| Natural killer(NK)cells contribute to the host's first line of tumor immunosurveillance with the ability to identify and eliminate transformed cells through the release of cytotoxic granules containing perforin and granzymes.Harnessing NK cell effector function represents a critical immunotherapeutic approach to cancer.Although NK cells are set up throughout development to be potent killers,their roles can be subverted at the tumor site.Tumor-infiltrating NK cells frequently exhibit a dysfunctional phenotype,express decreased levels of activating receptors and increased levels of inhibitory receptors,produce fewer cytokines such as interferon(IFN)-y,and exert low cytotoxicity.Both animal data and clinical evidence suggest the loss of functional NK cells and the accumulation of inactive and immature NK cells in tumor microenvironment.A better understanding of the molecular regulatory mechanisms that control NK cell maturation and survival is crucial for the development of NK cell-based immunotherapy.NK cells develop mainly in the bone marrow.After the acquisition of the interleukin(IL)-15 receptor ? chain(CD 122),followed by the expression of NK1.1,NK cells continue a late maturation program which can be further classified based on the surface expression of CD11b and CD27:CD11b-CD27+,CD11b+CD27+,and CD11b+CD27+.During maturation,NK cells maintain a balance between the expression of activating and inhibitory receptors and progressively increase their cytotoxic capacity but decrease their potential for homeostatic expansion and become prone to apoptosis.NK cell development and functional maturation are triggered by plenty of extracellular signals,among which IL-15 is critical for both NK cell lineage commitment and terminal maturation.In addition,NK cell development is dictated by a series of transcription factors(TFs)which promote the expression of genes coding for effector molecules and cell-surface maturation markers.For instance,Id2,STAT5,Tox,Ets1 and Nfil3 regulate early stage of NK development,while concerted actions of T-bet and Zeb2 determine terminal NK cell maturation and survival.Although large advances have been achieved in transcriptional regulation of NK development,negative regulators in the process of NK cell maturation are still largely unknown.Zinc fingers and homeoboxes 2(Zhx2),one member of the ZHX family,is identified as a ubiquitous transcriptional repressor.Zhx2 has attracted considerable attention due to its involvement in tumorigenesis,cell differentiation and metabolic related diseases,whereas its function in the immune system is mostly overlooked.Zhx2 is abundantly expressed in the thymus and spleen and has been reported to play a role in B cell development and macrophage polarization.By manipulating macrophages activation and differentiation,Zhx2 participates in inflammation related diseases,such as atherosclerosis and sepsis.Those findings indicate Zhx2 as a potential regulator which participates in immune cell differentiation.However,its function in NK cells is unclear.Here,we identified Zhx2 as a master regulator of NK cells which restricts NK cells maturation,survival programs,and effector functions.Mechanically,Zhx2 lowered NK cells responses to IL-15 and transcriptionally repressed Zeb2 expression.Deletion of Zhx2 in NK cells successfully enhanced the formation of mature NK cells and promoted curative antitumor immunity of adoptive NK cell immunotherapy.These data provide new insight into the regulatory mechanism of NK cell maturation and revealing novel opportunities for NK cell-based immunotherapy.The main results are as follows:1.Loss of Zhx2 leads to accumulation of matured NK cells in mice1.1.Zhx2 is highly expressed in tissue resident NK cells.We firstly detailed Zhx2 expression pattern in immune cells with the publicly available database,Notably,among members of the ZHX family,Zhx2 was highly expressed in lymphocytes and in tissue-resident NK cells1.2.Knockout of Zhx2 in NKp46+cells result in reduced maturation of NK cells.To study the role of Zhx2 in NK cells,we crossed Ncr1-cre mice with mice carrying floxed Zhx2 alleles,to generate NK cell-specific conditional knock-out mouse(Zhx2?