Pd l1 pe

PBMCs were incubated at 150,000 cells/well in 96-well, round-bottom plates for 24 h at 37°C and 5% CO₂ in the presence or absence of IL-27 (50 ng/ml; R&D Systems) or IFN-γ (100 ng/ml; R&D Systems). Cells were harvested and stained with Live/Dead Fixable Aqua viability dye (Thermo Fisher Scientific), CD3 AF700, CD4 PE-Cy7, CD27 PE-Cy5, CD45RO PE-Texas Red (BD Biosciences), and PD-L1 PE (BioLegend). Median fluorescence intensity of PD-L1 expression was assessed in the naive lymphocyte gate (CD3⁺CD4⁺CD27⁺CD45RO⁻).

The frequencies of PD-1 and PD-L1 expressions were assessed using a Beckman Coulter flow cytometer (FC500; Beckman Coulter, Inc., Brea, CA, USA) in all studied individuals according to the stain-lyse and wash protocol as previously described by Moniuszko et al (19 (link)). Briefly, 50 µl EDTA-anti-coagulated whole blood was stained with 5 µl of the following mouse anti-human monoclonal antibodies, CD4 fluorescein isothiocyanate (FITC; cat no. 317408), CD8a-FITC (cat no. 301006), CD14-FITC (cat no. 325604), PD-1-phycoerythrin (PE; cat no. 329906) and PD-L1-PE (cat no. 329706) to identify PD-1⁺CD4⁺, PD-1⁺CD8⁺ T cells and CD14⁺PD-L1⁺ monocytes, respectively (all used as supplied and obtained from BioLegend, Inc., San Diego, CA, USA). All blood samples were incubated for 30 min at 4°C in the dark. Thereafter, the cells were lysed using Optilyse C Lysis Solution (Immotech SAS, Marseille, MC, France) and washed twice with cold PBS. A minimum of 100,000 events was acquired. FlowJo version 7.6.1 (Tree Star, Inc., Ashland, OR, USA) software was used for the analysis of flow cytometry data.