*4.4. Isolation of Peripheral Blood Mononuclear Cells*

Peripheral blood mononuclear cells (PBMCs) were obtained from heparinized venous blood and were separated by Ficoll-Hypaque (LymphoprepTM, Axis-Shield, Oslo, Norway) gradient centrifugation [47]. They were then resuspended in RPMI-1640 with 10% heat-inactivated fetal calf serum (Gibco, Life Technologies Limited, Renfrew, UK), 25mM HEPES and 1% penicillin-streptomycin (Biowhittaker, Lonza, Barcelona, Spain). Cell enumeration was performed as previously described [48]. The PBMCs of each patients or control were adjusted to 1 106 cells/mL prior to antibody staining.

The cell number counts of lymphocytes subsets were calculated by the percentage of each subpopulation in the PBMCs determined by flow cytometry multiplied by the total number of lymphocytes per microliter obtained by a complete blood count from a conventional hemogram measured by Beckman Coulter, Inc (Brea, CA, USA).
