HD preculture of PBMCs allows the generation of CD8 T-cell lines with an improved representation of clones responding to low antigen concentrations. (A, left) IFN-γ responses of CD8 T cells from fresh or precultured PBMCs of 1 HLA–A0201-positive patient to titrated amounts of the peptide WT1_HUMAN 356-364 before expansion. (Right) Fresh or precultured PBMCs of the same patient were stimulated with 1 μg/ml of the peptide WT1_HUMAN 356-364 in a final cell density of 2 × 106 cells/ml and were expanded in the presence of IL-2, IL-7, and IL-15 for 2 weeks. After expansion, cells were re-stimulated with titrated amounts of the peptide WT1_HUMAN 356-364 or the control pool human actin in IFN-γ ELISPOT plates for 16 hours. Unpaired Student t test: **P < .005; ***P < .0005; ****P < .0001. (B, left) IFN-γ responses of CD8 T cells from fresh or precultured PBMCs of 1 representative healthy donor to titrated amounts of the HLA-class I restricted PepMix CEF standard and the control pool human actin before expansion. Fresh or precultured PBMCs of the same donor were stimulated with 0.1 μg/ml of the PepMix CEF standard (middle) or the PepMix Human Actin (right), and were expanded under the same conditions as presented in (A). After expansion, virus-specific CD8 T cells were re-stimulated with titrated amounts of the PepMix CEF standard or the control pool human actin in IFN-γ ELISPOT plates for 16 hours, respectively. Unpaired Student t test: *P < .05; **P < .005; ***P < .0005; ****P < .0001. Data represent mean ± SD for triplicate samples. Experiments were repeated 4 times.
