Generation And Application Of Tissue-resident NK-like Cells

Recently,researchers have shown an increased interest in tissue-resident natural killer(trNK)cells,which have been found in multiple tissues such as liver,uterus,skin,and salivary glands(SG).These trNK cells not only function to counter potential invading organisms,but also contribute to multiple processes.Uterine trNK cells can promote angiogenesis,fetus growth and immune homeostasis during early pregnancy.TrNK cells and cNK cells express similar markers such as NK1.1,NKp46,NKG2D,and T-bet,but can be distinguished by expressing high level of CD49a,CD69,and TRAIL on trNK cells,while cNK cells are CD49b+.Hepatic trNK cells differ from cNK cells by a lack of expression of Eomesodermin(Eomes),however,trNK cells in uterus and SG express high levels of Eomes.However,the regulation of Eomes expression in trNK cells remains unclear.Considering poor cell number and complex tissue separation processes,an in vitro generation system of trNK cells would contribute to developmental and functional research,as well as possible therapeutic applications.How to differentiate progenitor cells selectively into CD49a＋,as well as CD49a+Eomes trNK-like cells is still unknown.Major results of current work are summarized as follows:1.We established an in vitro cytokine-based feeder-free system in which bone marrow progenitor cells differentiate into CD49a+ NK cells.To investigate the developmental conditions of CD49a+ NK cell,an in vitro system was established in which BM cells differentiate into CD49a+NK1.1+ cells upon culture in multiple cytokine cocktails without feeders.These cells were further identified to be NK cells according to molecular expression and transcription factor dependency.2.Different roles of IL-15 and IL-4 signaling in the generation of CD49a+Eomes-and CD49a+Eomes+ NK cells.IL-15 has been well known to be critical to the development of NK cells from different organs,but not for all ILCls.With conditional cultures followed by flow cytometry analysis,we found that IL-15 is also the key factor for both the development and maintenance and of iNK(induced NK)cells while IL-2 contributes to their survival.We also found that iNK cells expressed high level of IL-4Ra and responded to IL-4-stimulation with upregulating Eomes expression.Moreover,Il4ra-/-mice failed to generate iNK cells from BM cells which indicated that not only does IL-4 induce Eomes,it is also necessary for the generation of CD49a+Eomes-iNK cells.By using an inhibitor of STAT6,we found that IL-4 is essential for the generation and expression of Eomes of CD49a+ iNK cells through STAT6.3.Generated CD49a+Eomes-and CD49a+Eomes+ NK cells share phenotypes with liver-resident and uterus-resident NK cells,respectively.To investigate the similarities between iNK cells in vitro and tissue NK cells in vivo,we compared the expression of key markers in CD49a+Eomes-and CD49a+Eomes+ iNK cells with tissue NK cells by flow cytometry analysis and quantitive PCR.The results showed that CD49a+Eomes-iNK cells share phenotypes with liver-resident NK cells,while CD49a+Eomes+ NK cells share phenotypes with uterus-resident NK cells.Furthermore,cNK cells from WT liver and uterus expressed low levels of IL-4Ra,while trNK cells and IILCls expressed higher levels than cNK cells.It was found that a decrease in trNK cells and ILCls proportion both in Il4ra-/-liver and uterus,compared with WT mice.Injection with IL-4 i.v.led to decrease of trNK cell proportion in liver but an increase in uterus.4.Generated CD49a-Eomes+ NK cells functioned alike uterus-resident NK cells and rescued fetal growth restriction.IL-4-stimulated iNK cells expressed Eomes and reprogrammed functionally.The expression of IFN-y by IL-4-stimulated iNK cells was increased and mainly contributed by Eomes+ cells.IL-4 promoted placental growth factor(PGF)secretion by iNK cells,which can be secreted by uterine NK cells and contributes to angiogenesis in early decidua.Subsequently,the killing assay against YAC-1 target cells showed a decrease of cytotoxicity in IL-4-stimulated iNK cells.Importantly,IL-4-stimulated iNK cells also expressed growth-promoting factors,such as PTN,OGN and OPN,similar to decidual NK cells during early pregnancy.Transferred Eomes+ iNK cells during early pregnancy can trafficked to decidua preferentially and rescue fetal growth restriction in Nfil3-/-mice and aged mice in a growth-promoting factor-dependent manner.Collectively,these studies show an approach to generate tissue-resident-like CD49a+Eomes-/+ NK cells,which shows potential for developmental research and therapeutic applications.