Role Of Vaccinia Virus-specific Memory T Cells In Vaccinia Virus Therapy Of Pancreatic Cancer In Mice

Backgrounds and aimsPancreatic cancer is one of the most malignant tumors with a very poor prognosis with a 5-year survival rate of less than 10%.In recent years,the incidence of pancreatic cancer has been on the rise.This situation means that a new treatment strategy that does not produce cross-resistance with traditional therapies is required.Vaccinia virus has become a therapeutic agent with strong potential to treat pancreatic cancer due to its inherent ability to specifically target and dissolve tumor cells and induce anti-tumor effects.Vaccinia virus(VV)can replicate in cancer cells,but cannot replicate in normal cells,leading to massive tumor lysis.In addition to this main effect,VV can also stimulate the immune system.The tumor microenvironment is an immunosuppressive environment in which the immune system is silent to avoid the body’s immune response to cancer cells.The entry of VV into the tumor can awaken the immune system and enable it to produce a strong and long-lasting immune response to the tumor.Among them,innate and adaptive immune response can promote the body to produce an immune response against tumor antigens and retain immune memory.For a pancreatic cancer,the level of tumor infiltrating lymphocytes(TIL),especially the level of CD8+T cells,has a strong correlation with the overall survival of the patient.We usually think that all T cells in tumors are TAA-specific,and we know very little about how viral infections affect the TIL population.In recent years,more and more studies have confirmed that these virus-specific T cells for virus infection can extend their surveillance range to tumors,thereby changing the prognosis of tumors.After injecting viral polypeptides into tumors,these antiviral memory T cells can be reactivated,and these virus-specific T cells can kill tumor cells.Vaccinia virus is an oncolytic virus that can be cleared by the host.We consider the role of memory T cells targeting vaccinia virus in vaccinia virus treatment of pancreatic cancer.Therefore,we studied the effect of vaccinia virus pre-immunization on vaccinia virus treatment of pancreatic cancer in mice and related mechanisms.Methods1.We evaluated the anti-tumor efficacy of vaccinia virus(VVLΔTK N1L)pre-immunization in a mouse model of subcutaneous pancreatic cancer.In this study,mice in the experimental group were pre-immunized with VVLΔTK N1L 1×10~6pfu,and then boosted again two weeks later.The control group was injected intramuscularly with PBS.Two weeks after the second pre-immunization of the experimental group,the experimental group and the control group were subcutaneously inoculated with DT6606 cell line(mouse pancreatic cancer cell line).When the subcutaneous tumor volume reached 80-100mm~3,the experimental group and In the control group,VVLΔTK N1L 1×10~8pfu was injected intratumorally for treatment,and intratumor injection was continued for 5 days.After the treatment,the tumor volume was measured every 3 days,and the tumor growth curve was drawn to explore whether VV immunization in advance is helpful for tumor treatment.And take mouse pancreatic carcinoma subcutaneous tumor,use immunohistochemical staining and flow cytometry to study the relevant treatment mechanism.2.Detect the antigen peptide with the strongest immunogenicity of vaccinia virus,and construct the DT6606 cell line stably expressing the strongest antigen peptide of VV.Take the spleens of C57 mice in the VVLΔTK N1L pre-immunization group and mice in the PBS group,collect spleen cells,make IFN-r in vitro,and label the stimulating peptide to verify the strongest antigen peptide of vaccinia virus.After verifying that the strongest antigenic peptide of vaccinia virus is B8R,the second-generation lentiviral packaging system was used to construct a DT6606 cell line stably expressing B8R,and then the optimal concentration of puromycin was used to screen positive cells stably expressing B8R.Real-time fluorescent quantitative PCR experiment identified the expression of B8R at the m RNA level,and obtained the DT6606 cell line with stable expression of B8R.The transformed stable cell line was named DT6606-B8R.Use Incu Cyte Zoom to dynamically monitor the proliferation of B8R-DT6606 and WT-DT6606 cells.3.Explore whether the therapeutic effect of VV early immunization on tumors is caused by VV-specific memory T cells.The mice in the VVLΔTK N1L immunization group and the PBS group were subcutaneously inoculated with DT6606-B8R cells,the tumor volume was measured regularly,the growth curve was drawn,and the relevant treatment mechanism was studied by immunohistochemical staining and flow cytometry experiments.Result1.The growth rate of subcutaneous tumors in the VVLΔTK N1L pre-immunization group was significantly slower than that of the PBS control group DT6606(P<0.01);the percentage of complete disappearance of DT6606subcutaneous tumors in the VVLΔTK N1L pre-immunization group was significantly higher than that in the control group(P<0.01).It is verified that VV immunization in advance contributes to the therapeutic effect of VV on mouse pancreatic cancer.2.Immunohistochemistry and flow cytometry have verified that VV pre-immunization followed by intratumoral injection of VV can induce more CD4and CD8 infiltrating lymphocytes inside the tumor than the PBS control group.3.The strongest immunogenic peptide for detecting VV is B8R,and DT6606cells stably expressing B8R have been constructed system.Real-time fluorescent quantitative PCR showed that the expression of B8R at the m RNA level was significantly increased,that is,the stable transgenic cell line DT6606-B8R was successfully constructed.4.Under VVLΔTK N1L pre-immunization,the growth rate of DT6606-B8R tumors was slower than that of DT6606-WT cells(P<0.05);the growth rate of DT6606-B8R cells in the VV pre-immunized mice group was slower than that of the PBS group(P<0.05)).5.Flow cytometry test verified that the proportion of B8R~+T cells infiltrating the tumor in the VV preimmunization group was higher in all infiltrating CD8~+T cells than in the PBS control group,thus verifying the reason why VV early immunization contributes to VV Treatment of tumors is related to stimulating the body to produce memory T cells targeting VV.ConclusionThese results suggest that VV advance immunization is beneficial to the treatment of pancreatic cancer in mice,which is related to inducing more CD4~+、CD8~+、Vaccina specific-CD8~+lymphocytes to infiltrate the tumor environment.