Figure 3. TKIs alone and with a cancer vaccine alter immune-cell function.

For cabozantinib studies, C57/BL6 CEA-Tg mice received cabozantinib beginning on day 0. Mice treated with vaccine received MVA-CEA/TRICOM on day 0 and rF-CEA/TRICOM on days 7 and 14. On day 35, spleens were harvested and a single cell suspension of splenocytes was obtained. (A) CEA peptide was incubated with the splenocytes for 7 days after which cells were incubated with fresh, irradiated, naïve splenocytes and either CEA or HIV-gag peptide for 24 hours at 37°C. After 24 hours, a cytometric bead array was used to analyze the supernatants for murine IFN-γ. CEA-specific cytokine production was determined by subtracting cytokine production induced by the HIV-gag peptide from that induced by the CEA peptide. Error bars depict the mean ± standard error. * = P < 0.05 relative to control and single agents as determined by Student’s t test. (B) Tregs (CD3⁺CD4⁺CD25⁺FoxP3⁺cells) were isolated from spleens using negative selection. Tregs were cultured with CD4⁺ T cells from naïve mice, antigen-presenting cells (APCs, irradiated allogeneic splenocytes) and soluble anti-CD3 for 72 hours. The background level of CD4⁺ T cell proliferation was determined by incubating the naïve CD4⁺ T cells with APCs and anti-CD3 in the absence of purified Tregs. Error bars depict the mean ± standard error. * = P < 0.01 compared to the proliferation of CD4⁺ T cells incubated with Tregs from untreated mice; ns = no significant difference between CD4⁺ T cell proliferation in the absence of Tregs and the proliferation of CD4⁺ T cells incubated with Tregs isolated from mice treated with the indicated therapy. Significance was determined Student’s t test. Data adapted from ^[12]. For sunitinib studies, C57/BL6 mice received sunitinib beginning on day 0. Mice treated with vaccine received MVA-CEA/TRICOM on day 7, and rF-CEA/TRICOM on day 14. On day 35, spleens were harvested and a single cell suspension of splenocytes was obtained. (C) CD4⁺ lymphocytes were isolated from spleens and cocultured with APCs and 6.25 g/mL CEA protein for 5 days at 37°C. One μCi [³H] thymidine was added to each well for the last 24 hours. Mean cellular proliferation was dictated by [³H] thymidine incorporation. Error bars depict the mean ± standard error. * = P < 0.01 relative to control and single agents as determined by Student’s t test. (D) Treg functional assay performed as in (B) using splenic Tregs purified from mice treated with sunitinib ± vaccine. Data adapted from ^[14].