th17-polarization), and treated with PNS (10 μg/mL) for another 72 h.

Isolation of naive CD4⁺ T cells from the mice spleen and differentiation into Th17/Treg cells in vitro were conducted according to our previously reported method with minor modifications (23 (link)). CD4⁺ T cells from splenic lymphocyte were magnetically enriched by a CD4⁺ T Cell Isolation Kit (Miltenyi, 130-104-454, Germany) with Columns (LS Column, Miltenyi, 130-042-401). Sorted naive CD4⁺ T cells were cultured for 5 days in 24-well plates in 1640 medium (including 10% fetal bovine serum and 1% penicillin–streptomycin solution) before supplemented with 10 μg/ml anti-CD3 (Bio X Cell, BE0001-1, USA) and 10 μg/ml anti-CD28 (Bio X Cell, BE0015-1). Th17 stimulations were complemented with 40 ng/ml IL-6 (Novoprotein, CG39), 1 ng/ml TGF-β1 (Novoprotein, CA59), 40 ng/ml IL-23 (Novoprotein, CS31), 20 μg/ml anti-IL-4 (Bio X Cell, BE0045), and 20 μg/ml anti-IFN-γ (Bio X Cell, BE0055). Treg stimulations were supplemented with 5 ng/ml TGF-β1 (Novoprotein, CA59), 20 ng/ml IL-2 (Novoprotein, CK24), 10 μg/ml anti-IL-4 (Bio X Cell, BE0045), and 10 μg/ml anti-IFNγ (Bio X Cell, BE0055).