Publications by authors named "Ofer Mandelboim"

194 Publications

Altered NKp46 Recognition and Elimination of Influenza B Viruses.

Viruses 2020 12 27;13(1). Epub 2020 Dec 27.

The Concern Foundation Laboratories at the Lautenberg Center for Immunology and Cancer Research, The Hebrew University Hadassah Medical School, Jerusalem 9112001, Israel.

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.
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http://dx.doi.org/10.3390/v13010034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7824211PMC
December 2020

Colon Cancer-Associated May Originate From the Oral Cavity and Reach Colon Tumors via the Circulatory System.

Front Cell Infect Microbiol 2020 7;10:400. Epub 2020 Aug 7.

The Institute of Dental Sciences, The Hebrew University-Hadassah School of Dental Medicine, Jerusalem, Israel.

is a common oral bacterium that is enriched in colorectal adenomas and adenocarcinomas (CRC). In humans, high fusobacterial CRC abundance is associated with chemoresistance and poor prognosis. In animal models, fusobacteria accelerate CRC progression. Targeting may reduce fusobacteria cancer progression and therefore determining the origin of CRC and the route by which it reaches colon tumors is of biologic and therapeutic importance. Arbitrarily primed PCR performed previously on matched same-patients CRC and saliva isolates, suggested that CRC may originate from the oral cavity. However, the origin of CRC fusobacteria as well as the route of their arrival to the tumor have not been well-established. Herein, we performed and analyzed whole genome sequencing of paired, same-patient oral, and CRC isolates and confirmed that CRC-fusobacteria originate from the oral microbial reservoir. Oral fusobacteria may translocate to CRC by descending via the digestive tract or using the hematogenous route during frequent transient bacteremia caused by chewing, daily hygiene activities, or dental procedures. Using the orthotropic CT26 mouse model we previously showed that IV injected colonize CRC. Here, we compared CRC colonization by gavage vs. intravenous inoculated in the MC38 and CT26 mouse orthotropic CRC models. Under the tested conditions, hematogenous fusobacteria were more successful in CRC colonization than gavaged ones. Our results therefore provide evidence that the hematogenous route may be the preferred way by which oral fusobacteria reach colon tumors.
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http://dx.doi.org/10.3389/fcimb.2020.00400DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426652PMC
August 2020

Human Metapneumovirus Escapes NK Cell Recognition through the Downregulation of Stress-Induced Ligands for NKG2D.

Viruses 2020 07 20;12(7). Epub 2020 Jul 20.

The Concern Foundation Laboratories at the Lautenberg Center for Immunology and Cancer Research, Institute for Medical Research Israel-Canada (IMRIC), Faculty of Medicine, Hebrew University Hadassah Medical School, Jerusalem 91120, Israel.

The Pneumoviridae family includes human metapneumovirus (HMPV) and human orthopneumovirus, which is also known as a respiratory syncytial virus (HRSV). These are large enveloped, negative single-strand RNA viruses. HMPV and HRSV are the human members, which commonly infect children. HMPV, which was discovered in 2001, infects most children until the age of five, which causes an influenza-like illness. The interaction of this virus with immune cells is poorly understood. In this study, we show that HMPV evades natural killer (NK) cell attack by downregulating stress-induced ligands for the activating receptor NKG2D including: Major histocompatibility complex (MHC) class I polypeptide-related sequences A and B (MICA, MICB), UL16 binding proteins ULBP2, and ULBP3, but not ULBP1. Mechanistically, we show that the viral protein G is involved in the downregulation of ULBP2 and that the viral protein M2.2 is required for MICA and MICB downregulation. These findings emphasize the importance of NK cells, in general, and NKG2D, in particular, in controlling HMPV infection, which opens new avenues for treating HMPV.
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http://dx.doi.org/10.3390/v12070781DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7412239PMC
July 2020

Molecular mechanisms of human herpes viruses inferring with host immune surveillance.

J Immunother Cancer 2020 07;8(2)

Institute for Medical Immunology, Martin-Luther-Universitat Halle-Wittenberg, Halle (Saale), Germany.

