Study On Anti-colorectal Cancer Effect Of NKG2D-CAR-T Cells By Down-regulating Of ACAT1 Gene Expression

CAR-T cell therapy has a history of more than thirty years since its inception,and it has a landmark significance in the history of human anti-cancer.Nowadays,a variety of CAR-T cell therapies and related researches are in full swing,and researchers are working to solve various challenges in CAR-T cell therapy.In many studies,the related functions of the ACAT1 gene have attracted attention.ACAT1participates in encoding cholesterol esterase,and cholesterol esterase can convert cholesterol to cholesterol esters,which regulate the level of cholesterol in cells,and then affect the function of cells.Therefore,on the basis of NKG2D-CAR-T cell therapy,this research further studied the effect of down-regulating the expression of ACAT1 gene on the ability of NKG2D-CAR-T cells against colorectal cancer cells and the related biological characteritics.First,the plasmids that constructed previously in our laboratory were used for packaging lentivirus.T cells were isolated from the human peripheral blood mononuclear cells?PBMCs?and infected by the concentrated viruses,the infection efficiency reached over 90%.Using NKG2D-CAR-T cells to kill colorectal cancer cells?HCT-8?,The anti-colorectal cancer effect of shACAT1-NKG2D-CAR-T cells was continued to increase,which proved that down-regulating the expression of ACAT1 gene could enhance the cytotoxicity of NKG2D-CAR-T cells.At the same time,the study also found that the expression level of CD69,CD107a,IFN-?and GzmB in shACAT1-NKG2D-CAR-T cells were also significantly increased,indicating that down-regulating the expression of ACAT1 gene could increase the activation level and the degranulation ability of NKG2D-CAR-T cells.In addition,the study also detected the exhaustion,proliferation and differentiation of NKG2D-CAR-T cells after the expression of ACAT1 gene was down-regulated.The expression levels of TIM-3 and PD-1 of shACAT1-NKG2D-CAR-T cells were lower than that of NKG2D-CAR-T cells.Compared with NKG2D-CAR-T cells,the cell proliferation of shACAT1-NKG2D-CAR-T cells had increased siganificantly.Moreover,the differentiation of shACAT1-NKG2D-CAR-T cells was also different from that of NKG2D-CAR-T cells,the proportion of T_CM was higher in shACAT1-NKG2D-CAR-T cells compared with NKG2D-CAR-T cells,and the proportion of CD8⁺T cells in shACAT1-NKG2D-CAR-T cells was also higher.Therefore,down-regulating of the expression of ACAT1 gene could improve the functions of NKG2D-CAR-T cells.Based on CAR-T cell therapy,the effect of down-regulating of ACAT1 gene expression on NKG2D-CAR-T cells was investigated in this study.It was found that down-regulation of ACAT1 gene expression could significantly enhance the ability of NKG2D-CAR-T cells against colorectal cancer.At the same time,it can be detected that the CAR-T cells have higher proliferation ability,degranulation ability,activation level,reduced depletion,and can differentiate more T_CM subpopulations.The study has opened up new ideas for the research of NKG2D-CAR-T cell therapy on colorectal cancer.