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-γ (IFN-γ) secretion from intratumoral NK cells. NKp46- and Ncr1-mediated IFN-γ 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-γ 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.

Trained Memory of Human Uterine NK Cells Enhances Their Function in Subsequent Pregnancies.

Natural killer cells (NKs) are abundant in the human decidua, regulating trophoblast invasion and angiogenesis. Several diseases of poor placental development are associated with first pregnancies, so we thus looked to characterize differences in decidual NKs (dNKs) in first versus repeated pregnancies. We discovered a population found in repeated pregnancies, which has a unique transcriptome and epigenetic signature, and is characterized by high expression of the receptors NKG2C and LILRB1. We named these cells Pregnancy Trained decidual NK cells (PTdNKs). PTdNKs have open chromatin around the enhancers of IFNG and VEGFA. Activation of PTdNKs led to increased production and secretion of IFN-γ and VEGFα, with the latter supporting vascular sprouting and tumor growth. The precursors of PTdNKs seem to be found in the endometrium. Because repeated pregnancies are associated with improved placentation, we propose that PTdNKs, which are present primarily in repeated pregnancies, might be involved in proper placentation.

Pregnancy trained decidual NK cells protect pregnancies from harmful Fusobacterium nucleatum infection.

Natural killer cells (NKs) found during pregnancy at the maternal-fetal interface named decidual (d)NKs, show signs of education following first pregnancy, resulting in better placentation and fetus-growth, hence termed pregnancy trained dNKs (PTdNKs). Here we show that PTdNKs provide increased protection of the fetus from Fusobacterium nucleatum (FN) infection. We demonstrate that PTdNKs secrete elevated amounts of the bacteriocidal protein granulysin (GNLY) upon incubation with FN compared to dNKs derived from first pregnancies, which leads to increased killing of FN. Furthermore, we showed mechanistically that the GNLY secretion is mediated through the interaction of the FN's Fap2 protein with Gal-GalNAc present on PTdNKs. Finally, we show in vivo, using GNLY-tg mice that enhanced protection of the fetuses from FN infection is observed, as compared to wild type and that this enhance protection is NK cell dependent. Altogether, we show a new function for PTdNKs as protectors of the fetus from bacterial infection.

Binding of the Fap2 protein of Fusobacterium nucleatum to human inhibitory receptor TIGIT protects tumors from immune cell attack.

Bacteria, such as Fusobacterium nucleatum, are present in the tumor microenvironment. However, the immunological consequences of intra-tumoral bacteria remain unclear. Here, we have shown that natural killer (NK) cell killing of various tumors is inhibited in the presence of various F. nucleatum strains. Our data support that this F. nucleatum-mediated inhibition is mediated by human, but not by mouse TIGIT, an inhibitory receptor present on all human NK cells and on various T cells. Using a library of F. nucleatum mutants, we found that the Fap2 protein of F. nucleatum directly interacted with TIGIT, leading to the inhibition of NK cell cytotoxicity. We have further demonstrated that tumor-infiltrating lymphocytes expressed TIGIT and that T cell activities were also inhibited by F. nucleatum via Fap2. Our results identify a bacterium-dependent, tumor-immune evasion mechanism in which tumors exploit the Fap2 protein of F. nucleatum to inhibit immune cell activity via TIGIT.

The inhibitory receptor CD300a is essential for neutrophil-mediated clearance of urinary tract infection in mice.

Neutrophils play a crucial role in immune defense against and clearance of uropathogenic Escherichia coli (UPEC)-mediated urinary tract infection, the most common bacterial infection in healthy humans. CD300a is an inhibitory receptor that binds phosphatidylserine and phosphatidylethanolamine, presented on the membranes of apoptotic cells. CD300a binding to phosphatidylserine and phosphatidylethanolamine, also known as the "eat me" signal, mediates immune tolerance to dying cells. Here, we demonstrate for the first time that CD300a plays an important role in the neutrophil-mediated immune response to UPEC-induced urinary tract infection. We show that CD300a-deficient neutrophils have impaired phagocytic abilities and despite their increased accumulation at the site of infection, they are unable to reduce bacterial burden in the bladder, which results in significant exacerbation of infection and worse host outcome. Finally, we demonstrate that UPEC's pore forming toxin α-hemolysin induces upregulation of the CD300a ligand on infected bladder epithelial cells, signaling to neutrophils to be cleared.