Several human herpes viruses (HHVs) exert oncogenic potential leading to malignant transformation of infected cells and/or tissues. The molecular processes induced by viral-encoded molecules including microRNAs, peptides, and proteins contributing to immune evasion of the infected host cells are equal to the molecular processes of immune evasion mediated by tumor cells independently of viral infections. Such major immune evasion strategies include (1) the downregulation of proinflammatory cytokines/chemokines as well as the induction of anti-inflammatory cytokines/chemokines, (2) the downregulation of major histocompatibility complex (MHC) class Ia directly as well as indirectly by downregulation of the components involved in the antigen processing, and (3) the downregulation of stress-induced ligands for activating receptors on immune effector cells with NKG2D leading the way. Furthermore, (4) immune modulatory molecules like MHC class Ib molecules and programmed cell death1 ligand 1 can be upregulated on infections with certain herpes viruses. This review article focuses on the known molecular mechanisms of HHVs modulating the above-mentioned possibilities for immune surveillance and even postulates a temporal order linking regular tumor immunology with basic virology and offering putatively novel insights for targeting HHVs.
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http://dx.doi.org/10.1136/jitc-2020-000841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7333871PMC
July 2020

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

Nat Commun 2020 06 26;11(1):3259. Epub 2020 Jun 26.

The Institute of Dental Sciences, The Hebrew University-Hadassah School of Dental Medicine, Jerusalem, Israel.

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.
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http://dx.doi.org/10.1038/s41467-020-16967-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7320135PMC
June 2020

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

J Immunother Cancer 2020 06;8(1)

The Concern Foundation Laboratories at the Lautenberg Center for Immunology and Cancer Research, Hebrew University Hadassah Medical School, Jerusalem, Israel

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.
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http://dx.doi.org/10.1136/jitc-2019-000266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7279670PMC
June 2020

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

Haematologica 2020 05 28. Epub 2020 May 28.

The Hebrew University Hadassah Medical School, Jerusalem, Israel;

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.
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http://dx.doi.org/10.3324/haematol.2019.238097DOI Listing
May 2020

Immunorthodontics: in vivo gene expression of orthodontic tooth movement.

Sci Rep 2020 05 18;10(1):8172. Epub 2020 May 18.

Department of Orthodontics, Faculty of Dental Medicine, The Hebrew University and Hadassah Medical Center, Jerusalem, Israel.

Orthodontic tooth movement (OTM) is a "sterile" inflammatory process. The present study aimed to reveal the underlying biological mechanisms, by studying the force associated-gene expression changes, in a time-dependent manner. Ni-Ti springs were set to move the upper 1-molar in C57BL/6 mice. OTM was measured by μCT. Total-RNA was extracted from tissue blocks at 1,3,7 and 14-days post force application, and from two control groups: naïve and inactivated spring. Gene-expression profiles were generated by next-generation-RNA-sequencing. Gene Set Enrichment Analysis, K-means algorithm and Ingenuity pathway analysis were used for data interpretation. Genes of interest were validated with qRT-PCR. A total of 3075 differentially expressed genes (DEGs) were identified, with the greatest number at day 3. Two distinct clusters patterns were recognized: those in which DEGs peaked in the first days and declined thereafter (tissue degradation, phagocytosis, leukocyte extravasation, innate and adaptive immune system responses), and those in which DEGs were initially down-regulated and increased at day 14 (cell proliferation and migration, cytoskeletal rearrangement, tissue homeostasis, angiogenesis). The uncovering of novel innate and adaptive immune processes in OTM led us to propose a new term "Immunorthodontics". This genomic data can serve as a platform for OTM modulation future approaches.
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http://dx.doi.org/10.1038/s41598-020-65089-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7235241PMC
May 2020

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

Pharmacol Res 2020 08 5;158:104682. Epub 2020 Feb 5.

Pharmacology & Experimental Therapeutics Unit, Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Israel. Electronic address:

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.
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http://dx.doi.org/10.1016/j.phrs.2020.104682DOI Listing
August 2020

A Unique Regulation Region in the 3' UTR of HLA-G with a Promising Potential.

Int J Mol Sci 2020 Jan 30;21(3). Epub 2020 Jan 30.

