Two milliliters of peripheral blood were collected from participants and placed in an ethylene diamine tetraacetic acid (EDTA) anticoagulant tube for routine blood tests during recruitment. Another 100 μL of peripheral blood was taken to be labeled with antibodies. After antibody labeling, the red blood cells were lysed and washed with phosphate-buffered saline. The antibody labels were then detected. The labeled antibodies were described in previous literature [25 (link)] as follows: anti-CD3-allophycocyanin (APC)-cyanine (Cy) 7 (clone SK7), anti-CD4-BB700 (clone SK3), anti-CD8-BV510 (clone SK1), anti-CD25-phycoerythrin (PE) (clone M-A251), anti-CD127-BV421 (clone HIL-7R-M21), anti-CD62L-APC (clone DREG-56), anti-CD45RO-BB515 (clone UCHL1), and anti-HLA-DR-PE-Cy7 (clone G46-6). One hundred microliters of peripheral blood were also collected from the homotypic controls for antibody labeling. Homotypic controls were tested using anti-CD3-APC-Cy7 (clone SK7), anti-CD4-BB700 (clone SK3), anti-CD8-BV510 (clone SK1), immunoglobulin 1 (IgG1) k-PE (clone MOPC-21), IgG1 k-BV421 (clone X40), IgG1 k-APC (clone MOPC-21), IgG2a k-BB515 (clone G155-178), and IgG2a k-PE-Cy7 (clone G155-178) (BD Biosciences).
Flow cytometry BD FACS Canto™ II was applied for data collection and FlowJo V10 software (Tree Star, Inc., Ashland, OR, USA) was utilized for data analysis.