| ObjectiveLymphocytes are classically viewed as continuously trafficking in blood,lymph nodes and other secondary lymphoid tissues.However,recent studies showed that there exist tissue-resident lymphocytes that usually reside in barrier sites(such as skin,lung,small intestine,and liver).These specialized tissue-resident lymphocytes include innate lymphoid cells(ILCs),natural killer(NK)cells,natural killer T(NKT)cells,mucosal associated invariant T(MAIT)cells,y8T cells,CD8aa+ intestinal intraepithelial lymphocytes(IELs),and tissue-resident memory T(TRM)cells.They act as sensors of perturbed tissue integrity stemming from infection and injury and are involved in the innate and adaptive immunity.They play an important role in defense of the infection and injury in barrier sites.It's worth noting that a multitude of studies have revealed that liver is not only an organ with metabolic activities,nutrient storage and detoxification,but also an immunologically complex organ.In terms of lymphocytic composition,the kinds of lymphocytes in the liver are highly distinct from that of the blood and lymphoid organs.The non-hepatocytes cells include liver sinusoidal endothelial(LSEC)cells,Kupffer cells,biliary cells,stellate cells and intrahepatic lymphocytes.Intrahepatic lymphocytes comprise enrichment of innate immune cells,such as macrophages,NK cells,NKT cells,and y8T cells.Recent studies showed a unique subpopulation of NK cells selectively residing in the murine liver and have differences in phenotype,cytokine production,cytotoxicity and developmental pathway compared with conventional NK(cNK)cells.Researchers showed that the proportion of ??T cells is higher in liver compared with spleen or thymus and they predominantly produce IL-17A.Considering the unique environment of liver,we surmised that whether a population of ??T cells that predominantly produce IL-17A reside in liver and have unique characteristics.We discuss those questions from three aspects.First of all,we found there are more CD44hiCD27-??T cells in liver than spleen and thymus.They predominantly produce IL-17A.Besides,we analysed the expression of resident markers and transcriptional factors by FACS of hepatic CD44hiCD27-??T cells.We confirmed the residency of CD44hiCD27-??T cells by parabiosis mice modle.Last but not least,we discussed the resident mechanism of these cells.Methods1.FACS was used to test the percentage of ??T cells in liver,spleen,thymus,IELs and LNs in C57BL/6J mouse and the production of IL-17A and IFN-y.2.CD44 and CD27 were performed to devided y8T cells into three subsets.Stimulated with PMA and ionomycin,FACS was used to test the level of IL-17A secreted by CD44hiCD27-??T cells.3.FACS was applied to test the expression of resident markers and transcription factors of CD44hiCD27-and CD44hiCD27+??T cells in liver,spleen and thymus.4.Parabiosis modle was established between CD45.1 mice and CD45.2 mice to confirm the residene of hepatic CD44hiCD27-??T cells.5.FACS was applied to test the proportion of pre-??T cells in liver,spleen and thymus.Parabiosis modle was used to examine the residence of hepatic pre-y8T cells.The ability for pre-y8T cells developing into ??T cells was observed through the incubation of pre-??T cells from liver with OP9-DL1 cells in vitro.6.FACS was used to test the expression of Notch1 receptor of hepatic CD44hiCD27-??T cells.Extracting the liver sinusoidal endothelial cells,RT-PCR was applied to test the gene expression of Notch ligands.7.FACS was performed to test the expression of CXCR3 and CXCR6 of hepatic CD44hiCD27-??T cells.Extracting the liver sinusoidal endothelial cells and culturing in vitro,ELISA was used to measure the CXCL16 level secreted by liver sinusoidal endothelial cells.8.Extracting the liver sinusoidal endothelial cells,FACS was used to test the expression of ICAM-1 and VCAM-1 of liver sinusoidal endothelial cells;FACS was used to test the expression of LFA-1 and VLA4 of hepatic CD44hiCD27-??T cells.Results1.CD44 and CD27 can devide ??T cells into three subsets,CD44hiCD27-??T cells,CD44hiCD27+??T cells and CD44lo/??T cells.The proportion of CD44hiCD27-??T cells in liver was significantly higher than in spleen and thymus.They predominantly secret IL-17A rather than IFN-y.2.Hepatic CD44hiCD27-?? T cells express high level of tissue-resident markers CD49a and CD69,whereas they express low level of lymphoid homing markers CD62L.3.Hepatic CD44hiCD27-?aT cells express high level of IL-17A related transcription factor RORyt and tissue-resident transcription factor PLZF,whereas they express low level of T-bet and Eomes.4.Hepatic CD44hiCD27-??T cells specially reside in liver.In the CD45.1 and CD45.2 parabiosis model,almost all the CD45.1+CD44hiCD27-??T cells existed in CD45.1 mice and the CD45.2+CD44hiCD27-??T cells existed in CD45.2 mice.5.Liver-resident CD44hiCD27-?? T cells may develop from hepatic pre-?? T cells.The proportion of pre-??T cells is higher than spleen and are resident in liver.They can develop into mature ?? T cells that predominantly secret IL-17A.6.Notch may play a role in the development and residence of hepatic CD44hiCD27-?? T cells.Hepatic CD44hiCD27-?? T cells has a higher expression level of Notch1 than spleen and thymus.Liver sinusoidal endothelial cells express Notch ligands JAG1,DLL1 and DLL4.7.Liver sinusoidal endothelial cells may promote the residence of CD44hiCD27-??T cells in liver.Hepatic CD44hiCD27-?? T cells express CXCR6,ICAM-1 and VCAM-1,and liver sinusoidal endothelial cells express high level of chemokine CXCL16,adhesion molecules LFA-1 and VLA-4.Conclusions1.The proportion of CD44hiCD27-??T cells in liver was significantly higher than in spleen and thymus.They predominantly secret IL-17A.2.The hepatic CD44hiCD27-??T cells have tissue-resident characteristics and specially reside in liver.3.Hepatic CD24hiCD73+ pre-??T cells are tissue resident and they can develop into mature ??T cells that predominantly secret IL-17A.4.Liver sinusoidal endothelial cells mediate adhesion and retention of liver-resident CD44hiCD27-?? T cells. |
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