Experimental Study Of γδT Cells And NKG2D Chimeric Antigen Receptor T Cells Immunotherapy Against Hepatocellular Carcinoma

Part Ⅰ Effect of γδT cells infiltration on prognosis of hepatocellular carcinomaObjective: To investigate the relationship between the distribution and abundance of infiltrating γδT cells and the overall survival of hepatocellular carcinoma(HCC).Methods: The expression profiles of γδT cells characteristic genes were extracted from the TCGA database and the corresponding clinical information were divided into high and low infiltration groups by calculating their infiltration degree.The relationship between the infiltration degree of γδT cells in HCC tissue and the prognosis of patients was analyzed,the differences of other immune cells in the two groups were compared.The HCC tissues of 12 patients with short survival period(< 5 years)and 15 patients with long survival period(≥ 5 years)after liver resection were selected for mutlti-color immunofluorescence.The distribution of γδT cells in liver cancer tissues of the two groups and the relationship between the infiltration degree and the prognosis of patients were analyzed.Results: Survival analysis showed that the overall survival time was significantly higher in the γδT high-infiltration group than in the γδT low-infiltration group(P<0.05).The proportion of CD8+ T cells and M1 macrophages in the γδT high infiltration group was higher than that in the low infiltration group,and the proportion of M2 macrophages was relatively lower(P<0.05).Significant differences in the distribution and degree of infiltration of γδT cells were found by immunofluorescence co-staining in the intra-tumour and tumour margin regions of patients with different prognosis of HCC.In the long-term survival group,γδT cells mainly infiltrated in the tumour tissue,while in the short-term survival group,γδT cells were abundantly distributed in the periphery region of the tumour.This study also found that CD8+T cell abundance was highly consistent with γδT cells,while γδT cell ligands expression was similarly higher in abundance within the tumours of patients with a better prognosis.Conclusion: The prognosis of HCC patients with high infiltration of γδT cells was significantly better than that of low infiltration group.γδT cells may be an anti-tumor effector T cells,the degree of infiltration can be used to predict the prognosis of HCC patients.Part Ⅱ A mplification,culture and functional detection of γδT cells in vitroObjective: The efficiency of γδT cells amplified and the expression of related receptors cultured by different populations and different cytokine combinations were analyzed,and the killing effect on HCC cell s were compared.Methods: The γδT cells were isolated from the peripheral blood of healthy people,cirrhotic patients and HCC patients respectively.The levels of purification and amplification of each group and the expression of related receptors and chemokines were tested by Flow cytometry.Peripheral blood mononuclear cells of healthy people were extracted and grouped by different combinations of cytokines.The purification and amplification efficiency of each group and the expression of related receptors and chemokines were detected by flow cytometry.Lactate dehydrogenase cytotoxicity assay kit and DELFIA (?) Eu TDA cytotoxicity assay kit were used to examine the killing effect of γδT cells stimulated by different groups and cytokines on HCC.Results: The amplification degree of healthy γδT cells was the highest in the three groups,and the expression of activated receptors and chemokines was also the highest in the three groups.In different cytokine groups,the amplification efficiency of IL-2,IL-7 combined with VC group was the highest.The proportion of Vγ9Vδ2T subgroup was the highest in IL-2+IL-7+VC group and IL-2 group.There was no significant difference in the expression of activated receptors and chemokines among all groups.The killing effect of healthy group γδT cells on five HCC cell lines was the stronges.The killing effect of γδT cells stimulated by different combinations of cytokines on five HCC cell lines was examined,and it was found γδT cells in each group had different degrees of killing effect on five HCC cell lines,among which IL-2+IL-15 and IL-2+IL-7 stimulated γδT cells had best killing effect on HCC cells.And the killing efficiency was positively correlated with the ratio of effector cells to target cells.Conclusion: The activity,amplification and killing effec t of γδT cells in healthy group were the best.The IL-2+IL-7+VC group had the highest expansion efficiency,which shortened the time of γδT cell expansion in vitro and might be a better in vitro expansion culture protocol.The killing effect of IL-2+IL-7 and IL-2+ IL-15 on HCC cell lines was better than that of the combined stimulation of other cytokines.Part Ⅲ Generation of chimeric antigen receptor T cells and evaluation of their killing effect on hepatocellular carcinoma cells in vitroObjective: Construction of CAR vectors and generating of chimeric antigen receptor T(CAR-T)cells with different structural designs against HCC cells in vitro.Methods: The experiments were divided into two groups.For the first group,electroporation method was used to transfer various CAR vectors into