Nivolumab Blockade of PD1 Expression on T-cells

PD1 expression on T-cells was blocked by incubating with 20 µg/mL Nivolumab (anti-PD1, MedChemExpress, NJ, USA) for 30 min at 37 °C. After incubation, cells were washed twice with PBSx1 supplemented with 0.5% BSA and 0.1% sodium azide, then washed once with PBSx1 before being centrifuged at 1500 rpm for 5 min. Next, cells were incubated with Fc Block (Milteny Biotech, Bergisch Gladbach, Alemania) for 30 min at 4 °C and then washed with PBSx1 supplemented with 0.5% BSA and 0.1% sodium azide before being centrifuged at 1500 rpm for 5 min. The cells were then subjected to surface staining using anti-Nivolumab PE (Anti-Human IgG4 pFc, Abcam, Cambridge, United Kingdom), anti-PD1 PE-Cy7 (Clone: EH12.1, BD Pharmingen, San Jose, CA, USA), and CD3 PERCP (Clone: HIT3a, BD Pharmingen) [27 (link)]. The corresponding isotypes and tube with cells without previous Nivolumab incubation were used as controls. PD1 expression and Nivolumab binding through human IgG4 were evaluated by flow cytometry using FACSCanto II (BD Biosciences) and analyzed using FlowJo (Tree Star, Ashland, OR, USA).