Research On The Specific Phenotypes And Functions Of Intrinsic Lymphocytes In Human Tissues And Mechanisms Of Hepatic Immune Tolerance

BACKGROUND:Liver transplantation is currently recognized as the most effective treatment for end-stage liver disease.Being a country with the highest incidence of end-stage liver disease globally,there are huge patient demands and broad application prospects for liver transplantation in China.Since the first case of liver transplantation was implemented in1963,thanks to the maturation of surgical techniques,the application of immunosuppressive agents,and the improvement of postoperative care,the survival rate of postoperative patients has been significantly improved,and the survival time greatly extended.Therefore,patients with end-stage liver disease are ready to relieve the pain through liver transplantation.However,the lack of donor liver has become an ever more difficult issue hindering the development of liver transplantation in China.In order to solve the problem,the introduction and development of DCD liver transplantation have alleviated this problem to some extent.Liver transplantation rejection is the biggest obstacle to improve the survival time after liver transplantation.It has been widely concerned by scholars.If the graft survives for a long time,it must be given an effective immunosuppressive agent to suppress rejection.In the early stage,ALG,Aza,hormones and other immunosuppressants were mainly used.Until the early 1980 s,the emergence and widespread use of Cs A only made a qualitative change in the effect of transplantation.At present,the survival rate of patients at well-known transplant centers at home and abroad can reach 90%,and a large number of patients can survive for a long time.Even so,most liver transplant recipients(52%-82%)still experience rejection and result in 5%-10% loss of graft liver function.The induction of immune tolerance and anti-rejection after liver transplantation is still a hot topic.The liver as an "immunologically excretive organ" usually has a lower probability and degree of rejection after transplantation than other organ transplants.This phenomenon indicates that there is a series of interactions between the transplanted liver and the host's immune system,which in turn forms an immune tolerant environment.In the combined transplantation of multiple organs such as liver and kidney,the liver can also induce the formation of immune tolerance of other organs in the recipient,thereby protecting other organs of the recipient from stable survival.Nowadays,major transplant centers and research institutes have devoted themselves to the study of the natural immune characteristics of the liver.The mechanism may be related to the anatomical and physiological characteristics of the liver itself,MHC-I molecules,APCs(such as DCs,macrophages,Kupffer cells,Activated B-cells and T-cells),micro-chimerism,clonal clearance,and special 40 k Da proteins.OBJECTIVE:To analyze the phenotype and functional differences of lymphocytes in the liver and the spleen,mesentery,and peripheral blood lymphocytes to study the mechanism of the microenvironment of the liver's natural immune tolerance.METHODS:Liver lavage fluid,spleen,mesenteric lymph nodes,and peripheral blood were collected from DCD liver donors.Lymphocytes(NK cells,CD4+ T cells,and CD8+ T cells)from different tissues and organs were isolated and purified.Then PHA and OKT-CD3/OKT-CD28 were used for in vitro stimulation and culture.After 12 hours,surface and intracellular staining were performed.FCM analysis was used to analyze the differences in specific phenotypes and functions of lymphocytes in different tissues and organs.By examining the expression of the intracellular inhibitory cytokine IL-10 and inflammatory cytokine IFN-?in the above lymphocytes,the mechanism of innate immune tolerance in the liver is explored.RESULTS:(1)Compared with the enzyme-digested liver tissue and the mechanically homogenized liver tissue,the method of isolating liver lavaged with 4°C UW solution can retain the phenotype and function of the intrahepatic lymphocyte better and more completely.(2)It is highly noted that the phenotypic frequencies of NK cells and NKT cells in intrahepatic lymphocytes were significantly higher than those in the spleen,mesenteric lymph nodes,and peripheral blood.The phenotypic frequencies of CD4+ T cells in intrahepatic lymphocytes and CD4+ T cells in spleens were significantly lower than those in mesenteric lymph nodes and peripheral blood.The phenotypic frequencies of CD8+ T cells in hepatic lymphocytes were markedly higher than those of CD8+ T cells in spleen and mesenteric lymph nodes,but not obviously different from CD8+ T cells in peripheral blood.(3)NK cells showed increased CD27 in the liver and mesenteric lymph nodes,while CD38 and CD69 increased in the liver,spleen,and lymph nodes.Compared with peripheral blood,CD62 L molecules in the liver,spleen,and lymph nodes were indicated as inhibitory.The detection of CD4+ T lymphocytes showed both the increase of expression of CD27 and CD38 in lymph nodes and of CD69 in liver,spleen,and lymph nodes.Compared with peripheral blood,the expression of CD62 L decreased in liver,spleen,and lymph nodes.The detection of CD8+ T cells found the increase of the expression of CD27 and CD38 in lymph nodes and the expression of CD38 in the spleen and liver;with the exception of peripheral blood,the expression of CD69 increased in three organs.In lymph nodes,spleen,and hepatocytes,the expression of CD62 L was suppressed.(4)By detecting the expression of NK cells,CD4+ T cells,and IL-10 and IFN-? in CD8+ T cells in the liver and peripheral blood,it is found that the expression of IL-10 in liver lymphocytes was significantly higher than that in peripheral blood lymphocyte.However,the expression of IFN-? was just the opposite:intrahepatic lymphocytes were significantly lower than peripheral blood lymphocytes.CONCLUSION:(1)DCD liver grafts isolated from livers with UW preservatives of 4 ? could obtain hepatocytes with relatively intact phenotype and function.(2)As one the noted characterstics of Intrahepatic Iymphocytes,there are an increase of NK cells and NKT cells in the intrahepatic lymphocytes and descrease of the CD4+ T cells in the intrahepatic lymphocytes,significantly less than peripheral blood and lymph nodes.(3)Intrahepatic Iymphocytes is of high immunocompetence with strong secretion function and weak migration.However,lymphocyte of peripheral blood has high migration but weak secretion function.Lymphocyte in mesenteric lymph nodes shows strong lymphocyte activation and cutotoxic function.(4)The expression of IL-10 in liver lymphocytes was significantly higher than that in peripheral blood lymphocytes,while the expression of IFN-? was significantly decreased.