Lymphocyte proliferation was performed in 96 well plates as already described [42] . Briefly, heparinized blood samples were diluted 1:15 in complete culture medium consisting of DMEM (Dulbecco's Modified Eagle Medium, Eurobio, France) supplemented with 5% fetal calf serum (Hyclone, Perbio, France), 2 mM L-glutamine, 100 U/mL penicillin and 50 mg/mL streptomycin (Eurobio, France). For detection of unspecific lymphocyte proliferation, the diluted blood samples were seeded in 96 well plates (200 mL/well) and mock-stimulated for 48 h by incubation in culture medium (control wells), or stimulated for 48 h by incubation with the culture medium supplemented with either 10 mg/mL ConA (Sigma, France), or 50 ng/mL of PMA (Sigma, France) and 1 mg/mL of ionomycin (Sigma, France), or 1 mg/ml LPS (Sigma, France). Control wells remained unstimulated. After 48 h of incubation at 39uC, 0.5 mCi of 3 H-methylthymidine (ICN, France) was added to each well. After another 24 h incubation period, the cells were harvested through glassfiber filters (Whatman, United Kingdom) by means of an automatic harvester (Titerteck-Skatron, Molecular Devices, France). Incorporation of tritiated thymidine was measured with a Liquid Scintillation Beta Counter (Kontron Instruments, France). Results were expressed as a stimulation index of lymphocyte proliferation calculated as mean counts per min (cpm) of the triplicate cultures in stimulated culture/mean cpm in control non-stimulated culture.