The Concern Foundation Laboratories at the Lautenberg Center for Immunology and Cancer Research, The Biomedical Research Institute Israel Canada of the Faculty of Medicine, The Hebrew University Hadassah Medical School, The Hebrew University of Jerusalem, 9112001 Jerusalem, Israel.

Human leukocyte antigen G (HLA-G) is a non-classical human leukocyte antigen (HLA) class I protein that interacts with inhibitory receptors and is commonly overexpressed in various cancers, thereby establishing itself as an inhibitory checkpoint immune ligand. It is also expressed in trophoblast cells during pregnancy and protects the fetus from immune rejection. Despite its crucial role and its intriguing expression pattern, the regulation of HLA-G's expression is only partially understood. HLA-G's mRNA is expressed in many tissues but the protein expression is restricted only to the cells mentioned above. Therefore, we suggest that HLA-G is post-transcriptionally regulated. Here, we reveal a distinctive site present only in the 3' Untranslated region (UTR) of HLA-G, which might explain its unique expression pattern. Consequently, we attempted to find binding factors such as RNA binding proteins (RBPs) and microRNAS (miRs) that regulate HLA-G expression by interacting with this distinct site present in its 3' UTR. Our research indicates that this site should be further studied in order to reveal its significance.
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http://dx.doi.org/10.3390/ijms21030900DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037441PMC
January 2020

Comprehensive annotations of human herpesvirus 6A and 6B genomes reveal novel and conserved genomic features.

Elife 2020 Jan 16;9. Epub 2020 Jan 16.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

Human herpesvirus-6 (HHV-6) A and B are ubiquitous betaherpesviruses, infecting the majority of the human population. They encompass large genomes and our understanding of their protein coding potential is far from complete. Here, we employ ribosome-profiling and systematic transcript-analysis to experimentally define HHV-6 translation products. We identify hundreds of new open reading frames (ORFs), including upstream ORFs (uORFs) and internal ORFs (iORFs), generating a complete unbiased atlas of HHV-6 proteome. By integrating systematic data from the prototypic betaherpesvirus, human cytomegalovirus, we uncover numerous uORFs and iORFs conserved across betaherpesviruses and we show uORFs are enriched in late viral genes. We identified three highly abundant HHV-6 encoded long non-coding RNAs, one of which generates a non-polyadenylated stable intron appearing to be a conserved feature of betaherpesviruses. Overall, our work reveals the complexity of HHV-6 genomes and highlights novel features conserved between betaherpesviruses, providing a rich resource for future functional studies.
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http://dx.doi.org/10.7554/eLife.50960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6964970PMC
January 2020

Cytomegalovirus protein m154 perturbs the adaptor protein-1 compartment mediating broad-spectrum immune evasion.

Elife 2020 Jan 13;9. Epub 2020 Jan 13.

Center for Proteomics, Faculty of Medicine, University of Rijeka, Rijeka, Croatia.

Cytomegaloviruses (CMVs) are ubiquitous pathogens known to employ numerous immunoevasive strategies that significantly impair the ability of the immune system to eliminate the infected cells. Here, we report that the single mouse CMV (MCMV) protein, m154, downregulates multiple surface molecules involved in the activation and costimulation of the immune cells. We demonstrate that m154 uses its cytoplasmic tail motif, DD, to interfere with the adaptor protein-1 (AP-1) complex, implicated in intracellular protein sorting and packaging. As a consequence of the perturbed AP-1 sorting, m154 promotes lysosomal degradation of several proteins involved in T cell costimulation, thus impairing virus-specific CD8 T cell response and virus control in vivo. Additionally, we show that HCMV infection similarly interferes with the AP-1 complex. Altogether, we identify the robust mechanism employed by single viral immunomodulatory protein targeting a broad spectrum of cell surface molecules involved in the antiviral immune response.
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http://dx.doi.org/10.7554/eLife.50803DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6957316PMC
January 2020

The integrated stress response promotes B7H6 expression.

J Mol Med (Berl) 2020 01 14;98(1):135-148. Epub 2019 Dec 14.

Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, PO Box 12065, 91120, Jerusalem, Israel.

