The activating receptor NKG2D is an anti-fungal pattern recognition receptor.

NKG2D is a central activating receptor involved in target recognition and killing by Natural Killer and CD8+ T cells. The known role of NKG2D is to recognize a family of self-induced stress ligands that are upregulated on stressed cells such as cancerous or virally infected cells. Fungal pathogens are a major threat to human health, infecting more than a billion patients yearly and becoming more common and drug resistant. Here we show that NKG2D plays a critical role in the immune response against fungal infections. NKG2D can recognize fungal pathogens from most major families including Candida, Cryptococcus and Aspergillus species, and mice lacking NKG2D are extremely sensitive to fungal infections in models of both invasive and mucosal infections, making NKG2D an anti-fungal pattern recognition receptor.

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.

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.

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.

The integrated stress response promotes B7H6 expression.

The B7 family member, B7H6, is a ligand for the natural killer cell receptor NKp30. B7H6 is hardly expressed on normal tissues, but undergoes upregulation on different types of tumors, implicating it as an attractive target for cancer immunotherapy. The molecular mechanisms that control B7H6 expression are poorly understood. We report that in contrast to other NK cell ligands, endoplasmic reticulum (ER) stress upregulates B7H6 mRNA levels and surface expression. B7H6 induction by ER stress requires protein kinase R-like ER kinase (PERK), one of the three canonical sensors of the unfolded protein response. PERK phosphorylates eIF2α, which regulates protein synthesis and gene expression. Because eIF2α is phosphorylated by several kinases following different stress conditions, the program downstream to eIF2α phosphorylation is called the integrated stress response (ISR). Several drugs were reported to promote the ISR. Nelfinavir and lopinavir, two clinically approved HIV protease inhibitors, promote eIF2α phosphorylation by different mechanisms. We show that nelfinavir and lopinavir sustainably instigate B7H6 expression at their pharmacologically relevant concentrations. As such, ER stress and ISR conditions sensitize melanoma targets to CAR-T cells directed against B7H6. Our study highlights a novel mechanism to induce B7H6 expression and suggests a pharmacological approach to improve B7H6-directed immunotherapy. KEY MESSAGES: B7H6 is induced by ER stress in a PERK-dependent mechanism. Induction of B7H6 is obtained pharmacologically by HIV protease inhibitors. Exposure of tumor cells to the HIV protease inhibitor nelfinavir improves the recognition by B7H6-directed CAR-T.

IFNG-AS1 Enhances Interferon Gamma Production in Human Natural Killer Cells.

Long, non-coding RNAs (lncRNAs) are involved in the regulation of many cellular processes. The lncRNA IFNG-AS1 was found to strongly influence the responses to several pathogens in mice by increasing interferon gamma (IFNγ) secretion. Studies have looked at IFNG-AS1 in T cells, yet IFNG-AS1 function in natural killer cells (NKs), an important source of IFNγ, remains unknown. Here, we show a previously undescribed sequence of IFNG-AS1 and report that it may be more abundant in cells than previously thought. Using primary human NKs and an NK line with IFNG-AS1 overexpression, we show that IFNG-AS1 is quickly induced upon NK cell activation, and that IFNG-AS1 overexpression leads to increased IFNγ secretion. Taken together, our work expands IFNG-AS1's activity to the innate arm of the type I immune response, helping to explain its notable effect in animal models of disease.

Human cytomegalovirus escapes immune recognition by NK cells through the downregulation of B7-H6 by the viral genes US18 and US20.

Human cytomegalovirus (HCMV) is a major human pathogen, causing serious diseases in immunocompromised populations and congenially infected neonates. One of the main immune cells acting against the virus are Natural Killer (NK) cells. Killing by NK cells is mediated by a small family of activating receptors such as NKp30 that interact with the cellular ligand B7-H6. The outcome of B7-H6-NKp30 interaction was, so far, mainly studied with regard to NK recognition and killing of tumors. Here, we demonstrated that the expression of B7-H6 is upregulated following HCMV infection and that HCMV uses two of its genes: US18 and US20, to interfere with B7-H6 surface expression, in a mechanism involving endosomal degradation, in order to evade NK cell recognition.

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.

HSV1 MicroRNA Modulation of GPI Anchoring and Downstream Immune Evasion.

