The Function And Mechanism Of Growth-promoting Factor Produced By NK Cells

Natural killer cells(NK cells)are named for their spontaneous killing ability.Since their accidental discovery 43 years ago,NK cells write a never-ending story of excitement and intrigue for immunologists and clinicians due to their unique fundamental properties and therapeutic promise for translational medicine.NK cells are not only "sentinel" in host defense,but also play a key role in maintaining tissue homeostasis by secreting cytokines and chemokines.Disturbance in the number of mature NK cells impact tumor formation and growth as well as autoimmune disease.However,at present,whether it is the study of NK cell functional molecules or the regulation factors for promoting to NK cell development and maturation,they are still limited to a few cytokines suc.h as IFN-? and IL-15.Growth-promoting factors are a class of cytokines that are widely expressed and have a great effect on cell development and maturation,but are rarely reported in the study of NK.At the same time,the function research of NK cells is mainly focused on the function of peripheral NK cells in host defense,but there are few reports on the tissue-specific functions of tissue-residue NK(trNK)cells.In human peripheral blood,NK cells account for about 10%of the total number of lymphocytes,but in the uterus,with the onset of pregnancy response,rapid decidualization of the endometrium,and the number of NK cells rapidly increases,up to 70%of the total number of decidual tissue lymphocytes.The number and optimal function of NK cells in decidual tissue are important for normal pregnancy and fetal health in women.CD56+CD3-NK cells in decidual tissue are generally considered to include a small amount of peripheral CD11b+ NK cells that still retain killing capacity and a small amount of CD27+ NK cells that produce IFN-? to inhibit Thl7 cells to maintain maternal-fetal immune tolerance.However,the function and mechanism of CD11b-CD27-trNK cells,which account for about 80%of the decidual NK cells,remain unknown.On the other hand,the research on the development and maturation of NK cells not only enhances our understanding of the formation and maintenance of mature NK cell pools in vivo,but also helps us to obtain mature NK cells with optimal function through differentiation and expansion in vitro.Bone marrow(BM)is generally considered to be the primary site of development and maturation of NK cells:NK cells derived from hematopoietic stem cells in the BM by programmed development and differentiation.After undergoing common lymphoid progenitor(CLP)cells,NK progenitor(NKP)cells,and immature NK cells,eventually mature NK cells migrate from the BM into the periphery and perform functions.The development and maturation of NK cells in the BM depends on the BM microenvironment,and the IL-15 and 1L-15 signaling pathways derived from the microenvironment are currently considered to be important factors for the development and maturation of NK cells.However,little is known about the key mechanisms of GPFs on the development and maturation of NK cells.This article focuses on exploring the role of growth-promoting factors in the tissue-specific functions of NK cells and in promoting the development and maturation of NK cells.The following results were obtained:1.NK cells promote fetal development through the secretion of growth-promoting factorsIn this section,we have identified a CD49a+Eomes+ subset of decidual NK(dNK)cells that secreted growth-promoting factors(GPFs),including pleiotrophin and osteoglycin,in both humans and mice.The crosstalk between HLA-G and ILT2 served as a stimulus for GPF-secreting function of this NK cell subset.Decreases in this GPF-secreting NK cell subset impaired fetal development,resulting in fetal growth restriction.These findings reveal properties of NK cells in promoting fetal growth.In addition,this research proposes approaches for therapeutic administration of NK cells in order to reverse restricted nourishments within the uterine microenvironment during early pregnancy.1.The differential analysis of dNK cells and peripheral NK cells(pNK)By gene ChIP,we identified that CD49a4+ dNK cells upregulated several GPFs,including pleiotrophin(PTN),osteoglycin(OGN)and osteopontin(OPN,encoded by SPP1),compared to these products in CD49a-NK cells.2.Uterine trNK cells secrete GPFsFurther protein analyses using both flow cytometry and immunofluorescence demonstrated that dNK cells had higher expressions of PTN,OGN,and OPN than pNK cells.All the PTN+ NK,OGN+ NK,and OPN+ NK cells primarily existed within the CD11b-CD27-NK cell subset.And,dNK cells,especially those in the CD49a+Eomes+ trNK cell subsets,secrete GPFs.3.HLA-G induces