CD40L is induced by either peptide or IL-12 and IL-18, but with independently regulated kinetics. Spleen cells from mice at day 8 (A-B) and day greater than 120 (B,D) after infection were activated as in Figure 2. IFNγ and CD40L were detected by intracellular staining. Dotplots (A-B) are gated on CD8⁺ T cells and show IFNγ and CD40L expression after 1, 3, and 12 hours of direct ex vivo stimulation with peptide or IL-12 and IL-18 in the presence of BFA for the last hour, or 12 hours of peptide stimulation with continuous BFA (12/12 h BFA). Graphs (C-D) show the percentage of virus-specific CD8⁺ T cells producing CD40L at each time interval. Values are averages ± SDs (n = 4). (E) The relative rates of CD40L production by day 8 and day greater than 120 (immune) virus-specific CD8⁺ T cells are represented as the percentage of maximum CD40L response at each time interval, with 100% maximum defined at 3 hours for peptide stimulation and 12 hours for IL-12 plus IL-18 stimulation.