FIGURE 1

Transduction efficiencies of antiviral CD8+ T cells versus bulk CD8+ T cells. Peripheral blood mononuclear cells from HLA-A*0201-positive, EBV and/or CMV seropositive healthy volunteer adult donors were activated by 10 ng/ml soluble CD3 mAb (OKT3, Jansen-Cilag, Levallois Perret, France) and 500 UI/ml recombinant human IL-2 (Cetus, Rueil Malmaison, France)^{3, 4} and transduced at day 3 by spin transduction or incubation with retroviral vector-containing supernatants harboring either the LNGFR-coding amphotropic SFCMM3 vector¹⁰ (dashed lines, black symbols) or the gibbon ape leukemia virus (GALV)-pseudotyped LNGFR-coding LNSN vector^{5, 11} (full lines, white symbols). Cells were stained at day 7, as previously described⁵ with an anti-human LNGFR mAb for 20 min at 4°C, then washed and incubated with goat-anti-mouse IgG-fluoroisothiocyanate (FITC) (Clinisciences, Montrouge, France) for 20 min at 4°C. Cells were then stained with phycoerythrin (PE)-labeled HLA-A*0201 tetramers complexed with EBV-derived peptides (lytic protein BMLF1: GLCTLVAML; latent protein EBNA-3C: VLEETSVML) or CMV-derived peptides (IE1 early antigen: LLDFVRFMGV; pp65 late antigen: NLVPMVATV) and CD8-phycocyanin-5 (PC5) mAb (Beckman-Coulter Immunotech, Marseille, France) for 30 min at room temperature.^{12, 13} After washing, cells were analyzed on a FACSCalibur flow cytometer using CellQuest software (Becton Dickinson). Analyses were performed after gating on the CD8⁺ lymphocyte population. For each individual staining, the percentage of LNGFR-positive cells is represented on the Y-axis for the bulk CD8+ cells (CD8+) and the tetramer-specific CD8+ cells (EBV: BMLF1+ and EBNA3C+; CMV: pp65+ and IE1+). Each symbol represents one independent experiment. CD3- CD56+ NK cells represented <5% of GMC^{6, 14} and were excluded from analysis by their low level of CD8 expression.