Intrahepatic Development Of Liver-resident NK Cells And Their Relationship With Mucosal ILC1s

As an organ with predominant innate immunity,the liver contain a large number of innate immune cells,such as NK cells,NKT cells,y8 T cells and Kupffer cells.Our lab previous study have shown that adult mouse liver NK cells are heterogeneous.According to different expression of CD49a and DX5,liver NK cells can be divided into two distinct subsets:CD49a-DX5+ conventional NK cells(cNKs)and CD49a+DX5-NK cells which rarely enter circulation and,thus,are referred to as 'liver-resident NK cells'(LrNKs).LrNKs predominate in fetal and neonatal liver NK cells.The proportion of LrNKs decrease with age and remain at around 50%until adulthood.However,the origin of LrNKs is not well understood.After bone morrow transplantation,LrNKs derived from BM cells are significantly reduced as compared with those in normal mice.Consistently,LrNKs from mice in parabiosis for more than two months still remain host-derived,suggesting that BM hematopoiesis is not the major origin of LrNKs.During mouse ontogeny,liver is the major site for the expansion and differentiation of HSCs during fetal life and maintains a few HSCs in adults.Moreover,lineage(Lin)-negative progenitor cells in adult liver can give rise to nomal LrNKs.these implying that LrNKs may have a unique intrahepatic development pathway.Because LrNKs express NCR and require IL-15 signaling for their development,they are considered as a sunbet of NK cells initially.LrNKs and mucosal ILC1s require T-bet while cNKs require Eomes for their development.Additionally,similar to LrNKs,mucosal ILC1s develop from Id2-expressing common helper-like progenitors(CHILPs)which cannot generate cNK cells.Thus,LrNKs are also currently called liver help-like ILC1s because of developmental similarities between them and mucosal ILC1s but it remains unclear whether they are similar to mucosal ILC1s in terms of phenotype,function and migration.In consideration of the above points,our study contain the following two parts:I.Intrahepatic development of liver-resident NK cells1.Adult mouse liver contains hematopoietic stem cellsWe found that adult mouse liver mononuclear cells(MNCs)were capable of forming hematopoietic myeloid-lineage and erythroid-lineage colonies by a colony-forming unit(CFU)assay.Further study showed that adult liver contained a small population of Lin-Sca-1+c-kit+ cells(LSK)that possessed long-term blood-repopulating potential and had the ability to rebuilt myeloid and lymphoid lineages,demonstrating that adult liver exists hematopoietic stem cells(HSCs).2.Adult mouse liver CD45+Lin-Mac-1+Sca-1+(LMS)hematopoietic progenitors are derived from fetal liver hematopoiesisAdult liver retains hematopoietic potential and contains a few CD45+Lin-Mac-1+Sca-1+(LMS)hematopoietic progenitor cells(HPCs),which are phenotypically distinct from BM lineage(Lin)-negative progenitor cells but resemble fetal liver counterparts.Further analyses reveal that the frequency and absolute number of LMS cells is significantly reduced after BM transplantation.Conversely,lethally irradiated mice adoptively transferred with fetal liver cells exhibit normal frequency and absolute number of LMS cells,suggesting adult liver LMS cells originate from fetal liver hematopoiesis.3.Adult liver CD45+Lin-Mac-1+Sca-1+ cells possess hematopoietic potential and give rise to liver-resident NK cellsAdoptive transfer of these adult liver LMS cells have the potential to differentiate into multiple hematopoietic lineages and predominantly give rise to CD3+T cells which similar to neonatal liver HSCs.LrNKs originate from fetal liver hematopoiesis,and predominate in fetal and neonatal liver NK cells.Moreover,adult liver LMS cells have the capacity to generate LrNKs but not cNKs in the liver of recipients.These hematopoietic properties of adult liver LMS cells are consistent with that of neonatal liver LMS cells.4.Liver CD49a+NKPs and preferentially develop into liver-resident NK cellsNK precursors(NKPs)appear at several distinct adult mice tissue sites.including the BM,liver and spleen,but only liver NKPs express high level of CD49a.We transferred purified hepatic CD49a+NKPs and found that NK cells generated from donor only can be detected in the liver of recipients,and all hepatic NK cells are CD49a+DX5-liver-resident NK cells.Conclusions ?:Adult mouse liver retains hematopoietic potential and contains a small population of hematopoietic progenitors characterized by CD45+Lin-Mac-1 +Sca-1+phenotype.The hematopoietic potential of adult hepatic LMS cells to generate CD49a+NKPs and then LrNKs,but not cNKs,is consistent with fetal liver HSCs.Thus,our studies suggest an alternative development pathway for LrNKs.?.Differential phenotypic and functional properties of liver-resident NK cells andmucosal ILC1s].A phenotypic comparison of liver-resident NK cells and mucosal ILC1s The frequency and absolute number of LrNKs were much higher than those ofsmall intestine lamina propria(siLP)ILC1s.However,ILC populations were more diverse in the siLP owing to the relative abundance of ILC2s and ILC3s.Moreover,we found that siLP ILCl s expressed IL-7R? and CD27,but LrNKs expressed intermediate levels of CD27 and IL-7Ra.Regarding homing-associated molecules,LrNKs expressed high level of CXCR6,which are involved in the migration of lymphocytes into the liver.However,siLP ILC1s expressed high level of CCR9,a chemokine receptor important for lymphocyte homing to the intestine,whereas LrNKs did not.2.Similar requirements for transcription factors in liver-resident NK cell and mucosal ILCldevelopmentIn Tbx21-/-mice,LrNKs and siLP ILC1s were nearly completely absent in both percentage and absolute cell number.indicating that the differentiation of LrNKs and siLP ILC1s is strictly T-bet-dependent.In Nfil3-/-mice,LrNKs were present.However,their absolute numbers were reduced and liver cNK cells were absent.suggesting that LrNKs are partially NFIL3-dependent.Moreover,NFIL3 deficiency led to significantly reduced numbers of cNK cells,ILC1s and NKp46+ ILC3s in the siLP,consistent with the previous finding that NFIL3 directs the differentiation of all ILC lineages.3.Mucosal ILCl s are tissue-residentParabiotic mice by surgically joining pairs of CD45.1 and CD45.2 congenic animals exhibited that siLP]LCls and NKp46+ILC3 failed to recirculate,suggesting tissue residency akin to liver CD49a+DX5-NK cells.4.Differential cytotoxic potential between liver-resident NK cell and mucosal ILC1sUpon stimulation with PMA and ionomycin,LrNKs expressed granzy'me B(GmB)and perforin at high levels,comparable to those of cNK cells,but siLP ILC1s expressed low levels of GmB and perforin.Moreover,TRAIL and FasL,which are known as initiators of target cell apoptosis,were expressed at higher levels on LrNKs than on siLP ILCIs and cNK cells,suggesting that LrNKs can initiate non-secretory apoptosis via TRAIL or FasL upon interaction with target cells.We found that LrNKs induced higher apoptosis of FasL-sensitive Yac-1 target cells than cNK cells,further confirming the strong cytotoxic effect of LrNKs.Conclusion ?:In conclusion,there were clear differences between LrNKs and mucosal ILCl s in expression of adhesion molecules and cytotoxic function.Notably,the finding that LrNKs but not mucosal ILC1s have relatively high cytotoxic potential contradicts the classification of these cells into non-cytotoxic ILCl subset.