Toxoplasma gondii is an obligate intracellular protozoan parasite that induces antitumor activity against certain types of cancers. However, little information is available regarding the immunologic mechanisms that regulate these effects. For this purpose, C57BL/6 mice were administered either the T. gondii Me49 strain orally or Lewis lung carcinoma (LLC) cells intramuscularly. Survival rates, tumor size, histopathology, and immune responses were determined for each group, and angiogenesis was evaluated by in vivo Matrigel plug assay. Toxoplasma-infected (TG-injected) mice survived the entire experimental period, whereas cancer cell-bearing (LLC-injected) mice died within six weeks. Mice injected with both T. gondii and cancer cells (TG/LLC-injected group) showed significantly increased survival rates, CD8+ T-cell percentages, IFN-gamma mRNA expression levels, serum IgG2a titers, and CTL responses as compared to the LLC-injected mice. In addition, angiogenesis in the TG/LLC-injected mice was notably inhibited. These effects in TG/LCC-injected mice were similar or were increased by the addition of an adjuvant, Quil-A. However, TG/LLC-injected mice showed decreased percentages of CD4+ and CD8+ T cells, IFN-gamma mRNA expression levels, and serum IgG1 and IgG2a titers as compared to TG-injected mice. Taken together, our results demonstrate that T. gondii infection inhibits tumor growth in the Lewis lung carcinoma mouse model through the induction of Th1 immune responses and antiangiogenic activity.
Animals ; Base Sequence ; CD4-Positive T-Lymphocytes/immunology ; CD8-Positive T-Lymphocytes/immunology ; Carcinoma, Lewis Lung/blood supply/genetics/immunology/*therapy ; Cell Line, Tumor ; Cytotoxicity, Immunologic ; DNA Primers/genetics ; Female ; Immunoglobulin G/blood ; Immunotherapy/*methods ; Interferon-gamma/genetics ; Mice ; Mice, Inbred C57BL ; Neovascularization, Pathologic ; RNA, Messenger/genetics/metabolism ; Th1 Cells/*immunology ; Toxoplasma/*immunology

OBJECTIVETo investigate the anti-metastatic effect of vascular endothelial growth factor receptor 2 extracellular domain gene-modified dendritic cell (DC-sVEGFR-2) vaccination.

METHODSDendritic cells (DC) were electroporated with pcDNA3. 1/sVEGFR-2 plasmid DNA. Expression of sVEGFR-2 was determined by ELISA. For immunization, C57BL/6 mice were intravenously injected three times with 1 x 10(5) cells per mouse of DC, pcDNA3. 1-transfected DC (DC-vector) , DC-sVEGFR-2, or 100 microl of PBS at 7-day intervals. At 10 days after the last immunization, the immunized mice were subjected to assessment of cytotoxic T lymphocyte ( CTL) response to VEGFR-2, alginate bead analysis of tumor cell-induced angiogenesis, and observation of the anti-metastatic effect in B16 melanoma metastasis model. CTL activity was determined by a standard 4-h 51Cr release assay against VEGFR-2 + vascular endothelial cell line H5V, 3LL cells stably transfected with pcDNA3. 1/sVEGFR-2 (3LL,-sVEGFR-2), and VEGFR-2- cell lines EL-4 and 3LL. Monoclonal antibodies GK1.5 anti-CD4 and 2.43 anti-CD8 were used to deplete in vivo CD4 + T cells and CD8' T cells, respectively.

RESULTSDC-sVEGFR-2 could effectively express sVEGFR-2, whereas DC-vector and DC could not. Immunization of mice with DC-sVEGFR-2 significantly induce CTL activity against VEGFR-2 + cell lines H5V and 3LL-sVEGFR-2, however, no significant CTL activity was observed when VEGFR-2- syngeneic cell lines EL-4 and 3LL. were used as target cells, implying this CTL activity was VEGFR-2 specific. Alginate bead analysis of in vivo neoangiogenesis showed that the inhibition reached 50% in mice vaccinated with DC-sVEGFR-2 compared with mice vaccinated with DC, DC-vector or PBS. Anti-metastatic experiment showed that profound reduction in pulmonary metastases was found in mice immunized with DC-sVEGFR-2, while mice immunized with PBS, DC, DC-vector developed extensive pulmonary metastases. The number of tumor nodules on lung surface decreased by 81.9% in mice immunized with DC-sVEGFR-2 when compared with mice immunized with DC-vector (49.7+/-12.7 vs. 9.0+/-3.2). In vivo T cell subset depletion experiments showed that the anti-metastatic effect of DC-sVEGFR-2 vaccination was abrogated in CD8 + T cell-depleted but not in CD4+ T cell-depleted mice.

