In contrast to CD40 monotherapy, CD40/TLR7* therapy rescues CD8⁺ memory T-cell function. Mice were immunized with 100 μg each of ΔV peptide, αCD40, and S-27609 in combinations as indicated. Memory CD8⁺ functionality was assessed 65 days later. (A) Representative dot plots of IFNγ secretion by memory CD8⁺ T cells isolated from spleens and lungs of vaccinated mice. Dot plots are gated on live CD8⁺ cells, and numbers indicate the percentage of cells positive for both IFNγ and CD44. (B) Memory CD8⁺ T-cell cytolytic activity was assessed by performing an in vivo cytotoxicity assay. Numbers reflect the percentage of antigen-specific lysis. (C,D) Quantification of relative and absolute numbers of memory CD8⁺ cells expressing IFNγ in the spleen (C) and lung (D). Absolute numbers of positive cells were determined by multiplying the relative percentage of each cell population by the total number of cells isolated from each tissue. (E) Quantification of the in vivo cytotoxicity assay presented in panel B. P ≤ .001 by one-tailed ANOVA. (F) CD127 expression on IFNγ⁺-memory CD8⁺ T cells derived from spleens or lungs of vaccinated mice. Isotype controls are shown as filled histograms. (G) Cytokine production by memory CD8⁺ T cells. Cells from panel F were analyzed for the ability to produce TNFα and IL-2. Numbers reflect the percentage of CD8⁺IFNγ⁺ cells that also are positive for TNFα or IL-2. In all cases, data are pooled from at least 2 independent experiments with 4 or more mice/group per experiment and plotted as means (± SEM).