| ObjectiveThe liver is an immune organ dominated by natural immunity,including NK cells,NKT cells,??T cells expressing TCR chain and mucosal-associated invariant T(MAIT)cells.Liver ??T cells ratio significantly higher than that of the spleen,lymph nodes and peripheral blood,many studies have shown that ??T cells exist extrathymic development pathway,the small intestine is one of its development site,our laboratory research confirmed that the liver is probably the organ of ??T cells extrathymic development preliminarily.What's more,how the ??T cells develop in the liver,and what is the difference between the thymus ??T cells,these problems is not clear.During mouse ontogenesis,embryonic liver is the main site for HSCs proliferation and differentiation.The majority of hematopoietic stem cells and hematopoietic process occurred in bone marrow after birth and adulthood,but several studies showed that there were still a small number of hematopoietic stem cells in the liver of adult mice.Through transplantation transfer experiment,it was found that these hematopoietic stem cells in adult liver retain certain hematopoietic capacity and can develop and differentiate into various mature immune cells,indicating that they have self-renewal ability.In recent years,it has been found that there is only a breed of hematopoietic stem cells Lineage-Mac-1+Sca-1+(LMS)cells in the adult liver,which comes from the hematopoietic embryo liver and can develop into liver resident NK cells(LrNKs).Can hepatic specific LMS cells develop into T cells in the liver?Is there a unique intrahepatic development of hepatic T cells?These questions have not yet been definitively studied.In order to solve the questions,our study explored the hepatic development process of liver ??T cells from the following aspects.First,we found that the proportion of liver ??T precursor cells were relatively high and had unique phenotypic characteristics compared with spleen,thymus and small intestine.Secondly,adoptive transfer experiment and co-culture experiment proved that liver ??T cell precursor cells can develop and differentiate into mature ??T cells.Finally,through the experiment of adoptive transfer liver LMS cells derived embryo,neonatal and adult life,we found that different stage of liver LMS cells can reconstruct ??T cells in the liver.The reconstructed ??T cells have unique expression of transcription factor and Vy subtype,and have ability of secretion cytokines IFN-y.Methods1.The mononuclear cells in liver,spleen,thymus and small intestine were isolated,and flow cytometry was used to analyse the proportion of ??T cells ??T precursor cells and phenotype of ??T precursor cells.2.The sorted liver and thymus ??T precursor cells were co-cultured with OP9-DL1 in vitro,and the development stage was observed.3.The CD45.1-derived liver or thymus ??T precursor cells were adoptive transferred to TCR?-/-mice exposed to half lethal irradiation(5Gy),and the ability of ??Tprecursor cells developing into mature ??T cells was observed after 3 weeks.4.The CD45.1-derived E16.5,neonatal,adult mice liver and thymus Lineage-cells or Lin-Mac-1+Sca-1+ were adoptive transferred to TCR?-/-mice exposed to half lethal irradiation(5Gy).Flow cytometry was performed to test the reconstructed ??T cells,the expression of transcription factors and Vy subtype,and the secretion of cytokines after 8 weeks.5.Flow cytometry was used to ananlyse the expressions of LMS cells in liver and thymus of E16.5-day,newborn and adult mice.Results1.The proportion of CD24+CD73+ ??T cell precursor cells in adult mouse liver was significantly higher than that of thymus and spleen.The phenotype was characterized by high expression of CD44 and low expression of CD27.The spleen and thymus showed medium or low expression of CD44 and high expression of CD27.2.CD24+CD73-??T progenitor cells and CD24+CD73+??T pregenitor cells in the liver of adult mice can be differentiated into mature T cells by co-cultured with OP9-DL1 cells and have the ability of secretion cytokines.3.CD24+CD73+ ??T cell precursor cells in adult mouse liver transferred to the TCR-/-mice can develop into mature y8T cells.4.Liver mononuclear cells were able to reconstruct ??T cells in various tissues,and the proportion of producing-IFN-?-??T cells was higher than that producing-IL-17A-??T17.Thymus-derived mononuclear cells were also able to reconstruct ??T cells in various tissues,and the proportion of producing-IL-17A-??T17 was higher than that producing-IFN-?-??T1 cells.5.The adoptive transfer experiment results showed that liver Lineage-cells from the newborn and adult mice could rebuilt ??T cells in the liver,spleen,thymus and intestine.And the reconstructed ??T cells were almost producing-IFN-?-??T1 cells.6.LMS cells were present in the liver of embryonic period of 16.5 days,newborn and adult,and the proportion gradually decreased,while LMS cells were not present at the stages of development in thymus.7.The adoptive transfer experiment results showed that the LMS cells derived embryonic period of 16.5 days,neonatal and adult mice liver can rebuild the ??T cells in the liver.And the reconstructed ??T cells were almost producing-IFN-?-??T1 cells with high expression of transcription factor T-bet and mainly V?1+ ??T cells.Conclusions1.Compared with thymus,spleen and small intestine,the proportion of ??T cell precursor cells in the liver is higher,with unique phenotypic and functional characteristics.2.In vitro and in vivo experiments verified that CD24+CD73-??T progenitor cells and CD24+CD73+ ??T pregenitor cells of adult mouse liver and thymus can develop into mature ??T cells.In addition,mature liver ??T cells mainly secreted cytokine IL-17A,while mature thymus ??T cells mainly secreted cytokine IFN-?.3.The liver LMS cells at different stages can reconstruct ??T cells in the liver and other tissues,which might develop into T cell precursor cells and then migrate to other tissues.The reconstructed ??T cells were mainly V?1+??T cells,and the cytokines were mainly IFN-?.Liver ??T cells have a unique intrahepatic development process. |
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