Cytokine-induced IFNγ production by virus-specific CD8⁺ T cells. (A) Spleen cells isolated from mice at 8 days (day 8) or greater than 120 days (immune) after LCMV infection were activated directly ex vivo with the combination of IL-12 and IL-18 for up to 12 hours. After gating on CD8⁺L^dNP118-Tetramer⁺ T cells, IFNγ production was detected by intracellular staining. Values are the average ± SD for 4 animals in each group. (B) Spleen cells from mice at day 8 after infection were activated directly ex vivo with IL-12 plus IL-18 for 5 hours to induce IFNγ production. Activated cells were then either maintained in cytokines, treated with the protein synthesis inhibitor CHX, or washed and replaced in medium without cytokines. After 5 hours of cytokine withdrawal (t = 10 hours), cells were restimulated with IL-12 plus IL-18 for 4 hours. IFNγ production by CD8⁺L^dNP118-Tetramer⁺ cells was detected by intracellular staining. Values are averages ± SDs (n = 3). (C) Spleen cells from mice at day 8 after infection were activated directly ex vivo with IL-12 and IL-18 for 5 hours to induce IFNγ production. Activated cells were then either maintained in IL-12 and IL-18 or washed and replaced in medium without cytokines. After 14 hours of cytokine withdrawal (t = 19 hours), cells were restimulated with IL-12 and IL-18 for an additional 8 hours. IFNγ production by CD8⁺L^dNP118-Tetramer⁺ cells was detected by intracellular staining. Values are averages ± SDs (n = 3).