Th9 differentiation assay

PBMCs obtained as mentioned above from 10 healthy donors were cultured in round-bottom 96-well plates and stimulated with anti-CD3 antibody (0.5 μg/mL, BioLegend) or anti-CD3/CD28 beads (4 μL/well, Thermo Fisher Scientific). Th9 cell induction with or without anti-PD-1 antibody (2 μg/mL, 10 μg/mL, or 50 μg/mL, J105, eBioscience) or anti-PD-L1 antibody (10 μg/mL, B7H1, BioLegend) was assessed by flow cytometry after 72 h of culture with recombinant IL-4 (10 ng/mL, BioLegend), recombinant TGF-β (10 ng/mL, BioLegend), both, or neither as control.