NK cells link obesity-induced adipose stress to inflammation and insulin resistance.

An important cause of obesity-induced insulin resistance is chronic systemic inflammation originating in visceral adipose tissue (VAT). VAT inflammation is associated with the accumulation of proinflammatory macrophages in adipose tissue, but the immunological signals that trigger their accumulation remain unknown. We found that a phenotypically distinct population of tissue-resident natural killer (NK) cells represented a crucial link between obesity-induced adipose stress and VAT inflammation. Obesity drove the upregulation of ligands of the NK cell-activating receptor NCR1 on adipocytes; this stimulated NK cell proliferation and interferon-γ (IFN-γ) production, which in turn triggered the differentiation of proinflammatory macrophages and promoted insulin resistance. Deficiency of NK cells, NCR1 or IFN-γ prevented the accumulation of proinflammatory macrophages in VAT and greatly ameliorated insulin sensitivity. Thus NK cells are key regulators of macrophage polarization and insulin resistance in response to obesity-induced adipocyte stress.

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.

2B4: A potential target in Staphylococcus aureus associated allergic inflammation.

Staphylococcus aureus (SA) and its exotoxins activate eosinophils (Eos) and mast cells (MCs) via CD48, a GPI-anchored receptor belonging to the signaling lymphocytes activation molecules (SLAM) family. 2B4 (CD244), an immuno-regulatory transmembrane receptor also belonging to the SLAM family, is the high-affinity ligand for CD48. 2B4 is expressed on several leukocytes including NK cells, T cells, basophils, monocytes, dendritic cells (DCs), and Eos. In the Eos and MCs crosstalk carried out by physical and soluble interactions (named the 'allergic effector unit', AEU), 2B4-CD48 binding plays a central role. As CD48 and 2B4 share some structural characteristics and SA colonization accompanies most of the allergic diseases, we hypothesized that SA exotoxins (e.g. Staphylococcus enterotoxin B, SEB) can also bind and activate 2B4 and thereby possibly further aggravate inflammation. To check our hypothesis, we used in vitro, in silico, and in vivo methods. By enzyme-linked immunosorbent assay (ELISA), flow cytometry (FC), fluorescence microscopy, and microscale thermophoresis, we have shown that SEB can bind specifically to 2B4. By Eos short- and long-term activation assays, we confirmed the functionality of the SEB-2B4 interaction. Using computational modeling, we identified possible SEB-binding sites on human and mouse 2B4. Finally, in vivo, in an SEB-induced peritonitis model, 2B4-KO mice showed a significant reduction of inflammatory features compared with WT mice. Altogether, the results of this study confirm that 2B4 is an important receptor in SEB-mediated inflammation, and therefore a role is suggested for 2B4 in SA associated inflammatory conditions.

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 human cytomegalovirus protein UL147A downregulates the most prevalent MICA allele: MICA*008, to evade NK cell-mediated killing.

Natural killer (NK) cells are innate immune lymphocytes capable of killing target cells without prior sensitization. One pivotal activating NK receptor is NKG2D, which binds a family of eight ligands, including the major histocompatibility complex (MHC) class I-related chain A (MICA). Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus causing morbidity and mortality in immunosuppressed patients and congenitally infected infants. HCMV encodes multiple antagonists of NK cell activation, including many mechanisms targeting MICA. However, only one of these mechanisms, the HCMV protein US9, counters the most prevalent MICA allele, MICA*008. Here, we discover that a hitherto uncharacterized HCMV protein, UL147A, specifically downregulates MICA*008. UL147A primarily induces MICA*008 maturation arrest, and additionally targets it to proteasomal degradation, acting additively with US9 during HCMV infection. Thus, UL147A hinders NKG2D-mediated elimination of HCMV-infected cells by NK cells. Mechanistic analyses disclose that the non-canonical GPI anchoring pathway of immature MICA*008 constitutes the determinant of UL147A specificity for this MICA allele. These findings advance our understanding of the complex and rapidly evolving HCMV immune evasion mechanisms, which may facilitate the development of antiviral drugs and vaccines.

SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Currently, as dangerous mutations emerge, there is an increased demand for specific treatments for SARS-CoV-2 infected patients. The spike glycoprotein on the virus envelope binds to the angiotensin converting enzyme 2 (ACE2) on host cells through its receptor binding domain (RBD) to mediate virus entry. Thus, blocking this interaction may inhibit viral entry and consequently stop infection. Here, we generated fusion proteins composed of the extracellular portions of ACE2 and RBD fused to the Fc portion of human IgG1 (ACE2-Ig and RBD-Ig, respectively). We demonstrate that ACE2-Ig is enzymatically active and that it can be recognized by the SARS-CoV-2 RBD, independently of its enzymatic activity. We further show that RBD-Ig efficiently inhibits in-vivo SARS-CoV-2 infection better than ACE2-Ig. Mechanistically, we show that anti-spike antibody generation, ACE2 enzymatic activity, and ACE2 surface expression were not affected by RBD-Ig. Finally, we show that RBD-Ig is more efficient than ACE2-Ig at neutralizing high virus titers. We thus propose that RBD-Ig physically blocks virus infection by binding to ACE2 and that RBD-Ig should be used for the treatment of SARS-CoV-2-infected patients.

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.

The human cytomegalovirus microRNA miR-UL112 acts synergistically with a cellular microRNA to escape immune elimination.

Although approximately 200 viral microRNAs are known, only very few share similar targets with their host's microRNAs. A notable example of this is the stress-induced ligand MICB, which is targeted by several distinct viral and cellular microRNAs. Through the investigation of the microRNA-mediated immune-evasion strategies of herpesviruses, we initially identified two new cellular microRNAs that targeted MICB and were expressed differently both in healthy tissues and during melanocyte transformation. We show that coexpression of various pairs of cellular microRNAs interfered with the downregulation of MICB, whereas the viral microRNAs optimized their targeting ability to efficiently downregulate MICB. Moreover, we demonstrate that through site proximity and possibly inhibition of translation, a human cytomegalovirus (HCMV) microRNA acts synergistically with a cellular microRNA to suppress MICB expression during HCMV infection.

The activating receptor NKp46 is essential for the development of type 1 diabetes.

The mechanism of action of natural killer (NK) cells in type 1 diabetes is still unknown. Here we show that the activating receptor NKp46 recognizes mouse and human ligands on pancreatic beta cells. NK cells appeared in the pancreas when insulitis progressed to type 1 diabetes, and NKp46 engagement by beta cells led to degranulation of NK cells. NKp46-deficient mice had less development of type 1 diabetes induced by injection of a low dose of streptozotocin. Injection of soluble NKp46 proteins into nonobese diabetic mice during the early phase of insulitis and the prediabetic stage prevented the development of type 1 diabetes. Our findings demonstrate that NKp46 is essential for the development of type 1 diabetes and highlight potential new therapeutic modalities for this disease.

Fusobacterium nucleatum and cancer.

Accumulating evidence demonstrates that the oral pathobiont Fusobacterium nucleatum is involved in the progression of an increasing number of tumors types. Thus far, the mechanisms underlying tumor exacerbation by F. nucleatum include the enhancement of proliferation, establishment of a tumor-promoting immune environment, induction of chemoresistance, and the activation of immune checkpoints. This review focuses on the mechanisms that mediate tumor-specific colonization by fusobacteria. Elucidating the mechanisms mediating fusobacterial tumor tropism and promotion might provide new insights for the development of novel approaches for tumor detection and treatment.

Anti-RhD antibody therapy modulates human natural killer cell function.

