collected in EDTA-containing tubes. The peripheral blood mononuclear cells (PBMCs) were
first isolated using Ficoll-Hypaque (Innotrain, Germany) density gradient centrifugation
(1000×g at room temperature) and then washed two times with phosphate-buffered saline
(PBS, Euroimmun, Germany). Centrifugation of samples was at 300 ×g and 4°C. Subsequently,
isolated PBMCs (106 cells/100 µl) were stained with anti-CD8-APC, CD45RO-PE,
CD27-Percp-eFlour780, CD28-Pe-Cy7, CD57-FITC, and CD56-PerCp-eFlour710 monoclonal
antibodies (mAbs) (all from eBioscience, USA) and incubated at 4°C for 30 minutes. The
samples were fixed with formaldehyde and analyzed within 24 hours by flow cytometer (BD
FACSAria, USA). At least 50,000 events were counted for each sample.