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.
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http://dx.doi.org/10.1007/s00109-019-01859-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952340PMC
January 2020

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

Cancer Immunol Immunother 2019 Oct 13;68(10):1721-1724. Epub 2019 Oct 13.

The Concern Foundation Laboratories at the Lautenberg Center for Immunology and Cancer Research, The Hebrew University Medical School, IMRIC, POB 12272, 91120, Jerusalem, Israel.

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.
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http://dx.doi.org/10.1007/s00262-019-02405-wDOI Listing
October 2019

Tumor-induced escape mechanisms and their association with resistance to checkpoint inhibitor therapy.

Cancer Immunol Immunother 2019 Oct 3;68(10):1689-1700. Epub 2019 Aug 3.

Institute of Medical Immunology, Martin Luther University Halle-Wittenberg, Magdeburger Straße 2, 06110, Halle (Saale), Germany.

Immunotherapy aims to activate the immune system to fight cancer in a very specific and targeted manner. Despite the success of different immunotherapeutic strategies, in particular antibodies directed against checkpoints as well as adoptive T-cell therapy, the response of patients is limited in different types of cancers. This attributes to escape of the tumor from immune surveillance and development of acquired resistances during therapy. In this review, the different evasion and resistance mechanisms that limit the efficacy of immunotherapies targeting tumor-associated antigens presented by major histocompatibility complex molecules on the surface of the malignant cells are summarized. Overcoming these escape mechanisms is a great challenge, but might lead to a better clinical outcome of patients and is therefore currently a major focus of research.
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http://dx.doi.org/10.1007/s00262-019-02373-1DOI Listing
October 2019

supresses anti-tumor immunity by activating CEACAM1.

Oncoimmunology 2019;8(6):e1581531. Epub 2019 Mar 27.

The Lautenberg Center of General and Tumor Immunology, The Hebrew University Hadassah Medical School, Institute for Medical Research Israel-Canada (IMRIC), Jerusalem, Israel.

) is an oral anaerobe found to be enriched in colorectal cancer (CRC). Presence of in CRC has been correlated with resistance to chemotherapy and poor prognosis. We previously demonstrated that the Fap2 outer-surface protein of binds and activates the human inhibitory receptor TIGIT which is expressed by T and Natural Killer (NK) cells, and inhibits anti-tumor immunity. Here we show that also binds and activates the human inhibitory receptor CEACAM1 leading to inhibition of T and NK cells activities. Our results suggest that using CEACAM1 and TIGIT inhibitors and specific targeting of fusobacteria should be considered for treating fusobacteria-colonized tumors.
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http://dx.doi.org/10.1080/2162402X.2019.1581531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6492956PMC
March 2019

The HopQ outermembrane protein inhibits immune cell activities.

Oncoimmunology 2019;8(4):e1553487. Epub 2019 Jan 29.

The Institute of Dental Sciences, The Hebrew University-Hadassah School of Dental Medicine, Jerusalem, Israel.

We previously showed that the colorectal cancer colonizing bacterium protects tumors from immune cell attack via binding of the fusbacterial Fap2 outer-membrane protein to TIGIT, a checkpoint inhibitory receptor expressed on T cells and NK cells. , the causative agent for peptic ulcer disease, is associated with the development of gastric adenocarcinoma and MALT lymphoma. The HopQ outer-membrane adhesin of was recently shown to bind carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) including CEACAM1, an inhibitory receptor expressed mainly by activated T and NK cells. Here we investigated the possibility that similar to Fap2, HopQ can also inhibit immune cell activities by interacting with CEACAM1. We used several approaches to confirm that HopQ indeed interacts with CEACAM1, and show that CEACAM1-mediated activation by HopQ, may inhibit NK and T cell functions.
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http://dx.doi.org/10.1080/2162402X.2018.1553487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6422397PMC
January 2019

Learning from experience: cellular and molecular bases for improved outcome in subsequent pregnancies.

Am J Obstet Gynecol 2019 09 22;221(3):183-193. Epub 2019 Feb 22.

