分化细胞经特定因子诱导重编程为多能干细胞

胚胎干细胞在再生医学领域有着十分诱人的应用前景。但是现有胚胎干细胞建系技术不能避开对卵细胞的操作, 成为ES细胞临床应用的障碍。通过反转录病毒载体系统, 在小鼠和人类高度分化细胞中表达干细胞因子Oct4, Sox2, Klf4和/或c-Myc等基因, 再经过干细胞标志因子Nanog或Oct4筛选, 可以获得与ES细胞特性十分近似的诱导多能干细胞系。这种不依赖于卵细胞的多能干细胞建系方法无疑是干细胞实验技术的重大进展, 也是对现有重编程理论假设的突破。综述了诱导多能干细胞系建系实验结果, 并对诱导重编程的机制和诱导多能干细胞系的临床应用前景进行了讨论。     

Natural Killer Lysis Receptor (NKLR)/NKLR-Ligand Matching as a Novel Approach for Enhancing Anti-Tumor Activity of Allogeneic NK Cells

Background

NK cells are key players in anti tumor immune response, which can be employed in cell-based therapeutic modalities. One of the suggested ways to amplify their anti tumor effect, especially in the field of stem cell transplantation, is by selecting donor/recipient mismatches in specific HLA, to reduce the inhibitory effect of killer Ig-like receptors (KIRs). Here we suggest an alternative approach for augmentation of anti tumor effect of allogeneic NK cells, which is founded on profile matching of donor NK lysis receptors (NKLR) phenotype with tumor lysis-ligands.

Methodology/Principal Findings

We show that an NKLR-mediated killing directly correlates with the NKLR expression intensity on NK cells. Considerable donor variability in the expression of CD16, NKp46, NKG2D and NKp30 on circulating NK cells, combined with the stability of phenotype in several independently performed tests over two months, indicates that NKLR-guided selection of donors is feasible. As a proof of concept, we show that melanoma cells are dominantly recognized by three NKLRs: NKG2D, NKp30 and NKp44. Notably, the expression of NKp30 on circulating NK cells among metastatic melanoma patients was significantly decreased, which diminishes their ability to kill melanoma cells. Ex vivo expansion of NK cells results not only in increased amount of cells but also in a consistently superior and predictable expression of NKG2D, NKp30 and NKp44. Moreover, expanded NK cultures with high expression of NKG2D or NKp30 were mostly derived from the corresponding NKG2D^high or NK30^high donors. These NK cultures subsequently displayed an improved cytotoxic activity against melanoma in a HLA/KIR-ligand mismatched setup, which was NKLR-dependent, as demonstrated with blocking anti-NKG2D antibodies.

Conclusions/Significance

NKLR/NKLR-ligand matching reproducibly elicits enhanced NK anti-tumor response. Common NKLR recognition patterns of tumors, as demonstrated here in melanoma, would allow implementation of this approach in solid malignancies and potentially in hematological malignancies, either independently or in adjunction to other modalities.     

Disruption of the Talin Gene Compromises Focal Adhesion Assembly in Undifferentiated but Not Differentiated Embryonic Stem Cells

We have used gene disruption to isolate two talin (−/−) ES cell mutants that contain no intact talin. The undifferentiated cells (a) were unable to spread on gelatin or laminin and grew as rounded colonies, although they were able to spread on fibronectin (b) showed reduced adhesion to laminin, but not fibronectin (c) expressed much reduced levels of β1 integrin, although levels of α5 and αV were wild-type (d) were less polarized with increased membrane protrusions compared with a vinculin (−/−) ES cell mutant (e) were unable to assemble vinculin or paxillin-containing focal adhesions or actin stress fibers on fibronectin, whereas vinculin (−/−) ES cells were able to assemble talin-containing focal adhesions. Both talin (−/−) ES cell mutants formed embryoid bodies, but differentiation was restricted to two morphologically distinct cell types. Interestingly, these differentiated talin (−/−) ES cells were able to spread and form focal adhesion-like structures containing vinculin and paxillin on fibronectin. Moreover, the levels of the β1 integrin subunit were comparable to those in wild-type ES cells. We conclude that talin is essential for β1 integrin expression and focal adhesion assembly in undifferentiated ES cells, but that a subset of differentiated cells are talin independent for both characteristics.     

