Whole peripheral blood (PB) samples (2 ml/subject) were collected in ethylenediamine tetra-acetic acid (EDTA) test tubes. The following monoclonal antibodies from BD Biosciences (San Jose, CA, USA) were used: CD3 FITC, CD4 PE-Cy7, CD5 FITC, CD8 APC Cy7, CD19 PE, CD45RA FITC, HLA-DR PE Cy7, CD16 + 56 PE and CD45 PerCP. Whole PB samples were labelled with appropriate volumes of conjugated MoAb for 20 min at room temperature, and then lysed with 2 ml of lysis solution (FACS Lysis Solution, BD). Cells were washed twice and analyzed on a standard FACSCanto instrument (BD) with DIVA software (BD). All results were expressed as percentages of each subset out of total lymphocytes.