CD40 and CD40L are both required for the induction of an H60-specific CD8 memory response. (A) Either 0.5 mg MR-1(anti-CD40L mAb) or hamster IgG was injected intraperitoneally into mice that had been previously immunized with male H60 cells at the time of boosting with male H60 cells and 2 times afterward at 3-day intervals. PBLs were stained on day 7 after boosting. FACs data obtained at the primary and secondary peaks are shown in parallel (gated for CD8⁺ cells). The fold-increase in peak size between the secondary and primary responses is plotted, and the error bars represent the standard deviation (SD). The data are representative of 3 independent experiments. (B) CD8 T cells purified from Ly5.1 (CD45.1⁺) congenic mice that had been previously immunized with male H60 cells were adoptively transferred to female B6, CD40L^−/−, and CD40^−/− hosts. The adoptive hosts were then primed with male H60 cells, and the PBLs were stained with H60-tetramer-PE, CD8-APC, and CD45.1-FITC. The FACs data shown are taken from 1 of 4 independent experiments and were obtained on day 7 after priming (gated for CD45.1⁺ cells). The percentage of H60-tetramer⁺ cells among the CD45.1⁺ CD8 T cells in the adoptive hosts is plotted and error bars represent the SD. (C) CD40^−/− or B6 adoptive hosts transferred with memory CD45.1⁺ CD8 T cells were primed with peptide (H60 + Gp61)–loaded splenocytes from CD40^−/− or normal B6 mice. Representative FACS data shown were obtained after staining of the PBLs on day 10 after priming with peptide-loaded cells. The percentage of H60-tetramer⁺ cells among the CD45.1⁺ CD8 T cells in the B6 or CD40^−/− adoptive hosts is plotted with error bars representing the SD (NS, not significant). Representative data were taken from 5 independent experiments.