/?).Western blot confirmed the deletion of Zhx2 in splenic NK cells.Compared to wild type(Zhx2+/+)mice,remarkable increment of NK cells was observed in Zhx2?/? mice in organs such as liver,spleen,and blood both in relative ratio and absolute quantity.Further flow cytometric analyses showed no difference of the number of Lin-CD122+NK progenitor.However,NK cell specific Zhx2 depletion led to an overall increased proportion of the most mature CD27-CD11b+NK population.The frequency of the KLRG1+mature subset was also significantly higher in the liver and spleen from Zhx2?/? mice compared to Zhx2+/+ mice.1.3.Zhx2-mediated a cell-intrinsic role in NK cell homeostasis.Both mixed bone marrow(BM)chimera experiment and non-competitive transplantation assay demonstrated a similar increase in NK1.1+ cells developing from the transferred Zhx2?/? BM in receipt mice.2.Enhanced effector functions of NK cells with Zhx2 deficiencyNK cells maturation accompanies with an enhancement of effector functions.We next assessed the functional effects of Zhx2?/? NK cells in vitro and in vivo.2.1.Reduced expression of inhibitory receptor NKG2A and increased expression of activating receptor NKG2D on splenic Zhx2?/? NK cells.Flow cytometry showed significantly reduced expression of inhibitory receptor NKG2A as well as the obviously increased expression of activating receptor NKG2D on splenic Zhx2?/? NK cells.2.2.Zhx2 deficiency renders NK cells enhanced effector functions.In order to access the function of NK cells,we used anti-NK1.1 antibody to stimulate Zhx2+/+and 2hx2?/? NK cells.Flow cytometry assay showed that hepatic and splenic CD3-NK1.1+NK cells from Zhx2?/? mice displayed markedly enhanced production of IFN-y,TNF-?,CD 107a,and granzyme than those in Zhx2+/+mice.RT RT-qPCR further verified the augmented production of effector molecules in purified murine splenic NK cells from Zhx2?/? mice.2.3.Better cytotoxic killing function of Zhx2?/? NK cellsFurther,splenic NK cells from control or Zhx2?/? mice were tested for their ability to kill mouse lymphoma Yac-1 cells.Supporting the notion that Zhx2 inhibits NK functions,NK cells from Zhx2?/? mice showed significant higher killing activities against Yac-1 cells than that from Zhx2+/+mice in different E:T ratio.2.4.Zhx2-deficience promotes NK cell antitumor function.We then evaluated the tumor elimination in vivo by subcutaneous injection of mice hepatoma cell line Hepal-6 cells into Zhx2?/? and Zhx2+/+ mice respectively.Larger tumors had evolved in Zhx2+/+ mice,whereas a pronounced reduction of tumor mass was observed in Zhx2?/? mice.2.5 Zhx2 inhibits NK92 cells'functions.To further confirm the role of ZHX2 in human NK cells,ZHX2 overexpression and knockdown were performed in human NK cell line NK92 cells.As expected,overexpression of ZHX2 decreased,while interference of ZHX2 expression enhanced expression of functional molecules IFN-y,TNF-?,granzyme,and perforin.Also,ZHX2 inhibited the cytotoxicity of NK92 cells against target K562 cells.3.Loss of Zhx2 enhance NK cells survival.We next investigated the mechanism of the increment of CD27low NK cells in Zhx2?/? mice.To address that,splenic NK cells were purified from both Zhx2+/+ or Zhx2?/? mice,and cell proliferation and survival were accessed.3.1.Zhx2 does not suppress IL-15 induced NK cells proliferation.Both EdU labelling assay and ex vivo Ki67 staining did not detect any significant difference between NK cells from two groups,indicating that Zhx2 does not influence NK cell proliferation.3.2.Loss of Zhx2 promotes NK cells survival.Annexin V/7-AAD staining assay showed obviously reduced number of apoptotic cells in NK cells isolated from Zhx2?/? mice than that from control Zhx2+/+mice.To confirl the apoptosis resistance of NK cells in Zhx2?/? mice,the apoptosis inducer Cycloheximide(CHX)was added to NK cell culture.Upon CHX treatment,in vitro cultured Zhx2?