Fully human igg4 s228p anti pd 1 receptor blocking monoclonal antibody

Proliferation was analyzed by flow cytometry of CFSE labeled cells as reported previously [22] . Briefly, PBMCs were isolated using Ficoll-Plus gradient (GE Healthcare Bio-Sciences) [16] . The PBMCs were then labeled and cultured in a 96-well plate with fresh RPMI medium and were stimulated or not with PWD (2.5 μg/mL) and treated with 5 μg/ mL of a fully human IgG4 (S228P) anti-PD-1 receptor-blocking monoclonal antibody (Bristol-Myers Squibb).

Roswell Park Memorial Institute (RPMI) medium (Invitrogen) was used for the cell cultures. The LPS, from Escherichia coli O111:B4, was obtained from Sigma-Aldrich. Carboxyfluorescein succinimidyl ester (CFSE) for the proliferation assays was purchased from Thermo Fisher. The CF-adapted ST395 P. aeruginosa clone was isolated from a patient who had been colonized during 13 years. LPS was obtained as previously described [23] . Colistimethate Sodium (polymyxin E) was purchased from Genéricos Españoles (GES) and dissolved in RPMI medium. The lymphocyte stimulus pokeweed (PWD) was purchased from Sigma-Aldrich. To inhibit PD-L1/PD-1 interaction, a fully human IgG4 (S228P) anti-PD-1 receptor-blocking monoclonal antibody was used (Bristol-Myers Squibb). All the reagents used for the cell cultures were endotoxin-free, as assayed with the Limulus amebocyte lysate test (Cambrex).