Anti pd l1 mab

Surface staining was performed using the following anti-mouse monoclonal antibodies (mAbs) or antibody controls: PE-conjugated IgG isotype and anti-MICA/B, anti-MICA, anti-MICB, anti-ULBP1, anti-ULBP2, anti-ULBP3, anti-HLA-ABC mAb (R&D Systems, Minneapolis, MN, USA), anti-PD-L1 mAb (eBioscience, San Diego, CA, USA), Percp-cy5.5-conjugated IgG isotype and anti-CD95 mAb . Cells were collected with

Freshly isolated naïve CD4⁺ T cells were seeded at a density of 5×10⁴ cells per well in 96-well plates and then cultured in complete RPMI 1640 medium (Gibco, Brazil). The purity of sorted cells was generally 90%, as shown in Supplemental Figure S1. All the cells were activated by a human CD3/CD28 activator (5 μg/ml) for 3 days with recombinant human interleukin 2 (IL-2) (0.5 U/ml) (STEMCELL, BC, Canada). After 3 days of activation, the cells were incubated with fresh complete RPMI 1640 medium for 2 days. For Treg differentiation, the cells were incubated with TCM in the presence or absence of anti-PD-L1 mAb (eBioscience, CA, USA) (10 ng/ml) for 2 days. At day 7 of the study, cells were collected and assessed for Treg by flow cytometry (BD Biosciences).