Characterization of the recognition of tumor cells by the natural cytotoxicity receptor, NKp44

NKp44 is a natural cytotoxicity receptor expressed by human NK cells upon activation. In this study, we demonstrate that cell surface heparan sulfate proteoglycans (HSPGs), expressed by target cells, are involved in the recognition of tumor cells by NKp44. NKp44 showed heparan sulfate-dependent binding to tumor cells; this binding was partially blocked with an antibody to heparan sulfate. In addition, direct binding of NKp44 to heparin was observed, and soluble heparin/heparan sulfate enhanced the secretion of IFNgamma by NK92 cells activated with anti-NKp44 monoclonal antibody. Basic amino acids, predicted to constitute the putative heparin/heparan sulfate binding site of NKp44, were mutated. Tumor cell recognition of the mutated NKp44 proteins was significantly reduced and correlated with their lower recognition of heparin. We previously reported that NKp44 recognizes the hemagglutinin of influenza virus (IV). Nevertheless, the ability of the mutated NKp44 proteins to bind viral hemagglutinin expressed by IV-infected cells was not affected. Thus, we suggest that heparan sulfate epitope(s) are ligands/co-ligands of NKp44 and are involved in its tumor recognition ability.     

Membrane-associated heparan sulfate proteoglycans are involved in the recognition of cellular targets by NKp30 and NKp46

Lysis of virus-infected and tumor cells by NK cells is mediated via natural cytotoxicity receptors (NCRs). We have recently shown that the NKp44 and NKp46 NCRs, but not the NKp30, recognize viral hemagglutinins. In this study we explored the nature of the cellular ligands recognized by the NKp30 and NKp46 NCRs. We demonstrate that target cell surface heparan sulfate proteoglycans (HSPGs) are recognized by NKp30 and NKp46 and that 6-O-sulfation and N-acetylation state of the glucose building unit affect this recognition and lysis by NK cells. Tumor cells expressing cell surface heparanase, CHO cells lacking membranal heparan sulfate and glypican-1-suppressed pancreatic cancer cells manifest reduced recognition by NKp30 and NKp46 and are lysed to a lesser extent by NK cells. Our results are the first clue for the identity of the ligands for NKp30 and NKp46. Whether the ligands are particular HSPGs, unusual heparan sulfate epitopes, or a complex of HSPGs and either other protein or lipid moieties remains to be further explored.     

Altered glycosylation of recombinant NKp30 hampers binding to heparan sulfate: a lesson for the use of recombinant immunoreceptors as an immunological tool

NKp30 is a natural cytotoxicity receptor expressed by human NK cells and involved in NK lytic activity. We previously published that membranal heparan sulfate serves as a coligand for human NKp30. In the present study, we complement our results by showing direct binding of recombinant NKp30 to immobilized heparin. The heparan sulfate epitope(s) on target tumor cells and the heparin epitope(s) recognized by NKp30 share similar characteristics. Warren and colleagues (Warren HS, Jones AL, Freeman C, Bettadapura J, Parish CR. 2005. Evidence that the cellular ligand for the human NK cell activation receptor NKp30 is not a heparan sulfate glycosaminoglycan. J Immunol. 175:207-212) published that NKp30 does not bind to membranal heparan sulfate on target cells and that heparan sulfate is not involved in NKp30-mediated lysis. In the current study, we examine the binding of six different recombinant NKp30s to membranal heparan sulfate and conclude that NKp30 does interact with membranal heparan sulfate. Yet, two of the six recombinant NKp30s, including the commercially available recombinant NKp30 (employed by Warren et al.) did not show heparan sulfate-dependent binding. We demonstrate that this is due to an altered glycosylation of these two recombinant NKp30s. Upon removal of its N-linked glycans, heparan sulfate-dependent binding to tumor cells and direct binding to heparin were restored. Overall, our results emphasize the importance of proper glycosylation for analysis of NKp30 binding to its ligand and that membranal heparan sulfate could serve as a coligand for NKp30. At the cellular level, soluble heparan sulfate enhanced the secretion of IFNgamma by NK-92 natural killer cells activated with anti-NKp30 monoclonal antibody. We discuss the involvement of heparan sulfate binding to NKp30 in NKp30-mediated activation of NK cells.     

