Preparation Ebv Specific CTL And Study Its Lethal Effect For EBV Positive Tumours

The Epstein-Barr virus is a gamma herpes virus that infected approximately90%of people by adulthood. It is the first human tumor virus recognized, and is involved in the oncogenesis of many diseases. Nasopharyngeal carcinoma and lymphoma are common malignant tumors, gradually formed by the participation and interaction of EBV latent infections, the environment and host genetic. They are complicated diseases which have a poor prognosis. Almost all of the nasopharyngeal carcinoma cells and majority lymphoma cells can detect the presence of EBV genome. Coding and expressing the Latent membrane protein1and2are involved in the occurrence、 development and metastasis of diseases and provided the specific antigens as a favorable condition for the adoptive cellular immunotherapy. Tumor specific cytotoxic T cell is the initial T cells activated by the antigen presenting cells which ingested and processed tumor specific antigen peptide under the coactions of IL-2in vitro. Because the low immunogenicity of LMP1、LMP2, it is a key to choose the suitable APC which can stable prepare abundant and activated CTL in the adoptive immunotherapy for two kinds of EBV positive tumors. ObjectiveThis study intends to use DC and LCLs as two kinds of APC stability prepare EBV specific CTL in vitro. To choose the optimal APC and provide the theoretical basis for further clinical research, a variety of methods used to detect the killing effect of EBV-CTL against EBV positive tumor cell. Methods 1. To culture T lymphocyte:The mononuclear cells were isolated from peripheral blood. After the stimulate of CD3%CD28monoclonal antibody, a large number of T lymphocytes were amplified under the action of IL-2; CCK8detected the influence of different culture conditions on cell proliferation and selected the optimal medium used in cell culture.2. The preparation of DC:In order to get mature DC, PBMC must be cultured with IL-4、GM-CSF and LMPs peptide. Detected the expression of cell surface molecules by Flow cytometry.3. To establish LCLs:PBMC cultured with the mixture of EBV virus supernatant and completely medium to induce the generation of LCLs. Detected the expression of cell surface molecules by Flow cytometry and amplified cell genome by RT-PCR.4. Inactivated LCLs by Mitomycin:CCK8detected the multiplication rate of LCLs which inactivated by different concentration mitomycin. Drawing the Proliferation curve.5. The Biological activities of EBV-CTL:As APC, mature DC and inactivated LCLs induced the T lymphocytes to generate EBV-CTL in vitro; CCK8、ELISA and flow cytometry analysised the ability of EBV-CTL to kill tumor cells and to secrete IFN-y.Results1. Analyzed the proliferation situation of T lymphocytes cultured with four kinds of condition:1640+10%FBS and GT-T551+2%plasma are two kinds of culture medium which proliferate cells80times in9days.2. The shape of prepared DC is irregular and there are obvious burr protrusions on the edge. Flow cytometry detected the increased expression of CD83, CD86. The expression quantity respectively is42.07%and45.09%. 3. Build LCLs Successfully, flow cytometry detected the height expression of cell surface molecules CD4、CD19、CD80、CD86。 respectively expressing quantity is97.23%,90.06%,85.97%and50.41%; LMP1and LMP2genes are successfully amplification by Rt-PCR.4. Cultured the LCLs which inactivated by100ug/ml or150ug/ml mitomycin, CCK-8detected the proliferation has been inhibited significantly. After24h, the reaction reached a steady state, cells stopped proliferate.5. DC-EBV-CTL kill nasopharyngeal carcinoma cells and lymphoma cells for24h in vitro, the killing rate respectively is77.15%±3.18%、58.72%±6.30%; at the same time, the amount of IFN-y secreted by DC-EBV-CTL is491.24%±23.80%pg/ml. The rate of LCLs-EBV-CTL destroyed nasopharyngeal carcinoma cells is72.72%±8.45%and75.6%for lymphoma cells. The proportion of LCLs-EBV-CTL which secretes IFN-y is25.04%, increased by12.3times.Conclusions1.1640+10%FBS and GT-T551+2%plasma are two kinds of culture medium that can promote cell proliferation. Choosed1640+10%FBS as a complete medium to culture T lymphocyte in the next experiment.2. Generated DC has the typical morphological characteristics and highly expressed CD83、CD86on cell surface. Such results declare that DC has been matured and owned the ability to present antigen.3. LCLs is Built successfully and highly expressed CD40. CD19. CD80. CD86on cell surface. Such results illustrate that cells derived from the B lymphocyte and owned the ability to present antigen; LMP1and LMP2genes amplified by RT-PCR proved that the transformation of cell is successful. LCLs can be used as the APC to present EBV specific antigen.4. This study generated DC-EBV-CTL and LCLs-EBV-CTL which have highly specific lethal effect for EBV positive nasopharyngeal carcinoma cells and lymphoma cells; when they faced second stimulus, a large number of IFN-y would be secreted which can affect the immune response indirectly.