Yotam Bar-On

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.

Killing of Avian and Swine Influenza Virus by Natural Killer Cells

ABSTRACT 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.

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.

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.

Dynamic Co-evolution of Host and Pathogen: HCMV Downregulates the Prevalent Allele MICA∗008 to Escape Elimination by NK Cells

SUMMARY Natural killer (NK) cells mediate innate immune responses against hazardous cells and are particularly important for the control of human cytomegalovirus (HCMV). NKG2D is a key NK activating receptor that recognizes a family of stress-induced ligands, including MICA, MICB, and ULBP1-6. Notably, most of these ligands are targeted by HCMV proteins and a miRNA to prevent the killing of infected cells by NK cells. A particular highly prevalent MICA allele, MICA*008, is considered to be an HCMV-resistant “escape variant” that confers advantage to human NK cells in recognizing infected cells. However, here we show that HCMV uses its viral glycoprotein US9 to specifically target MICA*008 and thus escapes NKG2D attack. The finding that HCMV evolved a protein dedicated to countering a single host allele illustrates the dynamic co-evolution of host and pathogen.

Natural Killer Cell-Mediated Host Defense against Uropathogenic E. coli Is Counteracted by Bacterial HemolysinA-Dependent Killing of NK Cells

Uropathogenic Escherichia coli (UPEC) are a common cause of urinary tract infections (UTIs) in humans. While the importance of natural killer (NK) cells in innate immune protection against tumors and viral infections is well documented, their role in defense against bacterial infections is still emerging, and their involvement in UPEC-mediated UTI is practically unknown. Using a systematic mutagenesis approach, we found that UPEC adheres to NK cells primarily via its type I fimbriae and employs its hemolysinA toxin to kill NK cells. In the absence of hemolysinA, NK cells directly respond to the bacteria and secrete the cytokine TNF-α, which results in decreased bacterial numbers in vitro and reduction of bacterial burden in the infected bladders. Thus, NK cells control UPEC via TNF-α production, which UPEC counteracts by hemolysinA-mediated killing of NK cells, representing a previously unrecognized host defense and microbial counterattack mechanism in the context of UTI.

Influenza Virus Uses Its Neuraminidase Protein to Evade the Recognition of Two Activating NK Cell Receptors

Natural Killer (NK) cells play a central role in the defense against viral infections and in the elimination of transformed cells. The recognition of pathogen-infected and tumor cells is controlled by inhibitory and activating receptors. We have previously shown that among the activating (killer) NK cell receptors the natural cytotoxicity receptors, NKp44 and NKp46, interact with the viral hemagglutinin (HA) protein expressed on the cell surface of influenza-virus-infected cells. We further showed that the interaction between NKp44/NKp46 and viral HA is sialic-acid dependent and that the recognition of HA by NKp44 and NKp46 leads to the elimination of the infected cells. Here we demonstrate that the influenza virus developed a counter-attack mechanism and that the virus uses its neuraminidase (NA) protein to prevent the recognition of HA by both the NKp44 and NKp46 receptors, resulting in reduced elimination of the infected cells by NK cells.

The viral KSHV chemokine vMIP-II inhibits the migration of Naive and activated human NK cells by antagonizing two distinct chemokine receptors.

Natural killer (NK) cells are innate immune cells able to rapidly kill virus-infected and tumor cells. Two NK cell populations are found in the blood; the majority (90%) expresses the CD16 receptor and also express the CD56 protein in intermediate levels (CD56Dim CD16Pos) while the remaining 10% are CD16 negative and express CD56 in high levels (CD56Bright CD16Neg). NK cells also reside in some tissues and traffic to various infected organs through the usage of different chemokines and chemokine receptors. Kaposis sarcoma-associated herpesvirus (KSHV) is a human virus that has developed numerous sophisticated and versatile strategies to escape the attack of immune cells such as NK cells. Here, we investigate whether the KSHV derived cytokine (vIL-6) and chemokines (vMIP-I, vMIP-II, vMIP-III) affect NK cell activity. Using transwell migration assays, KSHV infected cells, as well as fusion and recombinant proteins, we show that out of the four cytokine/chemokines encoded by KSHV, vMIP-II is the only one that binds to the majority of NK cells, affecting their migration. We demonstrate that vMIP-II binds to two different receptors, CX3CR1 and CCR5, expressed by naïve CD56Dim CD16Pos NK cells and activated NK cells, respectively. Furthermore, we show that the binding of vMIP-II to CX3CR1 and CCR5 blocks the binding of the natural ligands of these receptors, Fractalkine (Fck) and RANTES, respectively. Finally, we show that vMIP-II inhibits the migration of naïve and activated NK cells towards Fck and RANTES. Thus, we present here a novel mechanism in which KSHV uses a unique protein that antagonizes the activity of two distinct chemokine receptors to inhibit the migration of naïve and activated NK cells.

Loss of kindlin-3 alters the threshold for NK cell activation in human leukocyte adhesion deficiency-III

Recent evidence suggests that kindlin-3 is a major coactivator, required for most, if not all, integrin activities. Here we studied the function of kindlin-3 in regulating NK cell activation by studying a patient with kindlin-3 deficiency (leukocyte adhesion deficiency-III). We found that kindlin-3 is required for NK cell migration and adhesion under shear force. Surprisingly, we also found that kindlin-3 lowers the threshold for NK cell activation. Loss of kindlin-3 has a pronounced effect on NK cell-mediated cytotoxicity triggered by single activating receptors. In contrast, for activation through multiple receptors, kindlin-3 deficiency is overcome and target cells killed. The realization that NK cell activity is impaired, but not absent in leukocyte adhesion deficiency, may lead to the development of more efficient therapy for this rare disease.

Cytokine secretion and NK cell activity in human ADAM17 deficiency

Genetic deficiencies provide insights into gene function in humans. Here we describe a patient with a very rare genetic deficiency of ADAM17. We show that the patients PBMCs had impaired cytokine secretion in response to LPS stimulation, correlating with the clinical picture of severe bacteremia from which the patient suffered. ADAM17 was shown to cleave CD16, a major NK killer receptor. Functional analysis of patients NK cells demonstrated that his NK cells express normal levels of activating receptors and maintain high surface levels of CD16 following mAb stimulation. Activation of individual NK cell receptors showed that the patients NK cells are more potent when activated directly by CD16, albeit no difference was observed in Antibody Depedent Cytotoxicity (ADCC) assays. Our data suggest that ADAM17 inhibitors currently considered for clinical use to boost CD16 activity should be cautiously applied, as they might have severe side effects resulting from impaired cytokine secretion.