Magda and Richard Hoffman Center for Human Placenta Research, Department of Obstetrics and Gynecology, Hebrew University Hadassah Medical Center, Jerusalem, Israel. Electronic address:

The frequencies of preeclampsia, fetal growth restriction, fetal demise, and low birthweight are lower in subsequent pregnancies. Enhanced maternal cardiovascular adaptation, shorter first and second stages of labor, and more robust lactation also have been observed in subsequent as compared with first pregnancies. We sought to investigate the cellular and molecular bases for better outcomes in subsequent pregnancies. Based on the knowledge that specialized immune cells at the maternal-fetal interface, decidual natural killer cells, promote development of the placental bed and conversion of the spiral arteries by secreting a myriad of angiogenic and growth factors, we asked whether decidual natural killer cells differ in subsequent as compared with first pregnancies. This idea stemmed from recent studies suggesting that natural killer cells, although part of the innate immune system, possess some features of adaptive immunity, including a certain type of immune cell memory, termed trained immunity. We found that decidual natural killer cells from parous women "remember pregnancy" and differ from decidual natural killer cells of primigravidae. Compared with the decidual natural killer cells of first pregnancy, these cells, that we termed pregnancy-trained decidual natural killer cells, express greater levels of the natural killer receptors NKG2C and leukocyte immunoglobulin-like receptor B1, which interact with ligands expressed on invasive trophoblasts. Furthermore, they secrete greater levels of several growth factors, including vascular endothelial growth factor α as well as interferon-γ, augmenting remodeling of the placental bed. We propose that this pregnancy-trained memory dwells in the epigenome, where memory of stimuli is known to persist even when the stimulus is no longer present. This epigenetic memory apparently resides in endometrial natural killer cells between pregnancies. We suggest that this trained memory, which we coined pregnancy-trained decidual natural killer cells, may be the missing link in the immune basis for enhanced subsequent pregnancy. Epigenetic memory (chromatin modification) also may afford a global explanation for additional findings of enhanced maternal cardiovascular adaptation, shorter first and second stages of labor, and more robust lactation. Understanding the molecular and cellular bases of improved outcomes of subsequent pregnancy may lead to the development of treatment modalities designed for women at high risk for pregnancy disorders originating at the maternal-fetal interface.
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http://dx.doi.org/10.1016/j.ajog.2019.02.037DOI Listing
September 2019

A BW Reporter System for Studying Receptor-Ligand Interactions.

J Vis Exp 2019 01 7(143). Epub 2019 Jan 7.

The Lautenberg Center for General and Tumor Immunology, Department of Immunology and Cancer Research, Institute for Medical Research Israel Canada (IMRIC), Hadassah - Hebrew University Medical Center;

Interactions between receptors and ligands constitute a fundamental biological process. However, direct experiments with cells that express the native receptor and the ligand are challenging since the ligand of a specific receptor may be unknown and experimental procedures with the native ligand can be technically complicated. To address these obstacles, we describe a reporter system to detect the binding and activation of a specific receptor by a ligand of interest. In this reporter system, the extracellular domain of a specific receptor is conjugated to mouse CD3ζ and this chimeric protein is then expressed in mouse BW cells. These transfected BW cells can then be incubated with different targets (e.g., cells or antibodies). Activation of a transfected receptor leads to the secretion of mouse interleukin-2 (mIL-2) which can be detected by enzyme-linked immunosorbent assay (ELISA). This reporter system has the advantages of being sensitive and specific to a single receptor. In addition, the activation level of a specific receptor can easily be quantified and can be used even in cases where the ligand of the receptor is unknown. This system has been implemented successfully in many of our studies to characterize receptor-ligand interactions. We have recently employed this system to study the activation of human Fcγ receptors (FcγRs) by different monoclonal anti-CD20 antibodies in clinical use.
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http://dx.doi.org/10.3791/58685DOI Listing
January 2019

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

iScience 2019 Jan 3;11:466-473. Epub 2019 Jan 3.

The Lautenberg Center for General and Tumor Immunology, Institute for Medical Research Israel-Canada, The Hebrew University Hadassah Medical School, Jerusalem 9112001, Israel. Electronic address:

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.
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http://dx.doi.org/10.1016/j.isci.2018.12.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6354656PMC
January 2019

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

Eur J Immunol 2019 02 17;49(2):228-241. Epub 2018 Dec 17.

