Flow Cytometry analysis
All analyses were performed using fresh peripheral blood mononuclear
cell (PBMC) samples, isolated by density centrifugation using
Ficoll-Hypaque (Amersham Biosciences, Amersham, Buckinghamshire, United
Kingdom) from 20 mL EDTA and venous blood. The following monoclonal
antibodies were used for T cell immunophenotyping: CD4-APC-CY7,
CD8-FITC, CD28-APC, CD45RA-PE-CY7, CCR7-PERCP-CY5.5, CD27-AmCyan
HLA-DR-Pacific Blue, and CD57-PE (BD Biosciences, San Jose, CA).
Combinations of CD45RA, CCR7, CD28, and CD27 are commonly employed to
define four different T cell subsets [17]: 1)
CD28++CD27++CCR7+CD45RA+[naive T cells (TN)]; 2)
CD28+++CD27++CCR7+CD45RA-[central memory T cells (TCM)]; 3)
CD28+-CD27+-[effector memory T
cells (TEM)]; and CD28-CD27-[
(terminally differentiated memory T cells (TemRA)]. Cellular
activation in CD4 subsets was characterized by the expression of HLA-DR
[18]. Senescent CD4+ T cells were characterized by
CD57 expression [19]. Fluorescence was measured with a FACS Canto II
(BD Biosciences, Breda, the Netherlands). A total of 100,000 events were
collected in the lymphocyte gate using morphological parameters (forward
and side-scatter). The raw data were analysed using a fluorescence
activated cell sorter (FACS) Diva version 5 (BD Biosciences, Heidelberg,
Germany) and FlowJo software (TreeStar Inc., Ashland, OR, USA).