αβT or γδT lymphocytes to generate NKG2 D BBP CAR-T cells,The expression level was detected and further expanded in co-culture with engineered K562 cells.Flow cytometry was applied to detect the expression level of NKG2 D ligand in five HCC cell lines.Lactate dehydrogenase cytotoxicity assay kit and DELFIA(?) Eu TDA kit were applied to examine the in vitro killing effect of NKG2 D BBP CAR-γδT cells,NKG2 D BBP CAR-αβT cells,γδT cells and control CAR-T cells on the five HCC cell lines.Upon success of NKG2 D CAR-γδT on HCC model,various enhanced CARs were generated to improve the NKG2 D CAR-γδT efficiency in vitro,The killing effects of CAR-T cells with different structures on five liver cancer cell lines were compared.ELISA assay was used to detect IFN-γ secretion of CAR T cells in each group.Results: NKG2 D CAR vectors was successfully designed,constructed and NKG2 D CART cells were generated by obtained non-viral sleeping beauty transposons.After electroporation,the expression level of CAR-T cells was up to 30% by flow cytometry.NKG2 D ligands were expressed in different degrees in five HCC cell lines.In the first set of experiments,γδT cells and all the different types of NKG2 D CAR-T cells had killing effect on HCC cell lines,and the killing effect was proportional to the effective target ratio.In addition,the killing effect of NKG2 D BBP CAR-γδT cells was better than that of γδT cells.The second group of CAR-T cells with different design were found to have different degrees of killing effect on HCC cell lines,and the killing effect was proportional to the effective target ratio.NKG2 D BBZ CAR-γδT secreted the highest concentration of IFN-γ,Anti CD19 BBP CAR-γδT had the lowest secretion of IFN-γ.Conclusion: The NKG2 D CAR plasmid and second-generation NKG2 D CAR-T cells were successfully constructed,and was redesigned to produce potentially more potent CAR-T cells.NKG2 D BBP CAR-γδT cells were more effective than γδT cells.The cytotoxicity of NKG2 D BBP CAR-γδT cells was not weaker than that of NKG2 D BBZ CAR-γδT cells,but the cytokine secretion was reduced,which reduced the incidence of cytokine storm and other adverse reactions.Part Ⅳ Killing effect of chimeric antigen receptor T cells on hepatocellular carcinoma cells in vivoObjective: To establish an in situ transplantation tumour model and a subcutaneous transplantation tumour model in NCG mice to observe the killing effect of γδT cells and CAR-T cells against hepatocellular carcinoma cells in vivo.Methods: Establish of the orthotopic HCC xenograft model in mice,the infiltration and distribution of NKG2 D CAR-T were detected by in vivo imaging and immunohistochemistry.Establishment of subcutaneous HCC xenograft model in mice,the first batch of mice were randomly divided into 5 groups: PBS control group,γδT cell treatment group,NKG2 D BBP CAR-γδT cell treatment group,NKG2 D BBP αβT CAR-T cell treatment group and Anti CD19 BBP CAR-γδT cell treatment group.five mice in each group,the tumor volume and weight changes of mice were recorded,and the tumor growth curves of mice were plotted.The tumours and important organs were examined by hematoxylin and eosin(HE)staining.The second batch of mice were also randomly divided into 5 groups: NKG2 D BBZ CAR-γδT cell treatment group,NKG2 D BBP CAR-γδT cell treatment group,NKG2 D BBPKR CAR-γδT cell treatment group,C-JUN NKG2 D CAR-γδT cell treatment group and Anti CD19 BBP CAR-γδT cell treatment group.five mice in each group,the tumor volume and weight changes of mice were recorded,and the tumor growth curves of mice were plotted.The tumours and important organs were examined by HE staining.Results: The orthotopic HCC xenograft model and subcutaneous HCC xenograft model in mice were successfully constructed.In vivo imaging results of orthotopic liver transplantation tumor model in mice showed that NKG2 D CAR-γδT cells were mainly localized in the liver of mice after tail vein infusion.Immunohistochemistry showed T lymphocyte infiltration in the tumour tissues of the mice.The results of tumor volume measurement showed that the tumor volume of mice in PBS control group continued to increase rapidly during treatment.The tumor volume of mice in γδT cells,NKG2 D BBP CAR-γδT cells,NKG2 D BBP CAR-αβT cells and Anti CD19 BBP CAR-γδT treatment group grew slowly,but NKG2 D BBP CAR-γδT and NKG2 D BBP CAR-αβT cells had the best inhibitory effect.HE staining showed no significant morphological changes in the liver,spleen,lung and kidney of the mice in the NKG2 D CAR-T cell-treated group.During the second experiment,compared with C-JUN NKG2 D CAR-γδT cell and NKG2 D anti CD19 BBP CAR-γδT cell treatment group,the tumour volumes in the NKG2 D BBZ CAR-γδT cell-treated,NKG2 D BBP CAR-γδT cell-treated and NKG2 D BBPKR CAR-γδT cell-treated groups grew slowly,and there was no significant difference between the three groups.Conclusion: NKG2 D CAR-γδT cells can inhibit the growth of HCC cells in mice.The modified CAR-T cells had good tumor inhibitory effect and reduce the release of cytokines,which further improves the safety of CAR-T cell treatment.The improved NKG2 D BBPKR CAR-γδT cells may prolong the survival time of T cells.NKG2 D CAR-γδT cells are safe and effective,which lays the foundation for clinical transformation and application.