Cd279 pd 1 bv421

Expression of membrane and intracellular receptors of cancer stem cells and lymphocytes derived from the blood and the lungs was studied using mouse monoclonal antibodies following standard flow-cytometry protocols. Briefly, cell suspension was pre-incubated for 5 min with antimouse CD16/CD32 (BD FcBlock™). After pre-incubation, the cell suspension was stained with fluorophore-conjugated monoclonal antibodies: CD3 PerCP, CD8 BV510, CD44 APC-Cy™7, CD45 PerCP, CD45RA PerCP-Cy™5.5, CD69L APC, CD117 FITC, CD95 BV421, EGF (F4/80) Alexa Fluor^® 647, Axl BV421, CD279 (PD-1) BV421, CD197 (CCR7) PE (all—1/50 dilution, BD Biosciences, San Jose, CA, USA). The relevant isotype controls were used. Further, the cell suspension was stained with Sox2 PE intracellular antibodies (1/50 dilution, BD Biosciences, San Jose, CA, USA). FACS Canto II flow cytometer with FACS Diva software was used for analysis (BD Biosciences, Franklin Lakes, NJ, USA).

Cryopreserved PBMCs were thawed at 37°C, washed in RPMI/60% and RPMI/20% of fetal bovine serum (FBS), and incubated for 1 h at 37°C in RPMI/10%FBS. PBMCs were then stained with the Fixable Viability Stain-FVS780r (APC-H7 detect, BD Biosciences) for 15 mins. After PBMCs washing in PBS/1%FBS, cells were incubated in a U-bottom 96-well plate at a density of 1.5 million/well and stained with selected 14-color panel including: anti-CD19-AF700 (Clone HIB19), anti-IgD-Pe-Cy7 (Clone IA6-2), anti-IgM-BB515 (Clone G-20-127), polyclonal goat anti-IgA-Dylight^®649 (from Jackson Immunoresearch), CD10-BV650 (Clone H10a), CD21-PE-CF594 (Clone B-LY4), CD27-BV510 (Clone L128), CD38-BV786 (Clone HIT2), CD45-RB-PE (Clone MT4), CD86-PerCP-Cy5.5 (Clone 2331), CD279 (PD-1)-BV421 (Clone EH12.1), all from BD Biosciences unless indicated, for 15 mins. After washing twice in PBS/1% FBS, cells were fixed in PBS/1% formaldehyde, acquired in a BD LSRFortessa (BD Biosciences) cytometer using a plate HTS loader (BD Biosciences) and analyzed with FlowJo software (Tree Star). Gating strategy is described in Supplementary Figure 1. Lymphocyte gate was defined manually by morphological parameters excluding non-viable cells. B cells were identified as CD19+ CD21+/− cells and gated according to the expression of different markers to identify B-cell maturation stages as described in Supplementary Figure 1.