Human Induced Pluripotent Stem Cells Are Targets for Allogeneic and Autologous Natural Killer (NK) Cells and Killing Is Partly Mediated by the Activating NK Receptor DNAM-1

Human induced pluripotent stem cells (hiPSCs) could be used to generate autologous cells for therapeutic purposes, which are expected to be tolerated by the recipient. However, iPSC-derived grafts are at risk of giving rise to teratomas in the host, if residuals of tumorigenic cells are not rejected by the recipient. We have analyzed the susceptibility of hiPSC lines to allogeneic and autologous natural killer (NK) cells. IL-2-activated, in contrast to resting NK cells killed hiPSC lines efficiently (P=1.69x10^-39). Notably, the specific lysis of the individual hiPSC lines by IL-2-activated NK cells was significantly different (P=1.72x10^-6) and ranged between 46 % and 64 % in ⁵¹Cr-release assays when compared to K562 cells. The hiPSC lines were killed by both allogeneic and autologous NK cells although autologous NK cells were less efficient (P=8.63x10^-6). Killing was partly dependent on the activating NK receptor DNAM-1 (P=8.22x10^-7). The DNAM-1 ligands CD112 and CD155 as well as the NKG2D ligands MICA and MICB were expressed on the hiPSC lines. Low amounts of human leukocyte antigen (HLA) class I proteins, which serve as ligands for inhibitory and activating NK receptors were also detected. Thus, the susceptibility to NK cell killing appears to constitute a common feature of hiPSCs. Therefore, NK cells might reduce the risk of teratoma formation even after autologous transplantations of pluripotent stem cell-derived grafts that contain traces of pluripotent cells.     

Frozen Cord Blood Hematopoietic Stem Cells Differentiate into Higher Numbers of Functional Natural Killer Cells In Vitro than Mobilized Hematopoietic Stem Cells or Freshly Isolated Cord Blood Hematopoietic Stem Cells

Adoptive natural killer (NK) cell therapy relies on the acquisition of large numbers of NK cells that are cytotoxic but not exhausted. NK cell differentiation from hematopoietic stem cells (HSC) has become an alluring option for NK cell therapy, with umbilical cord blood (UCB) and mobilized peripheral blood (PBCD34⁺) being the most accessible HSC sources as collection procedures are less invasive. In this study we compared the capacity of frozen or freshly isolated UCB hematopoietic stem cells (CBCD34⁺) and frozen PBCD34⁺ to generate NK cells in vitro. By modifying a previously published protocol, we showed that frozen CBCD34⁺ cultures generated higher NK cell numbers without loss of function compared to fresh CBCD34⁺ cultures. NK cells generated from CBCD34⁺ and PBCD34⁺ expressed low levels of killer-cell immunoglobulin-like receptors but high levels of activating receptors and of the myeloid marker CD33. However, blocking studies showed that CD33 expression did not impact on the functions of the generated cells. CBCD34⁺-NK cells exhibited increased capacity to secrete IFN-γ and kill K562 in vitro and in vivo as compared to PBCD34⁺-NK cells. Moreover, K562 killing by the generated NK cells could be further enhanced by IL-12 stimulation. Our data indicate that the use of frozen CBCD34⁺ for the production of NK cells in vitro results in higher cell numbers than PBCD34⁺, without jeopardizing their functionality, rendering them suitable for NK cell immunotherapy. The results presented here provide an optimal strategy to generate NK cells in vitro for immunotherapy that exhibit enhanced effector function when compared to alternate sources of HSC.     

Monkeypox Virus Infection of Rhesus Macaques Induces Massive Expansion of Natural Killer Cells but Suppresses Natural Killer Cell Functions