Binding of natural cytotoxicity receptor NKp46 to sulfate- and α2,3-NeuAc-containing glycans and its mutagenesis

Natural cytotoxicity receptor 1 (NCR1, NKp46) binds to heparin and heparan sulfate; however, other natural ligands for NKp46 have yet to be elucidated. Using the recombinant extracellular region (coding for AA 22-258) of NKp46 tagged with 6× His (NKp46-H6), and mutants K136Q, R139Q, H142Q, R145Q, and K149Q, we determined their binding affinities to sulfate- and NeuAc-containing glycans-coated plates. NKp46-H6 directly bound to plates coated with heparin- and heparan sulfate-conjugated bovine serum albumin with K_d values of 770 and 850 nM, respectively. The binding of NKp46-H6 to heparin–BSA was suppressed by soluble heparin, herparan sulfate, fucoidan, λ-carrageenan, and dextran sulfate, but not by 2-O-, 6-O-, and N-desulfated heparin. NKp46-H6 also bound to multimeric sialyl Lewis X expressing transferrin secreted by human hepatoma HepG2 cells (HepTF) with a K_d value of 530 nM, but not to desialylated HepTF, commercially available TF, or 1-acid glycoprotein. Moreover, mutants R139Q, R145Q, and K149Q had significantly reduced binding to these sulfate-containing glycans, and K136Q and K149Q to HepTF, indicating that NKp46 binds to sulfate- and 2,3-NeuAc-containing glycans mainly via ionic interactions. However, the binding sites of NKp46 were different.     

Elucidating the mechanisms of influenza virus recognition by Ncr1

Natural killer (NK) cells are innate cytotoxic lymphocytes that specialize in the defense against viral infection and oncogenic transformation. Their action is tightly regulated by signals derived from inhibitory and activating receptors; the later include proteins such as the Natural Cytotoxicity Receptors (NCRs: NKp46, NKp44 and NKp30). Among the NCRs, NKp46 is the only receptor that has a mouse orthologue named Ncr1. NKp46/Ncr1 is also a unique marker expressed on NK and on Lymphoid tissue inducer (LTI) cells and it was implicated in the control of various viral infections, cancer and diabetes. We have previously shown that human NKp46 recognizes viral hemagglutinin (HA) in a sialic acid-dependent manner and that the O-glycosylation is essential for the NKp46 binding to viral HA. Here we studied the molecular interactions between Ncr1 and influenza viruses. We show that Ncr1 recognizes influenza virus in a sialic acid dependent manner and that N-glycosylation is important for this binding. Surprisingly we demonstrate that none of the predicted N-glycosilated residues of Ncr1 are essential for its binding to influenza virus and we thus conclude that other, yet unidentified N-glycosilated residues are responsible for its recognition. We have demonstrated that N glycosylation play little role in the recognition of mouse tumor cell lines and also showed the in-vivo importance of Ncr1 in the control of influenza virus infection by infecting C57BL/6 and BALB/c mice knockout for Ncr1 with influenza.     

生物肽SP对NK细胞活化性受体NCRs表达的影响

研究SP对NK92-MI细胞杀伤活性以及活化性受体NCRs(NKp46、NKp44和NKp30分子)的表达的影响,揭示SP对NK细胞杀伤功能的调节作用及其内在作用机制.MTT法测定NK92-MI细胞对K562细胞的杀伤活性;Real-Time PCR检测NCRs的mRNA表达;流式细胞术检测NCRs的膜表达.在10-14～10-8 mol/L浓度范围的SP作用24h,对NK92-MI细胞的杀伤活性有明显增强作用;10-14～10-8 mol/L的SP,均可增加NK92-MI细胞活化性受体NKp44、NKp46及NKp30的mRNA表达;该浓度范围的SP均可增加NKp46的膜表达水平,仅较低浓度( 10-14moL/L)的SP对NKp44的膜表达水平有增加作用,各浓度的SP对NKp30的膜表达水平均无明显影响.SP可通过上调活化性受体NCRs的表达水平来调节NK细胞的活性.     