/? NK cells undergoing less apoptosis than control at all detected time points.In accordance,RNA-seq and GSEA using the Broad Hallmark gene-set collection demonstrated the significant enrichment for gene set encoding apoptosis-related molecules in Zhx2?/? NK cells relative to their expression in Zhx2+/+NK cell subset.Congenially marked NK cells from Zhx2+/+ or Zhx2?/? mice were labelled with Cell Trace Violet(CTV)or CFSE respectively,co-transferred into recipient mice,a selective loss of Zhx2+/+NK cells in recipient mice.4.Zhx2 deficiency enhances NK cell response to IL-15 stimulation.IL-15 plays a crucial role in NK cell biology including survival,maturation,and function.We then came to examine whether Zhx2 participates in IL-15 signaling in NK cells.4.1.Zhx2-deficiency enhances IL-15 signaling in NK cell.GSEA was performed comparing RNA-seq data of splenic NK cells purified from Zhx2+/+ and Zhx2?/? mice and result demonstrate the enrichment of IL-15 signaling pathway in Zhx2?/? NK cells.Consistently,in vitro stimulation of IL-15 led to obvious increase in phosphorylated STAT5 and AKT in CD3-NK1.1+cells isolated from Zhx2?/? mice than that from Zhx2+/+ mice,although flow cytometry did not detect any significant difference in the expression of CD 122 in NK cells with the absence of Zhx2.4.2.Deletion of Zhx2 promotes IL-15-mediated NK cell function.With the IL-15 treatment,Zhx2?/? NK cells showed significantly enhanced effector functions displaying as increased levels of CD 107a and IFN-y.4.3.Zhx2 weakens IL-15 promoted NK cell function.To further verify the depressant function of Zhx2 in IL-15 pathway,the selective IL-15 signaling inhibitor STAT5-Inh was included.Treatment of STAT5-Inh not only largely rescued the IL-15 triggered up-regulation of phosphate STAT5 level in Zhx2?/?NK cells,but also eliminated the difference of IL-15 induced cell apoptosis between Zhx2+/+and Zhx2?/? NK cells.In accordance,STAT5-Inh almost completely destroyed the enhanced production of CD107a and IFN-y expression in Zhx2?/? NK cells.5.Zhx2 directly inhibits Zeb2 t.ranscription during NK cell development.To further identify the direct target of Zhx2 and elucidate the underlying mechanism by which Zhx2 regulate NK cells,RNA-seq and transposase-accessible chromatin using sequencing(ATAC-seq)was performed using Zhx2?/? and Zhx2?/?splenic NK cells.5.1.Zhx2 changes transcriptional network of NK cellsThe volcano plot from RNA-seq showed that 1642 genes significantly altered in Zhx2?/? NK cells compared with Zhx2+/+ NK cells(1325 upregulated and 317 downregulated,P<0.05).GSEA-was used to compare transcriptional profiles of Zhx2+/+NK cells and Zhx2?/? NK cells against previously defined signature genes of DP or CD27low NK cells.The set of Zhx2?/? NK cells was preferentially active in the CD27low subset.In addition,Gene Ontology(GO)assay showed that immune response and inflammatory response were significantly overrepresented in Zhx2?/?NK cells.5.2.Zhx2 regulates genes transcription related with NK cell maturationTo identify the direct target gene of Zhx2,we performed cluster analysis of sharing genes in ATAC-seq,RNA-seq data from Zhx2+/+ and Zhx2?/? splenic NK cells,and reported CD27low/DP NK represented genes.Results showed that most genes were specific to only one or two clusters.However,9 genes changed in all three clusters,which are believed to be Zhx2 regulated genes in NK cell maturation.The Zhx2 mediated regulation of these 9 genes were further verified with RT-qPCR with purified splenic CD3-NK1.1+NK cells from Zhx2?/? and Zhx2+/+ mice.Further comparing of the expression levels of these 9 genes during NK cell development with previoulsy reported data showed that Zhx2 upregulated genes are gradually up regulated during NK cell development,while Zhx2 down regulated genes showed their highest expression in the matured CD27low NK cells.Among 6 of Zhx2 down regulated targets,Zeb2 was the highest differentially expressed transcription factor.5.3.Zhx2 regulates transcription of Zeb2 by binding with its promoterRT-qPCR showed that the negative correlation of Zeb2 and Zhx2 mRNA in NK cells isolated from Zhx2?