The Concern Foundation Laboratories at the Lautenberg Center for Immunology and Cancer Research, The BioMedical Research Institute Israel Canada of the Faculty of Medicine (IMRIC), The Hebrew University Hadassah Medical School, Jerusalem, Israel.

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.
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http://dx.doi.org/10.1002/eji.201847611DOI Listing
February 2019

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

FASEB J 2019 03 19;33(3):3481-3495. Epub 2018 Nov 19.

Institute for Drug Research, School of Pharmacy, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem, Israel.

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.
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http://dx.doi.org/10.1096/fj.201801350RRDOI Listing
March 2019

Targeting PVR (CD155) and its receptors in anti-tumor therapy.

Cell Mol Immunol 2019 01 1;16(1):40-52. Epub 2018 Oct 1.

Center for Proteomics, Faculty of Medicine, University of Rijeka, Braće Branchetta 20, 51 000, Rijeka, Croatia.

Poliovirus receptor (PVR, CD155) has recently been gaining scientific interest as a therapeutic target in the field of tumor immunology due to its prominent endogenous and immune functions. In contrast to healthy tissues, PVR is expressed at high levels in several human malignancies and seems to have protumorigenic and therapeutically attractive properties that are currently being investigated in the field of recombinant oncolytic virotherapy. More intriguingly, PVR participates in a considerable number of immunoregulatory functions through its interactions with activating and inhibitory immune cell receptors. These functions are often modified in the tumor microenvironment, contributing to tumor immunosuppression. Indeed, increasing evidence supports the rationale for developing strategies targeting these interactions, either in terms of checkpoint therapy (i.e., targeting inhibitory receptors) or in adoptive cell therapy, which targets PVR as a tumor marker.
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http://dx.doi.org/10.1038/s41423-018-0168-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6318332PMC
January 2019

NKG2D Ligands-Critical Targets for Cancer Immune Escape and Therapy.

Front Immunol 2018 11;9:2040. Epub 2018 Sep 11.

The Lautenberg Center for General and Tumor Immunology, The BioMedical Research Institute Israel Canada of the Faculty of Medicine, The Hebrew University Hadassah Medical School, Jerusalem, Israel.

DNA damage, oncogene activation and excessive proliferation, chromatin modulations or oxidative stress are all important hallmarks of cancer. Interestingly, all of these abnormalities also induce a cellular stress response. By upregulating "stress-induced ligands," damaged or transformed cells can be recognized by immune cells and cleared. The human genome encodes eight functional "stress-induced ligands": MICA, MICB, and ULBP1-6. All of them are recognized by a single receptor, NKG2D, which is expressed on natural killer (NK) cells, cytotoxic T cells and other T cell subsets. The NKG2D ligand/NKG2D-axis is well-recognized as an important mediator of anti-tumor activity; however, patient data about the role of NKG2D ligands in immune surveillance and escape appears conflicting. As these ligands are often actively transcribed, tumor cells are urged to manipulate the expression of these ligands on post-transcriptional or post-translational level. Although our knowledge on the regulation of NKG2D ligand expression remains fragmentary, research of the past years revealed multiple cellular mechanisms that are adopted by tumor cells to reduce the expression of "stress-induced ligands" and therefore escape immune recognition. Here, we review the post-transcriptional and post-translational mechanisms by which NKG2D ligands are modulated in cancer cells and their impact on patient prognosis.We discuss controversies and approaches to apply our understanding of the NKG2D ligand/NKG2D-axis for cancer therapy.
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http://dx.doi.org/10.3389/fimmu.2018.02040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6141707PMC
September 2019

Spontaneous pulmonary hypertension in genetic mouse models of natural killer cell deficiency.

Am J Physiol Lung Cell Mol Physiol 2018 12 20;315(6):L977-L990. Epub 2018 Sep 20.

Departments of Biomedical and Molecular Sciences, Medicine, and Surgery, Queen's University Kingston , Ontario , Canada.

