Mechanism Of TIGIT Inhibition Of Immune Rejection In Rat Liver Transplantation In T Cells

Research Background:Liver transplantation(LT)is one of the effective means to treat end-stage liver disease,but immune rejection is still one of the main problems limiting its long-term effect and survival rate.Immune rejection can lead to liver failure and graft failure,seriously affecting the quality of life and survival of recipients.In recent years,with the continuous in-depth research on immune rejection of organ transplantation,we have gained more understanding of the mechanism of immune rejection of liver transplantation.However,it is still necessary to further explore the factors that affect the occurrence and development of immune rejection in liver transplantation,and seek new intervention strategies to improve the success rate and survival rate of liver transplantation,and how to effectively induce graft immune tolerance to reduce or even stop immune transplantation.Inhibitors are still a hot spot in the field of liver transplantation research.Part 1 Establishment of Rat Liver Transplantation ModelObjective:This part of the study aims to establish a reproducible and stable rat model of orthotopic liver transplantation(OLT),in order to further study the immune rejection of liver transplantation.Method:Allogeneic rats,Lewis,and Norwegian brown rats(BN)were selected for surgery using the"portal vein priority two cuff method",with Lewis→BN as the rejection group and BN→BN as the tolerance group.The survival condition and changes of serum biochemical indexes of recipient rats were observed after operation,and histopathology analysis was conducted to verify whether the model was successfully established after transplantation.Results:After transplantation,rats in the two groups showed decreased appetite and decreased activity from the 3rd to the 5th day after transplantation.The tolerance group began to gradually return to normal on the 7th day or so,while the rejection group aggravated symptoms.At the same time,serum alanine transaminase(ALT)and aspartate transaminase(AST)were significantly higher than those in the tolerance group,P<0.05.Pathological analysis showed that there were varying degrees of rejection reactions in the transplanted liver of the rejection group,while there was no rejection reaction in the tolerance group.There was a significant difference in rejection scores between the two groups,P<0.05.Conclusion:In this study,the Lewis→BN,BN→BN method was used to successfully establish the rat liver transplantation model with the "portal vein-first two-cuff method".Part 2 High expression of CD155-TIGIT gene and analysis of key differential genes in immune rejection of rat liver transplantationObjective:The immune rejection reaction after liver transplantation is mainly caused by the antigen presentation process and the activation of T lymphocytes.This study analyzes and explores the genetic changes in immune rejection after liver transplantation in rats through high-throughput and single-cell sequencing results.Method:The occurrence of immune rejection after liver transplantation occurs approximately 3-5 days after surgery.We selected rat liver tissue on the 7th day after liver transplantation for high-throughput and single-cell sequencing detection,Use Gene Ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)databases to find differentially expressed genes(DEGs)and their involved signal pathways.Use Gene Set Enrichment Analysis(GSEA)to identify key pathways in immune rejection.Meanwhile,an online String database was used to construct a Protein Protein Interaction(PPI)network for differentially expressed genes,and key genes were screened out.Then,single cell sequencing technology is used to compare and analyze the selected key genes and pathways,and locate the cell subpopulations where the key genes are located in the signaling pathway.Result:1.In this study,a total of 5252 differential genes were identified in liver tissue of rats with transplant rejection,of which 2962 genes were upregulated and 2290 genes were downregulated.2.GO enrichment analysis results showed that DEGs significantly enriched pathways such as lymphocyte activation regulation,adaptive immune response,and leukocyte cell adhesion in biological processes(BP);The cellular component(CC)significantly enriched cell locations such as the outer side of the plasma membrane,membrane rafts,and membrane microregions;Molecular function(MF)is significantly enriched in functions such as chemokines,cytokines,and immune receptors.3.KEGG analysis results suggest that DEGs are mainly enriched in cytokine receptor interaction,chemokine signaling pathway and cell adhesion molecule.4.GSEA analysis showed that cytokine receptor interaction,PI3K Akt signaling pathway and cell adhesion molecule were the main pathways in the process of liver transplantation immune rejection.Through comparative analysis,it was believed that the key genes in the cell adhesion molecule pathway of T cell immunoglobulin and ITIM domain protein(TIGIT)were quite different.5.A PPI network was constructed based on the functional connections between DEGs corresponding proteins,and five key genes,GZMB,CXCL2,CX3CL1,CCR1,and CCL19,were selected through enrichment analysis.6.The results of single cell sequencing indicate that when non parenchymal cells were grouped,monocytes and macrophages were found to be the largest group,while T cells were found to be the third largest group.Subsequently,T cells were divided into 6 groups,with Treg being the largest group in the tolerance group.Based on the characteristics of single cell expression profile and the expression of marker genes,it was found through annotation of cell population markers that the markers of cell populations 8 and 19 where the leukocyte differentiation antigen 155(CD155)-TIGIT signaling pathway is located are CD3e,belonging to the T lymphocyte population.Conclusion:1.402 immune related