The Study On Anti-colon Cancer Effect Of CTL Cells After Knocking Out The Immune Checkpoint Of CTLA-4

The morbidity and mortality of colon cancer ranks very high world widely.In our country,the morbidity of colon cancer keep increasing in recent years and is in the top 10 in terms of malignant cancer.At early stage of colon cancer,there may be no any symptoms,but when the symptoms such as bloating,dyspepsia,changing of the excrement character and bowel habits,the colon cancer usually get into middle or advanced stage,in hence,the difficulties in the treatment of colon cancer will increase.The conventional therapeutic methods for colon cancer are surgery and chemotherapy.The survival rate of the patients with stage IV cancer is slightly exceeded 10%.However,the rapid developing of the technology of immunotherapy brings the opportunity to the treatment of cancer.Our research is aim to increase the effect of the treatment of colon cancer with cellular immunotherapy.In recent years,the research on molecular immunology reveal the complicated mechanism of the regulation of cellular immune response in tumor immune microenvironment.They discovered that the inhibitory of immune checkpoint can initiate the immune response in anti-tumor T cells and produce longer-lasting immune response.The monoclonal antibody Ipilimumab to immune checkpoint CTLA-4 was appoved by FDA for the treatment of metastatic melanoma?lung cancer etc.We used the technology of CRISPR/Cas9 to knock out the immune checkpoint CTLA-4 of CTL,and focused on it is anti-tumor effect in vitro and in vivo.In the first part of the experiment,we designed CTLA-4-specific sgRNA and construct the plasmid of sgRNA/Cas9.The plasmid was transfected into CTL with lentivirus.After the CTLA-4 knocked out CTL cells(CTLA-4 KO CTL)were obtained,the transfection efficiency of the plasmid and the deleting efficiency of CTLA-4 were verified.In the second part of the experiment,the anti-tumor effect of CTLA-4 KO CTL on the targeted colon cancer HCT116 cell lines were assessed in vitro,as well as the effect on apoptosis.Meanwhile,the secretion of the TNF-? and IFN-?in the HCT116 cells after treatment was detected.The good results of the experiments in vitro are the basis of experiments in vivo,but the differences between the environment in vitro and in vivo may have distinct effects on the function of CTLA-4 KO CTL.Therefore,in the third part of the experiment,humanization NOD/SCID mice model was established to mimic the in vivo environment of cancer patients.After establishing the colon cancer xenograft mice model,CTLA-4 KO CTL treatment was administrated.The volume of the tumor and the lifetime of the mice were observed to evaluate the anti-tumor effect of the treatment in vivo.Part one Construct the LentiCRISPR-vector to transfect CTL cells for knocking out the immune checkpoint of CTLA-4Objective: Using CRSIPR/Cas9 knock out the immune checkpoint of CTLA-4 from CTL cells.To verify the transfection efficiency and deletion efficiency of CRSIPR/Cas9.Methods: CTLA-4-specific sgRNA was designed and the plasmid of sgRNA/Cas9 was constructed.The plasmid was packaged using lentivirus,and then verified the transfection efficiency and deleting efficiency of CTLA-4.Results:1.The sgRNA sequences were designed and the LentiCRISPR-vector was constructed successfully.2.CTL cells were transfected by using CRSIPR/Cas9 and the rate of transfection efficiency was(28.80±0.62)% detected by flow cytometry.3.Western blot and flow cytometry were used to detect the expression of CTLA-4 in CTL cells after CTLA-4 knockout.The results showed that the expression of CTLA-4 in CTLA-4 KO CTL group was significantly lower than that in CTL group(P<0.05).4.The nuclefection had temporary influence to the T cell proliferation,and the proliferation activity could recover when cultured with cytokines.Summary: LentiCRISPR-vector was constructed successfully.The system could efficiently transfect the CTL cells.CRSIPR/Cas9 could knock out the gene of CTLA-4 result in the expression of CTLA-4 decreasing significantly.Part two The study on anti-colon cancer effect of CTL cells after knocking out the immune checkpoint of CTLA-4 in vitroObjective: To observe the anti-tumor effect of CTLA-4 KO CTL cells in vitro.Methods: CTLA-4 KO CTL and CTL were co-cultured with colon cancer HCT116 respectively,and then the anti-tumor effect of the two groups of cells were detected by WST-1 method.The apoptosis of the tumor cell was analyzed by Annexin V/PI of flow cytometry.The expression of apoptotic protein in tumor cells was assessed by Western Blot method.Caspase activity kit assay was used to detect the activity of tumor cell apoptosis protein.ELISA was used to detect the secretion of cytokines TNF-? and IFN-?.Results:1.CTLA-4 KO CTL significantly increased the cytotoxicity of colon cancer cells compared with CTL(P<0.05).2.CTLA-4 KO CTL significantly increased the apoptosis rate of colon cancer cells compared with CTL(P<0.05).3.Compared with CTL,CTLA-4 KO CTL significantly increased the expression of apoptotic protein and the activity of apoptotic proteins in colon cancer cells(P<0.05).4.Compared with CTL,CTLA-4 KO CTL significantly increased the secretion of TNF-? and IFN-?(P<0.05).Summary: After the knockout of CTLA-4,the anti-tumor and apoptosis-induced activity of CTL was elevated,and the secretion of TNF-? and IFN-?increased.Part three The study on anti-colon cancer effect of CTL cells after knocking out the immune checkpoint of CTLA-4 in vivoObjective: To investigate the anti-tumor effect of CTLA-4 KO CTL cells to the colon cancer graft mouse in vivo.Methods: To build humanized mouse models with the NOD/SCID mouse by the means of tail vein injection of human peripheral blood mononuclear cells(PBMC).Flow cytometry was used to detect the expression of human CD3+T cells and human CD19+B lymphocytes in peripheral blood of mice.The colon cancer xenograft model was established by using humanization NOD/SCID mice.After the tumor was formed,mice were split into two groups,and administrated with CTLA-4 KO CTL(experimental group)and CTL(control group)treatment by mouse tail vein re-injection,the volume of the tumor?the lifetime of the mice and the secretion of TNF-?and IFN-? in mice were observed.Results:1.The expression of human CD3+ T lymphocytes and CD19+ B lymphocytes was detected in the peripheral blood of humanized NOD/SCID mice and the expression ratio was at normal adult level which indicating successful humanization.2.The tumor volume of mice in the CTLA-4 KO CTL group was significantly inhibited than the CTL group(P<0.05).3.The survival time of mice in the CTLA-4 KO CTL group was significantly prolonged than that the CTL group(P<0.05).4.Compared with CTL,CTLA-4 KO CTL significantly increased the secretion of TNF-?and IFN-? in mice(P<0.05).Summary: Humanized mouse models with the NOD/SCID mouse by the means of tail vein injection of human peripheral blood mononuclear cells were successful builthumanized,on which the colon cancer xenograft model was established successfully.CTLA-4 KO CTL could reduce the volume of tumor and prolong the lifetime of tumor-bearing mice significantly and increase the secretion of TNF-?and IFN-? in mice.Conclusion: The Lentiviral-CRISPR vector is an effective gene editing method and knocked out the immune checkpoint of CTLA-4 in CTL cells successfully.CTLA-4 KO CTL cells can significantly enhance the anti-tumor effect of CTL on colon cancer.