Natural killer (NK) cells play critical roles in innate immunity and in bridging innate and adaptive immune responses against viral infection. However, the response of NK cells to monkeypox virus (MPXV) infection is not well characterized. In this intravenous challenge study of MPXV infection in rhesus macaques (Macaca mulatta), we analyzed blood and lymph node NK cell changes in absolute cell numbers, cell proliferation, chemokine receptor expression, and cellular functions. Our results showed that the absolute number of total NK cells in the blood increased in response to MPXV infection at a magnitude of 23-fold, manifested by increases in CD56+, CD16+, CD16-CD56- double negative, and CD16+CD56+ double positive NK cell subsets. Similarly, the frequency and NK cell numbers in the lymph nodes also largely increased with the total NK cell number increasing 46.1-fold. NK cells both in the blood and lymph nodes massively proliferated in response to MPXV infection as measured by Ki67 expression. Chemokine receptor analysis revealed reduced expression of CXCR3, CCR7, and CCR6 on NK cells at early time points (days 2 and 4 after virus inoculation), followed by an increased expression of CXCR3 and CCR5 at later time points (days 7-8) of infection. In addition, MPXV infection impaired NK cell degranulation and ablated secretion of interferon-γ and tumor necrosis factor-α. Our data suggest a dynamic model by which NK cells respond to MPXV infection of rhesus macaques. Upon virus infection, NK cells proliferated robustly, resulting in massive increases in NK cell numbers. However, the migrating capacity of NK cells to tissues at early time points might be reduced, and the functions of cytotoxicity and cytokine secretion were largely compromised. Collectively, the data may explain, at least partially, the pathogenesis of MPXV infection in rhesus macaques.     

Detection of Calcium Transients in Embryonic Stem Cells and Their Differentiated Progeny

A central issue in stem cell biology is the determination of function and activity of differentiated stem cells, features that define the true phenotype of mature cell types. Commonly, physiological mechanisms are used to determine the functionality of mature cell types, including those of the nervous system. Calcium imaging provides an indirect method of determining the physiological activities of a mature cell. Camgaroos are variants of yellow fluorescent protein that act as intracellular calcium sensors in transfected cells. We expressed one version of the camgaroos, Camgaroo-2, in mouse embryonic stem (ES) cells under the control of the CAG promoter system. Under the control of this promoter, Camgaroo-2 fluorescence was ubiquitously expressed in all cell types derived from the ES cells that were tested. In response to pharmacological stimulation, the fluorescence levels in transfected cells correlated with cellular depolarization and hyperpolarization. These changes were observed in both undifferentiated ES cells as well as ES cells that had been neurally induced, including putative neurons that were differentiated from transfected ES cells. The results presented here indicate that Camgaroo-2 may be used like traditional fluorescent proteins to track cells as well as to study the functionality of stem cells and their progeny.     

Identification of Embryonic Neural Plate Border Stem Cells and Their Generation by Direct Reprogramming from Adult Human Blood Cells

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Natural Killer Cell Sensing of Infected Cells Compensates for MyD88 Deficiency but Not IFN-I Activity in Resistance to Mouse Cytomegalovirus

In mice, plasmacytoid dendritic cells (pDC) and natural killer (NK) cells both contribute to resistance to systemic infections with herpes viruses including mouse Cytomegalovirus (MCMV). pDCs are the major source of type I IFN (IFN-I) during MCMV infection. This response requires pDC-intrinsic MyD88-dependent signaling by Toll-Like Receptors 7 and 9. Provided that they express appropriate recognition receptors such as Ly49H, NK cells can directly sense and kill MCMV-infected cells. The loss of any one of these responses increases susceptibility to infection. However, the relative importance of these antiviral immune responses and how they are related remain unclear. In humans, while IFN-I responses are essential, MyD88 is dispensable for antiviral immunity. Hence, a higher redundancy has been proposed in the mechanisms promoting protective immune responses against systemic infections by herpes viruses during natural infections in humans. It has been assumed, but not proven, that mice fail to mount protective MyD88-independent IFN-I responses. In humans, the mechanism that compensates MyD88 deficiency has not been elucidated. To address these issues, we compared resistance to MCMV infection and immune responses between mouse strains deficient for MyD88, the IFN-I receptor and/or Ly49H. We show that selective depletion of pDC or genetic deficiencies for MyD88 or TLR9 drastically decreased production of IFN-I, but not the protective antiviral responses. Moreover, MyD88, but not IFN-I receptor, deficiency could largely be compensated by Ly49H-mediated antiviral NK cell responses. Thus, contrary to the current dogma but consistent with the situation in humans, we conclude that, in mice, in our experimental settings, MyD88 is redundant for IFN-I responses and overall defense against a systemic herpes virus infection. Moreover, we identified direct NK cell sensing of infected cells as one mechanism able to compensate for MyD88 deficiency in mice. Similar mechanisms likely contribute to protect MyD88- or IRAK4-deficient patients from viral infections.     

