Vaccination with dendritic cells transfected with BAK and BAX siRNA enhances antigen-specific immune responses by prolonging dendritic cell life

Abstract

Dendritic cell-based vaccines have become an important approach for the treatment of malignancies. Numerous techniques have recently been designed to optimize dendritic cell activation, tumor antigen delivery to dendritic cells, and induction of tumor-specific immune responses in vivo. Dendritic cells (DCs), however, have a limited life span because they are subject to apoptotic cell death mediated by T cells, hindering their long-term ability to prime antigen-specific T cells. Small interfering RNA targeting Bak and Bax antiapoptotic proteins can be used to allow transfected DCs to resist killing by T cells in vivo. In this study, we show that human papillomavirus E7-loaded dendritic cells transfected with BAK/BAX siRNA downregulate Bak and Bax protein expression and become resistant to killing by T cells, leading to enhanced E7-specific CD8+ T cell activation and antitumor effects in vivo. More importantly, we found that vaccination with E7-loaded DCs transfected with BAK/BAX siRNA was capable of generating a strong therapeutic effect in vaccinated mice, compared with DCs transfected with control siRNA. Our data indicate that transfection of dendritic cells with BAK/BAX siRNA represents a plausible strategy for enhancing dendritic cell-based vaccine potency.

FIG. 1

Western blot analysis to detect the expression of Bak and Bax protein in DC-1 cells transfected with the various siRNA constructs. DC-1 cells were transfected with either BAK/BAX siRNA or control siRNA. Western blot analysis was performed with 50 μg of cell lysates 24 and 48 hours after transfection. β-actin was used as a control to indicate that equal amounts of cell lysates were loaded. Untransfected DC-1 cells were used as a negative control.

FIG. 2

Intracellular cytokine staining and flow cytometry analysis to determine the number of IFN-γ-producing E7-specific CD8⁺ T cells in mice after immunization with E7 peptide-pulsed DCs transfected with the various siRNA constructs. Mice (five per group) were vaccinated with E7 peptide-loaded DCs transfected with BAK/BAX siRNA or control siRNA. Mice vaccinated with E7 peptide-loaded DCs without transfection were used as an additional control. (A) Representative flow cytometry data for pooled splenocytes harvested from the vaccinated mice and stimulated with E7 aa49-57 peptide or without peptide stimulation. (B) Bar graph depicting the number of IFN-γ-secreting E7-specific CD8⁺ T cell precursors/3 × 10⁵ splenocytes from mice after immunization with E7 peptide-loaded DCs transfected with control siRNA or BAK/BAX siRNA or from nontransfected mice (mean ± SE; t test, p < 0.001).

FIG. 3

Intracellular cytokine staining and flow cytometry analysis to determine the number of IFN-γ-producing E7-specific CD8⁺ T cells in mice after immunization with E7 peptide-pulsed BM-DCs transfected with the various siRNA constructs. Mice (five per group) were vaccinated with E7 peptide-loaded BM-DCs transfected with BAK/BAX siRNA or control siRNA. The bar graph depicts the number of IFN-γ-secreting E7-specific CD8⁺ T cell precursors/3 × 10⁵ splenocytes from mice after immunization with E7 peptide-loaded BM-DCs transfected with control siRNA or BAK/BAX siRNA or from nonimmunized mice (mean ± SE; t test, p < 0.002).

FIG. 4

In vivo tumor protection and treatment experiments. (A) Tumor protection experiment. Mice (five per group) were immunized with E7 peptide-loaded DCs transfected with either control siRNA or BAK/BAX siRNA and boosted after 1 week. Seven days after the last immunization, the mice were challenged with 5 × 10⁴ TC-1 tumor cells per mouse as described in the Materials and Methods section. The tumors were monitored twice a week. Mice without vaccinations were used as a negative control. (B) In vivo tumor treatment experiment. E7 peptide-loaded DCs transfected with BAK/BAX siRNA or control siRNA were administered 3 days after TC-1 tumor challenge (5 × 10⁴ TC-1 cells/mouse). Mice were boosted with the same dose and regimen of E7 peptide-loaded DCs 1 week later. Mice were sacrificed 28 days after tumor challenge to examine the growth of pulmonary nodules. Data are expressed as the mean number of lung nodules ± SE (t test, p < 0.001).

FIG. 5

Determination of the survival of E7 peptide-loaded BM-DCs transfected with BAK/BAX siRNA or control siRNA after administration of E7-specific CD8⁺ T cells in vivo. (A) Flow cytometry analysis to demonstrate the different level of CFSE-labeled E7 peptide-loaded BM-DCs transfected with either BAK/BAX siRNA (low CFSE) or control siRNA (high CFSE). BAK/BAX-transfected BM-DCs were labeled with 0.5 μM CFSE, while BM-DCs transfected with Control siRNA were labeled with 5 μM CFSE. The representative graph shows the presence of similar numbers of low CFSE-labeled E7 peptide-loaded BM-DCs transfected with BAK/BAX siRNA and high CFSE-labeled E7 peptide-loaded BM-DCs transfected with control siRNA before tail vein injection. (B) Flow cytometry analysis to demonstrate the ratio of low CFSE to high CFSE-labeled E7 peptide-loaded BM-DCs in the spleen and lungs of mice 16 hours after injection with a mixture containing equal numbers (2.5 × 10⁵/mouse) of low CFSE-labeled E7 peptide-loaded BM-DCs transfected with BAK/BAX siRNA and high CFSE-labeled E7 peptide-loaded BM-DCs transfected with control siRNA. These CFSE-labeled BM-DCs were injected into mice 3 days after the administration of 1 × 10⁶ E7-specific T cells per mouse. Note that the number of low CFSE-labeled cells was significantly higher than the number of high CFSE-labeled cells.

FIG. 6

Characterization of the surface molecules of E7 peptide-loaded DCs after transfection with BAK/BAX siRNA or control siRNA. Flow cytometry analysis was performed to determine the level of expression of CD11c, CD40, CD86, MHC I, and MHC II molecules in E7 peptide-loaded murine DC-1 cells transfected with BAK/BAX siRNA or control siRNA. E7 peptide-loaded DCs without transfection with siRNA were used as a negative control.