The Senescent Tumor Cell Vaccine Overexpressing IL-12 Enhance T Cell Homeostatic Proliferation-driven Anti-Tumor Immunity

BackgroundHigh-dose radiotherapy and chemotherapy can decrease the number of peripheral T lymphocytes in patients.After that,T cells can recognize autoantigen(including tumor-associated antigen)and undergo homeostatic proliferation.Homeostatically proliferating T cells have a powerful ability to recognize and kill cancer cells.However,in order to prevent autoimmune diseases,these T-cell are induced hyporesponsiveness in the later stage of homeostatic proliferation-driven antitumor responses by negative immune regulatory function.Because of that,few patients enhance the antitumor immune responses after high-dose radiotherapy and chemotherapy.Interleukin 12(IL-12)bridge innate resistance and antigen-specific adaptive immunity and can maintain function of autoreactive cells.In the present study,we intent to keep the antitumor function of homeostatically proliferating T cells and protect them from negative immune regulatory function by IL-12 stimulation.On the other hand,senescence tumor cells are in a state of permanent growth-arrest and still has the characteristics of secretory function.If we induce the tumor cell senescence and make senescence tumor cells to over-express IL-12,it will be an effective vaccine which can activate the homeostatically proliferating T cells and enhance the homeostatic proliferation-driven anti-tumor immunity.Our research would provide a new strategy to amplify anti-tumor response and prevent tumor recurrence after chemoradiotherapyMethods and Materials1.The establishment of IL-12 overexpressing B16 cell line:B16 cell were infected with IL-12 expression lentiviral vector.Using inverted fluorescence microscope to observe the infection efficiency.The mRNA expression of IL-12 was detected by qRT-PCR,and cell supernatants were collected for protein expression levels by ELISA.2.Detection of cell characteristics after lentiviral infection:EdU,plate cloning experiments and scratch-repair assay were used to measure proliferative ability and migration of B16/IL-12、B16/NC and B16 cell.3.The effect of interleukin-12 on antitumor immune function in mice with reconstitution of the lymphopenia environment was detected:We use 6.5Gy irradiation to induce lymphopenia in mice.After irradiation,C57BL/6 mice were received splenic T lymphocyte from another healthy C57BL/6 mice by tail intravenous injection and injected s.c.with B16/IL-12,B16/NC cells.The proportion of T cells with in tumor-draining lymph nodes were detected by flow cytometry.Tumor occurrence and survival were analyzed in mice.4.The senescence-associated secretory phenotype and tumorigenicity of senescence tumor cells were detected:B16/IL-12 and B16/NC cells were induced senescence by exposure to radiation and incubation in medium containing Veliparib.Cells were harvested for senescence test by SA-β-gal staining.Supernatants was collected from senescent tumor cell for IL-10,IL-12,IFN-γ,TNF-α,TGF-β measurements.The tumorigenicity was detected in the model of lymphopenia mice.5.The senescent tumor cell vaccine overexpressing IL-12 affect antitumor immunity in mice with reconstitution of the lymphopenia environment:The lymphopenia mice were injected the senescent tumor cell vaccine on the right back and then injected B16 cell in left back.The proportion of T cells with in tumor-draining lymph nodes were detected by flow cytometry.Tumor occurrence and survival were analyzed in mice.Conclusions:The senescent tumor cell vaccine overexpressing IL-12 protected homeostatically proliferating T cells from negative immune regulatory function in tumor microenvironment and enhance T cell homeostatic proliferation-driven anti-tumor immunity.