IL-12, but not IFN-α, is sufficient to program human CD8⁺ T-cell effector functions. (A) Intracellular expression of human IFN-γ and TNF-α from day 7 in vitro polarized human CD8⁺ T cells. Rested cells were reactivated for 4 hours with PMA and ionomycin in the presence of brefeldin A, and IFN-γ and TNF-α were assessed by intracellular stain and flow cytometric analysis. Data are gated on live, CD8⁺ cells. (B) Day 7 polarized cells were left unstimulated or stimulated with anti-CD3 for 24 hours, and supernatants were harvested for enzyme-linked immunosorbent assay. Error bars represent the SD of triplicate determinations of each condition. (C) Characterization of CTL activity by ⁵¹Cr release assay. Day 7 polarized CD8⁺ T cells were incubated for 4 hours with ⁵¹Cr-labeled THP-1 cells (target) at the E:T ratios shown. CTL activity was assessed by quantification of ⁵¹Cr released into the supernatant by β emission. These experiments were performed with 5 different healthy donors with similar results.