A Study On The Immunotherapy Of An Autologous Tumor-cell Vaccine To The Lewis Lung Carcinoma In C57BL/6 Mice

BackgroundImmunotherapy has always represented a very attractive fourth-modality therapeutic approach,especially in light of the many shortcomings of conventional surgery,radiation,and chemotherapies in the management of cancer. A major research effort amongst cancer immunologists during the latter half of the20th century was the identification and characterization of tumor specific molecules which are not typically present on the surface of neighboring normal cells, the so-called tumor antigens.Immune system can discriminate tumor antigen and make immune response to excluse it.The observations on animal model and tumor patients indicated that the immunologic rejection to tumor depends on tumor antigens-specific T lymphocytes.Therefore,the T lymphocytes were considered to be the ideal effect cell against tumor cells.T lymphocytes recognize tumor antigens by TCR interacted with HLA-antigen peptide compound.HLA-antigen peptide compound is composited with fragments derived from antigen protein and presented on specific HLA.Antigen protein expressed by tumor cell was cut to be 9-20 amino acids,that is antigen peptide.CD4~+T cells and CD8~+T cells are the most important cells that kill tumor cells.CD4~+T cells can reject tumor cells by recognization to the soluble antigen which secreted by tumor cells with the participation of APC and activation to B cells,macrophage cells,NK cells and CTLs.However,CD8~+T cells are the key cells that reject tumor.Cytotoxic T-lymphocyte can be activated through the recognization with MHCⅠ- antigen peptide.Then the CTLs proliferate and differentiate to be the CTLs with the activity killed tumor cells.The cancer vaccine approach to therapy is based on the notion that the immune system could possibly mount a rejection strength specific response against the neoplastically transformed cell conglomerate.Today,various approaches have been explored ranging from the use of irradiation inactivated whole-cell vaccines derived from both autologous and allogeneic tumors (even tumor cell lines),and genetically modified versions of such cellular vaccines which aim at correcting costimulatory dysfunction or altering the in situ humoral milieu to aid immune recognition and activation.The theoretical basis for all of these approaches is very well founded.Animal models,albeit highly artificial,have yielded promising results.Clinical trials in humans,however,have been somewhat disappointing.Although general immune activation directed against the target antigens contained within the cancer vaccine has been documented in most cases, reduction in tumor load has not been frequently observed,and tumor progression and metastasis usually ensue,possibly following a slightly extended period of remission.Whether tumor cell vaccine could provoke specific immunity response,and could the specific immunity response resolute tumor to raised survival rate? We carried out experiments to approach this question.Autologous vaccines have the advantages of containing all potentially relevant tumor-associated antigens and individual specific human leucocyte antigen.Furthermore,autologous vaccines have more safety and utility than allogeneic vaccines.We adopted C57BL/6 mice and Lewis lung carcinoma cell as our objects.Objective and purpose To approach the efficacy and feasibility of autologou-tumor-eell being a tumor vaccine and to supply evidence for Autologous tumor cell vaccines therapy.Materials and methodsⅠ.An empirical study on the immunogenicity of the Lewis lung carcinoma in C57BL/6 mice.Grouping in experiment:1.There were two groups in tumor forming experiment:experimental group(EG) and control group(CG),28 mice per group.2.There were two groups in detection of immunity response:tumor bearing group and normal group,10 mice per group.Empirical methods:A tumor-bearing C57BL/6 mice model was established.The tumor was removed at day 12 after inoculation with Lewis lung carcinoma cells for the first time.At the same time the mouse was again inoculated with Lewis lung carcinoma cells.We observed the time of tumor-formation,the size of tumor and the rate of tumor-formatiom.Then we did histological study on the tumor tissue formed at the first and second inoculation with the conventional pathologic technique.We assayed the transformation efficiency of the splenocytes of the two groups of mice with MTT chromatometry.We also assayed the subsets of the lymphocytes in peripheral blood of the two groups of mice(one group is normal,the other is tumor-bearing) with flow cytometry.Ⅱ.An empirical study on the efficacy of the immunotherapy to C57BL/6 mice with the Lewis lung carcinoma by autologou-tumor-cell vaccine.Grouping in experiment:There were three groups in experiment:LLC vaccine group,control group and normal group,10 mice per group.Experimentl methods:Three groups of mice(LLC vaccine group,control group and normal group) were administered intramuscular injection,totally 4 times.When the forth vaccine was given the mice were received injections of Lewis lung tumor tissue suspension at right axillary subcutaneously region at the same time.tumor formation was observed.Then we did histological study on the tumor tissue with the conventional pathologic technique.We assayed the subsets of the lymphocytes (CD4-T cell,CD8-T cell and CD19-B cell) in peripheral blood of the three groups of mice with flow cytometry.We assayed antibody IgG in the sura of the mice and assayed the transformation efficiency of the splenocytes of the three groups of mice with MTT chromatometry.We also observeExperimental resultⅠ.An empirical study on the immunogenicity of the Lewis lung carcinoma cell line in C57BL/6 mice.1.The rate of the tumor-formatiom is 100%when the C57BL/6 mice were inoculated the Lewis lung cancer cells for the first time.And the rate is 89.3%when the mice were inoculated for the second time.However,there is no significant difference between them(P=0.236).2.There is no significant histological difference between the first and second time formed tumor tissue and scanty infiltrated lymph-cells were observed in the tumor tissue.3.Contrasted with the normal group,the tumor-bearing group has no significant difference in the CD4~+T celI,CD8~+T cell and CD19~+B cell subsets of the lymphocytes in peripheral blood(P＞0.05).4.Contrasted with the normal group,the tumor-bearing group has no significant difference in efficiency of the splenocytes(P＞0.05).Ⅱ.An empirical study on efficacy of immunotherapy to the C57BL/6 mice with Lewis lung carcinoma by autologou-tumor-cell vaccine.1.Three groups of mice have the same rate of tumor-formation(100%).The time of tumor formation of LLC vaccine group,control group and normal group respectively are 8.7d,8.1d and 7.3d,and there is a significant difference among them(P=0.030).But the significant difference only exist between LLC vaccine group and normal group(P=0.009),there is no significant difference between LLC vaccine group and control group(P=0.236).The medium survival time of LLC vaccine group,control group and normal group respectively are 33d,33d and 36d,there is no significant difference among them(P=0.254).2.There is no significant histological difference among three groups of formed tumor tissue and scanty infiltrated lymph-cells were observed in the tumor tissue through the observation on the section stained by HE.3.There is no significant difference in efficiency of the splenocytes among the three groups of mice(P=0.063).The efficiency of the splenocytes of LLC group and control group are higher than that of normal group,but there is no significant difference.4.There is no significant difference in the CD4~+T cell and CD8~+T cell subsets of the lymphocytes in peripheral blood.However,there is significant difference in CD19~+B cell subsets of the lymphocytes in peripheral blood(P=0.000),the percentage of CD19~+B cell subset of normal group is highest,next LLC vaccine group,the last one control group.5.There is no significant difference in Cytotoxic activity of T lymphocyte to LLCs amoung three groups of mice(P=0.535).There is also no significant difference in Cytotoxic activity of T lymphocyte to 782 cells amoung three groups of mice(P=0.931).And there is also no significant difference between Cytotoxic activity ofT lymphocyte to LLCs and 782 cells(P=0.214).ConclusionsⅠ.The Lewis lung carcinoma cell line has low immunogenic properties in C57BL/6 mice.Ⅱ.The Lewis autologous tumor-cell vaccine can enhance B-cell but not T-cell immune responses and can not protect C57BL/6 mice from Lewis lung carcinoma cells challenge.