Natural Killer Cells Improve Hematopoietic Stem Cell Engraftment by Increasing Stem Cell Clonogenicity In Vitro and in a Humanized Mouse Model

Cord blood (CB) is increasingly used as a source of hematopoietic stem cells (HSC) for transplantation. Low incidence and severity of graft-versus-host disease (GvHD) and a robust graft-versus-leukemia (GvL) effect are observed following CB transplantation (CBT). However, its main disadvantages are a limited number of HSC per unit, delayed immune reconstitution and a higher incidence of infection. Unmanipulated grafts contain accessory cells that may facilitate HSC engraftment. Therefore, the effects of accessory cells, particularly natural killer (NK) cells, on human CB HSC (CBSC) functions were assessed in vitro and in vivo. CBSC cultured with autologous CB NK cells showed higher levels of CXCR4 expression, a higher migration index and a higher number of colony forming units (CFU) after short-term and long-term cultures. We found that CBSC secreted CXCL9 following interaction with CB NK cells. In addition, recombinant CXCL9 increased CBSC clonogenicity, recapitulating the effect observed of CB NK cells on CBSC. Moreover, the co-infusion of CBSC with CB NK cells led to a higher level of CBSC engraftment in NSG mouse model. The results presented in this work offer the basis for an alternative approach to enhance HSC engraftment that could improve the outcome of CBT.     

Susceptibility of Human Melanoma Cells to Autologous Natural Killer (NK) Cell Killing: HLA-Related Effector Mechanisms and Role of Unlicensed NK Cells

Background

Despite Natural Killer (NK) cells were originally defined as effectors of spontaneous cytotoxicity against tumors, extremely limited information is so far available in humans on their capability of killing cancer cells in an autologous setting.

Methodology/Principal Findings

We have established a series of primary melanoma cell lines from surgically resected specimens and here showed that human melanoma cells were highly susceptible to lysis by activated autologous NK cells. A variety of NK cell activating receptors were involved in killing: particularly, DNAM-1 and NKp46 were the most frequently involved. Since self HLA class I molecules normally play a protective role from NK cell-mediated attack, we analyzed HLA class I expression on melanomas in comparison to autologous lymphocytes. We found that melanoma cells presented specific allelic losses in 50% of the patients analyzed. In addition, CD107a degranulation assays applied to NK cells expressing a single inhibitory receptor, revealed that, even when expressed, specific HLA class I molecules are present on melanoma cell surface in amount often insufficient to inhibit NK cell cytotoxicity. Remarkably, upon activation, also the so called “unlicensed” NK cells, i.e. NK cells not expressing inhibitory receptor specific for self HLA class I molecules, acquired the capability of efficiently killing autologous melanoma cells, thus additionally contributing to the lysis by a mechanism independent of HLA class I expression on melanoma cells.

Conclusions/Significance

We have investigated in details the mechanisms controlling the recognition and lysis of melanoma cells by autologous NK cells. In these autologous settings, we demonstrated an efficient in vitro killing upon NK cell activation by mechanisms that may be related or not to abnormalities of HLA class I expression on melanoma cells. These findings should be taken into account in the design of novel immunotherapy approaches against melanoma.     

MHC Class I Expression by Donor Hematopoietic Stem Cells Is Required to Prevent NK Cell Attack in Allogeneic,but Not Syngeneic Recipient Mice

NK cells resist engraftment of syngeneic and allogeneic bone marrow (BM) cells lacking major histocompatibility (MHC) class I molecules, suggesting a critical role for donor MHC class I molecules in preventing NK cell attack against donor hematopoietic stem and progenitor cells (HSPCs), and their derivatives. However, using high-resolution in vivo imaging, we demonstrated here that syngeneic MHC class I knockout (KO) donor HSPCs persist with the same survival frequencies as wild-type donor HSPCs. In contrast, syngeneic MHC class I KO differentiated hematopoietic cells and allogeneic MHC class I KO HSPCs were rejected in a manner that was significantly inhibited by NK cell depletion. In vivo time-lapse imaging demonstrated that mice receiving allogeneic MHC class I KO HSPCs showed a significant increase in NK cell motility and proliferation as well as frequencies of NK cell contact with and killing of HSPCs as compared to mice receiving wild-type HSPCs. The data indicate that donor MHC class I molecules are required to prevent NK cell-mediated rejection of syngeneic differentiated cells and allogeneic HSPCs, but not of syngeneic HSPCs.     

