Human tgf β

WT and Stat3^WT/GOF naïve CD4⁺ T cells were purified using the “Naïve CD4+ T Cell Isolation Kit” (Miltenyi Biotec, Cat. 130-104-453), cultured in supplemented RPMI (Th0, Th1, and Treg) or supplemented IMDM (Th17) media, and stimulated with plate-bound anti-CD3 (2.5ug/mL; BioXCell, 145-2C11) and soluble anti-CD28 (1ug/mL; SouthernBiotech, PV-1). T cell differentiation conditions: Th1, anti-IL-4 (10ug/mL; BioXCell, 11B11), murine IL-2 (50ng/mL; PeproTech, Cat. 212–12), and murine IL-12 (10ng/mL; R&D Sysemts, Cat. 419-ML-010); Treg, anti-IL-4, anti-IFNγ (10ug/mL; BioXCell, R4-6A2), murine IL-2, and human TGF-β (2.5ng/mL; PeproTech, Cat. 100–21); classical Th17, anti-IL-4, anti-IFNγ, human TGF-β, and murine IL-6 (30ng/mL; PeproTech, Cat. 216–16); pathogenic Th17, anti-IL-4, anti-IFNγ, human TGF-β, murine IL-1β (20ng/mL; Miltenyi Biotec, Cat. 130-094-053), and murine IL-23 (20ng/mL; R&D Systems, Cat. 1887-ML-010); Th0, anti-IL-4, anti-IFNγ, and IL-2. On day 5, cells were stimulated with phorbol-12-myristate-13-acetate (PMA; 50ng/mL; Sigma-Aldrich, Cat. 5.00582), ionomycin (1ug/mL; MilliporeSigma, Cat. 407950), and GolgiPlug Protein Transport Inhibitor (1:1000; BD Biosciences, Cat. 555029) and then stained for flow cytometry.

Naïve CD4 T cells were isolated from the spleens of mice using magnetic enrichment following direction of the supplier (Miltenyi Biotec, Auburn, CA, USA). These cells were cultured in complete RPMI 1640 media at 1×10⁶ cells per ml on tissue culture plates coated with 2μg/ml of anti-CD3 (145-2C11, BioXCell, Labanon, NH, USA) and soluble anti-CD28 (5μg/ml, 37.51, BioXCell) for Th0/unpolarized conditions. In addition to CD3/CD28 stimulation, Th2 cells were cultured with murine IL-4 (20ng/ml, Peprotech, Rocky Hill, NJ, USA) and anti-IFN-γ (XMG1.2, 10μg/ml, BioXCell); Th9 cells received IL-4 and anti-IFN-γ as per Th2 cells, but also were treated with 2ng/ml of human TGF-β (Miltenyi Biotec); Th17 cells were cultured with murine IL-6 (50ng/ml, Peprotech), human TGF-β (2ng/ml), anti-IFN-γ (10μg/ml) and anti-IL-4 (11B11, 10μg/ml, BioXCell); iTregs were cultured in the presence of human TGF-β ( 2ng/ml) and human IL-2 (50U/ml). After three days of initial polarization, cells were expanded in 3 volumes fresh media containing half concentrations of the cytokines listed above.
In some experiments, cells were also treated with various STAT3 agonist cytokines including IL-6, IL-10, IL-21. IL-23, IL-27 and GM-CSF (all from Peprotech), or with IL-2 blocking reagents (anti-IL-2: 10ug/ml, JES6-5H4, Biolegend, anti-CD25: 10ug/ml, PC61, Biolegend).