Natural killer cells control metastasis via structural editing of primary tumors in mice.

Natural killer (NK) cells are innate immune lymphocytes which express an array of activating and inhibitory receptors. These receptors bind a large spectrum of ligands, which are expressed on stressed, malignantly transformed or virally infected cells, as well as on bacterial, fungal, and parasitic pathogens. The decision on whether or not to kill the target is based on the integration of activating and inhibitory signals sent downstream from NK cell receptors. One of the most prominent NK cell activating receptor families is the family of natural cytotoxicity receptors (NCRs) which includes NKp30, NKp44, and NKp46. NKp46 is the only NCR to have a fully functional mouse orthologue denoted Ncr1. Despite a large body of evidence highlighting its importance in the clearance of both solid and liquid tumors, the membrane-bound tumor ligand for NKp46 and its mouse orthologue Ncr1 is still unknown. Here we review the discovery of a novel role for NKp46/Ncr1, not only in tumor clearance but also in prevention of metastasis by structural editing of primary tumors.

NK-cell receptors NKp46 and NCR1 control human metapneumovirus infection.

Natural killer (NK) cells are capable of killing various pathogens upon stimulation of activating receptors. Human metapneumovirus (HMPV) is a respiratory virus, which was discovered in 2001 and is responsible for acute respiratory tract infection in infants and children worldwide. HMPV infection is very common, infecting around 70% of all children under the age of five. Under immune suppressive conditions, HMPV infection can be fatal. Not much is known on how NK cells respond to HMPV. In this study, using reporter assays and NK-cell cytotoxicity assays performed with human and mouse NK cells, we demonstrated that the NKp46-activating receptor and its mouse orthologue Ncr1, both members of the natural cytotoxicity receptor (NCR) family, recognized an unknown ligand expressed by HMPV-infected human cells. We demonstrated that MHC class I is upregulated and MICA is downregulated upon HMPV infection. We also characterized mouse NK-cell phenotype in the blood and the lungs of HMPV-infected mice and found that lung NK cells are more activated and expressing NKG2D, CD43, CD27, KLRG1, and CD69 compared to blood NK cells regardless of HMPV infection. Finally, we demonstrated, using Ncr1-deficient mice, that NCR1 plays a critical role in controlling HMPV infection.

NKp46 Recognizes the Sigma1 Protein of Reovirus: Implications for Reovirus-Based Cancer Therapy.

The recent approval of oncolytic virus for therapy of melanoma patients has increased the need for precise evaluation of the mechanisms by which oncolytic viruses affect tumor growth. Here we show that the human NK cell-activating receptor NKp46 and the orthologous mouse protein NCR1 recognize the reovirus sigma1 protein in a sialic-acid-dependent manner. We identify sites of NKp46/NCR1 binding to sigma1 and show that sigma1 binding by NKp46/NCR1 leads to NK cell activation in vitro Finally, we demonstrate that NCR1 activation is essential for reovirus-based therapy in vivo Collectively, we have identified sigma1 as a novel ligand for NKp46/NCR1 and demonstrated that NKp46/NCR1 is needed both for clearance of reovirus infection and for reovirus-based tumor therapy.IMPORTANCE Reovirus infects much of the population during childhood, causing mild disease, and hence is considered to be efficiently controlled by the immune system. Reovirus also specifically infects tumor cells, leading to tumor death, and is currently being tested in human clinical trials for cancer therapy. The mechanisms by which our immune system controls reovirus infection and tumor killing are not well understood. We report here that natural killer (NK) cells recognize a viral protein named sigma1 through the NK cell-activating receptor NKp46. Using several mouse tumor models, we demonstrate the importance of NK cells in protection from reovirus infection and in reovirus killing of tumors in vivo Collectively, we identify a new ligand for the NKp46 receptor and provide evidence for the importance of NKp46 in the control of reovirus infections and in reovirus-based cancer therapy.