Herpes simplex virus 1 (HSV1) is a ubiquitous human pathogen that utilizes variable mechanisms to evade immune surveillance. The glycosylphosphatidylinositol (GPI) anchoring pathway is a multistep process in which a myriad of different proteins are covalently attached to a GPI moiety to be presented on the cell surface. Among the different GPI-anchored proteins there are many with immunological importance. We present evidence that the HSV1-encoded miR H8 directly targets PIGT, a member of the protein complex that covalently attaches proteins to GPI in the final step of GPI anchoring. This results in a membrane down-modulation of several different immune-related, GPI-anchored proteins, including ligands for natural killer-activating receptors and the prominent viral restriction factor tetherin. Thus, we suggest that by utilizing just one of dozens of miRNAs encoded by HSV1, the virus can counteract the host immune response at several key points.

NK Cell Recognition of Candida glabrata through Binding of NKp46 and NCR1 to Fungal Ligands Epa1, Epa6, and Epa7.

Natural killer (NK) cells form an important arm of the innate immune system and function to combat a wide range of invading pathogens, ranging from viruses to bacteria. However, the means by which NK cells accomplish recognition of pathogens with a limited repertoire of receptors remain largely unknown. In the current study, we describe the recognition of an emerging fungal pathogen, Candida glabrata, by the human NK cytotoxic receptor NKp46 and its mouse ortholog, NCR1. Using NCR1 knockout mice, we observed that this receptor-mediated recognition was crucial for controlling C. glabrata infection in vitro and in vivo. Finally, we delineated the fungal ligands to be the C. glabrata adhesins Epa1, Epa6, and Epa7 and demonstrated that clearance of systemic C. glabrata infections in vivo depends on their recognition by NCR1. As NKp46 and NCR1 have been previously shown to bind viral adhesion receptors, we speculate that NKp46/NCR1 may be a novel type of pattern recognition receptor.

BREX is a novel phage resistance system widespread in microbial genomes.

The perpetual arms race between bacteria and phage has resulted in the evolution of efficient resistance systems that protect bacteria from phage infection. Such systems, which include the CRISPR-Cas and restriction-modification systems, have proven to be invaluable in the biotechnology and dairy industries. Here, we report on a six-gene cassette in Bacillus cereus which, when integrated into the Bacillus subtilis genome, confers resistance to a broad range of phages, including both virulent and temperate ones. This cassette includes a putative Lon-like protease, an alkaline phosphatase domain protein, a putative RNA-binding protein, a DNA methylase, an ATPase-domain protein, and a protein of unknown function. We denote this novel defense system BREX (Bacteriophage Exclusion) and show that it allows phage adsorption but blocks phage DNA replication. Furthermore, our results suggest that methylation on non-palindromic TAGGAG motifs in the bacterial genome guides self/non-self discrimination and is essential for the defensive function of the BREX system. However, unlike restriction-modification systems, phage DNA does not appear to be cleaved or degraded by BREX, suggesting a novel mechanism of defense. Pan genomic analysis revealed that BREX and BREX-like systems, including the distantly related Pgl system described in Streptomyces coelicolor, are widely distributed in ~10% of all sequenced microbial genomes and can be divided into six coherent subtypes in which the gene composition and order is conserved. Finally, we detected a phage family that evades the BREX defense, implying that anti-BREX mechanisms may have evolved in some phages as part of their arms race with bacteria.

tRNA genes rapidly change in evolution to meet novel translational demands.

Changes in expression patterns may occur when organisms are presented with new environmental challenges, for example following migration or genetic changes. To elucidate the mechanisms by which the translational machinery adapts to such changes, we perturbed the tRNA pool of Saccharomyces cerevisiae by tRNA gene deletion. We then evolved the deletion strain and observed that the genetic adaptation was recurrently based on a strategic mutation that changed the anticodon of other tRNA genes to match that of the deleted one. Strikingly, a systematic search in hundreds of genomes revealed that anticodon mutations occur throughout the tree of life. We further show that the evolution of the tRNA pool also depends on the need to properly couple translation to protein folding. Together, our observations shed light on the evolution of the tRNA pool, demonstrating that mutation in the anticodons of tRNA genes is a common adaptive mechanism when meeting new translational demands. DOI: http://dx.doi.org/10.7554/eLife.01339.001.