IL-15 during priming results in the long-lasting high-avidity CD8+ CTLs. (a) Spleen cells from three to four mice in each group were pooled, CD8+ T cells were positively purified and stimulated with splenocytes loaded with different concentrations of P18-I10, and proliferation was measured as described in Materials and Methods. Data are representative of three repeated experiments with consistent results. Each data point indicates mean ± SEM of triplicate assay. (b and c) Spleen CD8+ T cells from three to four mice in each group were pooled and stimulated with 1.0 (c Left) or 0.001 μM(b and c Right) P18-I10-pulsed splenocytes for 1 week. Lytic activity of CD8+ CTLs against P815 cells pulsed with different concentrations of the peptide was measured. Data represent mean ± SEM of triplicate assay at an effector-to-target cell ratio of 20:1. Similar kinetics of response were observed at all effector-to-target cell ratios tested, 80:1, 40:1, 20:1, and 10:1 (data not shown) in three repeat experiments. Data presented in b were normalized to the maximum lysis to compare avidity independent of magnitude of lysis and show mean ± SEM of triplicate assays. (d) Spleen CD8+ T cells from mice immunized as indicated were restimulated with 1.0 or 0.001 μM soluble P18-I10 overnight in the presence of brefeldin A. Cells were stained for intracellular IFN-γ by the manufacturer's protocol.