The Distinction between Dematiaceous Molds and Non-Dematiaceous Fungi in Clinical and Spiked Samples Treated with Hydrogen Peroxide Using Direct Fluorescence Microscopy.

Dematiaceous fungi are pigmented molds with a high content of melanin in their cell walls that can cause fatal infections in immunocompromised hosts. Direct microscopy is the main method for the rapid diagnosis of dematiaceous fungi in clinical specimens. However, it is often difficult to distinguish their hyphae from non-dematiaceous hyphae and yeast pseudohyphae. Our aim was to develop a fluorescence staining method that targets melanin for the detection of dematiaceous molds in clinical specimens. Glass slide smears of clinical samples and sterile bronchoalveolar lavage spiked with dematiaceous and non-dematiaceous fungi were treated with hydrogen peroxide, and digital images were recorded using direct microscopy with different fluorescent filters. The images of fungi were compared for their fluorescence intensity using the NIS-Elements software. The fluorescent signal between dematiaceous and non-dematiaceous fungi demonstrated a markedly increased mean intensity for dematiaceous molds following hydrogen peroxide treatment (7510.3 ± 10,427.6 vs. 0.3 ± 3.1, respectively, p < 0.0001). No fluorescent signal was detected in the absence of hydrogen peroxide. "Staining" fungal clinical specimens with hydrogen peroxide, followed by fluorescence microscopy examination, can differentiate between dematiaceous and non-dematiaceous fungi. This finding can be used for the detection of dematiaceous molds in clinical specimens and enables the early and appropriate treatment of infections.

Eradication of CD48-positive tumors by selectively enhanced YTS cells harnessing the lncRNA NeST.

Natural killer (NK) cells are currently used in clinical trials to treat tumors. However, such therapies still suffer from problems such as donor variability, reproducibility, and more, which prevent a wider use of NK cells therapeutics. Here we show a potential immunotherapy combining NK cell-mediated tumor eradiation and long non-coding (lnc) RNAs. We overexpressed the interferon (IFN) γ secretion-enhancing lncRNA nettoie Salmonella pas Theiler's (NeST) in the NK cell-like cell line YTS. YTS cells express the co-stimulatory receptor 2B4 whose main ligand is CD48. On YTS cells, 2B4 functions by direct activation. We showed that NeST overexpression in YTS cells resulted in increased IFNγ release upon interaction with CD48 (selectively enhanced (se)YTS cells). Following irradiation, the seYTS cells lost proliferation capacity but were still able to maintain their killing and IFNγ secretion capacities. Finally, we demonstrated that irradiated seYTS inhibit tumor growth in vivo. Thus, we propose seYTS cells as off-the-shelve therapy for CD48-expressing tumors.

Candida albicans evades NK cell elimination via binding of Agglutinin-Like Sequence proteins to the checkpoint receptor TIGIT.

Candida albicans is the most common fungal pathogen and a prevalent cause of deadly bloodstream infections. Better understanding of the immune response against it, and the ways by which it evades immunity, are crucial for developing new therapeutics against it. Natural Killer (NK) cells are innate lymphocytes best known for their role against viruses and tumors. In recent years it became clear that NK cells also play an important role in anti-fungal immunity. Here we show that while NK cells recognize and eliminate C. albicans, the fungal cells inhibit NK cells by manipulating the immune checkpoint receptor TIGIT (T cell immunoreceptor with Ig and ITIM domains) in both humans and mice. We identify the responsible fungal ligands as members of the Als (Agglutinin-Like Sequences) protein family. Furthermore, we show that blocking this interaction using immunotherapy with a TIGIT-blocking antibody can re-establish anti-Candida immunity and serve as a potential therapeutic tool.

Fusobacterium nucleatum CbpF Mediates Inhibition of T Cell Function Through CEACAM1 Activation.