CONCLUSIONImmunization of mice with DC-sVEGFR-2 could break self-tolerance and induce a significant CTL response to VEGFR-2, leading to profound inhibition of tumor-cell induced angiogenesis and metastasis. This anti-metastatic effect is mainly mediated by CD8+ T cells.

Animals ; CD8-Positive T-Lymphocytes ; immunology ; Cancer Vaccines ; immunology ; Carcinoma, Lewis Lung ; blood supply ; immunology ; pathology ; Cell Line, Tumor ; Dendritic Cells ; immunology ; metabolism ; Electroporation ; Female ; Immunotherapy, Adoptive ; methods ; Lung Neoplasms ; secondary ; therapy ; Mice ; Mice, Inbred C57BL ; Neovascularization, Pathologic ; immunology ; therapy ; T-Lymphocytes, Cytotoxic ; immunology ; Vascular Endothelial Growth Factor Receptor-2 ; biosynthesis ; genetics

BACKGROUNDGene-radiotherapy, the combination of gene therapy and radiation therapy, is a new paradigm for cancer treatment. To enhance anti-tumor effect of gene-radiotherapy, in this study we construct a radiation-inducible dual-gene co-expression vector pEgr-interferon (IFN)-gamma-endostatin and studied the anti-tumor effect of pEgr-IFN-gamma-endostatin gene-radiotherapy in mice bearing Lewis lung carcinoma and its mechanism.

METHODSGene recombinant technique was used to construct dual-gene co-expression plasmid pEgr-IFN-gamma-endostatin, and single-gene expression plasmid pEgr-IFN-gamma and pEgr-endostatin. The plasmids packed by liposome were injected locally into the tumors of the mice, and the tumors were irradiated with 5 Gy X-ray 36 hours later. The tumor growth rate at different time and mean survival period of the mice were observed. Cytotoxic activity of splenic cytotoxic T-lymphocyte (CTL), natural killer (NK) cell and tumor necrosis factor (TNF)-alpha secretion activity of peritoneal macrophages of the mice in various groups were evaluated 15 days after irradiation. The intratumor micro-vessel density was evaluated by immunohistochemical staining 10 days after irradiation.

RESULTSThe tumor growth rate of the mice in dual-gene-radiotherapy group was significantly lower than those in control group, 5 Gy group and single-gene-radiotherapy group at different time after gene-radiotherapy, and the mean survival period of which was longer. Cytotoxic activity of splenic CTL, NK and TNF-alpha secretion activity of peritoneal macrophages of the mice in dual-gene-radiotherapy group were significantly higher than those in control group, 5 Gy X-ray irradiation group and pEgr-endostatin gene-radiotherapy group 15 days after irradiation. The intratumor micro-vessel density of the mice in dual-gene-radiotherapy group was significantly lower than those in control group, 5 Gy X-ray irradiation group and pEgr-IFN-gammagene-radiotherapy group.

CONCLUSIONThe anti-tumor effect of dual-gene-radiotherapy was significantly better than that of single-gene-radiotherapy by combining the enhancement of anti-tumor immunologic function induced by IFN-gamma with the anti-angiogenesis function of endostatin.

Animals ; Carcinoma, Lewis Lung ; blood supply ; immunology ; therapy ; Cell Line, Tumor ; Combined Modality Therapy ; DNA-Binding Proteins ; genetics ; Early Growth Response Protein 1 ; Endostatins ; genetics ; Female ; Genetic Therapy ; Immediate-Early Proteins ; genetics ; Interferon-gamma ; genetics ; Mice ; Mice, Inbred C57BL ; Neovascularization, Pathologic ; therapy ; Plasmids ; Transcription Factors ; genetics ; X-Ray Therapy