Natural killer (NK) cells are cytotoxic innate lymphoid cells with an established role in the regulation of vascular structure in pregnancy and cancer. Impaired NK cell function has been identified in patients with pulmonary arterial hypertension (PAH), a disease of obstructive vascular remodeling in the lungs, as well as in multiple rodent models of disease. However, the precise contribution of NK cell impairment to the initiation and progression of PAH remains unknown. Here, we report the development of spontaneous pulmonary hypertension in two independent genetic models of NK cell dysfunction, including Nfil3 mice, which are deficient in NK cells due to the absence of the NFIL3 transcription factor, and Ncr1-Gfp mice, which lack the NK activating receptor NKp46. Mouse models of NK insufficiency exhibited increased right ventricular systolic pressure and muscularization of the pulmonary arteries in the absence of elevated left ventricular end-diastolic pressure, indicating that the development of pulmonary hypertension was not secondary to left heart dysfunction. In cases of severe NK cell impairment or loss, a subset of mice failed to develop pulmonary hypertension and instead exhibited reduced systemic blood pressure, demonstrating an extension of vascular abnormalities beyond the pulmonary circulation into the systemic vasculature. In both mouse models, the development of PAH was linked to elevated interleukin-23 production, whereas systemic hypotension in Ncr1-Gfp mice was accompanied by a loss of angiopoietin-2. Together, these results support an important role for NK cells in the regulation of pulmonary and systemic vascular function and the pathogenesis of PAH.
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http://dx.doi.org/10.1152/ajplung.00477.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6337009PMC
December 2018

Sweet Killers: NK Cells Need Glycolysis to Kill Tumors.

Cell Metab 2018 08;28(2):183-184

The Concern Foundation Laboratories at the Lautenberg Center for Immunology and Cancer Research, Hebrew University Medical School, IMRIC, Jerusalem, Israel. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cmet.2018.07.008DOI Listing
August 2018

The Ebola-Glycoprotein Modulates the Function of Natural Killer Cells.

Front Immunol 2018 2;9:1428. Epub 2018 Jul 2.

The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel.

The Ebola virus (EBOV) uses evasion mechanisms that directly interfere with host T-cell antiviral responses. By steric shielding of human leukocyte antigen class-1, the Ebola glycoprotein (GP) blocks interaction with T-cell receptors (TCRs), thus rendering T cells unable to attack virus-infected cells. It is likely that this mechanism could promote increased natural killer (NK) cell activity against GP-expressing cells by preventing the engagement of NK inhibitory receptors; however, we found that primary human NK cells were less reactive to GP-expressing HEK293T cells. This was manifested as reduced cytokine secretion, a reduction in NK degranulation, and decreased lysis of GP-expressing target cells. We also demonstrated reduced recognition of GP-expressing cells by recombinant NKG2D and NKp30 receptors. In accordance, we showed a reduced monoclonal antibody-based staining of NKG2D and NKp30 ligands on GP-expressing target cells. Trypsin digestion of the membrane-associated GP led to a recovery of the recognition of membrane-associated NKG2D and NKp30 ligands. We further showed that membrane-associated GP did not shield recognition by KIR2DL receptors; in accordance, GP expression by target cells significantly perturbed signal transduction through activating, but not through inhibitory, receptors. Our results suggest a novel evasion mechanism employed by the EBOV to specifically avoid the NK cell immune response.
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http://dx.doi.org/10.3389/fimmu.2018.01428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6036185PMC
July 2018

The Human Cytomegalovirus Protein UL148A Downregulates the NK Cell-Activating Ligand MICA To Avoid NK Cell Attack.

J Virol 2018 09 16;92(17). Epub 2018 Aug 16.