F. nucleatum is an anaerobic bacterium that is associated with several tumor entities and promotes tumorigenesis. Recent evidence suggests that F. nucleatum binds the inhibitory receptor carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) via the trimeric autotransporter adhesin CbpF. However, whether this binding is functional or whether other fusobacterial trimeric autotransporter adhesins are involved in CEACAM1 activation is unknown. In this study, using F. nucleatum mutants lacking the type 5c trimeric autotransporter adhesins fvcA (CbpF), fvcB, fvcC, and fvcD, we show that F. nucleatum CbpF binds and activates CEACAM1 and also binds carcinoembryonic antigen (CEA), a tumor-associated protein. We further find that CEACAM antibodies directed against the CEACAM N-terminal domain block the CbpF-CEACAM1 interaction. In functional assays, we demonstrate CbpF-dependent inhibition of CD4+ T cell response. Thus, we characterize an immune evasion mechanism in which F. nucleatum uses its surface protein CbpF to inhibit T cell function by activating CEACAM1.

Altered NKp46 Recognition and Elimination of Influenza B Viruses.

Every year, millions of people worldwide are infected with influenza, causing enormous health and economic problems. The most common type of influenza is influenza A. It is known that Natural Killer (NK) cells play an important role in controlling influenza A infection, mostly through the recognition of the viral protein hemagglutinin (HA) by the activating receptor, NKp46. In contrast, little is known regarding NK cell recognition of influenza B viruses, even though they are responsible for a third of all pediatric influenza deaths and are therefore included in the seasonal vaccine each year. Here we show that NKp46 also recognizes influenza B viruses. We show that NKp46 binds the HA protein of influenza B in a sialic acid-dependent manner, and identified the glycosylated residue in NKp46, which is critical for this interaction. We discovered that this interaction has a binding affinity approximately seven times lower than NKp46 binding of influenza A's HA. Finally, we demonstrated, using mice deficient for the mouse orthologue of NKp46, named NCR1, that NKp46 is not important for influenza B elimination. These findings enable us to better understand the interactions between the different influenza viruses and NK cells that are known to be crucial for viral elimination.

Nectin4 is a novel TIGIT ligand which combines checkpoint inhibition and tumor specificity.

BACKGROUND: The use of checkpoint inhibitors has revolutionized cancer therapy. Unfortunately, these therapies often cause immune-related adverse effects, largely due to a lack of tumor specificity. METHODS: We stained human natural killer cells using fusion proteins composed of the extracellular portion of various tumor markers fused to the Fc portion of human IgG1, and identified Nectin4 as a novel TIGIT ligand. Next, we generated a novel Nectin4 blocking antibody and demonstrated its efficacy as a checkpoint inhibitor in killing assays and in vivo. RESULTS: We identify Nectin4 to be a novel ligand of TIGIT. We showed that, as opposed to all other known TIGIT ligands, which bind also additional receptors, Nectin4 interacts only with TIGIT. We show that the TIGIT-Nectin4 interaction inhibits natural killer cell activity, a critical part of the innate immune response. Finally, we developed blocking Nectin4 antibodies and demonstrated that they enhance tumor killing in vitro and in vivo. CONCLUSION: We discovered that Nectin4 is a novel ligand for TIGIT and demonstrated that specific antibodies against it enhance tumor cell killing in vitro and in vivo. Since Nectin4 is expressed almost exclusively on tumor cells, our Nectin4-blocking antibodies represent a combination of cancer specificity and immune checkpoint activity, which may prove more effective and safe for cancer immunotherapy.

Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression.

