Enhanced sensitivity of human CD8 T cells to defined viral antigens by HD preculture of PBMCs. (A) IFN-γ responses of CD8 T cells from fresh or precultured PBMCs from a representative healthy donor were directly compared by ELISPOT assay after stimulation with titrated PepMix influenza A. Unpaired Student t test: ***P < .0005; ****P < .0001. (B) Compiled data from 6 healthy donors tested as in (A). Data represent the mean fold-increase in IFN-γ responses of CD8 T cells from precultured PBMCs relative to CD8 T cells from fresh PBMCs after stimulation with the PepMix influenza A. Mann–Whitney U test: *P < .05; **P < .01. Data represent median with IQR. Raw data: supplemental Figure 5A. (C) IFN-γ responses of CD8 T cells from fresh or precultured PBMCs from an HLA–A0201-positive, CMV–sero-positive healthy donor after stimulation with titrated PP65_HCMV 495-503. (C) Unpaired Student t test: ***P < .0005; ****P < .0001. (D) Compiled data from 5 donors tested as in (C). Data presentation as in (B). Fold increase could not be calculated for the concentrations 0.0001 and 0.001 μg/ml where no response was detectable in fresh PBMCs (supplemental Figure 5B). (E) CD8 T-cell responses in fresh and precultured PBMCs to 2 μg/ml of 3 different ADV-derived peptides. (F) Compiled data from 7 HLA-A01 (HEX_ADE02 901-910), HLA-A02 (E1A_ADE02 19-27), or HLA-A24 (HEX_ADE02 37-45) positive healthy individuals. Data presentation as in (B). Mann-Whitney U test: **P < .01. Data represent median with IQR. n.s., not significant.