Purified PBMCs were washed in 50 mL D-PBS without MgCl 2 and CaCl 2 , incubated with 2 mL pig serum at 4uC for 20 min, washed again in 50 mL D-PBS without MgCl 2 and CaCl 2 and then washed in 50 mL S/W buffer (1 g/L NaN 3 , 10 g/L bovine serum albumin in PBS, pH 7.3) at a final concentration of 5.10 6 cells/mL. 10 6 cells were used for each antibody labelling. Single, double or triple staining was performed using monoclonal antibodies (mAbs) directed against i) CD2 (MSA4, isotype IgG2a, VMRD) and CD16 (MCA1971, isotype IgG1, Serotec), ii) CD4 (PT90A, isotype IgG2a, VMRD) and CD8a (PT81B, isotype IgG2b, VMRD) iii) TCRcd (MAC320, isotype IgG2a, BD Biosciences Pharmingen), iv) IgM (PIG45A, isotype IgG2b, VMRD) and v) MHCII (MSA3, isotype IgG2a, VMRD), CD16 (MCA1971, isotype IgG1, Serotec) and CD172a (74-22-15A, isotype IgG2b, BD Biosciences Pharmingen). Briefly, PBMCs were stained with primary mAbs for 25 min at 4uC, washed in S/W buffer and stained with allophycocyanin-conjugated anti-mouse IgG1 (BD Biosciences, France), phycoerythrin-conjugated antimouse IgG2a (Southern Biotech, France), FITC-conjugated antimouse IgG2b (Southern Biotech, France), APC-conjugated antimouse IgG1 (BD Biosciences, France), phycoerythrin-conjugated anti-rat IgG2a (BD Pharmingen, France), or phycoerythrinconjugated anti-mouse IgG2b (Southern Biotech, France). After washing in S/W buffer, cells were fixed in BD Cellfix solution (Becton Dickinson, Germany). Data acquisition and analysis were carried out with the FACScan and CELLQuest software (Becton Dickinson, UK).