Dimerization of NKp46 receptor is essential for NKp46-mediated lysis: characterization of the dimerization site by epitope mapping

NKp46 is a primary activating receptor of NK cells that is involved in lysis of target cells by NK cells. Previous studies showed that the membrane-proximal domain of NKp46 (NKp46D2) retained the binding of NKp46 to its ligands and is involved in lysis. We studied NKp46D2 by using a peptide-based epitope mapping approach and identified an NKp46D2-derived linear epitope that inhibited NKp46-mediated lysis. The epitope, designated as pep4 (aa 136-155), interacted with NKp46, and lysis by NK cells was inhibited by the presence of pep4. Through modeling and mutagenesis, we showed that pep4 could be involved in NKp46 homodimerization. R145 and D147 contribute to the function of pep4, and R145Q mutation in recombinant NKp46 reduced its binding to target cells. At the cellular level, fluorescent resonance energy transfer analysis revealed that pep4 is indeed involved in dimerization of cell membrane-associated NKp46. We suggest that the NKp46-derived pep4 site is part of the dimerization surface of NKp46 and that NKp46 dimerization contributes to NKp46-mediated lysis by NK cells.     

Harnessing soluble NK cell killer receptors for the generation of novel cancer immune therapy

The natural cytotoxic receptors (NCRs) are a unique set of activating proteins expressed mainly on the surface of natural killer (NK) cells. The NCRs, which include three members; NKp46, NKp44 and NKp30, are critically involved in NK cytotoxicity against different targets, including a wide range of tumor cells derived from various origins. Even though the tumor ligands of the NCRs have not been identified yet, the selective manner by which these receptors target tumor cells may provide an excellent basis for the development of novel anti-tumor therapies. To test the potential use of the NCRs as anti-tumor agents, we generated soluble NCR-Ig fusion proteins in which the constant region of human IgG1 was fused to the extracellular portion of the receptor. We demonstrate, using two different human prostate cancer cell lines, that treatment with NKp30-Ig, dramatically inhibits tumor growth in vivo. Activated macrophages were shown to mediate an ADCC response against the NKp30-Ig coated prostate cell lines. Finally, the Ig fusion proteins were also demonstrated to discriminate between benign prostate hyperplasia and prostate cancer. This may provide a novel diagnostic modality in the difficult task of differentiating between these highly common pathological conditions.     

Recognition and prevention of tumor metastasis by the NK receptor NKp46/NCR1

NK cells employ a variety of activating receptors to kill virally infected and tumor cells. Prominent among these receptors are the natural cytotoxicity receptors (NCRs) (NKp30, NKp44, and NKp46), of which only NKp46 has a mouse ortholog (NCR1). The tumor ligand(s) of NKp46/NCR1 is still unknown, but it was shown that the human NKp46 and the mouse NCR1 are involved in tumor eradication both in vitro and in vivo. Whether any of the NK activating receptors is involved in the prevention of tumor metastasis is unknown. To address this question, we studied the activity of the NK cell receptor NKp46/NCR1 in two spontaneous metastasis models, the B16F10.9 melanoma (B16) and the Lewis lung carcinoma (D122) in the NCR1 knockout mouse that was generated by our group, in various in vitro and in vivo assays. We demonstrated that all B16 and D122 tumors, including those generated in vivo, express an unknown ligand(s) for NKp46/NCR1. We have characterized the properties of the NKp46/NCR1 ligand(s) and demonstrated that NKp46/NCR1 is directly involved in the killing of B16 and D122 cells. Importantly, we showed in vivo that NKp46/NCR1 plays an important role in controlling B16 and D122 metastasis. Thus, to our knowledge, in this study we provide the first evidence for the direct involvement of a specific NK killer receptor in preventing tumor metastasis.     

Umbilical cord blood T cells express multiple natural cytotoxicity receptors after IL-15 stimulation, but only NKp30 is functional

The natural cytotoxicity receptors (NCRs) NKp30, NKp44, and NKp46 are thought to be NK lineage restricted. Herein we show that IL-15 induces NCR expression on umbilical cord blood (UCB) T cells. NCRs were mainly on CD8(+) and CD56(+) UCB T cells. Only NKp30 was functional as demonstrated by degranulation, IFN-gamma release, redirected killing, and apoptosis. Since NCRs require adaptor proteins for function, the expressions of these adaptors were determined. The adaptors used by NKp30 and NKp46, FcepsilonR1gamma and CD3zeta, were detected in UCB T cells. There was a near absence of DAP12, the adaptor for NKp44, consistent with a hypofunctional state. NKp46 was on significantly fewer UCB T cells, possibly accounting for its lack of function. Adult peripheral blood (PB) T cells showed minimal NCR acquisition after culture with IL-15. Since UCB contains a high frequency of naive T cells, purified naive T cells from adult PB were tested. Although NKp30 was expressed on a small fraction of naive PB T cells, it was nonfunctional. In contrast to UCB, PB T cells lacked FcepsilonR1gamma expression. These results demonstrate differences between UCB and PB T cells regarding NCR expression and function. Such findings challenge the concept that NCRs are NK cell specific.     

