Anti human pdl1 antibody

Flow cytometry was performed as previously described.^{24 (link)} in brief, HN4 and HN30 cells were incubated with the indicated agent for 48 h. The cells were collected and incubated with anti-human PDL1 antibody at 1:100 (BD Biosciences, Franklin Lakes, NJ) for 30 min on ice. Then, the cells were resuspended in 100 μl fluorescence-activated cell sorting buffer and analysed on BD Fortessa flow cytometer. The final results were analysed with FlowJo software. Signal intensity was calculated as the ratio of the median fluorescence of the PDL1 antibody to that of the isotype control antibody (SFI: specific fluorescence index). CD4-FITC antibody, CD8-PerCP-Cy5.5 antibody, CD56-APC antibody, and PD1-PE antibody (all purchased from BD Biosciences) were applied to detect the PD1 expression on the surface of immune cells from peripheral blood of HNSCC patients and healthy controls. The IFNAR1 antibody (Abcam, Cambridge, MA, UK) and PE-conjugated secondary antibody (Proteintech, Rosemont, IL, USA) were used to analyse the surface IFNAR1 expression on immune cells.

To examine the effect of tumor cells on lymphocyte apoptosis, 5×10⁶ MDA-MB-231 SCR cells and KD cells were cocultured with 5×10⁵ Jurkat leukemia T cells in the presence of mouse IgG (2 or 5 μg/ml; 17314; IBL Co., Ltd., Gunma, Japan), anti-human PD-L1 antibody (5 μg/ml; MIH1; BD) or anti-PD-1 antibody (2 μg/ml; EH12.2H7; BioLegend) in a 6-well plate for 24 hours. To block the PD-1/PD-L1 pathway, SCR cells were preincubated with anti-human PD-L1 antibody (5 μg/ml; BD) for 30 min or Jurkat cells were preincubated with anti-PD-1 antibody (2 μg/ml; BioLegend) for 30 min. Extent of apoptosis in Jurkat cells was determined by flow cytometry using FITC-Annexin V (11858777001; Roche) according to the manufacturer's instructions.