Hepatocellular Carcinoma With High Expression Of COX-2 Promotes CD8~+ T Cell Exhaustion Through M2 Macrophages And Studies On The Generation Of Type 1 Conventional Dendritic Cells With Human Induced Pluripotent Stem Cells And Differentiational Optimizatio

Background and purpose:Cellular immunotherapy of tumors has become one of the important methods of tumor treatment.However,the current immunotherapy is not very effective.One of the main reasons is the existence of the tumor microenvironment,which leads to the weakening of the killing ability of cytotoxic T cells to tumors,or the lack of targeting,which makes it impossible to accurately identify tumor cells.As one of the main immunosuppressive cells in the tumor microenvironment,M2 macrophages can not only induce immune escape of tumor cells,but also inhibit the function of cytotoxic T cells through the TGF-β pathway.Therefore,one of the main goals of this study is to confirm the high expression of COX-2 hepatocellular carcinoma cell line can induce the transformation of monocytes into M2 macrophages through PGE2,thereby affecting the ability of CD8+T cells to produce Granzyme B and IFN-γ after activation.In addition,the DC vaccine is one of the important methods of cellular immunotherapy.Its main function is to enhance the targeting of cytotoxic T cells by presenting tumor antigens to cytotoxic T cells.However,because of the existence of the tumor microenvironment and the inability of dendritic cells to expand in vitro,the number of patient-derived DC is limited and the ability to present tumor antigens is insufficient.Therefore,another purpose of this study is to apply in vitro Model,in which HPC derived from iPSCs were induced to differentiate into DC and its subgroup cDCl in vitro;at the same time,from the phenotype,key transcription factors and important In terms of genes and other aspects,it detects the difference of cDC1 from different sources;finally,it lays a solid foundation for the clinical application of iPSCs-derived cDC1 in tumor immunotherapy.Methods:(1)First,use immunofluorescence technology to detect the expression of COX-2,CD163,CD206 in liver cancer patient tissue sections,use PCR technology to detect the expression level of COX-2 gene in liver cancer patient tissue specimens,and use ELISA technology to detect liver cancer patient tissues The content of PGE-2 in the specimens;then a co-culture model was constructed,and flow cytometry was used to detect the co-expression of CD163 and CD206 on CD 14+monocytes before and after co-cultivation.CD28,CD25,and CD 107A before and after CD8+T cell activation The expression of Granzyme B and IFN-γ in CD8+T cells after activation before and after co-cultivation.In addition,the use of ELISA and flow detection technology to detect PGE2,TGF-β,Granzyme B and IFN-γ in the culture medium.The levels of cytokines were detected by automated western technology to detect the expression of related proteins in the TGF-β pathway such as Smad2,Smad3,pSmad2,pSmad3 and FoxPl;finally,an in vivo animal model was constructed and verified accordingly.(2)Use the differentiation kit or the existing differentiation medium to induce iPSCs into hematopoietic progenitor cells,and use the differentiation kit to induce primary HSC and iPSCs-derived HPC into CD14+ monocytes,using GM-CSF,IL4 and TNF-α to induce CD14+monocytes from different sources into mature DC,using Flt3L,SCF,GM-CSF,IL4 and other four transcription factors to convert the hematopoietic stem/The progenitor cells are induced as cDC1.The tissue lysis kit was used to extract the primary DC and cDC1 from the spleen tissue.Lentiviral transfection technology was used to construct iPSCs cell line with PU.1-IRF8-BATF3 sequence.Use flow cytometry to analyze the co-expression level of CD34/CD45 on the surface of hematopoietic stem/progenitor cells,analyze the expression level of CD 14 on the surface of monocytes,analyze the co-expression level of CD11c/HLA-DR on the surface of DC,and analyze CD11c,CD141 on the surface of cDC1/CLEC9A expression level,analyze the expression levels of 5 key transcription factors in cDC1 from different sources.Use ordinary transcriptome sequencing technology to analyze primary DC derived from spleen,primary peripheral blood CD14+monocytes,primary CD14+monocytes derived DC,primary hematopoietic stem cells derived monocytes,primary hematopoietic stem cells derived DC cells,and iPSCs Source hematopoietic progenitor cell isogenic profile expression.Use luciferase detection kit and PCR relative fluorescence quantitative technology to detect whether the iPSCs cell line is successfully constructed.Micro-PCR relative fluorescence quantitative technology was used to detect the transcription levels of 31 important genes in cDC1 from different sources.Results:(1)The expression of COX-2 in paraffin sections of HCC patients is positively correlated with the expression of M2 macrophage markers-CD163/CD206.HCC cell lines with high COX-2 expression can induce human primary CD14+ monocytes to M2 Macrophage polarization,M2 macrophages induced by HCC cell line can inhibit the production of IFN-γ and Granzyme B by activated CD8+T cells,M2 macrophages induced by HCC cell line are inhibited through the TGF-β pathway The activated CD8+T cells produce IFN-y and Granzyme B.(2)Peripheral blood primary CD14+monocytes can be induced into DC cells,primary hematopoietic stem cells can be induced into CD 14+monocytes,and further into DC cells,iPSCs can be induced into hematopoietic progenitor cells,CD14+ monocytes,and It was further induced into DC;the sequencing results showed:comparing the two sets of data of primary peripheral blood CD14+monocytes and primary CD14+monocyte-derived DC cells,LILRB2,AZI2,NLRP12,DCSTAMP,BATF3,SLAMF1 these 6 genes The changes are significantly different;comparing the two sets of data of primary hematopoietic stem cell-derived monocytes and primary hematopoietic stem cell-derived DC cells,NLRP12,SLC11A1,ZBTB46,TMEM176B,TMEM176A,TREM2,BATF3,DCSTAMP,SLAMF1,TRPM4,IRF8 11 There are significant differences in the changes of these genes;comparing the two sets of data of primary DC cells derived from spleen and primary CD14+monocytes,there are significant differences in the changes of these three genes:BCL3,TREM2,and DCSTAMP.(3)The spleen contains primary cDC1 cells of CD11clow CD141+CLEC9A+;in an in vitro model,using Flt3L,SCF,GM-CSF,IL4 and other four cytokines,primary hematopoietic stem cells can be induced to differentiate into cDC1 cells;iPSCs The source of HPC has poor ability to induce differentiation into cDC1,and the expression of related transcription factors has obvious defects.(4)In an in vitro model,iPSCs cell lines with PU.1-IRF8-BATF3 sequence(collectively referred to as PIB cells)can be successfully induced to cDCl and the expression of related transcription factors is significantly increased;at the same time,HSC-derived The transcription level of genes in the presentation of tumor antigens in cDC1 is more prominent,and the transcription level of genes in cDC1 derived from PIB is more prominent in activating CD4+T cells and optimizing the anti-tumor immunity of CD8+ T cells.Conclusions:(1)For HCC patients with high COX-2 expression,selective COX-2 inhibitors can be used as adjuvants together with cellular immunotherapy to reduce the inhibitory effect of the tumor microenvironment on cytotoxic T cells.(2)Our in vitro induction program is feasible and can induce hematopoietic stem/progenitor cells from different sources into DC and cDC1 subgroups in DC;at the same time,the transcription level of genes presenting tumor antigens in cDC1 derived from HSC is more prominent,PIB-derived cDC1 activates CD4+ T cells,and optimizes the transcription level of genes in CD8+T cell anti-tumor immunity.