Study Of The Adoptive Immunotherapy On Breast Cancer With Dendritic Cells Loaded With Tumor Antigen

Study of the Adoptive Immunotherapy on Breast Cancer with Dendritic Cells Loaded with Tumor AntigenFan Ping (student), Wu Zhengyan (tutor)ObjectiveDendritic cells are important antigen presenting cells in human being. They can specifically present antigen to T lymphocytes and initiate specific antitumor immunity reaction in vitro and in vivo. Their remarkable effectiveness is in large part due to their efficiency in capturing, processing, and presenting antigens along with costimulatory signals. Therefore, dendritic cells have become one of the hot spots in the field of tumor immuno-adoptive therapy since 90s. In order to determine immuno-status of breast cancer patients in this paper, we study the changes of immunocytes such as T lymphocytes, B lymphocytes, and natural killer ect. in the peripheral blood of breast cancer patients. We also study the molecules such as CD80, CD86, and MHC ect. expressing on the breast cancer cell lines and primary breast cancer cells. We expect that tumor antigen loaded dendritic cells could make up for the immunodeficiency of breast cancer patients. For this purpose, we first research on adoptive immunotherapy to breast cancer animal model using tumor antigen loaded dendritic cells.Materials and Methods1.We selected 30 admission patients with breast cancer between March and October in 2001 at random . Using flow cytometery analysis, we examined CD3, CD4, CD8, CD 16, CD 19 and CD28 molecules expressing on the lymphocytes and CD40, CD80 and CD86 molecules expressing on the monocytes in the peripheral blood samples from breast cancer patients.2.We detected these molecules expressing on the five breast cancer cell lines using flow cytometery analysis. The cell lines include MCF-7, SK-BR-3, T47D, MDA-MB-435s, and ZR-75-30. The expression levels were compared with that of normal mammary cell line HBL-100 . At the same time, we examined those molecules expressing on the 35 primary breast cancer cells and compared with that of benign breast diseases.3.Fifty cases peripheral blood were drawn from 20 healthy adult persons and 30 breast cancer patients. Every person was drawn 10 milliliter blood and the blood was anticoagulated by heparin (5unit per milliliter). We use Ficoll Hypaque centrifugation to isolate the mononuclear cells in the peripheral blood. These cells are plated in six-well culture plates(106/ml,2ml/well) in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 100ng/ml GM-CSF, 20ng/ml IL-4, and/or 20ng/ml TNF- a .After 2 hours, nonadherent cells were gently removed and fresh medium was added. Culture medium was refreshed every other day. Cultured cells were taken a picture using focus scanning microscope (Zeiss LSM510)and analyzed by flow cytometry with labeled monoclonal antibodies. 4.Under sterile condition, lower limbers of Balb/c mice were cut with scissors. In the dish, the marrow was flushed out using 2ml of RPMI 1640 with a syringe. The tissue was suspended and red cells were lysed with 0.8% ammonium chloride. After washed twice with PBS, these cells are plated in six-well culture plates(106/ml,2ml/well) in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 3.3ng/ml GM-CSF, 20ng/ml IL-4. Three days later, the cultures were fed by gently swirling the plates, aspirating all of the medium, and adding back fresh medium with the same concentration cytokines as before. An object of this wash was to remove nonadherent granulocytes. On 6th day, the aggregates of loosely attaching to culture plate cells were dislodged with pipettes. Those cells were removed to a new culture plate and cultured 24 hours. Cultured cells were analyzed by flow cytometry with labeled monoclonal antibodies, cytokines secretion was detected with RT-PCR, and immunological activity was searched with mixed lymphocytes reaction.5. Female Balb/c mice were 4-6 weeks old. Breast cancer cell line TM40D derived from Balb/c mice. Digested by 0.25% trypsin , washed twice with PBS, exponentially growing TM40D cells (...