Crystal structures of NK1-heparin complexes reveal the basis for NK1 activity and enable engineering of potent agonists of the MET receptor

NK1 is a splice variant of the polypeptide growth factor HGF/SF, which consists of the N-terminal (N) and first kringle (K) domain and requires heparan sulfate or soluble heparin for activity. We describe two X-ray crystal structures of NK1-heparin complexes that define a heparin-binding site in the N domain, in which a major role is played by R73, with further contributions from main chain atoms of T61, K63 and G79 and the side chains of K60, T61, R76, K62 and K58. Mutagenesis experiments demonstrate that heparin binding to this site is essential for dimerization in solution and biological activity of NK1. Heparin also comes into contact with a patch of positively charged residues (K132, R134, K170 and R181) in the K domain. Mutation of these residues yields NK1 variants with increased biological activity. Thus, we uncover a complex role for heparan sulfate in which binding to the primary site in the N domain is essential for biological activity whereas binding to the K domain reduces activity. We exploit the interaction between heparin and the K domain site in order to engineer NK1 as a potent receptor agonist and suggest that dual (positive and negative) control may be a general mechanism of heparan sulfate-dependent regulation of growth factor activity.     

The abundant NK cells in human secondary lymphoid tissues require activation to express killer cell Ig-like receptors and become cytolytic

Natural killer cells are important cytolytic cells in innate immunity. We have characterized human NK cells of spleen, lymph nodes, and tonsils. More than 95% of peripheral blood and 85% of spleen NK cells are CD56(dim)CD16(+) and express perforin, the natural cytotoxicity receptors (NCRs) NKp30 and NKp46, as well as in part killer cell Ig-like receptors (KIRs). In contrast, NK cells in lymph nodes have mainly a CD56(bright)CD16(-) phenotype and lack perforin. In addition, they lack KIRs and all NCR expression, except low levels of NKp46. The NK cells of tonsils also lack perforin, KIRs, NKp30, and CD16, but partially express NKp44 and NKp46. Upon IL-2 stimulation, however, lymph node and tonsilar NK cells up-regulate NCRs, express perforin, and acquire cytolytic activity for NK-sensitive target cells. In addition, they express CD16 and KIRs upon IL-2 activation, and therefore display a phenotype similar to peripheral blood NK cells. We hypothesize that IL-2 can mobilize the NK cells of secondary lymphoid tissues to mediate natural killing during immune responses. Because lymph nodes harbor 40% and peripheral blood only 2% of all lymphocytes in humans, this newly characterized perforin(-) NK cell compartment in lymph nodes and related tissues probably outnumbers perforin(+) NK cells. These results also suggest secondary lymphoid organs as a possible site of NK cell differentiation and self-tolerance acquisition.     

Novel Interaction between Proliferating Cell Nuclear Antigen and HLA I on the Surface of Tumor Cells Inhibits NK Cell Function through NKp44

NK cell function is closely regulated by numerous inhibitory and activating receptors binding corresponding ligands on the surface of target cells, providing vital first line defenses against infections and cancer. NKp44, originally discovered as an activating NK cell receptor, was recently found to elicit inhibitory effects on NK cell effector function through recognition of cell surface PCNA. Other reports have pointed to potential associations between NKp44 and HLA I molecules, as well as HLA I and Damage Associated Molecular Pattern molecules (DAMPs) on the surface of tumor cells. In this report, we have identified novel interaction between HLA I and PCNA on the surface of human tumor cells by confocal microscopy and immunoprecipitation. In addition to previous reports, we show PCNA on the cell surface where novel association with HLA I does not require the presence of NKp44 expressing NK cells and occurs with endogenous PCNA. The association of HLA I and PCNA forms the inhibitory ligand for NKp44, resulting in inhibition of NK cell cytotoxicity. We further postulate NCR ligands are composed of DAMP molecules localized to the cell surface, colocalizing with HLA I, and potentially heparin sulfate proteoglycans.     

