Anti cd3 mab

Peripheral blood cells were collected and isolated with Ficoll-Paque^TM (GE healthcare, Uppsala, Sweden) as described previously.19 (link) Briefly, peripheral blood (3 mL) diluted with sterile PBS (3 mL) was added to equal volume Ficoll. The tubes were vortexed for 30 minutes under 2,000 rpm at 20°C. PBMCs were collected and incubated with CD4⁺ microbeads for 30 minutes at 4°C. CD4⁺ T-cell clones were expanded after being stimulated with plate-bound anti-CD3 mAb (5 mg/mL), and anti-CD28 mAb (2 mg/mL, eBioscience, San Diego, CA), followed by being stimulated with recombined human (rh) TGFβ (20 ng/mL), rh IL-6 (30 ng/mL), anti-IFNγ (20 µg/mL), and anti-IL-4 (20 µg/mL) with PBS or TWP (1.25 mg/mL) for 72 hours. Differentiated CD4⁺ T-cells were collected for qRT-PCR. CD4⁺ T-cells were stimulated with mentioned cocktails for 5 days followed by being stimulated with PMA (50 ng/mL, Sigma-Aldrich, St Louis, MO, USA), ionomycin (750 ng/mL, Sigma-Aldrich), and Brefeldin A (3  μg/mL, eBioscience) for 5 hours. Differentiated CD4⁺ T-cells were collected for cell membrane and intracellular staining, eg, CD25, IFNγ, IL-17A, and FOXP3 for flow cytometer analysis.

Liver naïve CD4+ T cells (CD4⁺ CD25⁻ CD44⁻ CD62L⁺) were sorted from the infected WT mice as previously described.31 (link) Naïve CD4+ T cells were then cultured (3×10⁵ cells/well) in 96-well plates coated with anti-CD3 mAb (5 μg/mL, eBioscience) and soluble anti-CD28 mAb (3 μg/mL, eBioscience) and kept in the presence of IL-2 (100 U/mL, eBioscience). Exogenous rmIL-33 (10 ng/mL) was added to investigate its ability to polarize Treg and PBS used as the control. Cells were cultured in RPMI-1640 Medium, 10% fetal calf serum, 2 mM L-glutamine, 100 U/mL of Penicillin/Streptomycin for 5 days.