Enhanced recall responses of HD-precultured CD8 T cells to virus-derived peptide pools. (A) IFN-γ ELISPOT responses of fresh or precultured PBMCs of 1 representative healthy donor to titrated amounts of the HLA-class I restricted PepMix CEF standard. Unpaired Student t test: ***P < .0005. (B) Response of the same donor as shown in (A) to titrated amounts of the negative control pool PepMix Human Actin. Data represent mean ± SD for triplicate samples. (C-D) Identification of memory CD8 T cells as source for IFN-γ production by intracellular cytokine staining of fresh and precultured PBMCs of 1 representative donor that is different from (A-B). Results are shown as percentage of IFN-γ–positive cells among CD8⁺CD45R0⁺ cells and CD8⁺CD45R0⁻ cells. (E) Compiled data from 22 healthy donors tested as in Figure 2A and presented as in Figure 2B. Mann-Whitney U test: ****P < .0001. Data represent median with IQR. Raw data: supplemental Figure 5C. n.s., not significant; US, unstimulated.