Hepatic toxicity associated with αCD40 monotherapy is reversed with TLR7 agonism. (A,B) Kinetic analysis of serum transaminases. Mice were treated with PBS, 100 μg αCD40, 100 μg TLR7*, or both intravenously. Serum was isolated at various time points afterward, and serum levels of alanine transaminase (A) or aspartate transaminase (B) were measured as described. Data are representative of 3 independent experiments, with n = 3 to 8 mice per group, per time point. (C-F) Histologic analysis of livers treated with PBS (C), 100 μg αCD40 (D), 100 μg TLR7* (E), or 100 μg αCD40 and 100 μg TLR7* (F) for 48 hours. (G) Semiquantitative assessment of histopathologic changes in livers from mice treated as above for 48 hours. Data are pooled from 2 independent experiments, with n = 6 mice in each treatment group. P = .026 by Mann-Whitney nonparametric test.

Kinetic analysis of lung-infiltrating lymphocytes. (A) Experimental design. (B) Representative dot plots of lymphocytes isolated from metastatic target organs at day 10 or 21 after tumor challenge. Cells were isolated from tumor-bearing lungs as described in “Methods” and subjected to an in vitro restimulation with tumor peptide. Plots are gated on live, CD8⁺ cells. Numbers in the upper right-hand quadrant reflect the frequency of CD8⁺ T cells that are positive for both IFNγ and the activation marker CD44. Data are representative of 3 independent experiments with 4 mice per group in each experiment. (C,D) Quantification of lung infiltrates at either 10 (C) or 21 (D) days after tumor challenge. Data are plotted as means (± SEM) and represent pooled data from either 2 (C, n = 8 mice/group) or 3 (D, n = 12 mice/group) independent experiments, with 4 mice/group in each experiment. (E) Effector phenotype of CD8⁺ T cells isolated from lungs of mice vaccinated with tumor antigen plus αCD40/TLR7* at either 10 or 21 days following tumor inoculation. The dot plots are first gated on live CD8⁺ cells and then further gated on IFNγ⁺CD44⁺ populations. Data are representative of at least 2 independent experiments, with 4 mice/group in each experiment.

Concomitant signaling through CD40 and TLR7 drives expansion of self-antigen–specific CD8⁺ T cells with enhanced cytolytic activity. C57BL/6 mice were immunized intravenously with 100 μg of the tumor-associated antigen ΔV, 100 μg αCD40 FGK45, and 100 μg S-27609 in combinations as indicated. Seven days later, mice were bled and cells were restimulated in vitro with TRP2_(180-188) to assess the ability to produce IFNγ and translocate CD107a as described in “Methods.” Lymphocytes were identified by forward and side scatter and subsequently gated on all CD8⁺ events. (A) Representative dot plots from vaccinated mice. The numbers in the upper right corners indicate the frequency of CD8⁺ T cells that are positive for IFNγ and CD44 (top row) or IFNγ and CD107a (bottom row). (B) Percentage of peripheral blood lymphocytes expressing the CD8 antigen. P ≤ .001 by one-tailed ANOVA (C) Quantification of the percentages of CD8⁺ cells that degranulated in response to peptide restimulation. In all cases, data presented are representative of at least 3 independent experiments. Data are plotted as means plus or minus SEM (n = 8 in each group). P ≤ .001 by one-tailed ANOVA.

αCD40/TLR7* therapeutic intervention slows progression of metastatic melanoma. C57BL/6 mice were challenged with 10⁵ metastatic B16.F10 melanoma cells intravenously. Four days later, mice were vaccinated with 100 μg of the tumor-associated antigen ΔV, 100 μg αCD40 FGK45, and 100 μg S-27609 in combinations as indicated. After 24 days, mice were killed, lungs were removed, and metastatic surface tumor nodules were enumerated with the aid of a dissecting microscope. (A) Photograph of macroscopically visible tumor nodules on lungs of mice, 24 days after tumor challenge. Numbers below the lungs reflect the mean survival time and long-term survival rate of mice monitored for therapeutic efficacy. Data are pooled from 3 to 4 independent experiments with greater than 8 mice per group in each experiment. (B) Enumeration of lung metastases. Data are pooled from 2 independent experiments and are presented as means plus or minus SEM (n = 16 mice in each group). Data are representative of more than 4 separate experiments with at least 6 mice in each group. (C) Enumeration of lung metastases after effector cell depletion. Mice were treated as above except for the depletion of effector cell populations prior to tumor challenge as described in “Methods.” The data are expressed as means plus or minus SEM (n = 8 mice in each group) and are representative of 3 independent experiments.

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).