TNFα production is transient following peptide stimulation and is not induced by IL-12 and IL-18. Spleen cells from mice at day 8 (A,C) or day greater than 200 (B,D) after infection were activated directly ex vivo with NP118 peptide and/or IL-12 and IL-18. IFNγ and TNFα production by CD8⁺ T cells was detected by intracellular staining. Dotplots (A-B) are gated on CD8⁺ cells and depict IFNγ and TNFα production after 1, 3, and 8 hours of stimulation with peptide or IL-12 plus IL-18 in the presence of BFA added for the last hour, or 8 hours of peptide stimulation with continuous BFA (8 of 8 hours BFA) to fully sequester intracellular cytokines for comparison. Graphs (C-D) depict the percentage of CD8⁺ T cells producing TNFα versus time. Values are averages ± SDs (n = 4 day 8, n = 8 day> 200). (E) The relative rates of TNFα production by day 8 and day greater than 200 (immune) CD8⁺ T cells are represented as the percentage of maximum TNFα response at each time interval, with 100% maximum set at 2 hours.