| Background and Objective:Liver cancer exosomes can regulate the anti-tumor immune response of CD8~+T cells by inducing tumor-associated macrophages(TAM),regulatory B cells(Breg)and other immunosuppressive cells,but its direct regulatory effect on CD8~+T cells is still unclear.The purpose of this study is to clarify the role of liver cancer exosomes on the activation,proliferation and tumor cell killing function of CD8~+T cells.Methods:1.Determination of the extraction method of exosomes and identification of exosomes:Exo Quick kit method,qEV size exclusion column method,and ultracentrifugation method were used to extract exosomes from150 m L cell supernatants.The total amount of exosomes,the percentage of exosomes to total particles,and total exosomes were compared.For the number of exosomal particles,select an extraction method that can obtain exosomes with a low total protein,a high percentage of exosomes in the total number of particles,and a large number of total exosomes particles.Electron microscopy,nanoparticle tracking analyzer and Western Blot were used to identify the shape and size of exosomes,particle size distribution and the expression of characteristic protein molecules,respectively.2.Study on the activation and proliferation of CD8~+T cells by exosomes of hepatocarcinoma cells(1)Effect of exosomes of hepatocellular carcinoma cells(Hep G2,Hep3B,PLC/PRF/5)on the activation and proliferation of resting CD8~+T cells:Density gradient centrifugation was used to separate human peripheral blood mononuclear cells(PBMCs),and resting CD8~+T cells were separated from PBMCs by immunomagnetic bead method and labeled with CFSE.After co-cultivating resting CD8~+T cells with exosomes of liver cancer cells or normal liver cells WRL-68 for 4 hours,stimulate CD8~+T cells with anti-human CD3/CD28 antibody or allogeneic dendritic cells(DC)Activation and proliferation.Detect CD8~+T cell activation(ELISA to detect the secretion level of IFN-γ,flow cytometry to detect the expression level of CD107a on the cell surface)and proliferation(flow cytometry to detect CFSE fluorescence intensity)respectively on the 3rd and 7th days.(2)Activation and proliferation of liver cancer cells(Hep G2,Hep3B,PLC/PRF/5)exosomes on activated CD8~+T cells:After obtaining resting CD8~+T cells from human peripheral blood with the same method,use anti-human CD3/CD28 antibody or allogeneic DC to stimulate CD8~+T cell activation and add liver cancer cell exosomes or WRL-68 exosomes to the cell fluid as comparison.Detect CD8~+T cell activation(ELISA to detect the secretion level of IFN-γ,flow cytometry to detect the expression level of CD107a on the cell surface)and proliferation(use flow cytometry to detect CFSE fluorescence intensity)respectively on the 3rd and 7th days.3.Study on the effect of exosomes of liver cancer cells(Hep G2,Hep3B,PLC/PRF/5)on the tumor-killing function of activated CD8~+T cells(1)Obtain resting CD8~+T cells from human peripheral blood in the same way as before,co-culture them with liver cancer cell exosomes or WRL-68 exosomes for 4 hours,and then add anti-human CD3/CD28 antibody to stimulate CD8~+T cell activation.Two days later,the activated CD8~+T cells and Hep G2 liver cancer cells were co-cultured.After 24 hours,the LDH release method was used to detect the killing ability of activated CD8~+T cells on Hep G2.(2)Obtain resting CD8~+T cells from human peripheral blood in the same way as before,and add anti-human CD3/CD28 antibody to stimulate CD8~+T cell activation.Two days later,the activated CD8~+T cells and Hep G2liver cancer cells were co-cultured with liver cancer exosomes or WRL-68exosomes as controls.After 24 hours,the LDH release method was used to detect the ability of CD8~+T cells to kill Hep G2.Results:1.Exo Quick kit method,qEV size exclusion column method and ultracentrifugation method extracted exosomes from 150 m L Hep3B supernatant.The number of exosomes particles were 7.52×10~8,3.48×10~7and 4.86×10~6,and the total protein content was 552μg,38.68μg,54μg,respectively.Compared with Exo Quick kit method,qEV size exclusion column method and ultracentrifugation method’s number of exosomal particles and total protein obtained are lower.In the exosomes extracted by the qEV size exclusion column method and ultracentrifugation method,the percentage of exosomes particles to the total number of particles is 57%and50%,respectively.Compared with the ultracentrifugation method,the qEV size exclusion column method has a higher percentage of the total number of exosomes in the exosomes extracted.The cell exosomes extracted by the qEV size exclusion column method have a double-layer membrane structure,the size is between 40-160 nm,and they express Alix,TSG101 and other characteristic proteins of exosomes,which are in line with the characteristics of exosomes.2.(1)Compared with the control group of CD8~+T cells not treated with exosomes and WRL68 exosomes,resting CD8~+T cells treated with exosomes of liver cancer cells(Hep G2,Hep3B,PLC/PRF/5)were activated by anti-human CD3/CD28 monoclonal antibody.The IFN-γsecreted by CD8~+T cells increased,the expression level of CD107a on CD8~+T cells increased,and the percentage of proliferating cells increased;and after the stimulation of allogeneic DC,the secretion of IFN-γdecreased,the expression level of CD107a decreased,and the percentage of proliferating cells decreased.(2)Compared with the control group of CD8~+T cells that were not treated with exosomes and WRL68 exosomes,activated CD8~+T cells stimulated by anti-human CD3/CD28 antibody or allogeneic DC were treated with liver cancer cell exosomes.The secretion of IFN-γ,the expression level of CD107a and the percentage of proliferating cells did not change significantly.3.(1)Compared with the control group of CD8~+T cells without exosomes and WRL68 exosomes,resting CD8~+T cells treated with hepatocarcinoma cells Hep G2 and Hep3B exosomes were activated by anti-human CD3/CD28 antibody.The activated CD8~+T cells were co-cultured with liver cancer cells Hep G2,and it was found that the cell damage rate of Hep G2 increased(p=0.0134,p=0.0318,p=0.0453).However,compared with the control group,the Hep G2 cell damage rate of the PLC/PRF/5 exosome treatment group did not change,and the difference was not statistically significant.(2)Compared with the control group of CD8~+T cells not treated with exosomes and WRL68 exosomes,activated CD8~+T cells stimulated by anti-human CD3/CD28 antibody were treated with exosomes of liver cancer cells,which were co-cultured with Hep G2.The injury rate of Hep G2 did not change significantly,and the difference was not statistically significant..Conclusion(s):1.The qEV size exclusion column method extracts exosomes,which can remove a large amount of soluble protein components in the exosomes and obtain more exosomes with higher purity than the ultracentrifugation method.2.(1)Pretreatment of liver cancer cell exosomes can enhance the activation and proliferation of resting CD8~+T cells stimulated by anti-CD3/CD28 antibodies;The pretreatment of liver cancer cell exosomes can inhibit the activation and proliferation of resting CD8~+T cells stimulated by allogeneic DC.(2)Post-treatment of liver cancer cell exosomes had no effect on the activation and proliferation of CD8~+T cells stimulated by anti-CD3/CD28 antibodies and allogeneic DC.3.(1)Hep G2 and Hep3B exosomes pretreatment can increase the killing activity of anti-human CD3/CD28 antibody-activated CD8~+T cells,while PLF/PRF/5 exosome treatments failed to change the killing activity of CD3/CD28 antibody-activated CD8~+T cells.(2)All post-treatment of liver cancer cell exosomes failed to change the killing activity of CD3/CD28antibody activated CD8~+T cells. |
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