CpG-ODN-stimulated dendritic cells act as a potent adjuvant for E7 protein delivery to induce antigen-specific antitumour immunity in a HPV 16 E7-associated animal tumour model

Abstract

We previously reported that both E7 and CpG-oligodeoxynucleotide (ODN) are required for protecting animals from human papillomavirus (HPV) 16 E7-associated tumour challenge. Here we investigate dendritic cells (DC)-based approach in this protection. In the study, we isolated bone marrow-derived DC and stimulated DC with E7 and ODN. In vitro stimulation of DC with E7 plus ODN resulted in more production of interleukin-12, as compared to that with E7 or ODN alone. Further injection with E7+ODN-stimulated DC resulted in more significant tumour protection, as compared to stimulation with E7 or ODN alone. We further evaluated the levels of immune responses induced by DC stimulated with E7+ODN. We observed little enhancement of E7-specific antibody and T helper cell proliferative responses by E7+ODN stimulation, as compared to E7 stimulation. However, there was some enhancement of interferon-gamma (IFN-gamma) production from CD4+ T cells and a more significant production of IFN-gamma from CD8+ T cells by E7+ODN stimulation, as compared to E7 stimulation alone. This was consistent with intracellular IFN-gamma staining levels of CD8+ T cells. Tumour protection further appeared to be mediated by CD8+ T cells, as determined by in vivo T-cell depletion. Thus, these data suggest that upon ODN stimulation DC might function as a potent adjuvant for E7 protein delivery for induction of protective cellular immunity against HPV E7-associated tumour challenge.

Figure 1

Production of murine dendritic cells from bone marrow stem cells. Murine bone marrow cells were stimulated with GM-CSF and IL-4. Non-adherent cells were harvested after 7 days and expression of surface markers were analysed using flow cytometry. Filled lines show isotype controls.

Figure 2

Production of IL-12 upon stimulation of DC with E7+ODN. Bone marrow-derived DC were prepared as described in Materials and Methods. DC (2·5 × 10⁶ cells/ml) were stimulated with 10 µg of E7 and/or ODN per ml for 2 days. Cell supernatants were obtained for detecting IL-12 levels.

Figure 3

Antitumour effects of ODN application for DC pulsing. Each group of mice (n = 5−10) was immunized i.p. with 5 × 10⁵ DC previously pulsed with E7 and/or ODN at 0 and 2 weeks. Three weeks after the second injection, mice were challenged s.c. with 5 × 10⁴ (a) and 2 × 10⁵ (b) TC-1 cells. The number of animals showing tumour growth was counted over time. *Statistically significant at P < 0·05 using anova compared to DC + E7 treated groups.

Figure 4

E7-specific IgG (a) and T helper cell proliferative responses (b) were unaffected by injection with DC pulsed in the presence of ODN. Each group of mice (n = 5) was immunized i.p. with 5×10⁵ DC previously pulsed with E7 and/or ODN at 0 and 2 weeks. Three weeks after the second injection, Mice were bled and sera were tested for ELISA (a). Mice were killed and spleens were pooled for subsequent T helper cell proliferation studies (b). Samples were assayed in triplicate. This was repeated twice with similar results. *Statistically significant at P < 0·05 using the paired Student's t-test compared to control groups.

Figure 5

IFN-γ production was enhanced from CD4⁺ T cells and more significantly from CD8⁺ T cells by pulsing DC in the presence of ODN. Animals were immunized as shown in Fig. 4. Three weeks after the last injection, mice were killed and spleen cells were pooled. Splenocytes were stimulated with 1 µg of E7 proteins/ml (a) and with mitomycin C-treated TC-1 cells (b) for 3 days. Samples were assayed in triplicate. Values and bars represent mean of released IFN-γ concentrations and the standard deviation. The experiments were repeated twice more with similar results. *Statistically significant at P < 0·05 using the paired Student's t-test compared to control groups. **Statistically significant at P < 0·05 using the paired Student's t-test compared to DC + E7 pulsed groups.

Figure 6

Intracellular IFN-γ staining of splenocytes after stimulation in vitro with E7 CTL epitopes. Animals were immunized as shown in Fig. 4. Three weeks after the last injection, mice were killed and spleen cells were pooled. Splenocytes were stimulated in vitro with 1 µg/ml E7 CTL epitopes for 5–6 h and then tested for FACS analysis. (a) Representative figure of the FACS data in three experiments performed. (b) Bar graphs depicting the percent of double positive (CD8⁺/IFN-γ⁺) cells in total cells tested (mean ± SD).

Figure 7

Effects of CD8⁺ T cells on tumour growth. Each group of mice (n = 4) was immunized i.p. with 5 × 10⁵ DC previously pulsed with E7 plus ODN at 0 and 2 weeks. Three weeks after the second injection, mice were depleted in vivo of CD8⁺ T cells, as described in Materials and Methods. Animals were challenged s.c. with 5 × 10⁴ TC-1 cells and then the number of animals showing tumour growth was counted over time.