Anti-RhD antibodies are widely used in clinical practice to prevent immunization against RhD, principally in hemolytic disease of the fetus and newborn. Intriguingly, this disease is induced by production of the very same antibodies when an RhD negative woman is pregnant with an RhD positive fetus. Despite over five decades of use, the mechanism of this treatment is, surprisingly, still unclear. Here we show that anti-RhD antibodies induce human natural killer (NK) cell degranulation. Mechanistically, we demonstrate that NK cell degranulation is mediated by binding of the Fc segment of anti-RhD antibodies to CD16, the main Fcγ receptor expressed on NK cells. We found that this CD16 activation is dependent upon glycosylation of the anti-RhD antibodies. Furthermore, we show that anti-RhD antibodies induce NK cell degranulation in vivo in patients who receive this treatment prophylactically. Finally, we demonstrate that the anti-RhD drug KamRho enhances the killing of dendritic cells. We suggest that this killing leads to reduced activation of adaptive immunity and may therefore affect the production of anti-RhD antibodies.

Inhibitory and Coactivating Receptors Recognising the Same Ligand: Immune Homeostasis Exploited by Pathogens and Tumours.

Coactivating and inhibitory receptors that share at least one ligand interact with a wide variety of ligands, indicating their importance in a range of situations. Here, we discuss principles of mainly human paired receptor function and ligand recognition, and possible therapeutic implications of targeting these receptors in cancer, autoimmune diseases, and allergy. We summarise and emphasise the idea that these receptors, which have evolved in part in response to pathogen pressure, fine-tune the immune response, preserve homeostasis, and that pathogens and tumours use the dominance of the inhibitory receptors over the coactivating receptors to avoid immune elimination. Finally, we discuss the options of using paired receptors and their ligand for immune cell education and therapy.

Human anti-NKp46 antibody for studies of NKp46-dependent NK cell function and its applications for type 1 diabetes and cancer research.

Natural killer (NK) cells are innate lymphocytes that efficiently eliminate cancerous and infected cells. NKp46 is an important NK activating receptor shown to participate in recognition and activation of NK cells against pathogens, tumor cells, virally infected cells, and self-cells in autoimmune conditions, including type I and II diabetes. However, some of the NKp46 ligands are unknown and therefore investigating human NKp46 activity and its critical role in NK cell biology is problematic. We developed a unique anti-human NKp46 monocloncal antibody, denoted hNKp46.02 (02). The 02 mAb can induce receptor internalization and degradation. By binding to a unique epitope on a particular domain of NKp46, 02 lead NKp46 to lysosomal degradation. This downregulation therefore enables the investigation of all NKp46 activities. Indeed, using the 02 mAb we determined NK cell targets which are critically dependent on NKp46 activity, including certain tumor cells lines and human pancreatic beta cells. Most importantly, we showed that a toxin-conjugated 02 inhibits the growth of NKp46-positive cells; thus, exemplifying the potential of 02 in becoming an immunotherapeutic drug to treat NKp46-dependent diseases, such as, type I diabetes and NK and T cell related malignancies.

Human microRNAs regulate stress-induced immune responses mediated by the receptor NKG2D.

MICA and MICB are stress-induced ligands recognized by the activating receptor NKG2D. A microRNA encoded by human cytomegalovirus downregulates MICB expression by targeting a specific site in the MICB 3' untranslated region. As this site is conserved among different MICB alleles and a similar site exists in the MICA 3' untranslated region, we speculated that these sites are targeted by cellular microRNAs. Here we identified microRNAs that bound to these MICA and MICB 3' untranslated region sequences and obtained data suggesting that these microRNAs maintain expression of MICA and MICB protein under a certain threshold and facilitate acute upregulation of MICA and MICB during cellular stress. These microRNAs were overexpressed in various tumors and we demonstrate here that they aided tumor avoidance of immune recognition.

Transcription of the NKG2D ligand MICA is suppressed by the IRE1/XBP1 pathway of the unfolded protein response through the regulation of E2F1.

