Anti hla a2 fitc antibody clone bb7.2

HLA-A2 typing was performed as previously described [22 (link)]. Briefly, 100 μL of peripheral blood was incubated with 0.25 μg of anti-HLA-A2 FITC antibody (clone BB7.2; BD Pharmingen, San Diego, CA, USA) for 20 minutes at 4°C [25 (link)]. After adding 500 μL of 1X lysis buffer (BD Bioscience, San José, CA, USA), the cells were incubated for 15 minutes at room temperature. Finally, the cells were washed with 1X phosphate-buffered saline (PBS; Eurobio, Les Ulis, France), and data were collected using a FACSAria II flow cytometer (BD Immunocytometry Systems, San José, CA, USA). The data were analyzed with FlowJo 9.3.2 software (Tree Star, Inc., Ashland, OR, USA).

Blood samples from 3 healthy donors and 35 cancer patients [10 lung cancers, 11 head & neck cancers, 14 metastatic renal cell carcinomas (mRCC)] were collected at the Hôpital Européen Georges Pompidou (Paris, France) after approval by the local ethics committee (CPP Ile de France, II N°2013-06-03). Blood samples were also collected from 12 healthy donors at the Etablissement Français du Sang (EFS, Rungis, France) after informed consent and following EFS guidelines. PBMCs were isolated by density gradient (Ficoll-Paque PLUS, GE Healthcare). The HLA-DR or A^*02:01 genotypes of all donors were determined using the Gold SSP DRB1 typing kit (Invitrogen) after DNA extraction of PBMCs with NucleoSpin Blood L Kit (Macherey Nagel) or by staining with the monoclonal anti-HLA-A2-FITC antibody (Clone BB7.2, BD Biosciences).