DEGs are involved in immune rejection reactions,among which GZMB,CXCL2,CX3CL1,CCR1,CCL19,and TIGIT are key genes.2.The regulation of lymphocyte activation outside the plasma membrane,including chemokines,cytokine receptor interactions,PI3K Akt signaling pathway,cell adhesion molecule and other related mechanisms or pathways,may play an important role in the immune rejection after liver transplantation.3.CD155-TIGIT signaling pathway may play an important role in inhibiting immune rejection in T lymphocyte adhesion molecule pathway after liver transplantation.Part 3 The expression and possible role of TIGIT and its signal pathway in the immune rejection of rat liver transplantationObjective:Through differential gene analysis,we found significant differences in TIGIT and its signaling pathway genes,located in the T lymphocyte subgroup.However,the expression of this pathway at the protein level is not yet clear.This study aims to explore the expression intensity and location of TIGIT and its signaling proteins in liver transplant immune rejection through immune methods,providing a theoretical basis for the role of TIGIT in liver transplant immune rejection.Method:Establish an animal model,select rat liver tissue on the 7th day after liver transplantation,and perform immunohistochemistry and immunofluorescence protein molecular validation.Result:1.In the tolerance group,a small amount of inflammatory cell infiltration was observed in the hepatic sinusoids,with scattered TIGIT positive cells.Oil microscopy(100×)Observe the lymphocytes that are TIGIT+in morphology.Compared with the tolerance group,the rejection group showed a large number of infiltrating inflammatory cells around the hepatic sinusoids,with most TIGIT+cells infiltrating in inflammatory cells,and most TIGIT+lymphocytes in positive cells observed under oil microscopy.2.CD155 is highly expressed on the surface of TIGIT+T cells in the CD155 TIGIT signaling pathway.Both the chemokine ligand CXCR3 and the co stimulator ligand GITRL are expressed in positive cells.Morphologically,it is believed that CD 155 and CXCR3 are located on lymphocytes,while GITRL is located on macrophages.3.Immunofluorescence shows that the CD 155-TIGIT signaling pathway is mainly located on CD4+T cells,indicating that T cells affect liver transplant immune rejection.Conclusion:In rat liver transplantation,TIGIT may inhibit immune rejection after liver transplantation by regulating its signaling pathway CD 155-TIGIT.Part4 Functional proteins such as TIGIT and their pathways can inhibit the activation and functional expression of T cellsObjective:In the first two parts,we investigated the effect of TIGIT on T lymphocytes,which may inhibit T cell function through its signaling pathway CD 155-TIGIT,thereby inhibiting liver transplant immune rejection.This study aims to investigate the effects of TIGIT and its pathways on T cells through in vitro cell experiments,providing theoretical basis for the role and mechanism of TIGIT in liver transplantation.Method:In the immune rejection reaction of liver transplantation,among the inflammatory factors secreted by T cells after activation,IL-6,IL-23,and TNF play a pro-inflammatory role-α、INF-γ;The anti-inflammatory effects are:IL-10,TNF-β。In order to verify the impact of changes in the CD155-TIGIT signaling pathway on T cell function,we conducted in vitro cell co culture experiments using peritoneal macrophages from Lewis rats and CD4+T cells from the spleen of BN rats to simulate the immune rejection environment of in vitro liver transplantation.We constructed a TIGIT silenced cell line and activated the TIGIT pathway by CD 155 stimulation,Verify the protein expression of TIGIT and CD226,as well as the expression of inflammatory factors after T cell activation.Group as follows:T+M group:CD4+T cells+macrophagesT+M shTIGIT group:CD4+T cells+macrophages after silencing TIGIT expressionT group:CD4+T cellsT+CD155 group:CD155+CD4+T cellsT+M+CD155 group:CD155+CD4+T cells+macrophagesResult:1.The inflammatory factors IL-6,IL-23,and TNF in T+M shTIGIT group were co cultured for 24 hours-α、INF-γ The level is significantly higher than that of the T+M group(P<0.05),and the anti-inflammatory factors IL-10 and TNF-β The level slightly increased compared to the T+M group(P<0.05);2.Comparing T group with T+CD155 group,after adding CD155,IL-10 in T+CD155 group was significantly upregulated,and INF-γ There is a significant decrease,P<0.05;3.Comparing T+CD155 group with T+M+CD155 group,IL-10 was significantly upregulated in T+M+CD155 group,and INF-γ Downregulation,P<0.05;4.Comparing the T+M group with the T+M+CD155 group,after receiving CD155 stimulation,the anti-inflammatory factor IL-10 in the T+M+CD155 group was significantly upregulated compared to the T+M group,P<0.05,and the inflammatory factor INF-γ Significantly inhibited,P<0.05.Conclusion:The CD155-TIGIT pathway can inhibit T cell activation in cell experiments,manifested by increased secretion of anti-inflammatory factors(such as IL-10)and pro-inflammatory factors(such as INF)γ)The decrease in secretion of CD 155 TIGIT suggests that the inhibition of liver transplant rejection by CD 155 TIGIT may be achieved by inhibiting the activation of T cells.Full text conclusion:1.This study successfully established a rat orthotopic liver transplantation model using the Lewis→ BN,BN→BN method,and the "portal vein priority two cuff method".2.High throughput and single cell sequencing showed that T lymphocytes are the key cell subsets in the immune rejection after liver transplantation,and CD 155-TIGIT signaling pathway in the cell adhesion molecule pathway may play an important role in the immune rejection after liver transplantation.3.In the immune rejection response of rat liver transplantation,TIGIT can inhibit the activation of T cells,inhibit the immune rejection response after liver transplantation,and induce immune tolerance by regulating its signaling pathway CD155-TIGIT.