Treatment with proteasome inhibitor bortezomib enhances antigen-specific CD8+ T-cell-mediated antitumor immunity induced by DNA vaccination

Abstract

There is an urgent need to develop new innovative therapies for the control of cancer. Antigen-specific immunotherapy and the employment of proteasome inhibitors have emerged as two potentially plausible approaches for the control of cancer. In the current study, we explored the combination of the DNA vaccine encoding calreticulin (CRT) linked to human papillomavirus type 16 E7 antigen (CRT/E7) with the proteasome inhibitor, bortezomib, for their ability to generate E7-specific immune responses and antitumor effects in vaccinated mice. We found that the combination of treatment with bortezomib and CRT/E7(detox) DNA generated more potent E7-specific CD8+ T cell immune responses and better therapeutic effects against TC-1 tumors in tumor-bearing mice compared to monotherapy. Furthermore, we found that treatment with bortezomib led to increased apoptosis of TC-1 tumor cells and could render the TC-1 tumor cells more susceptible to lysis by E7-specific CD8+ T cells. Our data have significant implications for future clinical translation.

Fig. 1

In vivo tumor treatment experiments. Groups of C57BL/6 mice (five per group) were subcutaneously challenged with 7 × 10⁴ per mouse of TC-1 tumor cells on day 0. Tumor-bearing mice were treated with bortezomib at a dose of 1 mg/kg intraperitoneally four times with 3-day intervals starting from day 2. Starting from day 9, mice were vaccinated with DNA encoding 4a-CRT/E7(detox) DNA via gene gun in the amount of 2 μg/mouse three times with 4-day intervals. a Schematic diagram of the treatment regimen of bortezomib and the 4a-CRT/E7(detox) DNA vaccine. b Line graph depicting the tumor volume in TC-1 tumor-bearing mice treated with bortezomib followed by the 4a-CRT/E7(detox) DNA vaccine (mean + SE; p < 0.001). c Kaplan and Meier survival analysis of TC-1 tumor-bearing mice treated with bortezomib followed by the 4a-CRT/E7(detox) DNA vaccine. Data shown are representative of two experiments performed

Fig. 2

Intracellular cytokine staining followed by flow cytometry analysis to determine the number of E7-specific CD8⁺ T cells in tumor-bearing mice treated with bortezomib and/or CRT/E7(detox) DNA. Groups of C57BL/6 mice (five per group) were challenged with TC-1 tumor cells and treated with bortezomib and/or 4a-CRT/E7(detox) DNA using the regimen as illustrated in Fig. 1a. One week after the last vaccination, splenocytes from tumor-bearing mice were harvested and characterized for E7-specific CD8⁺ T cells using intracellular IFN-γ staining followed by flow cytometry analysis. a Representative data of intracellular cytokine staining followed by flow cytometry analysis showing the number of E7-specific IFNγ+ CD8+ T cells in the various groups (right upper quadrant). b Bar graph depicting the numbers of E7-specific IFN-γ-secreting CD8⁺ T cells per 3 × 10⁵ pooled splenocytes (mean + SE). Data shown are representative of two experiments performed

Fig. 3

Intracellular cytokine staining followed by flow cytometry analysis to determine the number of E7-specific CD8⁺ T cells in mice treated with bortezomib and/or CRT/E7(detox) DNA. a Schematic diagram of the immunization regimen of bortezomib and the 4a-CRT/E7(detox) DNA vaccine. Groups of C57BL/6 mice (five per group) were injected with bortezomib at a dose of 1 mg/kg intraperitoneally four times with 3-day intervals. Starting from day 7, mice were vaccinated with CRT/E7(detox) DNA via gene gun in the amount of 2 μg/mouse three times with 4-day intervals. One week after the last vaccination, splenocytes from mice were harvested and stained for CD8 and intracellular IFN-γ and then characterized for E7-specific CD8⁺ T cells using intracellular IFN-γ staining followed by flow cytometry analysis. b Representative data of intracellular cytokine staining followed by flow cytometry analysis showing the number of E7-specific IFNγ+ CD8+ T cells in the various groups (right upper quadrant). c Bar graph depicting the numbers of E7-specific IFN-γ-secreting CD8⁺ T cells per 3 × 10⁵ pooled splenocytes (mean ± SE). Data shown are representative of two experiments performed

Fig. 4

Flow cytometry analysis to determine the apoptotic cell death in tumors isolated from mice treated with or without bortezomib. Groups of C57BL/6 mice (five per group) were subcutaneously challenged with 1 × 10⁵ per mouse of TC-1 tumor cells on day 0. One week later, tumor-bearing mice were treated with bortezomib 1.5 mg/kg intraperitoneally four times with 3-day intervals. Twenty-four hours later, tumor cells from mice were harvested and stained for 7-AAD and annexin V followed by flow cytometry analysis. a Representative flow cytometry data demonstrating the percentage of apoptotic tumor cells isolated from tumor-bearing mice treated with or without bortezomib (right upper quadrant). b Bar graph depicting the percentage of apoptotic cells per 1 × 10⁴ TC-1 cells (mean ± SE). Data shown are representative of two experiments performed

Fig. 5

In vitro cytotoxicity assay. Luciferase-expressing TC-1 tumor cells were added to 24-well plates at a dose of 1 × 10⁵ per well. Eighteen hours later, TC-1 tumor cells were treated with 16 nM bortezomib overnight followed by incubation with or without 1 × 10⁶ E7-specific cytotoxic T cells (CTL). The E7-specific CD8+ T cells have been previously described [23]. Untreated TC-1 tumor cells and TC-1 cells treated with 1 × 10⁶ E7-specific cytotoxic T cells (CTL) alone were used as controls. The degree of CTL-mediated killing of the tumor cells was indicated by the decrease of luminescence activity using the IVIS luminescence imaging system series 2000. Bioluminescence signals were acquired for 15 s. a Representative luminescence images of 24-well plates showing lysis of the tumor cells. b Bar graph depicting the quantification of luminescence intensity in tumor cells treated with bortezomib and/or E7-specific cytotoxic T cells (mean ± SE). Data shown are representative of two experiments performed