/? mice and Zhx2+/+ mice.In accordance,co-transfection and dual luciferase assay demonstrated that ZHX2 overexpression greatly upregulated the promoter activity of ZEB2 in 293T cells,which is consistent with our ATAC-seq data showing the more accessibility of Zeb2 loci in Zhx2?/? NK cells.Furthermore,ChIP assay performed with anti-ZHX2 and NK92 cell lysate demonstrated that ZHX2 significantly occupied with ZEB2 promoter in NK cells.The Zhx2 mediated transcription inhibition of Zeb2 was further confirmed with GSEA analysis comparing our RNA-seq data and reported data.Zeb2 up-regulated transcripts were decreased in Zhx2?/? subsets.These results suggested that Zhx2 is capable of binding to the Zeb2 locus,thus regulates Zeb2 transcription.5.4.Zeb2 involved inzhx2-mediated NK cell maturation and function.Zeb2 interference was performed by lentivirus in splenic NK cells from Zhx2+/+and Zhx2?/? mice and then the splenic NK cells were transferred to irradiated Zhx2+/+mice.Zeb2 knockdown significantly dampened the enrichment of CD27low NK subset and the augment of CD 107a expression in the Zhx2?/? group.The interference efficiency of Zeb2 was confirmed by western blot.6.Better control of tumor growth by NK cells with Zhx2 deletion.It is well established in previous studies that blockade of NK cell maturation impairs their antitumor potential.Now we wonder whether Zhx2 participates in this process.6.1.The expression of Zhx2 is negatively correlated with NK cell infiltration in the tumor.We analyzed TCGA hepatocellular carcinoma(HCC)dataset for NK cell abundance using specific gene sets for identifying NK cells.High ZHX2 expression in NK subsets correlated with the low frequency of tumor infiltrating NK(TINK)cells and vice versa,tumor with more infiltration of TINK cells displayed less ZHX2 expression in NK cells.Furthermore,patients with low ZHX2 expression in NK cells showed better survival than that with high ZHX2 in NK cells.This was further verified in mice orthotopical HCC model.Results of flow cytometry showed that the number of tumor infiltrating CD3-NK1.1+NK cells from Zhx2?/? mice was significantly higher than that of Zhx2+/+group.Terminal maturation NK cell subsets(CD27low)displayed an increase in tumors of Zhx2+/+mice.Also,TINK fro1 the Zhx2?/? mice had a marked increase in expression of the effector molecular CD 107a.6.2.ZhX2-deficient NK cells show better control of tumor growth.To address whether the deletion of Zhx2 would generate more functional and long-survival NK cells to protect against cancer,both subcutaneous hepatoma homograft and lung metastasis model were performed to assess the antitumor capacity of Zhx20?/? NK cells.Transfer of splenic NK cells from Zhx2?/? mice greatly suppressed the growth of Hepa1-6 subcutaneous homograft.Also,Zhx2-deficient NK cells significantly reduced lung metastasis of B16-F10 melanoma.The number of metastatic nodules in the lung was significantly reduced in mice transferred with Zhx2?/? splenic NK cells while the overall survival of the mice treated with Zhx2?/?NK cells was also significantly longer.6.3.Zhx2 interfere promotes NK cell-based tumor therapy against human cancerIn order to verify whether ZHX2 could be a potential intervention target aiming to enhancing NK cells immunosurveillance in tumor therapy,two human HCC models were set up in NSG mice.HepG2-luciferase cells were intraperitoneally injected and,in the meantime,NK92 cells infected with LV-shNC or LV-shZHX2 lentivirus were transferred.Results of d-luciferin-based bioluminescence assay showed that transfer of NK92 cells infected with LV-shZHX2 lentivirus greatly suppressed the growth of peritoneal hepatoma xenografts along the time.The overall survival of the LV-shZHX2 group was also significantly longer.Similar results were got with subcutaneous HCC xenograft model.Significantly smaller tumors were found in the mice received LV-shZHX2 NK92 cells treatment. |
PDF Full Text Request