OBJECTIVETo observe the changes in the activity of dendritic cells (DCs) after carcino-embryonic antigen (CEA) gene transfection mediated by recombinant adeno-associated virus type2 (rAAV) and tumor cell lysate.

METHODSImmature DCs isolated from peripheral blood monocytes of HLA-A11-positive healthy volunteers were infected with the rAAV carrying CEA gene or loaded with tumor cell lysate. The surface markers of the DCs such as CD40, CD 1alpha, and CD86 were analyzed by flow cytometry. Interleukin-12 (IL-12) in the supernatants of DCs and interferon-gamma (IFN-gamma) released by the cytotoxic T lymphocytes (CTLs) were determined by ELISA detection kit. The specific killing activity of CTL against LoVo cells was assessed by MTT assay.

RESULTSThe DCs following antigen loading with the two methods both highly expressed CD40, CD86 and IL-12, and induced specific CTL that specifically recognized and killed LoVo cells, but the killing effect resulting from rAAV infection of the DCs was much better than that induced by tumor cell lysate loading.

CONCLUSIONBoth methods of antigen loading can induce mature DCs from peripheral blood monocyte cells, but rAAV infection of the DCs can be more effective than tumor cells lysate loading. DCs infected with rAAV may have the potential to serve as an adjuvant immunotherapy for patients with colorectal carcinoma.

B7-2 Antigen ; metabolism ; CD40 Antigens ; metabolism ; Cancer Vaccines ; biosynthesis ; immunology ; Carcinoembryonic Antigen ; genetics ; Cell Line, Tumor ; Colorectal Neoplasms ; therapy ; Dendritic Cells ; immunology ; metabolism ; Dependovirus ; genetics ; Genetic Vectors ; Humans ; Interleukin-12 ; metabolism ; Transfection

OBJECTIVETo study the feasibility of preparing a therapeutic lung cancer vaccine by transfecting dendritic cells (DCs) with adeno-associated virus vector carrying carcino-embryonic antigen gene (rAAV/CEA).

METHODSAdherent cells (monocytes) isolated from the peripheral blood of a healthy donor were infected with rAAV/CEA virus stock or pulsed with CEA peptide (control). The monocytes in both groups were induced into mature DCs with recombinant human GM-CSF, IL-4 and TNF-α. At day 7 of induction, the mature DCs were harvested and mixed with T lymphocytes. T cell proliferation stimulated by the DCs was assessed with (3)H-thymidine uptake, and the expression of IL-4, IFN-γ, CD8, CD4, CD25 and CD69 in cytotoxic T lymphocytes (CTL) was analyzed with flow cytometry. The cytotoxicity of the CTL against the target CEA-positive lung cancer A549 cells was tested by (51)Cr releasing assay.

RESULTSThe DCs transfected with rAAV/CEA strongly stimulated the proliferation of the T cell populations, and the induced CTL showed high expressions of CD8, CD69 and IFN-γ. The transfected DCs exhibited a high killing ability of CEA-positive lung cancer cells, and the killing showed a CEA antigen specificity and was limited by MHC I. These results suggested the ability of rAAV/CEA-transfected DCs in generating specific cellular immunity in vitro.

CONCLUSIONIt is feasible to prepare therapeutic lung cancer vaccines by transfecting DCs with rAAV/CEA.

Cancer Vaccines ; Carcinoembryonic Antigen ; genetics ; Cell Line ; Dendritic Cells ; immunology ; Dependovirus ; genetics ; Genetic Vectors ; Humans ; Monocytes ; immunology ; Transfection

OBJECTIVETo study the feasibility of transfecting breast cancer BA46 gene into dendritic cells (DCs) using adeno-associated virus (AAV) to induce specific cellular immunity.

METHODSMononuclear cells (DC precursor) were isolated from the peripheral blood of healthy donors by density gradient centrifugation and infected with rAAV/BA46/Neo virus stock (transfection group) or pulsed with 293 cell lysate (control group). In both groups, maturation of the DC precursor was induced by granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4) and tumor necrosis factor-alpha(TNF-alpha). On day 7, the DCs were collected and mixed with T cells at the ratio of 1 to 20 to induce cytotoxic T lymphocytes (CTL). The capacity of the DCs in stimulating T lymphocyte proliferation was assessed using (3)H-thymidine incorporation assay. The expressions of interferon-gamma (IFN-gamma), IL-4, CD4, CD8, CD25 and CD69 in the CTLs were analyzed with cytometry, and the cytotoxicity of the CTLs was evaluated with (51)Cr-release assay using BA46-positive breast cancer cell line Hs578T as the target.

RESULTSThe DCs transfected with BA46 gene exhibited potent capacity to stimulate T lymphocyte proliferation. The CTL population induced by the transfected DCs expressed high levels of CD8, CD69 and IFN-gamma, and showed strong cytotoxicity against BA46-positive breast cancer cell line Hs578T, which was BA46 antigen-specific and MHC-limited.

CONCLUSIONThe success in BA46 gene transfer in the DCs that induce specific cellular immunity provides the experimental basis for breast cancer immunotherapy using genetically modified cells.

Antigens, Surface ; genetics ; metabolism ; Breast Neoplasms ; genetics ; immunology ; Cells, Cultured ; Dendritic Cells ; immunology ; metabolism ; Dependovirus ; genetics ; metabolism ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Immunity, Cellular ; Immunotherapy ; Interleukin-4 ; pharmacology ; Milk Proteins ; genetics ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection