Mechanism And Anlitumor Effect Of Combined Treatment With Gene Modification In Adoptive T Cell Therapy In Malignant Melanoma

Objective:To increase the immunogenicity of tumor vaccines and to break the body's immune tolerance to endogenous tumor antigens.Transduction of a heterologous protein gene into a tumor cell can increase the immunogenicity of the tumor cell.Although our previous studies showed that the immunoprotective effect induced by live vaccine in animal experiments,the live vaccine has a great limitation because it has the possibility of transplanting tumor in vivo.Therefore,this study aims to find a way to use of live tumor vaccines and to exert the efficacy of live tumor vaccines.Methods1?Construction of ESAT6 and IL-21 shuttle vectors.The inserts were confirmed by Sanger sequencing.2?Generation of recombinant adenoviral vector.First,an adenoviral backbone was prepared.The PAD simple vector was extracted by using the QiaGa-MaXi pretreatment kit.Then pStuth-ESAT-6 vector,pStuthTy-IL-21 vector and pStuthTel-ESAT-6IRES-IL-21 vector were added.3?The production of adenovirus:(1)Generation of primary adenovirus stock.A bottle of 293A cells was thawed from liquid nitrogen in a 37?.(2)Propagation of adenovirus.A bottle of 293A cells was thawed from liquid nitrogen in a 37?.Cells were transduced with 5 MOI primary adenovirus antigens.Place the virus in the medium and gently shake the dish to spread the virus evenly.The virus was then extracted and maintained for 2 to 4 days,until CPE appeared.(3)Purification of adenovirus.The virus supernatant was transferred to an ultracentrifuge tube and the adenovirus was concentrated by centrifugation and stored at-80?.4?Titration of adenovirus.The adenovirus prepared in this study was immunocytochemically titrated with anti-hexagonal antibody.Calculate infectious units(PFU)/ml for each well as follows:(?)5?Transduction of B16F10 cells with adenovirus.Different MOI were used to transduce B16F10 cells.After 48 hours,the expression of GFP was observed under fluorescence microscope.Western and blot were used to detect the expression of IL-21 and ESAT-6 protein in transfected cells.6?ESAT-6-T cell production:Mouse DC was isolated from dendritic cells extracted from rat spleen by kits.Adenovirus vector containing ESAT-6 transduced mouse DC cells.At the same time,T cells were isolated from mice.Finally,ESAT-6 loaded DC was used to start T cell test.The number of T cells and DC was calculated in proportion to 10:1 and ESAT-6 loaded DC mixed cells.All T cells were harvested for detection.7?ESAT-6-T cleaved target cells:ESAT-6,IL-21 or ESAT-6/IL-21 transduced B16F10 were used as target cells.ESAT-6 primer T cells were added in different proportions to observe the target cell lysis and the secretion of cytokines was determined by ELISA.8?Mouse melanoma cell line B16F10 was subcutaneously inoculated into C57BL/6 mice.IL-21 and ESAT-6 recombinant adenoviruses were injected into the tumor and ESAT-6-T cells were transfused into mice.The growth of tumor cells and the secretion of different cytokines were monitored daily.Serum levels of IL-2 and IFN-gamma were measured by ELISA.Results1?Immunocytochemical titration of the adenovirus prepared in this study was performed by using anti-hexanexin immunocytochemical method after purification with ultralong centrifuge tube.The titer of adenovirus was above 1010PFU/mL.2?Adenovirus vector expressing GFP was used to transduce B16F10 cell line to determine the optimal transduction of MOI.Adenovirus with MOI less than 20 failed to transduce B16F10 cells.When MOI reached 200,almost all B16F10 cells showed GFP expression.All downstream transduction experiments using MOI were 200.B16F10-ESAT-6?3?ESAT-6 modified T cells can effectively target recombinant B16F10 cells expressing ESAT-6,but have little killing effect on B16F10-IL-21 cells.4?When ESAT-6 modified T cells combined with target tumor cells,they could produce IL-2 and IFN-gamma,and the secretion levels of IL-2 and IFN-gamma increased with the increase of E:T ratio.5?.The levels of cytokines in serum of mice injected with recombinant adenovirus IL-21 and ESAT-6 were significantly higher than those of other mice.After injection of adenovirus,the secretion of IFN-gamma in serum continued to increase,while adenovirus carrying IL-21 or ESAT-6 stimulated a stronger immune response.After 22 days of intratumoral injection,serum IL-2 level decreased significantly.6?Injecting ESAT-6 modified T cells can inhibit the proliferation of tumor cells expressed by ESAT-6.Comparing the survival rates of mice,tumor cells grew rapidly in mice injected with GFP adenovirus.Compared with GFP-adenovirus-injected mice,tumor size is sl after intratumoral injection of interleukin-21(IL-21),while IL-21+ESAT-6 or ESAT-6 obviously led to increase slowly.Twenty-two days after intratumoral injection of GFP adenovirus,the mice gradually died.The mice receiving IL-21 or ESAT-6 recombinant adenovirus had longer survival time and slower tumor growth.ConclusionB16F10 cells transduced by heterologous ESAT-6 and IL-2 through adenovirus have high immunogenicity.It can effectively activate the specific anti-tumor immune response in vivo.Breaking the body's immune tolerance to tumor vaccines.At the same time,the adoptive and transfused ESAT-6 specific T cells were modified.In situ tumor modified by in situ gene modification,it play a better role in immunotherapy,.It will probably provide an advanced technology solution for melanoma immunotherapy.