The Effect Of Liver Microenviroment On Development Of Liver Resident NK Cells

The immune composition of liver is quite different from other organs,containing a large number of resident innate immune cells,such as liver-resident NK(LrNK)cells(Peng et al.,2013;Sojka et al.,2014),y8T cells(Li et al.,2017)and NKT cells(Thomas et al.,2011).Recently,our studies have shown that adult mouse liver NK cells can be divided into two distinct subsets based on mutually exclusive expression of CD49a and CD49b.Liver CD49a-CD49b+NK cells that are similar to the splenic NK cells are termed conventional NK(cNK)cells.The other subset characterized as CD49a+ CD49b-is liver resident NK(LrNK)cells,which reside in the hepatic sinusoid and can confer the memory-like response(Peng et al.,2013).After adoptive transplantation,adult liver cells can give rise to normal liver LrNK cells as compared with those in normal mice.However,LrNK cells reconstructed by BM cells are significantly reduced,implying that the LrNK cells may have a unique intrahepatic development pathway.Our previous studies have shown that adult liver hematopoietic progenitors characterized by Lin-Sca-l+Mac-1+(LSM)phenotype resemble fetal liver HSC properties and generate LrNK cells via a specific CD49a+NK precursor(NKP)stage.Suggesting that the intrahepatic microenvironment,such as cytokine signaling and cell cross-talk,is an essential part for the development of LrNK cells.In our study,we provide evidence that the development and maturation of liver LrNK cells is dependent on hepatic microenvironment,containing the following two parts:?.Interferon-y Signaling Directs the Intrahepatic Development of Tissue-resident NK Cells1.IFN-y signaling orchestrates the homeostasis of intrahepatic hematopoietic progenitorsWe observed that the IFN-? receptor CD 119 was expressed by liver LSM cells and CD49a+ NKPs,but negative on BM HSCs that are characterized as Lin-Sca-l+c-kit+(LSK),suggesting that intrahepatic hematopoietic progenitors have the potential to directly respond to environmental IFN-y.Furthermore,the number of liver LSM cells decreased in GKO mice,compared to WT controls.The proliferation of liver LSM cells and CD49a+ NKPs were inhibited by IFN-? deficiency.As expected,hydrodynamic injection of IFN-y plasmid into GKO mice induced a significant increase of liver LSM cells and CD49a+ NKPs.These results indicate that IFN-y signaling promotes the expansion of these intrahepatic hematopoietic progenitors,which might thereby increases the production of LrNK cells.2.IFN-y signaling is required for LrNK cell developmentFollowing adoptive transfer a mixture of equal numbers of GRKO and WT fetal liver cells into lethally irradiated recipient mice,there was a deficiency in the production of LrNK cells derived from GRKO donor cells,compared to those from WT donor cells.Overall,these results demonstrate that signaling through IFN-y and its receptor promotes the production of LrNK cells.3.Reduced LrNK cells in the absence of IFN-y signalingHere,by using GKO and GRKO mice,we consistently found that the percentage and number of NK cells declined significantly in the liver,but not in the spleen,BM and thymus.Phenotypic analysis of hepatic NK cells further revealed that IFN-y deficiency reduced the frequency and number of CD49a CD49b-NK cells,and similar observations were obtained in IFN-?R1-deficient mice.Thus,these results suggest that the absence of IFN-y signaling preferentially reduces LrNK cells.4.Overexpression of IFN-? increases LrNK cell productionTo further confirm the role of IFN-y in LrNK cell development,plasmid DNA encoding IFN-y were delivered into GKO mice via hydrodynamic tail vein injection.IFN-y was detected in the serum or liver and stably maintained at a high level for long periods of time after injection,which was accompanied by the increased frequency and number of LrNK cells in GKO mice.However,liver cNK cell numbers exhibited a transient increase after injection and then returned to normal levels.Following adoptive transfer a mixture of equal numbers of GKO and WT fetal liver cells into lethally irradiated WT recipient mice,the proportion and number of GKO-derived LrNK cells were similar to that of WT-derived counterparts,suggesting that environmental IFN-? produced by WT hematopoietic cells restored the number of GKO-derived LrNK cells.5.IFN-y signaling promotes intrahepatic development of LrNK cells in a T-bet-dependent mannerWe cultured T-bet-deficient and WT fetal liver cells with or without the presence of IFN-y.We found that addition of IFN-y in the culture system could significantly promoted WT fetal liver cells to generate CD49a+CD49b-LrNK cells,and had no effect on CD49a-CD49b+cNK cell production,which was in accordance with the results obtained in vivo.However,T-bet-deficient fetal liver cells failed to generate CD49a+CD49b-LrNK cells,even upon IFN-y stimulation,suggesting that IFN-y-promoted LrNK cell development is dependent on T-bet expression.Furthermore,over expression of IFN-y in T-bet-deficient mice via hydrodynamic injection did not increase the frequency and number of CD49a+Eomes"LrNK cells,further confirming that T-bet is required for IFN-?-promoted LrNK cell development in the liver.Conclusions ?:In conclusion,we found that the absence IFN-y signaling reduced the number of LSM cells and CD49a+ NKP cells in adult mouse liver.The IFN-y signaling promote the development of LrNK cells and increase the number of LrNK cells depend on T-bet.?.CD8+ T Cells Promote the Maturation of Liver Tissue-resident NK Cells1.CD27+ LrNK cells can give rise to CD27-LrNK cellsThe expression of CD27 on LrNK cell is heterogeneous.We found that the frequency of CD27+ LrNK cells was decreased during' the ontogenesis of mice.CD27-LrNK cells were more mature than CD27+ LrNK cells.CD27+ LrNK cells could give rise to CD27-LrNK cells in vitro.However,CD27-LrNK cells could not generate CD27+ LrNK cells,further confirming that CD27-LrNK cells are more mature than CD27+ LrNK cells.2.The expression of CD27 on LrNK cells are increased in CD8-/-miceThe percentage and number of LrNK cells were not affected whereas the frequency of CD27+ LrNK cells was increased in Ragl-/-mice and CD8-/-mice,but not in CD4-/-mice,CD1d-/-mice and ?M-/-mice.Thus,these results suggest that the absence of CD8+ T cells preferentially increases the expression of CD27 on LrNK cells.3.CD8+ T cells promote the maturation of LrNK cellsTo further confirm the role of CD8+ T cells in LrNK cell maturation,splenic CD8+ T cells were transferred into CD8-/-mice.The frequency of CD27 on LrNK cells of CD8-/-recipient mice were similar to that of WT mice,suggesting that CD8+ T cells promote CD27+ LrNK cells to generate CD27-LrNK cells.In vitro experiment indicate that CD8+ T cells induce CD27 expression on LrNK cells in a cell-contact dependent manner.Collectively,these results suggest that CD8+ T cells promote the maturation of LrNK cells.Conclusions ?:CD27-LrNK cells are more mature than CD27+ LrNK cells.CD8+T cells promote CD27+ LrNK cells to give rise to CD27-LrNK cells,suggesting that CD8+ T cells promote the maturation of LrNK cells.