Human tgf β

Teff cells (3 × 10⁵ cells/well) were cultured in complete RPMI-1640 medium and stimulated with Dynabeads^® Mouse T-Activator CD3/CD28 (8 μl/well) for 72 h along with cytokines and neutralizing antibodies for the desired polarization as follows: human-IL-6 (100 ng/ml; PeproTech. INC.), mouse-IL-23 (10 ng/ml; Biolegend), human-TGF-β (5 ng/ml), anti-IL4 (10 μg/ml; Biolegend) and anti-IFN-γ (10 μg/ml; Biolegend) for Th17; mouse IL-2 (10 ng/ml), human-TGF-β (2 ng/ml), anti-IL4 (10 μg/ml) and anti-IFN-γ (10 μg/ml) for inducible Treg (iTreg) cell polarization. Cells were restimulated with PMA (50 ng/ml), ionomycin (1 μg/ml) and brefeldin A (1 μg/ml) for 6 h. Cells were harvested and stained with anti-IL-17 or anti-Foxp3 antibodies and analyzed by flow cytometry.

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).

EW-7197 was purchased from Selleck Chemicals. Roscovitine (Sigma-Aldrich) was solubilized in DMSO and stored in 10 mmol/L aliquots at −20°C. Human TGFβ was ordered from BioLegend. The following Western blot antibodies were purchased from Cell Signaling Technology: p44/42 MAPK (MAPK3/1, or ERK1/2) #9102; phospho-p44/42 MAPK (ERK1/2; Thr202/Tyr204) #9101; CDK5 (1H3) #12134; p35/p25 (C64B10) #2680; p39 #3275; phospho-SMAD2 (Ser465/467; 138D4) #3108; phospho-SMAD3 (Ser423/425; C25A9) #9520. The following Western blot antibodies were purchased from Santa Cruz Biotechnology: GAPDH (0411) #sc-47724 and β-ACTIN (ACTB) (C4) #sc-47778. All flow cytometry antibodies were purchased from BioLegend: CD11b (ITGAM)-FITC (M1/70); CD27-PE (LG.3A10); NK1.1 (KLRB1C)-APC (PK136); CD3 (CD3ε)-PerCP (145-2C11); NKG2D (KLRK1)-PE (CX5); 2B4 (CD244a)-FITC [m2B4 (B6)458.1]; LY6C-PE (HK1.4); LY49C/F/I/H (KLRA3/KLRA6/KLRA9/KLRA8)-PE (14B11); NKp46 (NCR1)-PE (29A1.4); NKG2A (KLRC1)-PE (16A11).