Fusobacterium nucleatum is an oral anaerobe recently found to be prevalent in human colorectal cancer (CRC) where it is associated with poor treatment outcome. In mice, hematogenous F. nucleatum can colonize CRC tissue using its lectin Fap2, which attaches to tumor-displayed Gal-GalNAc. Here, we show that Gal-GalNAc levels increase as human breast cancer progresses, and that occurrence of F. nucleatum gDNA in breast cancer samples correlates with high Gal-GalNAc levels. We demonstrate Fap2-dependent binding of the bacterium to breast cancer samples, which is inhibited by GalNAc. Intravascularly inoculated Fap2-expressing F. nucleatum ATCC 23726 specifically colonize mice mammary tumors, whereas Fap2-deficient bacteria are impaired in tumor colonization. Inoculation with F. nucleatum suppresses accumulation of tumor infiltrating T cells and promotes tumor growth and metastatic progression, the latter two of which can be counteracted by antibiotic treatment. Thus, targeting F. nucleatum or Fap2 might be beneficial during treatment of breast cancer.

Sweet Killers: NK Cells Need Glycolysis to Kill Tumors.

Natural killer (NK) cells are cytotoxic innate lymphocytes that play a major role in the immune battle against cancer. In this issue of Cell Metabolism, Cong et al. (2018) demonstrate that progressing tumors reduce NK cell glycolytic capacity, which leads to reduced cytotoxicity and an overall dysfunctional NK phenotype.

Quantification of Bacterial Attachment to Tissue Sections.

Here we describe a method to test bacterial adhesion to paraffin embedded tissue sections. This method allows examining binding of different bacterial strains, transfected with a fluorescent protein reporter plasmid to various tissues, to better understand different mechanisms such as colonization. This assay provides a more physiological context to bacterial binding, than would have been achieved using adhesion assays to cell lines. The sections can be imaged using fluorescent microscopy and adhesion of various bacterial strains can be quantified and tested, simultaneously.

Expression and function of NKp46 W32R: the human homologous protein of mouse NKp46 W32R (Noé).

Natural killer (NK) cells eradicate infected cells and tumors following the triggering of activating receptors, like the Natural Cytotoxicity Receptors (NCRs), which include NKp30, NKp44 and NKp46. NKp46 is the only NCR expressed in mice (mNKp46), and except for some Innate Lymphoid Cell (ILC) populations (ILC1/3 subsets), its expression is restricted to NK cells. Previously, a mouse named Noé was generated in which a random point mutation (W32R) impaired the cell surface expression of mNKp46. Interestingly, the Noé mice NK cells expressed twice as much of the transcription factor Helios, and displayed general non-NKp46 specific hyperactivity. We recently showed that the mNKp46 W32R (Noé) protein was expressed on the surface of various cells; albeit slowly and unstably, that it is aberrantly glycosylated and accumulates in the ER. Interestingly, the Tryptophan (Trp) residue in position 32 is conserved between humans and mice. Therefore, we studied here the human orthologue protein of mNKp46 W32R, the human NKp46 W32R. We demonstrated that NKp46 W32R is aberrantly glycosylated, accumulates in the ER, and is unstable on the cell surface. Furthermore, we showed that overexpression of NKp46 W32R or Helios resulted in augmented NK cell activation, which may be applied to boost NK activity for therapeutic applications.

The role of microglia and their CX3CR1 signaling in adult neurogenesis in the olfactory bulb.

Microglia play important roles in perinatal neuro- and synapto-genesis. To test the role of microglia in these processes during adulthood, we examined the effects of microglia depletion, via treatment of mice with the CSF-1 receptor antagonist PLX5622, and abrogated neuronal-microglial communication in CX3C receptor-1 deficient (Cx3cr1-/-) mice. Microglia depletion significantly lowered spine density in young (developing) but not mature adult-born-granule-cells (abGCs) in the olfactory bulb. Two-photon time-lapse imaging indicated that microglia depletion reduced spine formation and elimination. Functionally, odor-evoked responses of mitral cells, which are normally inhibited by abGCs, were increased in microglia-depleted mice. In Cx3cr1-/- mice, abGCs exhibited reduced spine density, dynamics and size, concomitantly with reduced contacts between Cx3cr1-deficient microglia and abGCs' dendritic shafts, along with increased proportion of microglia-contacted spines. Thus, during adult neurogenesis, microglia regulate the elimination (pruning), formation, and maintenance of synapses on newborn neurons, contributing to the functional integrity of the olfactory bulb circuitry.