Figure 5.

Intratumoral treatment with the pIC+R848 combination activates systemic and memory adaptive antitumor immune responses in mice rejecting tumors. (A, B) pIC+R848 treatment in a two-tumor model. (A) Schematic representation of the experimental protocol. CMT167 cells were subcutaneously injected in the right (A, in blue) and left (B, in red) flanks. From day 9 to day 21, mice received intratumoral injection of pIC+R848 combination (25 µg of each drug) only in one tumor (A). Control mice received only saline. (B) Evolution of each tumor growth (right tumor in blue (A) and left tumor in red (B)) in mice treated with pIC+R848 versus control. n=5 per group. (C, D) Tumor growth in mice depleted for CD4⁺ and CD8⁺ T cells or NK cells and treated with pIC+R848. Mean±SEM. n=7 per group. Statistical comparison was performed using one-way analysis of variance followed by Tukey’s multiple comparison test. Statistically significant differences are represented as ***p<0.001. (E) CMT167 tumors intratumorally treated with two injections of pIC+R848 were analyzed with flow cytometry for the proportion of CD69⁺ and PD1⁺ cells in CD4⁺ and CD8⁺ T cells. n=5 per group. Bars represent mean±SEM. Statistical comparison was performed using a t-test. Statistically significant differences are represented as **p<0.01. (F) Evolution of tumor growth in mice treated with six intratumoral injections of pIC+R848 combination, as indicated in figure 2A. Mice with no sign of tumor growth (tumor rejected mice) were rechallenged with CMT167 tumor cells at day 70 and did not receive any treatments afterward. Tumor growth of individual mice (inset) up to day 105 and comparison with a control group of naïve mice receiving tumor cell injection. (G) In vitro cytotoxicity of spleen macrophages derived from tumor-rejected or tumor-bearing naïve mice in coculture experiments with CMT167 lung cancer cells. Bars represent mean±SEM. Statistical comparison was performed using a t-test. Statistically significant differences are represented as **p<0.01. (H, I) Proliferation of (H) CD8⁺ or (I) CD4⁺ T cells from CellTrace stained splenocytes from tumor-bearing naïve or tumor-rejected mice, cocultured for 72 hours with syngeneic healthy naïve mice-derived DCs, which were previously exposed to CMT167 cell lysate with or without pIC+R848. Untreated splenocytes and CD3/CD28 beads were used as controls. DC, dendritic cell; pIC, poly(I:C); R848, resiquimod.