Anti il 4 antibody

Naïve CD4+ T cells were cultivated in a 96-well plate (1 × 10⁵ cells/well) containing plate-bound anti-CD3 antibody (2.5 µg/ml) and anti-CD28 antibody (5 µg/ml) (Tonbo Biosciences). To induce Treg cell differentiation, TGF-β1 (3 ng/ml) and IL-2 (2 ng/ml) were added. To induce Th17 cell differentiation, recombinant human TGF-β1 (3 ng/ml), IL-23 (25 ng/ml), anti-IL-4 antibody (5 µg/ml), and IFN-γ antibody (5 µg/ml) (PeproTech) were added. Naïve CD4+ T cells were treated with either butyrate alone (0.2 mM) or both butyrate (0.2 mM) and GW9662 (10 µM) for 5 days. Next, the cells were used for flow cytometry analysis, western blotting, glycolytic rate analysis and a mitochondrial stress assay.

CD4+ T cells were isolated from spleens harvested from female DBA1/1 mice using adverse magnetic bead-based selection and a mouse naïve CD4+ T-cell isolation kit (Miltenyi Biotec, CA, USA; Cat # 130-104-453) according to the manufacturer’s procedure. Cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (HyClone, Carlsbad, CA, USA), 100 U/ml penicillin, and 100 g streptomycin (all from Invitrogen-Gibco).
To stimulate Th17 cell differentiation, 1 µg/ml plate-bound anti-CD3, 1 µg/ml soluble anti-CD28, 50 ng/ml IL-6 (PeproTech), 10 ng/ml TGF-1 (PeproTech), 2 g/ml anti-IFN-γ antibody (clone R4-6A2, eBioscience), and 2 µg/ml anti-IL-4 antibody were added to cells to promote differentiation. IL-6/IL-23/TGF-β was added to each culture well, followed by erianin treatment (1 and 5 nM), incubation for 72 h, and determination of cytokine levels in the culture supernatant using ELISA.

CD4⁺ T cells were purified from spleens of naïve DBA/1 mice by positive selection using microbeads against CD4 (Miltenyi Biotec) following the protocol provided by the manufacturer. Then, the purified naïve CD4⁺ T cells were seeded at 2 × 10⁶ per well into 96-well U-bottom microplates with RPMI 1640 medium (Hyclone) supplemented with 10% fetal bovine serum (Hyclone, Carlsbad, CA), 100 units/ml penicillin, and 100 μg of streptomycin (all from Invitrogen-Gibco). Th1 differentiation was driven by the of naïve CD4⁺ T cells with 1 μg/ml plate-bound anti-CD3 (clone 2C11, eBioscience), 1 μg/ml soluble anti-CD28 (clone PV1.17, eBioscience), 10 ng/ml IL-12 (PeproTech), 5 ng/ml IL-2 (PeproTech), and 2 μg/ml anti-IL-4 antibody (clone 11B11, eBioscience). Th17 differentiation was driven by the stimulation of naïve CD4⁺ T cells with 1 μg/ml plate-bound anti-CD3, 1 μg/ml soluble anti-CD28, 50 ng/ml IL-6 (PeproTech), 10 ng/ml TGF-β1 (PeproTech), 2 μg/ml anti-IFN-γ antibody (clone R4-6A2, eBioscience) and 2 μg/ml anti-IL-4 antibody. DXM was used at the dose indicated at the beginning of the Induction. Cells were harvested on day 4 of DXM treatment and analyzed for intracellular cytokines while culture supernatants were examined for cytokine levels by ELISA assay.

PBMCs were isolated from peripheral blood using Ficoll density-gradient centrifugation. Naïve CD4⁺T cells were purified from PBMCs according to the manufacturer’s instruction (Miltenyi Biotec, Bergisch Gladbach, Germany). These purified naïve CD4⁺T cells (1 × 10⁶/well) were differentiated into Tfh cells under Tfh cell-polarizing condition (3 μg/ml soluble anti-CD3/28 (eBioscience, San Diego, CA, USA), 50 ng/ml recombinant IL-6 (rIL-6, PeproTech Inc, Rocky Hill, NJ, USA), 50 ng/ml rIL-21 (Abcam, Cambridge, MA, USA), 10 μg/ml anti-IL-4 antibody (eBioscience), 10 μg/ml anti-IFNγ antibody (eBioscience) and 10 μg/ml anti-TGF-β antibody (R&D, Minneapolis, MN, USA)) for 3 days. After initial culture, these differentiating Tfh cells were washed with PBS for 2 times and further expanded alone or cocultured with UC-MSCs (1 × 10⁵/well) in the presence of 3 μg/ml soluble anti-CD3/28 for another 2 days. To detect the factors involved in UC-MSCs-mediated suppression, UC-MSCs were collected after 2 days’ coculture with differentiating Tfh cells and then were fixed by Trizol. Furthermore, 100 μM 1-methyl-DL-tryptophan (1-MT, Sigma), the inhibitor of IDO enzyme activity or 10 μg/ml anti-IL-10 antibody (eBioscience) or 10 μg/ml anti-HLA-G antibody (Biolegend, San Diego, CA, USA) was added to the MSCs-Tfh cells coculture system to block their effects on Tfh cells.