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. 2016 Mar 29;7(13):16855-65.
doi: 10.18632/oncotarget.7806.

Exceptional antineoplastic activity of a dendritic-cell-targeted vaccine loaded with a Listeria peptide proposed against metastatic melanoma

Ricardo Calderon-Gonzalez  1 , Lucia Bronchalo-Vicente  1   2 , Javier Freire  3 , Elisabet Frande-Cabanes  1 , Lidia Alaez-Alvarez  1 , Javier Gomez-Roman  3 , Sonsóles Yañez-Diaz  2 , Carmen Alvarez-Dominguez  1
Affiliations

Exceptional antineoplastic activity of a dendritic-cell-targeted vaccine loaded with a Listeria peptide proposed against metastatic melanoma

Ricardo Calderon-Gonzalez et al. Oncotarget. .

Abstract

Vaccination with dendritic cells (DCs) is proposed to induce lasting responses against melanoma but its survival benefit in patients needs to be demonstrated. We propose a DC-targeted vaccine loaded with a Listeria peptide with exceptional anti-tumour activity to prevent metastasis of melanoma. Mice vaccinated with vaccines based on DCs loaded with listeriolysin O peptide (91-99) (LLO91-99) showed clear reduction of metastatic B16OVA melanoma size and adhesion, prevention of lung metastasis, enhanced survival, and reversion of immune tolerance. Robust innate and specific immune responses explained the efficiency of DC-LLO91-99 vaccines against B16OVA melanoma. The noTable features of this vaccine related to melanoma reduction were: expansion of immune-dominant LLO91-99-specific CD8 T cells that helped to expand melanoma-specific CD8+ T cells; high numbers of tumour-infiltrating lymphocytes with a cytotoxic phenotype; and a decrease in CD4+CD25high regulatory T cells. This vaccine might be a useful alternative treatment for advanced melanoma, alone or in combination with other therapies.

Keywords: Listeria peptides; dendritic cells; immunotherapy; melanoma; vaccines.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1. B16OVA model of metastatic melanoma and DC vaccination
(A) B16OVA was transplanted intraperitoneally into mice for 7 (7D) or 14 (14D) days. Melanoma was recovered post-transplantation and measured immediately with a calliper (lower images) (n = 10). We also quantified post-transplantation the number of metastases as visual nodules in the lungs or after histological analysis of the peritoneum and liver (right images). (B) Mice were transplanted with B16OVA for 0, 7, 14 or 23 days (n = 10/time) (left plot) and the number of surviving mice counted. Results are expressed as the percentages of surviving mice (P < 0.05). At the end of transplantation, mice were killed and spleens immediately removed and processed for histological analysis (right images) or cell populations by FACS. Results are expressed as the mean of percentages of positive cells ± SD (right values plot) (P < 0.05). (C) Mice were transplanted with B16OVA for 14 days, killed, and spleens were removed (n = 5). Intracellular cytokine staining was performed immediately in isolated spleen homogenates stimulated with B16OVA extract (B16OVAext) in the presence of brefeldin A (left histograms showing the percentages of B16OVAext-CD4+ and B16OVAext-CD8+ and IFN-γ producers). Frequencies of CD8+-OVA257–264 were examined using dimers of recombinant dimeric H-2Kb: Ig fusion protein loaded with OVA257–264 peptide (right plots) (P < 0.05). (D) Mice were vaccinated with DC-LLO91–99, DC-LLO296–304, DC-GAPDH1–15 or DC-GAPDH1–22 vaccines or left unvaccinated (NV) for 7 days. Mice were transplanted with B16OVA for 7 (7D, black bars) or 14 (14D, white bars) days and killed. Size of recovered melanoma was measured with a calliper (Ø mm) (n = 5). (E) Mice were vaccinated with different DC vaccines for 7 days and transplanted with B16OVA for 14 days. Post-transplantation, we quantified the number of melanoma metastases in the lungs (left plot) and adherence activity of recovered melanoma. Results are expressed as the mean of lung metastases ± SD (left plot) or the percentage of cells adhered to plates (right plot) (P < 0.05).
Figure 2
Figure 2. (A) Early and late apoptosis examined in NV or DC-vaccinated mice and transplanted with B16OVA for 14 days (14D)
Apoptosis was analysed by FACS after double staining with 7-AAD (7-AAD-PE) and annexin V (annexin V-APC) and expressed as percentages. Q2 area shows late apoptotic cells, and Q4 area shows early apoptotic cells. (B) Mice were vaccinated with different DC vaccines or left unvaccinated (NV) for 7 days (n = 10). All mice were transplanted with B16OVA and the number of surviving animals was counted at 1, 7, 14 or 23 days. All groups of mice were compared to the NV group. Results correspond to the mean ± SD number of surviving mice (P < 0.05). (C) Left plot shows spleen cell populations analysed by FACS that corresponded to NV mice or mice vaccinated with DC-LLO91–99 or DC-GAPDH1–22 for 7 days and transplanted with B16OVA for 14 days (14D). Results expressed as percentages of positive cells ± SD. Right plots show cytokines levels from mice sera, expressed as cytokine concentrations (pg/ml of mean ± SD, P < 0.05). (D) Intracellular IFN-γ staining of spleens from DC-LLO91–99-vaccinated mice and stimulated with OVA257–264 or LLO91–99 peptides in the presence of brefeldin A (left lower plots). Right plots show the frequencies of CD8+–OVA257–264 or CD8+-LLO91–99 using dimers of recombinant H-2Kb: Ig fusion protein loaded with peptides. Results are the mean ± SD (P < 0.05). (E) Model of action of DC-LLO91–99 vaccines against advanced melanoma.
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