CCL3 and CCL20-recruited dendritic cells modified by melanoma antigen gene-1 induce anti-tumor immunity against gastric cancer ex vivo and in vivo

Abstract

Background: To investigate whether dendritic cell (DC) precursors, recruited by injection of chemokine ligand 3 (CCL3) and CCL20, induce anti-tumor immunity against gastric cancer induced by a DC vaccine expressing melanoma antigen gene-1 (MAGE-1) ex vivo and in vivo.

Methods: B6 mice were injected with CCL3 and CCL20 via the tail vein. Freshly isolated F4/80-B220-CD11c+ cells cultured with cytokines were analyzed by phenotype analysis and mixed lymphocyte reaction (MLR). For adenoviral (Ad)-mediated gene transduction, cultured F4/80-B220-CD11c+ cells were incubated with Ad-MAGE-1. Vaccination of stimulated DC induced T lymphocytes. The killing effect of these T cells against gastric carcinoma cells was assayed by MTT. INF-gamma production was determined with an INF-gamma ELISA kit. In the solid tumor and metastases model, DC-based vaccines were used for immunization after challenge with MFC cells. Tumor size, survival of mice, and number of pulmonary metastatic foci were used to assess the therapeutic effect of DC vaccines.

Results: F4/80-B220-CD11c+ cell numbers increased after CCL3 and CCL20 injection. Freshly isolated F4/80-B220-CD11c+ cells cultured with cytokines were phenotyically identical to typical DC and gained the capacity to stimulate allogeneic T cells. These DCs were transduced with Ad-MAGE-1, which were prepared for DC vaccines expressing tumor antigen. T lymphocytes stimulated by DCs transduced with Ad-MAGE-1 exhibited specific killing effects on gastric carcinoma cells and produced high levels of INF-gamma ex vivo. In vivo, tumor sizes of the experimental group were much smaller than both the positive control group and the negative control groups (P < 0.05). Kaplan-Meier survival curves showed that survival of the experimental group mice was significantly longer than the control groups (P < 0.05). In addition, MAGE-1-transduced DCs were also a therapeutic benefit on an established metastatic tumor, resulting in a tremendous decrease in the number of pulmonary metastatic foci.

Conclusions: CCL3 and CCL20-recruited DCs modified by adenovirus-trasnsduced, tumor-associated antigen, MAGE-1, can stimulate anti-tumor immunity specific to gastric cancer ex vivo and in vivo. This system may prove to be an efficient strategy for anti-tumor immunotherapy.

Figure 1

CCL3 and CCL20 injection recruites F4/80^-B220^-CD11c⁺ cells into the peripheral blood in mice. B6 mice were injected via the tail vein with 1 mg of CCL3 and CCL20 or with PBS (control). Peripheral blood was obtained by cardiac puncture at the different time intervals (0 h, 8 h, 16 h, 24 h, 48 h, 72 h, 120 h). F4/80^-B220^-CD11c⁺ cells were sorted from PBMNCs and analyzed by FACS. Results are given as means ± SD with 10 mice per group from three independent experiments.

Figure 2

Morphological characteristics of CCL3 and CCL20-recruited F4/80^-B220^-CD11c⁺ cells before and after culture. (A), Fresh CCL3 and CCL20-recruited F4/80^-B220^-CD11c⁺ cells were sorted from PBMNCs of mice by FACS and observed by light microscopy (original magnification ×200). (B), These cells were cultured with GM-CSF and TNFα for 5~6 days, then were observed by light microscopy (Giemsa staining was performed, original magnification ×400).

Figure 3

Immunophenotypic analysis of CCL3 and CCL20-recruited F4/80^-B220^-CD11c⁺ cells. CCL3 and CCL20-recruited F4/80^-B220^-CD11c⁺ cells cultured for 5~8 days were incubated with PE or FITC-labeled MAbs. The phenotype of these cells was analyzed by immunofluorescence staining as described in the Materials and Methods. Results are given as means ± SD from three independent experiments.

Figure 4

The capacity of CCL3 and CCL20-recruited F4/80^- B220^-CD11c⁺ cells to enhance allogeneic MLR. Allogeneic MLR were performed using splenic T cells purified from B6 mice as responder cells. Fresh and cultured F4/80^-B220^-CD11c⁺ cells were treated with MMC to arrest cell proliferation and were used as stimulator cells at the indicated cell numbers, respectively. Macrophage were used as controls. T cell proliferation was determined with MTT after 5 days of culture. Results are expressed as the mean ± SD of triplicate cultures. All data are representative of three independent experiments.

Figure 5

Generation of tumor-specific CTLs ex vivo. Splenic CD3⁺ T cells were isolated from B6 mice with MACS. T cells were primed with MAGE-1-modified DCs as described in Materials and Methods. DC-Ad-LacZ and untreated DCs were used as controls. Primed T cells (effector cells) were titrated by serial dilution, then mixed with MFC or B16F10 target cells, and their lytic activity was assayed. Results are given as means ± SD from three independent experiments.

Figure 6

Inhibition of tumor growth in tumor-bearing mice by immunization with MAGE-1-modified, CCL3 and CCL20-recruited DC vaccine. (A), Each of 10 mice in a group was challenged s.c. with 1 × 10⁵ viable MFC tumor cells. Mice were subsequently injected s.c. with DC-Ad-MAGE-1 5 days later. As controls, tumor-bearing mice were injected with DC-Ad-LacZ, DC-MFC Ag, or untreated DCs. Survival was observed over time after immunization of mice harboring preexisting tumors. Survival rate was compared with a long-rank test of Kaplan-Meier curves. (B), Tumor growth was measured every 2~3 days after the second immunization. Data are given as means ± SD of 10 mice per group from three independent experiments.

Figure 7

Assay for tumor-specific, CTL activity and IFN-γ secretion in immunized mice. (A), Splenic T cells from immunized mice were restimulated ex vivo by culturing with MMC-treated, MFC tumor cells. The restimulated T cells (effector cells) were incubated with target MFC or B16F10 cells for 20 h. Cytolytic activity (lysis) was determined. (B), Supernatants were collected for IFN-γ assay. All data are shown as means ± SD for 10 mice per group and are representative of three independent experiments. * P < 0.05.