Tgf β

Spleen cells were excised from mice and single-cell suspensions of splenocytes were prepared in RPMI 1640 by teasing the organ through a sterile nylon mesh. Naive CD4⁺ T cells were purified using an EasySep mouse naive CD4⁺ T cell isolation kit (Stemcell Technologies, Vancouver, BC, Canada) and cultured with plate-bound anti-CD3 (5 µg/ml; Bio X Cell), anti-CD28 (5 µg/ml; Bio X Cell), and cytokines used alone, including IL-12 (R&D Systems), IFN-γ (R&D Systems), IL-1 (R&D Systems), IL-4 (R&D Systems), IL-6 (Cell Signaling Technology), IL-23 (eBioscience), and TGF-β (Invitrogen), or in combination with Abs (Bio X Cell) for Th17-promoting (2.5 ng/ml TGF-β, 20 ng/ml IL-6, 10 µg/ml anti–IFN-γ, 10 µg/ml anti–IL-4, 10 µg/ml anti–IL-2), Th1-promoting (10 ng/ml IL-12, 5 µg/ml anti–IL-4), Th2-promoting (10 ng/ml IL-4, 5 µg/ml anti–IFN-γ), or regulatory T cell (Treg)–promoting (5 ng/ml TGF-β, 5 µg/ml anti–IFN-γ, 5 µg/ml anti–IL-4) conditions. In some experiments, naive CD4⁺ T cells were cultured in Th17-promoting conditions and IL-21 (20 ng/ml; eBioscience) or IL-23 (50 ng/ml) was added to cultures after 24 h. Cells were harvested after 48 h for real-time PCR analysis and after 72 h for cytokine expression by flow cytometry as previously described (23 (link)).

The ST2 stromal cell line was cultured in α-minimum essential medium (α-MEM; Sigma-Aldrich, St. Louis MO) with L-glutamine, antibiotics, and 10% fetal bovine serum. 5×10⁴ cells were seeded into 6 wells plates prior to cytokine stimulation. Recombinant IL-17F.S65L and IL-17F were synthesized by Bon Opus Biosciences (Millburn, NJ) by expression in Expi293 cells (ThermoFisher). Mouse TNFα was from Peprotech and used at 2 ng/ml (Rocky Hill, NJ).
Naïve splenic CD4⁺ T cells were purified by magnetic separation (Miltenyi Biotec). T cells were activated with α-CD28 (5 ug/ml; BioXCell) and plate-bound α-CD3 (clone 145-TC11, 5 ug/ml; BioXCell) in RPMI supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 50 mM 2-β-mercaptoethanol, and sodium pyruvate) for 4 d with IL-1β (50 ng/ml), IL-6 (50 ng/ml), IL-23 (50 ng/ml), and TGFβ (5 ng/ml). Cytokines were from R&D Systems.