NK cells to secrete GPFsDecidual NK cells secreted more GPFs,including PTN,OGN,and OPN,upon co-stimulation with extravillous trophoblast(EVT)cells.HLA-G from EVT cells,but not HLA-C,promotes GPFs secretion in dNK cells.Further functional experiments prove that HLA-G-ILT2-KIR2DL4 axis is essential in GPFs secretion from dNK cells in early pregnancy.4.Impaired GPF+ NK cells in patients with fetal lossDecidual NK cells from recurrent spontaneous abortion(RSA)patients exhibited a significantly decreased percentage of CD11b-CD27-and CD49a+Eomes+ trNK cell subsets.Furthermore,contrary to the high GPF expression in normal dNK cells,significantly decreased expression of GPFs,including PTN,OGN,and OPN,were confirmed in dNK cells from RSA patients.5.Fetal growth restriction in Ptn-/-Ogn-/-Spp1-/-micePtn-/-Ogn-/-Sph1-/-mice were generated using CRISPR-Cas9 technology.These Ptn-/-Ogn-/-Spp1-/-mice showed significantly decreased uterine weight and fewer live fetuses,as well as impaired GPF expression.?.PBX1 programs natural killer cell development and maturation in the bone marrowIn this section,we have identified that PBX1,a conserved transcription factor,programs NK cell development and maturation.In progenitor cells,ablation of Pbx1 reduced NK cell numbers.During the maturing of NK cells in the BM,PBX1 promoted the transcription of growth-promoting factors pleiotrophin(Pin)and osteoglycin(Ogn)through direct promoter binding.Pbx1-deficient mice exhibited impaired NK cell maturation in the BM due to reduced expression of PTN and OGN.Furthermore,PTN and OGN supported mature NK cell homeostasis in vivo with upregulating the expression of T-bet.This study demonstrates the PBX1-PTN/OGN axis to be intrinsic to mature NK cell pool maintenance.Further,exploitation of this axis may be a means by which to improve NK cell-based immunotherapies.1.PBX1 is a highly conserved transcription factor in NK cellsThe phylogeny of NK cells contains clues to the regulation of NK cell development.Analyzing the conservation of key genes of NK cells in different species,it was found that PBX1 is a highly conserved transcription factor of NK cells.Hman BM NK cells express PBXl with high specificity relative to T cells,and that PBX1 is highly expressed in early and mature NK cells.2.PBX1 regulates the transcription of PTN and OGN in NK cellsBioinformatics prediction analysis showed that gene promoter regions of Pin and Ogn are rich in multiple binding sites of PBX1.Both knockdown and overexpression of PBX1 showed that PBX1 promoted the expression of PTN and OGN in NK cells.CHIP-seq with anti-PBX1 antibody showed that PBX1 had direct binding activity in the PTN and OGN promoter regions.Both luciferase reporter and DNA-pulldown experiment of cells demonstrated that PBX1 in human NK cells binds to specific sequences of the PTN and OGN promoter regions.3.Selective Pbxl ablation impairs NK cell developmentThe results by the mouse BM chimera model showed that the percentage and number of NK cells decreased significantly with Pbxl knockdown,indicating that a reduction in PBX1 impaired the development of NK cells.And,the results by Phx1f/f,Vav1Cre and Phx1f/f Ncr1Cre mice indicate that PBXl not only regulates the early development of NK cells,but also promotes late-stage maturation of NK cells.4.PBX1 supports BM NK cell maturationDue to the significant reduction in the expression levels of PTN and OGN in BM NK cells of Pbx1f/f:Vav1Cre and Phx1f/f;NcrlCre mice,an overall reduction in the late-stage mature CD11b+CD27-NK cells was observed in the BM and spleen.PTN and OGN rescued NK cell terminal maturation and restored the number of mature NK cells in the BM and periphery of Phx1f/f;NcrlCre mice,while IL-15 did not perform as well.These findings suggest that a deficiency in PBX1 results in a loss of growth-promoting factors,reducing intrinsic motility,late-stage NK cell BM,maturation,and selective cell depletion.5.The PBX1-PTN/OGN axis replenishes the pool of mature N K cells in vivoTaken together results from Pbx1f/f;Ncr1Cre mice and Ptn-/-Ogn-/-mice identify a PBX1-PTN/OGN axis in which PBX1 regulates NK cell autocrine PTN and OGN to promote late-stage NK cell maturation,and that PBX1 is the intrinsic motivation factor for terminal maturation of BM NK cells.6.PBX1-PTN/OGN axis upregulates T-bet expressionT-bet is an important checkpoint transcription factor that controls the final maturation of BM NK cells.PTN and OGN upregulated T-bet expression in BM NK cells during the rescue of BM NK cells late-stage maturation in Phxl1f/f;NcrlCre and Ptn-/-Ogn-/-mice by injection of soluble PTN and OGN.PBX1-PTN/OGN axis promotes the terminal maturation of NK cells in the BM may be through the up-regulation of T-bet expression.