Activation of Natural Killer Cells by Newcastle Disease Virus Hemagglutinin-Neuraminidase

The avian paramyxovirus Newcastle disease virus (NDV) selectively replicates in tumor cells and is known to stimulate T-cell-, macrophage-, and NK cell-mediated responses. The mechanisms of NK cell activation by NDV are poorly understood so far. We studied the expression of ligand structures for activating NK cell receptors on NDV-infected tumor cells. Upon infection with the nonlytic NDV strain Ulster and the lytic strain MTH-68/H, human carcinoma and melanoma cells showed enhanced expression of ligands for the natural cytotoxicity receptors NKp44 and NKp46, but not NKp30. Ligands for the activating receptor NKG2D were partially downregulated. Soluble NKp44-Fc and NKp46-Fc, but not NKp30-Fc, chimeric proteins bound specifically to NDV-infected tumor cells and to NDV particle-coated plates. Hemagglutinin-neuraminidase (HN) of the virus serves as a ligand structure for NKp44 and NKp46, as indicated by the blockade of binding to NDV-infected cells and viral particles in the presence of anti-HN antibodies and by binding to cells transfected with HN cDNA. Consistent with the recognition of sialic acid moieties by the viral lectin HN, the binding of NKp44-Fc and NKp46-Fc was lost after desialylation. NKp44- and NKp46-CD3ζ lacZ-inducible reporter cells were activated by NDV-infected cells. NDV-infected tumor cells stimulated NK cells to produce increased amounts of the effector lymphokines gamma interferon and tumor necrosis factor alpha. Primary NK cells and the NK line NK-92 lysed NDV-infected tumor cells with enhanced efficiency, an effect that was eliminated by the treatment of target cells with the neuraminidase inhibitor Neu5Ac2en. These results suggest that direct activation of NK cells contributes to the antitumor effects of NDV.Virulent strains of Newcastle disease virus (NDV) infect domestic poultry and other birds, causing a rapidly spreading viral disease that affects the alimentary and respiratory tracts as well as the central nervous system (55). In humans, however, NDV is well tolerated (17, 18). Other than mild fever for a day, only a few adverse effects have been reported. NDV, also known as avian paramyxovirus 1, is an enveloped virus containing a negative-sense, single-stranded RNA genome which codes for six proteins in the order (from 3′ to 5′) of nucleoprotein, phosphoprotein, matrix protein, fusion (F) protein, hemagglutinin-neuraminidase (HN), and large polymerase protein (19). There are many different strains of NDV, classified as either lytic or nonlytic for different types of cells. Lytic and nonlytic NDV strains both replicate much more efficiently in human cancer cells than they do in most normal human cells (43). Viruses of both strain types have been investigated as potential anticancer agents (30, 49, 52). The NDV strains that have been evaluated most widely for the treatment of cancer are 73-T, MTH-68, and Ulster (1, 7, 11, 17, 18, 53, 54, 56, 71).Initial binding of NDV to a host cell takes place through the interaction of HN molecules in the virus coat with sialic acid-containing molecules on the cell surface (31). NDV neuraminidase has strict specificity for the hydrolysis of the NeuAc-α2,3-Gal linkage, with no hydrolysis of