Enhanced immune response induced by P5 HER2/neu-derived peptide-pulsed dendritic cells as a preventive cancer vaccine

Abstract

Dendritic cells are special and powerful antigen-presenting cells that can induce primary immune responses against tumour-associated antigens. They can present antigens via both MHC-I and MHC-II, so they have the ability to stimulate both cytotoxic T lymphocytes and T helper cells. Furthermore, CD8⁺ cytotoxic T lymphocytes require activation by CD4⁺ T cells. This requires a CD4⁺ T cell activator molecule, of which PADRE is one of the best. We chose an approach to use both of these important arms of the immune system. We prepared dendritic cells from mouse bone marrow, loaded them with our target peptides (P5 peptide alone or P5 + PADRE), and then injected these pulsed dendritic cells alone or in combination with CpG-ODN (as adjuvant) into BALB/C mice. After the last boosting dose, mice were inoculated with TUBO cells, which overexpress HER2/neu. Two weeks after the tumour cell injection, immunological tests were performed on splenocyte suspensions, and the remaining mice were evaluated for tumour growth and survival. Our data indicate the formulation that contains PADRE plus P5 loaded onto DC in combination with CpG-ODN was the most effective formulation at inducing immune responses. Interferon production in CD4⁺ and CD8⁺ gated cells, cytotoxicity rates of target cells and mice survival were all significantly greater in this group than in controls, and all the mice in this group were tumour-free throughout the experiment. Based on our results and the role of HER2/neu as a candidate in human immunotherapy, this approach may be an effective cancer treatment.

Keywords: cancer; dendritic Cells; pan HLA-DR epitope peptide; peptide vaccine.

Figure 1

CD11c, CD40, CD80 (B7‐1), CD86 (B7‐2) and MHC IA/IE expression on immature and mature DCs. Expression of these markers is greater in mature than in immature DCs. Dendritic cells were generated from mouse bone marrow and matured by the addition of LPS. The dot plot represents cell percentages in the mature and immature states.

Figure 2

Uptake of OVA‐FITC by DCs. The uptake assay was performed to analyse the endocytic ability of prepared DCs. Dendritic cells took up more OVA‐FITC 37°C than at 4°C. The fluorescent intensity corresponds to the uptake.

Figure 3

Geometric mean fluorescence intensity (MFI) of IFN‐γ in gated CD4⁺ cells (A), IL‐4 in gated CD4⁺ cells (B) and IFN‐γ in gated CD8⁺ cells (C). Cytokine profile frequency was analysed by FACS analysis to determine the immune response rate. Splenocytes were stained by anti‐CD4 and anti‐CD8 antibodies. After stimulation with PMA and ionomycin, cells were stained with anti‐IFN‐γ and anti‐IL‐4 antibodies. Data are expressed as the mean ± S.E.M. (n = 3). Results were analysed by one‐way anova, and statistically significant differences are designated as follows: ns: P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 4

In vitro IFN‐γ production was analysed in vaccinated mice 2 weeks after the last booster by ELISpot assay. Splenocytes were harvested and treated with (+peptide) or without peptides (‐peptide) and the IFN‐γ secretion was determined. The data indicate the mean ± S.E.M. (n = 3). Statistical significances were designated as follows: ns: P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Figure 5

Induction of specific CTL potency for tumour cell killing by the different formulations was assessed by in vitro CTL activity assay. Five different effector to target cell ratios were tested. HER2/neu‐expressing TUBO cells and CT26 cells (as a negative control) were labelled by Calcein AM and co‐incubated with different ratios of splenocytes. The data are expressed as means ± S.E.M.s (n = 3). E: effector cells and T: target cells. One‐way anova was employed and statistically significant differences are shown as follows: ns: P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 6

Results of the formulations on TUBO tumour growth in mice. Two weeks after the final boosting dose, TUBO cells were injected into mice (n = 5). Tumours were measured and volumes recorded weekly. The values are means. Mice were monitored for 100 days. Data were analysed by the two‐way anova test and statistical significances were designated as follows: Ns: P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 7

The tumour mass scale based on tumour size in inoculated mice on their last survival days (A), and their survival (B, C and D) were monitored by the multiple comparison log‐rank (Mantel–Cox) test. Statistical significances were designated as follows: ns: P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.