Evidence that the cellular ligand for the human NK cell activation receptor NKp30 is not a heparan sulfate glycosaminoglycan

NKp30 (NCR3, CD337) is a natural cytotoxicity receptor, expressed on subsets of human peripheral blood NK cells, involved in NK cell killing of tumor cells and immature dendritic cells. The cellular ligand for NKp30 has remained elusive, although evidence that membrane-associated heparan sulfate (HS) proteoglycans are involved in the recognition of cellular targets by NKp30 was recently reported. The data presented in this report show conclusively that HS glycosaminoglycans (GAG) are not ligands for NKp30. We show that removing HS completely from the cell surface of human 293-EBNA cells with mammalian heparanase does not affect binding of rNKp30/human IgG1 Fc chimera complexes or binding of multimeric liposome-rNKp30 complexes. Removing HS from 293-EBNA cells, culture-generated DC, MM-170 malignant melanoma cells, or HeLa cells does not affect the NKp30-dependent killing of these cells by NK cells. We show further that the GAG-deficient hamster pgsA-745 cells that lack HS and the GAG-expressing parent CHO-K1 cells are both killed by NK cells, with killing of both cell lines inhibited to the same extent by anti-NKp30 mAb. From these results we conclude that HS GAG are not ligands for NKp30, leaving open the question as to the nature of the cellular ligand for this important NK cell activation receptor.     

Tumor-induced apoptosis of human IL-2-activated NK cells: role of natural cytotoxicity receptors

We provide evidence that tumor cells can induce apoptosis of NK cells by engaging the natural cytotoxicity receptors (NCR) NKp30, NKp44, and NKp46. Indeed, the binding between NCR on NK cells and their putative ligands on tumor target cells led to NK cell apoptosis, and this event was abolished by blocking NCR/NCR-ligand interaction by anti-NCR-specific mAbs. The engagement of NCR induced up-regulation of Fas ligand (FasL) mRNA, FasL protein synthesis, and release. In turn, FasL interacting with Fas at NK cell surface causes NK cell suicide, as apoptosis of NK cells was inhibited by blocking FasL/Fas interaction with specific mAbs. Interestingly, NK cell apoptosis, but not killing of tumor target cells, is inhibited by cyclosporin A, suggesting that apoptosis and cytolysis are regulated by different biochemical pathways. These findings indicate that NCR are not only triggering molecules essential for antitumor activity, but also surface receptors involved in NK cell suicide.     

The Stalk Domain and the Glycosylation Status of the Activating Natural Killer Cell Receptor NKp30 Are Important for Ligand Binding

The natural cytotoxicity receptors are a unique set of activating proteins expressed mainly on the surface of natural killer (NK) cells. The human natural cytotoxicity receptor family comprises the three type I membrane proteins NKp30, NKp44, and NKp46. Especially NKp30 is critical for the cytotoxicity of NK cells against different targets including tumor, virus-infected, and immature dendritic cells. Although the crystal structure of NKp30 was recently solved (Li, Y., Wang, Q., and Mariuzza, R. A. (2011) J. Exp. Med. 208, 703-714; Joyce, M. G., Tran, P., Zhuravleva, M. A., Jaw, J., Colonna, M., and Sun, P. D. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 6223-6228), a key question, how NKp30 recognizes several non-related ligands, remains unclear. Therefore, we investigated the parameters that impact ligand recognition of NKp30. Based on various NKp30-hIgG1-Fc fusion proteins, which were optimized for minimal background binding to cellular Fcγ receptors, we identified the flexible stalk region of NKp30 as an important but so far neglected module for ligand recognition and related signaling of the corresponding full-length receptor proteins. Moreover, we found that the ectodomain of NKp30 is N-linked glycosylated at three different sites. Mutational analyses revealed differential binding affinities and signaling capacities of mono-, di-, or triglycosylated NKp30, suggesting that the degree of glycosylation could provide a switch to modulate the ligand binding properties of NKp30 and NK cell cytotoxicity.     

