Fig. 4.

Simultaneous detection of multiple T-cell epitopes using HCMV pp65-MHCI RNA. a PBMCs from a healthy HCMV sero-positive HLA-A*0201⁺ donor were tested in CFC for CD8⁺ reactivity against the control peptide, pp65 peptide 495–503, NY-ESO-I-MHCI RNA as an irrelevant control, and pp65-MHCI RNA (10 μg each). Numbers represent the percentage of IFNγ secreting cells. Data are representative for three independent experiments. b CD8⁺ and CD4⁺ lymphocytes of a HCMV⁺ healthy donor were co-cultivated with autologous DCs loaded with four different sub pools of overlapping pp65 peptides (each peptide 1.75 μg/ml; see schematic drawing). c After expansion for 7 days each effector cell population (4 × 10⁴/well) was tested in IFN(ELISPOT on autologous DCs (3 × 10⁴/well) either loaded with the whole pp65 peptide pool, a pool of irrelevant peptides spanning the WT1 protein sequence (1.75 μg/ml), or transfected with pp65-MHCI RNA (20 μg). Each bar represents the mean spot number of duplicates. Due to the lack of adequate discrimination spot numbers were only quantified to a maximum of 800 per well