the NeuAc-α2,6-Gal linkage (41).NDV infection of tumor cells not only improves T-cell responses (53, 58, 68), but has also been reported to vigorously stimulate innate immune responses. In the course of NDV infection, large amounts of alpha interferon (IFN-α) are released (68) and in turn activate dendritic cells and NK cells and polarize, in concert with interleukin-12 (IL-12), toward a Th1 T-cell response (33, 44, 47). In addition, NDV induces antitumor cytotoxicity in murine macrophages which produce increased amounts of tumor necrosis factor alpha (TNF-α) and nitric oxide (51, 60) and in human monocytes through the induction of TRAIL (64). Little is known about the NDV-mediated activation of NK cells. The coincubation of peripheral blood mononuclear cells with NDV was shown previously to stimulate NK-mediated cytotoxicity (70). Enhanced cytotoxicity correlates with the induction of IFN-α (70). It is not known, however, whether NDV-infected cells can directly activate NK cells and, if so, which molecular interactions are involved.The cytolytic activity of NK cells against virus-infected or tumor cells is regulated by the engagement of activating or inhibitory NK cell surface receptors, the actions of cytokines, and cross talk with other immune cells (32, 39). Most inhibitory receptors recognize particular major histocompatibility complex (MHC) class I alleles and thereby ensure the tolerance of NK cells against self antigens (38). Activating receptors on human NK cells include CD16; NKG2D; the natural cytotoxicity receptors (NCR) NKp30, NKp44, and NKp46; as well as NKp80; DNAM-1; and various stimulatory coreceptors (32).NCR are important activating receptors for the antitumor and antiviral activities of NK cells (5, 32, 37). Heparan sulfate has been discussed previously as a cellular ligand for NKp46, NKp44, and NKp30 (9, 26, 27), and nuclear factor BAT3, which can be released from tumor cells under stress conditions, has been described as a cellular ligand for NKp30 (42). Ligands for NKp30 and NKp44 can be detected on the surfaces and in the intracellular compartments of several kinds of tumor cells (10). Moreover, a number of pathogen-derived NCR ligands have been reported. The hemagglutinin protein of influenza virus and the HN of Sendai virus can bind to NKp46 and NKp44 and activate NK cells (3, 24, 34). The pp65 protein of human cytomegalovirus has been shown to bind NKp30 and inhibit its function (4). Human immunodeficiency virus, vaccinia virus, and herpes simplex virus have also been shown to upregulate the expression of cellular NCR ligands in infected cells (13, 14, 62). The Plasmodium falciparum erythrocyte membrane protein 1 is involved in the NCR-mediated NK cell attack against infected erythrocytes (36). Furthermore, NKp46 recognizes cells infected with mycobacteria (22, 61), and NKp44 was recently reported to directly bind to the surfaces of mycobacteria and other bacteria (21).In this study, we investigated the expression of ligand structures for NCR and NKG2D on NDV-infected cells. We demonstrate that NDV HN proteins which are strongly expressed on NDV-infected tumor cells function as activating ligand structures for NKp44 and NKp46 but that cellular ligands for NKG2D are partially downregulated during NDV infection.     

