Enhanced antiviral IFN-γ response in human CD8 memory T cells by HD preculture of PBMCs. (A) Representative IFN-γ ELISPOT assay of fresh or precultured PBMCs. Cytokine release after stimulation with 1 μg/ml CD28 superagonist TAB08 (previously: TGN1412) and 0.1 μg/ml PepMix CEF standard was assessed after 16 hours. Unstimulated fresh and precultured PBMCs were used as negative controls. (B) Intracellular IFN-γ staining of fresh and precultured PBMCs after stimulation with 0.1 μg/ml PepMix CEF standard for 16 hours. Gating was performed on viable lymphocytes (left) and CD8 T cells (right). (C) Functional maturation of CD8 T cells and monocytes contributes to the HD preculture effect. CD8 T cells and monocytes were purified from fresh and precultured PBMCs of the same donor by magnetic sorting (magnetic-activated cells sorting) and cocultured under standard conditions at a 1:1 ratio in the presence of either 0.1 μg/ml PepMix CEF standard or 1 μg/ml OKT3, and were assessed by IFN-γ ELISPOT 16 hours after stimulation. Experiments were repeated ≥3 times. Data represent mean ± SD for triplicate samples for 1 representative donor out of 3. F, fresh; P, precultured; US, unstimulated.