The unfolded protein response (UPR) is an adaptive signaling pathway activated in response to endoplasmic reticulum (ER) stress. The effectors of the UPR are potent transcription activators; however, some genes are suppressed by ER stress at the mRNA level. The mechanisms underlying UPR-mediated gene suppression are less known. Exploration of the effect of UPR on NK cells ligand expression found that the transcription of NK group 2 member D (NKG2D) ligand major histocompatibility complex class I polypeptide-related sequence A/B (MICA/B) is suppressed by the inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1) pathway of the UPR. Deletion of IRE1 or XBP1 was sufficient to promote mRNA and surface levels of MICA. Accordingly, NKG2D played a greater role in the killing of IRE1/XBP1 knockout target cells. Analysis of effectors downstream to XBP1s identified E2F transcription factor 1 (E2F1) as linking UPR and MICA transcription. The inverse correlation between XBP1 and E2F1 or MICA expression was corroborated in RNA-Seq analysis of 470 primary melanoma tumors. While mechanisms that connect XBP1 to E2F1 are not fully understood, we implicate a few microRNA molecules that are modulated by ER stress and possess dual suppression of E2F1 and MICA. Because of the importance of E2F1 and MICA in cancer progression and recognition, these observations could be exploited for cancer therapy by manipulating the UPR in tumor cells.-Obiedat, A., Seidel, E., Mahameed, M., Berhani, O., Tsukerman, P., Voutetakis, K., Chatziioannou, A., McMahon, M., Avril, T., Chevet, E., Mandelboim, O., Tirosh, B. Transcription of the NKG2D ligand MICA is suppressed by the IRE1/XBP1 pathway of the unfolded protein response through the regulation of E2F1.

Activation of Siglec-7 results in inhibition of in vitro and in vivo growth of human mast cell leukemia cells.

Advanced systemic mastocytosis is a rare and still untreatable disease. Blocking antibodies against inhibitory receptors, also known as "immune checkpoints", have revolutionized anti-cancer treatment. Inhibitory receptors are expressed not only on normal immune cells, including mast cells but also on neoplastic cells. Whether activation of inhibitory receptors through monoclonal antibodies can lead to tumor growth inhibition remains mostly unknown. Here we show that the inhibitory receptor Siglec-7 is expressed by primary neoplastic mast cells in patients with systemic mastocytosis and by mast cell leukemia cell lines. Activation of Siglec-7 by anti-Siglec-7 monoclonal antibody caused phosphorylation of Src homology region 2 domain-containing phosphatase-1 (SHP-1), reduced phosphorylation of KIT and induced growth inhibition in mast cell lines. In SCID-beige mice injected with either the human mast cell line HMC-1.1 and HMC-1.2 or with Siglec-7 transduced B cell lymphoma cells, anti-Siglec-7 monoclonal antibody reduced tumor growth by a mechanism involving Siglec-7 cytoplasmic domains in "preventive" and "treatment" settings. These data demonstrate that activation of Siglec-7 on mast cell lines can inhibit their growth in vitro and in vivo. This might pave the way to additional treatment strategies for mastocytosis.

Decay of the Stress-Induced Ligand MICA Is Controlled by the Expression of an Alternative 3' Untranslated Region.

Recognition of the human stress-induced ligand MHC class I polypeptide-related sequence A (MICA) by the receptor NKG2D expressed on NK cells leads to NK cell-mediated killing of the target cells. Hence, the expression of MICA must be tightly regulated, and its cell surface expression needs to be quickly downregulated to avoid inappropriate activation of immune cells. In this article, we describe a transcript variant of human MICA that has not yet been studied, which contains a 3' untranslated region of 119 nt instead of 174. We identify its polyadenylation signal and demonstrate that, upon stresses, such as heat shock, butyrate treatment, and some oxidative and DNA-damaging treatments, the balance between the two MICA variants changes in favor of the less stable, longer variant. Mechanistically, we showed that this change is linked to microRNA activity and that poly-ADP ribose polymerase 1 is involved in the induction of the longer variant following stress. Thus, to our knowledge, we identify the first regulatory mechanism of a stress ligand's decay and also provide one of the first physiological examples for the biological function of a longer 3' untranslated region of a particular gene.