Effector cell activity of the T-cell lines generated from seronegative donors after stimulation with CD40-B cells pulsed with a mixture of antigenic peptides. CD8⁺ T cells from CMV-seronegative donors were stimulated 3 times with autologous CD40-B cells pulsed with a mixture of 2 to 4 peptides and tested for effector activity. CMVpp65-derived synthetic peptides used are listed in Table 3. The antigenicity of these peptides was proven in the experiments whose results are shown in Figure 5. (A) ELISPOT assay was conducted using LCL/pp65, LCL/EGFP (□), or LCL/EGFP pulsed with 1 μM of each peptide indicated (▪). Each bar represents the number of spots per 10³ cells. (B) Peptide titration was conducted using LCL/EGFP pulsed with various concentrations of the peptides indicated by ELISPOT assay. Percent spot was calculated for individual T-cell lines by dividing the number of spots at indicated peptide concentrations by the maximal number of spots × 100%. (C) Cytolysis of CTL lines was assessed against autologous LCL/pp65 (•), LCL/EGFP (○), HLA-matched dermal fibroblasts infected with CMV supernatant (▪), with mock supernatant (□), or with 1 μM peptide mixture showing spots in the ELISPOT assay (▴) over a range of E/T ratios by ⁵¹Cr release assay.