Study On Tumor-Killing Cells Induced By Tumor Soluble Antigen And Staphylococcus Enterotoxin Superantigen

Tumor antigen expressed by tumor cells is the key to inducing immune response to tumor,and the base of recognizing and activating the immune system. But most of tumor antigens have weak immunogenicity.The host immune response to tumor antigens leads to reduction or loss of surface antigen of tumor cells.Tumor cells are not recognized by the immune system, which can not induce effective anti-tumor immune response. Based on this understanding, the use of basic methods of enhancing immunogenicity of tumor antigens induce anti-tumor immune responses in the body in order to achieve the purpose of reducing and eliminating tumors. In recent years, there are many ways to improve the immunogenicity of tumor antigens, such as the use of adjuvants, mitogen, superantigen and so on. Staphylococcus enterotoxin (SE) is a highly biologically active proteins, is currently studied as bacterial superantigens. SE can be directly related to the outside the antigen binding groove of MHC-Ⅱmolecules of APC membrane,without processing of antigen presenting cells (APC). Tthe complete form of protein molecules is presented onto T cells to activate T cells. The aquantity of the cells activated by SE is thousands times more than that of the common antigen. SE have many immunological activity, including stimulation of a large number of T cells, and activation of NK cells, and promotion of a large number of cytokine secretion, such as: interleukin-2, interferon, tumor necrosis factor, and even colony stimulating factor. SE is the strongest stimulant of human lymphocytes and the most powerful inducer of cytokines at present, which may directly or indirectly kill and inhibit tumor cells.ObjectiveBased on improving the immunogenicity of tumor antigen and targeting of superantigen, superantigen SEC and tumor soluble antigen were used to jointly act on T lymphocyte cells to induce the killing cells for us to explore the new cell immunotherapy on tumor.Methods1. Preparation of Peripheral Blood Lymphocytes:lymphocytes were isolated by density gradient centrifugation through Ficoll-Hypaque from Peripheral blood of normal donors. The cells were washed for 3 times and then incubated 60min in 37℃, 5% CO2 incubator. Non-adherent cells were collected as peripheral blood lymphocytes.2. Preparation of Soluble Tumor Antigen:Esophageal Eca109 cells were cultured. In the phase of logarithmic growth, the cells were taken out and made up in suspension of 10~7 -10~8 cells /ml, freezed and thawed for 4 times under the conditions of -80℃/ 37℃. centrifuged for 40min under the condition of 5000r/min. The supernatant was collected as the tumor soluble antigen (TSA). Concentration of protein were examined by Bradford method. TSA was filted with 0.22μm of membrane filter to eliminate microbials, then placed in -20℃of freezing.3. Proliferation of the Cells Stimulated by SEC and TSA :The cell concentration was adjusted to 10~6/ml with complete RPMI-1640 medium. The cells were inoculated into flat-bottomed culture plate of 96-wells. And then same volume of complete RPMI1640 culture medium were added with different concentrations of SEA or TSA, each with three duplicate wells, while the negative control group were set (no SEC,TSA). Proliferation of the lymphocytes were determined by CCK-8 at 24h, 48h, 72h, 96h. Optimal stimulation concentration by the SEC / TSA were determine. Group A containe lymphocetes(L); group B L + SEC; group C L + TSA; group D L + SEC + TSA. Lymphocyte proliferation of experimental groups were determined by CCK-8 at 24h, 48h, 72h, 96h.4. Analysis of Cell Phenotype by Flow Cytometry:According to the above experimental groups, the cell concentration was adjust to 10~6/ml with complete RPMI-1640 medium, then inoculated into 6-wells plate, 4-wells in per group. After 3 days, the cells were collected and were added with FITC labeled CD19/CD3/CD4/CD8 to each of the wells, tested by flow cytometry.5. Test of the Levels of TNF-αin Cell Culture Supernatant:The cell concentration was adjust to 10~6/ml with complete RPMI-1640 medium, according to experimental groups inoculated in 24-wells of plate, and then added with the same volume of complete RPMI1640 culture medium, containing different concentrations of SEA (0.1ng/ml)or TSA (50μg / ml), each with three duplicate wells. Supernatants of each group were collected at the 1d, 3d, 5d, and tested with ELISA kit.6. Test of Cytotoxic Activity:Esophageal Eca109 and cervical Hela cells were taken as target cells and the cells in experiment groups taken as effector cells. Effect / target is 20:1. Cells were stimulated by esophageal TSA with superantigen SEC as effector cells. After 24h culture, killing rates of each group were measured by CCK-8 method.7. Test of Apoptosis by Wright Staining:Experiments was divided into control group (only cells) and experimental group (SEC + TSA group),cultured 3 days in vitro. The cells were adjusted to the concentration to 1×10~6/ml. Eca109 cells were collected (1×10~5/ml), placed in six-well plate with coverslips, cultured after 1 day, mixed with the control group and experimental group cells. After 24h, the cells were 8. washed with PBS for three times to remove suspended cells and non-adherent tumor cells. The cells on the cover- slips were stained and examined under microscopy.Results1. Proliferation of peripheral blood lymphocytes stimulated by different concentrations of SEC / TSA are different in vitro. proliferation of peripheral blood lymphocyte was the most significant when the concentration of SEC was 0.1ng/ml and TSA was 50μg/ml. Within the 96h, the lymphocyte proliferation is differences in the different time points.in the time between 24h-72h, Lymphocyte proliferation increased with the time increase in each group. The proliferation of experimental group cells were the most significant at 72h (P <0.05). Lymphocyte proliferation is different in each group in the same time. The group D stimulating by the esophageal TSA combined superantigen SEC was the highest proliferation activity compared with groupA, groupB, groupC and peaked at 72h.2. Phenotype analysis of the cells shown that: CD3 were positive in 4 groups, CD8 +T of the group A were 26.31%, but that of group B, group C, group D were significantly higher than that of the group A (P <0.05), CD8 + of group D was the highest( 39.19%), so indicating that the induced cells were CD8 + CTL cells.3. Compared with the group A, TNF-αin culture supernatant in groups B,groups C,groups D begun to increase on 1d, peaked on 3d. Among them, the group D stimulated by TSA and SEC was the significantly difference compared with the group A and group B(P <0.01).4. Cytotoxicity of CTLs induced by the esophageal TSA combined superantigen SEC was 97.36%, which significantly higher than that of group A( 63.04%). Cytotoxicity of effector cells induced by TSA of Eca109 was significantly higher than that of Hela, the difference was significant (P <0.05).5. When target cells and effecter cells were mixed for 3h, which shown a large number of lymphocyte adhere around tumor cells under the microscope. By Wright's stain, Normal nucleus stained dark blue or blue-purple, color uniformity, apoptotic cell pyknosis, fragmentation, staining became deeper, membrane shrinkage, appearance of apoptotic bodies。Conclusions1.SEC and TSA can improve the co-stimulatory signals to enhance lymphocyte activation and proliferation, which are CD8 + CTL.And can stimulate lymphocytes to secrete high levels of TNF-α.2. The CTL induced by TSA of esophageal Eca109 have selective and targeting effect on tumor cells.3. CTLs induced by the esophageal TSA with superantigen SEC can induce tumor cells to produce apoptotic bodies.