Be0015 5

T cells were isolated from spleen and lymph nodes of naïve C57BL6/J mice as previously described (6 (link)). Naïve CD4⁺ T cells were purified using the Naive CD4+ T Cell Isolation Kit (Miltenyi, #130-104-453) according to the manufacturer’s instruction. Naive cells were cultured at a concentration of 1.0–1.5 × 106/ml. Cells were stimulated in the presence of plate bound anti-CD3 Ab (1–3 μg/ml, BioXCell #BE0002) and anti-CD28 Ab (1–2 μg/ml, BioXCell #BE0015-5). For the generation of T_H17 cells, naive T cells were cultured with IL-6 (30 ng/ml, R&D #406-ML-005), TGF-β (3 ng/ml), anti-IFNγ (10 µg/ml, BioXCell #BE0055-1MG), and anti-IL-4 (10 µg/ml, BioXCell #BE0045-1MG). After 48h, T_H17 cells were supplemented with 10 ng/ml IL-23. For T_H1 cells, naïve CD4⁺ T cells were culture in the presence of IL-12 (10 ng/ml, R&D #419-ML-010) and anti-IL-4 (10 µg/ml). For T_Reg, cells were cultured in the presence of TGF-β (5 ng/ml), anti-IFNγ (10 µg/ml), and anti-IL-4 (10 µg/ml). After 3 days, recombinant mouse PTN or vehicle was added. After 4 days, cytokine was measured by intracellular cytokine staining (using the Foxp3/Transcription Factor Staining Buffer Set, eBiosciences #00-5523-00) and subsequent flow cytometry.