Isolation and Characterization of PBMCs

Peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood samples using Ficoll density gradients as described previously^{34 (link)}. Isolated PBMCs were stained for surface markers, fixed, permeabilized with IntraPreReagent (Beckman Coulter, Fullerton, CA), and further stained with antibodies directed against intracellular markers. Leukocytes were stimulated with Leukocyte Activation Cocktail (BD Bioscience, San Jose, CA, USA) at 37 °C for 4 h prior to the intracellular staining using Pharmingen’s staining protocol. Anti-human mAbs against CD3-PE-CF594, CD56-FITC, IFN-γ-PE, and TNF-α-PE with corresponding isotype-matched controls were purchased from BD Biosciences (San Jose, CA, USA). Data were acquired on a Gallios instrument (Beckman Coulter, Brea, CA, USA) and analysed with FlowJo software (FlowJo, LCC, USA).