The Regulatory Mechanism Of KIR Gene Expression And Its Effect On NK Cell Function

Background and aims:Natural killer(NK) cells as a component of the innate immune system provide a first line of defense against viral infections and malignancies.Killer immunoglobulin-like receptors(KIRs) are a polymorphic gene family expressed on NK cells.In humans,a major component of NK cell target recognition depends mainly on the surveillance of human leukocyte antigen (HLA) classⅠmolecules by KIRs.Different KIR can transmit inhibitory or activating signal to the cell,and effector function is considered to result from the balance of these contributing signals.Inhibitory KIRs are important not only for tumor and virus-infection surveillance,but also for immunological tolerance to discriminate between normal and abnormal cells.Clarifying the relationship between inhibitory KIRs regulation and NK cell function will facilitate the potential manipulation of these immunocytes both in vitro and in vivo.In this study,we investigated the regulation of DNA methylation and transcription factor E2F on KIR gene expression and analyzed the direct effect of DNA demethylating treatment on human NK cell cytolytic activity.Methods:(1) To analyze the promoter methylation patterns of KIR3DL1 gene and its relationship with gene expression,the CpG methylation pattern of KIR3DL1 promoter region in KIR3DL1-positive human NK cell line NK-92MI and KIR3DL1-negative human leukemic cell line K562 was detected by bisulfite sequencing technique.Then NK-92MI cells were treated with 5-azacytidine (5-Aza) to induce the demethylation of CpG islands.The level of KIR3DL1 gene expression was determined by RT-PCR.To study the role of transcription factor E2F in the transcriptional activity of KIR3DL1 promoter,the mutant and wild-type core promoter fragment of KIR3DL1 gene was respectively amplified from K562 genomic DNA and a plasmid containing the wild-type KIR3DL1 promoter sequence by PCR.PCR products were cloned into pGL3-basic reporter vector to construct KIR3DL1 promoter-luciferase reporter plasmids.The binding of E2F1 to KIR3DL1 promoter was detected by chromatin immunoprecipitation (CHIP) assay.KIR3DL1 promoter-luciferase reporter construct was transfected into K562 cells using polycationic compound SuperFect.The binding of E2F1 to the construct was detected by CHIP assay and reporter activity was quantitated using the dual-luciferase reporter assay system.Finally,the mammalian expression vector containing E2F1 cDNA was co-transfected into K562 cells with KIR3DL1 promoter-luciferase reporter construct and reporter activity was quantitated using the dual-luciferase reporter assay system.(2) To investigate the direct effect of DNA demethylating treatment on human NK cell cytolytic activity, NK-92MI cells and freshly isolated human primary NK cells were respectively treated with 5-Aza.The KIR3DL1-positive and -negative NK cell subsets were sorted by flow cytometry.The cytotoxicity of NK cells against human K562 leukemic cells was detected by the lactate dehydrogenase release assay.The promoter methylation patterns of inhibitory KIR genes in NK-92MI cell line were detected by bisulfite sequencing technique.The levels of inhibitory KIRs expression in 5-Aza-treated and untreated NK cells were determined by RT-PCR and flow cytometry.The granzyme B and perforin release by NK cells was detected by enzyme linked immunosorbent assay.Results:(1) The core promoter region of KIR3DL1 gene exhibits densely methylated in NK-92MI and K562 cells.Treatment of NK-92MI and K562 cells with 5-Aza significantly increased the expression level of KIR3DL1 gene.DNA sequencing revealed a naturally occurring point mutation(TT(?)GGCGC→TT(?)GGCGC) within a putative E2F binding site in the KIR3DL1 promoter in K562 cells.Interestingly,this mutation introduced a new methylation site.E2F1 could bind to the KIR3DL1 promoter in NK-92MI cells,and the point mutation in the E2F binding site absolutely abolished their binding in K562 cells.The binding of E2F1 to the mutant KIR3DL1 promoter-reporter construct was partially abolished compared to its binding to the wild-type construct.The mutation in E2F binding site significantly decreased the relative luciferase activity of the mutant construct,yielding an average of 50%of activity of the wild-type one. Co-transfection of E2F1 expression plasmid transactivated the wild-type KIR3DL1 promoter,yielding about 2.4-fold higher values than the non-cotransfected control.(2) Inhibitory KIR genes including KIR2DL1, KIR2DL2,KIR2DL3 and KIR3DL1 exhibited characteristic epigenetic repression,whose promoter regions are densely methylated.Treatment of NK-92MI cells with 5-Aza significantly increased the expression levels of these inhibitory KIRs.But the cytolytic activity of NK-92MI cells and freshly isolated human NK cells against human K562 leukemic cells was strongly suppressed upon 5-Aza-treatment.Furthermore,granzyme B and perforin release by NKs cells was also decreased.Among the 5-Aza-treated NK cells,both cytolytic activity and level of granzyme B and perforin release in KIR3DL1-negative NK subsets were higher than those in KIR3DL1-positive ones.Conclusion:The KIR3DL1 gene expression in NK cells is determined by promoter methylation.E2F1 contributes to the transcriptional activation of the KIR3DL1 gene.Demethylating treatment suppresses NK cell cytolytic activity. The suppression is associated with 5-Aza-induced overexpression of inhibitory KIRs and impaired cytotoxic granules release by these cells.