Reprogramming Adult Schwann Cells to Stem Cell-like Cells by Leprosy Bacilli Promotes Dissemination of Infection

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Highlights? Leprosy bacteria reprogram adult Schwann cells by altering host-gene expression ? Bacterially reprogrammed cells resemble progenitor/stem-like cells (pSLC) of mesenchymal trait ? pSLC promote bacterial spread to mesenchymal tissues by redifferentiation ? pSLC secrete immune factors, recruit macrophages, transfer bacteria, form granulomas, and disseminate infection     

Killing of Avian and Swine Influenza Virus by Natural Killer Cells

Today, global attention is focused on two influenza virus strains: the current pandemic strain, swine origin influenza virus (H1N1-2009), and the highly pathogenic avian influenza virus, H5N1. At present, the infection caused by the H1N1-2009 is moderate, with mortality rates of less <1%. In contrast, infection with the H5N1 virus resulted in high mortality rates, and ca. 60% of the infected patients succumb to the infection. Thus, one of the world greatest concerns is that the H5N1 virus will evolve to allow an efficient human infection and human-to-human transmission. Natural killer (NK) cells are one of the innate immune components playing an important role in fighting against influenza viruses. One of the major NK activating receptors involved in NK cell cytotoxicity is NKp46. We previously demonstrated that NKp46 recognizes the hemagglutinin proteins of B and A influenza virus strains. Whether NKp46 could also interact with H1N1-2009 virus or with the avian influenza virus is still unknown. We analyzed the immunological properties of both the avian and the H1N1-2009 influenza viruses. We show that NKp46 recognizes the hemagglutinins of H1N1-2009 and H5 and that this recognition leads to virus killing both in vitro and in vivo. However, importantly, while the swine H1-NKp46 interactions lead to the direct killing of the infected cells, the H5-NKp46 interactions were unable to elicit direct killing, probably because the NKp46 binding sites for these two viruses are different.Natural killer (NK) cells, which comprise 5 to 15% of peripheral blood lymphocytes, are a key frontline defense against a number of pathogens, including intracellular bacteria, parasites, and most importantly with respect to the present study, viruses (6, 40). The antiviral mechanisms by which NK cells operate include both cytotoxic activity and cytokine/chemokine secretion (21). The NK killing activity is executed by numerous receptors, including NKG2D, NKp80, CD16, and the natural cytotoxic receptors (NCRs): NKp30, NKp44, and NKp46 (7, 10, 25).Although the cellular ligands for NKG2D were identified (31, 38), the identity of several of the cellular ligands for the human NCRs is still unknown, except for BAT3 and B7-H6, which are ligands for NKp30 (8, 30). In contrast, viral ligands were identified for the NCRs, and we demonstrated that pp65 of HCMV interacts with NKp30 (3) and that various influenza virus hemagglutinins (HAs) are ligands for the NKp44 and NKp46 receptors (5, 22). Supporting these observations, it was recently shown that the HA-neuraminidase of Newcastle disease virus could also interact with NKp46 and NKp44 but not with NKp30 (17). Furthermore, we have shown in vivo that in the absence of NCR1 (the mouse homologue of NKp46), A/PR8 influenza virus infection is lethal (14).Human influenza virus (H1 and H3 subtype) infections pose a major threat to the entire population, as exemplified by the three major influenza pandemics that occurred during the 20th century. The Asian (A/H2N2) in 1957 to 1958 and the Hong Kong (A/H3N2) pandemics in 1968 to 1969 resulted in the deaths of 1 to 2 million people and the 1918 “Spanish flu” (A/H1N1) pandemic killed around 50 million people (18). At present, the worldwide concern regarding influenza pandemics concentrates mainly on two viruses: the A/H1N1 swine origin influenza virus (H1N1-2009), which currently causes only a moderate pandemic (the mortality rates are ca. 1%) but is more pathogenic than a regular seasonal influenza virus (19, 26, 27), and the avian influenza virus carrying the unique H5 HA (20). The avian influenza virus is quite deadly and, although it remains a zoonotic infection, ca. 60% of infected humans died due to the infection (28).The unique properties of the H5 protein of the avian influenza virus are one of the main reasons for the virulence of the virus. The H5 of the avian influenza virus binds to cell surface glycoproteins or glycolipids containing terminal sialyl-galactosyl residues linked by 2-3-linkage [Neu5Ac(α2-3)Gal] that are found in the human conjunctiva and ciliated portion of the respiratory columnar epithelium (33). In contrast, human viruses (including all three strains that caused the pandemics described above and the H1N1-2009) bind to receptors that mostly contain terminal 2-6-linked sialyl-galactosyl moieties [Neu5Ac(α2-6)Gal]. Such glycosylations are predominant on epithelial cells in the nasal mucosa, paranasal sinuses, pharynx, trachea, and bronchi (33, 37). It has been suggested that the lack of human-to-human transmission of avian influenza viruses is due to their α2,3-SA receptor binding preference, and the concern is that genetic changes in H5 might alter its preference from α2,3-SA to α2,6-SA, allowing human-to-human transmission.In our previous studies (4, 22) we showed that the interaction between NKp46 and influenza virus HAs depends on the sialylation of the NKp46 receptor. We further demonstrated that the sialic acid residues, which are linked via α2,6 to the threonine 225 residue of NKp46, are crucial for the NKp46 interactions with the various influenza virus HAs (4).We show that, both in vitro and in vivo, the killing of H1N1-2009-infected cells is correlated with the degree of NKp46 binding. Surprisingly, we observed that although NKp46 efficiently recognized the avian H5 HA, such interactions were unable to elicit the direct killing of the infected cells. By using mutagenesis analysis experiments and killing assays we demonstrate that NKp46 interacts with H1 and H5 at distinct sites, since we show that the sugar carrying residue at position 225 is crucial for the NKp46-H1N1-2009 interactions, whereas the interaction of H5 with NKp46 depends on both residues 216 and 225.