Characterization of endostatin binding to heparin and heparan sulfate by surface plasmon resonance and molecular modeling: role of divalent cations

Endostatin (20 kDa) is a C-terminal proteolytic fragment of collagen XVIII that is localized in vascular basement membrane zones in various organs. It binds zinc, heparin/heparan sulfate, laminin, and sulfatides and inhibits angiogenesis and tumor growth. Here we determined the kinetics and affinity of the interaction of endostatin with heparin/heparan sulfate and investigated the effects of divalent cations on these interactions and on the biological activities of endostatin. The binding of human recombinant endostatin to heparin and heparan sulfate was studied by surface plasmon resonance using BIAcore technology and further characterized by docking and molecular dynamics simulations. Kinetic data, evaluated using a 1:1 interaction model, showed that heparan sulfate bound to and dissociated from endostatin faster than heparin and that endostatin bound to heparin and heparan sulfate with a moderate affinity (K(D) approximately 2 microm). Molecular modeling of the complex between endostatin and heparin oligosaccharides predicted that, compared with mutagenesis studies, two further arginine residues, Arg(47) and Arg(66), participated in the binding. The binding of endostatin to heparin and heparan sulfate required the presence of divalent cations. The addition of ZnCl(2) to endostatin enhanced its binding to heparan sulfate by approximately 40% as well as its antiproliferative effect on endothelial cells stimulated by fibroblast growth factor-2, suggesting that this activity is mediated by the binding of endostatin to heparan sulfate. In contrast, no increase in the antiangiogenic and anti-proliferative activities of endostatin promoted by vascular endothelial growth factor was observed upon the addition of zinc.     

Phenotypic and cytolytic activity of Macaca nemestrina natural killer cells isolated from blood and expanded in vitro

Natural killer (NK) cells from nonhuman primates have not been completely characterized, and methods for expanding nonhuman primates NK cells in vitro have been described only in rhesus species. The purpose of this report was to characterize NK cells in pigtail macaques (Macaca nemestrina), a species that is frequently used in studies of transplantation biology/immunology, virology, vaccine development, and reproductive biology. NK cells from Macaca nemestrina peripheral blood were best defined by the expression of CD16 and CD8alpha, and the absence of CD3. Subsets of these cells express CD56, NKp30, and NKp46. An enhanced ability to kill K562 cells was not present in fluorescence activated cell sorted (FACS)-purified CD16-/CD3+ and CD16-/CD56+ cells isolated from fresh peripheral blood. However, FACS-purified CD16+/CD3- and CD16+/CD56- cells were highly efficient killers of K562 cells. Macaca nemestrina NK cells can be expanded by in vitro culturing of FACS-purified CD16+/CD2-/CD3-/CD56- cells, or from peripheral blood cells depleted of cells expressing CD3, CD4, and HLA-DR. Cells in these cultures expand 70-fold after 21 days of culturing. After culturing, these cells express high levels of natural cytotoxicity receptors (NCRs) NKp30 and NKp46. NK cell populations obtained from FACS-purified CD16+/CD3-, CD16+/CD56- cells and CD3/CD4/HLA-DR-depleted cells were highly efficient killers of K562 cells. These data suggest that a population of highly enriched cytolytic NK cells can be obtained from purified CD16+/CD3- and CD16+/CD56- cells obtained from peripheral blood, as well as from cells that have been cultured and expanded from peripheral blood that is depleted of CD3/CD4/HLA-DR-expressing cells.     

NKp46

Combating diseases such as cancer and virus infection is one of the major goals of the next millennium. Remarkably, our immune system has developed a unique cell type called Natural Killer (NK) cells, able to kill cancer and virus-infected cells. The nature of the lysis/killer receptors expressed on NK cells, mediating this killing, and their ligands, is only little understood. Four lysis receptors for human NK cells were recently identified, including the NKp30, NKp44, NKp46 and CD16, all of which are capable of mediating direct killing of virus-infected and tumor cells. Among these, the NKp46 receptor is considered to be the major lysis receptor for NK cells. The viral hemagglutinin protein was recently identified as the ligand for the NKp46 receptor. However, the cellular (non-viral) ligands recognized by all of the lysis receptors (NKp30, NKp44, NKp46 and CD16) are currently unknown.