The Lautenberg Center for General and Tumor Immunology, The Faculty of Medicine, The Hebrew University Medical School, IMRIC, Jerusalem, Israel

Natural killer (NK) cells are lymphocytes of the innate immune system capable of killing hazardous cells, including virally infected cells. NK cell-mediated killing is triggered by activating receptors. Prominent among these is the activating receptor NKG2D, which binds several stress-induced ligands, among them major histocompatibility complex (MHC) class I-related chain A (MICA). Most of the human population is persistently infected with human cytomegalovirus (HCMV), a virus which employs multiple immune evasion mechanisms, many of which target NK cell responses. HCMV infection is mostly asymptomatic, but in congenitally infected neonates and in immunosuppressed patients it can lead to serious complications and mortality. Here we discovered that an HCMV protein named UL148A whose role was hitherto unknown is required for evasion of NK cells. We demonstrate that UL148A-deficient HCMV strains are impaired in their ability to downregulate MICA expression. We further show that when expressed by itself, UL148A is not sufficient for MICA targeting, but rather acts in concert with an unknown viral factor. Using inhibitors of different cellular degradation pathways, we show that UL148A targets MICA for lysosomal degradation. Finally, we show that UL148A-mediated MICA downregulation hampers NK cell-mediated killing of HCMV-infected cells. Discovering the full repertoire of HCMV immune evasion mechanisms will lead to a better understanding of the ability of HCMV to persist in the host and may also promote the development of new vaccines and drugs against HCMV. Human cytomegalovirus (HCMV) is a ubiquitous pathogen which is usually asymptomatic but that can cause serious complications and mortality in congenital infections and in immunosuppressed patients. One of the difficulties in developing novel vaccines and treatments for HCMV is its remarkable ability to evade our immune system. In particular, HCMV directs significant efforts to thwarting cells of the innate immune system known as natural killer (NK) cells. These cells are crucial for successful control of HCMV infection, and yet our understanding of the mechanisms which HCMV utilizes to elude NK cells is partial at best. In the present study, we discovered that a protein encoded by HCMV which had no known function is important for preventing NK cells from killing HCMV-infected cells. This knowledge can be used in the future for designing more-efficient HCMV vaccines and for formulating novel therapies targeting this virus.
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http://dx.doi.org/10.1128/JVI.00162-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6096798PMC
September 2018

Obinutuzumab activates FcγRI more potently than other anti-CD20 antibodies in chronic lymphocytic leukemia (CLL).

Oncoimmunology 2018;7(6):e1428158. Epub 2018 Feb 12.

The Lautenberg Center for General and Tumor Immunology, Department of Immunology and Cancer Research, Institute for Medical Research Israel Canada (IMRIC), Hebrew University-Hadassah Medical School, Jerusalem, Israel.

Treatment with monoclonal antibodies has revolutionized clinical medicine, especially in the fields of cancer and immunology. One of the oldest antibodies, which is widely used for the treatment of lymphomas and autoimmune diseases, is the anti-CD20 antibody rituximab. In recent years, new antibodies against CD20 have been developed including ofatumumab and obinutuzumab. An important mechanism of action of therapeutic monoclonal antibodies is activation of immune cells via Fc receptors (FcγRs). However, surprisingly, little is known about triggering of FcγRs by different therapeutic antibodies in general and anti-CD20 antibodies in particular. Here we establish a reporter assay to assess whether a particular antibody activates a certain Fc receptor. Using this assay we corroborated previous reports demonstrating obinutuzumab's ability to highly activate FcγRIIIa (CD16a). Importantly, we discovered that obinutuzumab also activates FcγRI (CD64) significantly more than rituximab and ofatumumab in response to chronic lymphocytic leukemia (CLL) cells obtained from patients. Mechanistically we show that this is due to the lack of FcγRIIb-mediated internalization of obinutuzumab following binding to CD20. Moreover, we show that obinutuzumab induces increased phagocytosis by primary macrophages in an FcγRI-dependent manner. Beyond the discovery of a new mechanism of obinutuzumab activity, the reporter assay can be applied to other therapeutic antibodies and may assist in developing antibodies with improved immunological properties.
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http://dx.doi.org/10.1080/2162402X.2018.1428158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5980409PMC
February 2018

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

Immunity 2018 05;48(5):951-962.e5

The Concern Foundation Laboratories at the Lautenberg Centre for Immunology and Cancer Research, IMRIC, Hebrew University Faculty of Medicine, Jerusalem, Israel. Electronic address:

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.
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http://dx.doi.org/10.1016/j.immuni.2018.03.030DOI Listing
May 2018