NKp46 Receptor-Mediated Interferon-γ Production by Natural Killer Cells Increases Fibronectin 1 to Alter Tumor Architecture and Control Metastasis

Natural killer (NK) cells are innate lymphoid cells, and their presence within human tumors correlates with better prognosis. However, the mechanisms by which NK cells control tumors in vivo are unclear. Here, we used reflectance confocal microscopy (RCM) imaging in humans and in mice to visualize tumor architecture in vivo.We demonstrated that signaling via the NK cell receptor NKp46 (human) and Ncr1 (mouse) induced interferon-g (IFN-g) secretion from intratumoral NK cells. NKp46- and Ncr1-mediated IFN-g production led to the increased expression of the extracellular matrix protein fibronectin 1 (FN1) in the tumors, which altered primary tumor architecture and resulted in decreased metastases formation. Injection of IFN-g into tumor-bearing mice or transgenic overexpression of Ncr1 in NK cells in mice resulted in decreased metastasis formation. Thus, we have defined a mechanism of NK cell-mediated control of metastases in vivo that may help develop NK cell-dependent cancer therapies

Identification of putative novel O-glycosylations in the NK killer receptor Ncr1 essential for its activity

Natural killer (NK) cells kill tumor and virus-infected cells using activating NK cell receptors. One of the major NK-activating receptors is NKp46 and its mouse ortholog Ncr1. NKp46/Ncr1 is expressed exclusively on NK cells and on a subset of innate lymphoid cells. NKp46/Ncr1 was shown to be involved in a myriad of pathologies and immunological settings. Specifically, NKp46/Ncr1 was shown to interact with the viral hemagglutinin (HA) protein and with an unknown tumor/cellular ligand. NKp46 and Ncr1 are structurally similar; however, they are substantially different in their glycosylation patterns. Although the human NKp46 carries both O- and N-glycosylations that are essential for its activity, the mouse Ncr1 was predicted to have N-linked glycosylations only. Here we discovered using prediction algorithms and high-performance liquid chromatography analysis that Ncr1 carries two putative novel O-glycosylations, one of which (Thr 225) is conserved in NKp46. We next used surface plasmon resonance, biochemical, mutational and functional in vitro and in vivo assays to demonstrate that the putative O-glycosylations of Ncr1 are critical for its function

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

Stromal Cell-Derived Factor 1 Mediates Immune Cell Attraction upon Urinary Tract Infection

Urinary tract infection (UTI) is the most common type of bacterial infection in humans. Fifty percent of all women will experience at least one UTI in their lifetime, with uropathogenic Escherichia coli (UPEC) accounting for 80% of reported cases. UTI evokes a complex, well-timed immune response that is crucial for bacterial clearance. The majority of immune cells participating in the immune response are absent from the healthy bladder, and the mechanisms used to recruit them upon UTI are not fully understood. Here, we show that immediately after UPEC infection, bladder epithelial cells secrete stromal cell-derived factor 1 (SDF-1), initiating immune cell accumulation at the site of infection. SDF-1 blockade significantly reduced immune cell migration to the infected bladder, resulting in severe exacerbation of infection. We also show that FimH, the adhesin of type 1 fimbria, one of UPEC’s virulence factors, is directly involved in the secretion of SDF-1 upon UTI

Mouse TIGIT

The activity of natural killer (NK) cells is controlled by a balance of signals derived from inhibitory and activating receptors. TIGIT is a novel inhibitory receptor, recently shown in humans to interact with two ligands: PVR and Nectin2 and to inhibit human NK-cell cytotoxicity. Whether mouse TIGIT (mTIGIT) inhibits mouse NK-cell cytotoxicity is unknown. Here we show that mTIGIT is expressed by mouse NK cells and interacts with mouse PVR (mPVR). Using mouse and human Ig fusion proteins we show that while the human TIGIT (hTIGIT) cross-reacts with mPVR, the binding of mTIGIT is restricted to mPVR. We further demonstrate using surface plasmon resonance (SPR) and staining with Ig fusion proteins that mTIGIT binds to mPVR with higher affinity than the co-stimulatory PVR-binding receptor mDNAM1. Functionally, we show that triggering of mTIGIT leads to the inhibition of NK-cell cytotoxicity, that IFN-γ secretion is enhanced when mTIGIT is blocked and that the TIGIT-mediated inhibition is dominant over the signals delivered by the PVR-binding co-stimulatory receptors. Additionally, we identify the inhibitory motif responsible for mTIGIT inhibition. In conclusion we show that TIGIT is a powerful inhibitory receptor for mouse NK cells

Mouse TIGIT inhibits NK-cell cytotoxicity upon interaction with PVR

The activity of natural killer (NK) cells is controlled by a balance of signals derived from inhibitory and activating receptors. TIGIT is a novel inhibitory receptor, recently shown in humans to interact with two ligands: PVR and Nectin2 and to inhibit human NK‐cell cytotoxicity. Whether mouse TIGIT (mTIGIT) inhibits mouse NK‐cell cytotoxicity is unknown. Here we show that mTIGIT is expressed by mouse NK cells and interacts with mouse PVR. Using mouse and human Ig fusion proteins we show that while the human TIGIT (hTIGIT) cross‐reacts with mouse PVR (mPVR), the binding of mTIGIT is restricted to mPVR. We further demonstrate using surface plasmon resonance (SPR) and staining with Ig fusion proteins that mTIGIT binds to mPVR with higher affinity than the co‐stimulatory PVR‐binding receptor mouse DNAM1 (mDNAM1). Functionally, we show that triggering of mTIGIT leads to the inhibition of NK‐cell cytotoxicity, that IFN‐γ secretion is enhanced when mTIGIT is blocked and that the TIGIT‐mediated inhibition is dominant over the signals delivered by the PVR‐binding co‐stimulatory receptors. Additionally, we identify the inhibitory motif responsible for mTIGIT inhibition. In conclusion, we show that TIGIT is a powerful inhibitory receptor for mouse NK cells

Neuraminidase-Mediated, NKp46-Dependent Immune-Evasion Mechanism of Influenza Viruses

Natural killer (NK) cells play an essential role in the defense against influenza virus, one of the deadliest respiratory viruses known today. The NKp46 receptor, expressed by NK cells, is critical for controlling influenza infections, as influenza-virus-infected cells are eliminated through the recognition of the viral hemagglutinin (HA) protein by NKp46. Here, we describe an immune-evasion mechanism of influenza viruses that is mediated by the neuraminidase (NA) protein. By using various NA blockers, we show that NA removes sialic acid residues from NKp46 and that this leads to reduced recognition of HA. Furthermore, we provide in vivo and in vitro evidence for the existence of this NA-mediated, NKp46-dependent immune-evasion mechanism and demonstrate that NA inhibitors, which are commonly used for the treatment of influenza infections, are useful not only as blockers of virus budding but also as boosters of NKp46 recognition

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