Dendritic cell vaccination with a toll-like receptor agonist derived from mycobacteria enhances anti-tumor immunity

Abstract

Dendritic cell (DC)-based vaccines are considered useful in cancer immunotherapy, and the interaction of DC and adjuvants is important in the design of the next generation vaccines. In this study, whether DC combined with Rv2299c derived from mycobacteria could improve anti-tumor immune responses in a colon cancer mouse model was evaluated. MC38 cell lines were injected subcutaneously to establish colon-cancer-bearing mice and the following four groups were evaluated: PBS control, tumor antigen (TA) loaded-DC, Rv2299c, and a combination of TA-loaded-DC and Rv2299c. The combination treatment with TA-loaded-DC and Rv2299c exhibited greater inhibition of tumor growth compared to other groups. These effects were associated with the reduction of suppressor cells, such as myeloid-derived suppressor cells and regulatory T cells, and the induction of effector cells, such as CD4+ T cells and CD8+ T cells, in spleen, and with the activation of cytotoxic T Lymphocytes and NK cells. These results suggest that TA-loaded-DC vaccination with Rv2299c derived from mycobacteria enhanced anti-tumor immunity in a mouse colon cancer model by inhibiting the generation of immune-suppressive cells and recovering numbers of effector cells, and demonstrated superior polarization of the Th1/Th2 balance in favor of the Th1 immune response.

Keywords: dendritic cells; mouse colon cancer; mycobacterial heat shock protein 90.

Figure 1. Characteristics of bone-marrow-derived CD11c⁺ dendritic cells (DC)

A. The phenotype of DC were analyzed for expression levels of MHC I, MHC-II, CD80, CD86, CD40, and CCR7 using flow cytometry; mature DC (mDC) expressed higher levels of maturation molecules than immature DC (iDC). Representative histograms show marker expression levels (shaded) compared to those of the isotype controls (black line). B, C. An ELISA revealed that mDC produced higher levels of IL-12p70 and lower levels of IL-10 in culture supernatants after subsequent CD40L stimulation, compared to iDC (P < 0.05). The data are shown as the means (pg/mL) ± standard deviation (SD) of triplicate cultures from three independent experiments.

Figure 2. Rv2299c injection results in enhanced anti-tumor activity against MC-38 colon cancer

A. Schematic experimental schedule of in vivo animal vaccinations with Rv2299c. Experiments consisted of five mice per group. B. Mice treated with 5 and 10 μg of Rv2299c showed significantly greater inhibition of tumor growth compared to those receiving 1 μg of Rv2299c or the PBS control (*P < 0.05), as measured by the monitoring tumor volume. C, D. Mice in the 5- and 10-μg Rv2299c treatment groups had an increased proportion of CD4⁺ T cells and CD8⁺ T cells among splenocytes compared to the 1-μg Rv2299c or PBS control group, as measured by flow cytometry in the left panel and compared by quantitated bar graphs in the right panel. Data are representative of more than 3 experiments.

Figure 3. The proportions of CD4⁺ CD25⁺ regulatory T cells (Tregs), CD4⁺ Foxp3⁺ Tregs, and myeloid-derived suppressor cells (MDSCs) were measured by flow cytometry in the left panel and compared by quantitated bar graphs in the right panel

A, B. The percentages of CD4⁺ CD25⁺ Tregs and CD4⁺ Foxp3⁺ Tregs were decreased in the 5- and 10-μg Rv2299c treatment groups compared to the 1-μg Rv2299c and PBS control groups. C. The percentages of MDSCs were not significantly different among the four groups. D. The phenotypes of DC in the spleens of tumor-bearing mice measured by flow cytometry (left panel) and compared by quantitated bar graphs in the right panel. The DC from splenocytes isolated from the mice injected with 5- and 10-μg Rv2299c showed increased expressions of MHC class I and II, CD80, CD86, CD40, and CCR7 compared to the 1-μg Rv2299c and PBS control groups. Representative histograms show marker expression (shaded) compared to the isotype controls (black line). Data are representative of more than 3 experiments.

Figure 4. In vivo animal vaccination

Four vaccination groups were established: 1) PBS control, 2) Tumor antigen (TA)-loaded DC vaccination, 3) Rv2299c injection, and 4) TA-loaded DC vaccination plus Rv2299c injection. On day 0, MC-38 cells (5 × 10⁵/mouse) were injected subcutaneously into the right flank of C57BL6 mice. A. The DC were administered subcutaneously into the left flank of C57BL6 mice on days 7, 9, 11 and 13, and Rv2299c was injected intraperitoneally on days 8, 10, 12, 14, 16 and 18. B. Mice vaccinated with TA-loaded DC plus Rv2299c showed significant inhibition of tumor growth (*P < 0.05 on day 19) and induced a long-term systemic anti-colon cancer immunes response (Supplemental Fig. 4) compared to TA-loaded DC or Rv2299c alone. Experiments consisted of eight mice per group.

Figure 5. Activation of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, the proportion of CD4⁺ T cells, CD8⁺ T cells, memory T cells, and cytokine production induced by vaccination with TA-loaded DC plus Rv2299c

A. The number of IFN-γ-secreting lymphocytes in spleens of mice treated with PBS, TA-loaded DC, Rv2299c and DC plus Rv2299c were counted in an IFN-γ ELISPOT assay. A combination of DC vaccination and Rv2299c injection significantly increased the number of IFN-γ-secreting lymphocytes against MC-38 and YAC-1 cells compared to other groups (*P < 0.05). B. IL-10 and C. IFN-γ production from splenocytes of vaccinated mice was evaluated by ELISA. The culture supernatants of splenocytes from the mice vaccinated with TA-loaded DC plus Rv2299c showed lower level of IL-10 and higher level of IFN-γ compared to other mice (*P < 0.05). Data are shown as means (pg/mL) ± SD of triplicate cultures from three independent experiments. The proportions of D. CD4⁺ T cells, E. CD8⁺ T cells, and F. memory T cells were measured by flow cytometry in the left panel and compared by quantitated bar graphs in the right panel. The results revealed that there were significant increases of effector cells in the TA-loaded DC groups with or without Rv2299c compared to the group with Rv2299c alone. The data are representative of more than 3 experiments. Statistical comparisons were performed using the one-way nonparametric ANOVA.

Figure 6. The proportions of

A. MDSCs, B. CD4⁺ CD25⁺ Tregs and C. CD4⁺ Foxp3⁺ Tregs in the spleens of vaccinated tumor-bearing mice were measured by flow cytometry in the left panel and compared by quantitated bar graphs in the right panel. The proportions of MDSCs and Tregs were significantly increased in the PBS control group after tumor inoculation. Rv2299c alone increased the generation of MDSCs compared to TA-loaded DC alone or TA-loaded DC plus Rv2299c, while the percentages of Tregs were lower in the Rv2299c adjuvant and TA-loaded DC plus Rv2299c groups compared to the TA-loaded DC alone group. And the data are representative of more than 3 experiments. Statistical comparisons were performed using the one-way nonparametric ANOVA (*P < 0.05).