PBMCs were purified by density gradient centrifugation. A volume of 13 mL heparinised blood was added to Leucosept tubes (Greiner Bio-one, France) pre-filled with 17 mL Ficoll (Lymphocytes Separation Medium, Eurobio, France) and centrifuged at 1200 rpm for 35 min. PBMCs were collected at the ficoll interface and washed in 50 mL D-PBS without MgCl 2 and CaCl 2 (GIBCO, Invitrogen, France). Cells were then incubated in 2 mL BD Pharmlyse 1X (BD Biosciences, France) at room temperature.