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1.
Eur J Immunol ; 49(2): 228-241, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30536875

RESUMO

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.


Assuntos
Anticorpos Monoclonais/química , Antígenos Ly/metabolismo , Diabetes Mellitus Tipo 1 , Células Matadoras Naturais/metabolismo , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias , Animais , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/metabolismo , Humanos , Células Jurkat , Células K562 , Camundongos , Neoplasias/diagnóstico , Neoplasias/metabolismo
3.
Immunity ; 48(1): 107-119.e4, 2018 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-29329948

RESUMO

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.


Assuntos
Antígenos Ly/metabolismo , Fibronectinas/metabolismo , Interferon gama/metabolismo , Células Matadoras Naturais/metabolismo , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Neoplasias/metabolismo , Animais , Western Blotting , Feminino , Citometria de Fluxo , Imunofluorescência , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Camundongos , Microscopia Confocal , Metástase Neoplásica/genética , Neoplasias/patologia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética
4.
Cell Rep ; 17(4): 949-956, 2016 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-27760325

RESUMO

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.


Assuntos
Glicosilfosfatidilinositóis/metabolismo , Herpesvirus Humano 1/genética , Evasão da Resposta Imune , MicroRNAs/metabolismo , Aciltransferases/metabolismo , Antígenos CD/metabolismo , Citotoxicidade Imunológica , Regulação para Baixo/genética , Proteínas Ligadas por GPI/metabolismo , Humanos , Células Matadoras Naturais/metabolismo , Ligantes , MicroRNAs/genética
5.
Cell Rep ; 15(11): 2331-9, 2016 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-27264178

RESUMO

Cells in our body can induce hundreds of antiviral genes following virus sensing, many of which remain largely uncharacterized. CEACAM1 has been previously shown to be induced by various innate systems; however, the reason for such tight integration to innate sensing systems was not apparent. Here, we show that CEACAM1 is induced following detection of HCMV and influenza viruses by their respective DNA and RNA innate sensors, IFI16 and RIG-I. This induction is mediated by IRF3, which bound to an ISRE element present in the human, but not mouse, CEACAM1 promoter. Furthermore, we demonstrate that, upon induction, CEACAM1 suppresses both HCMV and influenza viruses in an SHP2-dependent process and achieves this broad antiviral efficacy by suppressing mTOR-mediated protein biosynthesis. Finally, we show that CEACAM1 also inhibits viral spread in ex vivo human decidua organ culture.


Assuntos
Antígenos CD/metabolismo , Moléculas de Adesão Celular/metabolismo , Citomegalovirus/fisiologia , Orthomyxoviridae/fisiologia , Animais , Infecções por Citomegalovirus/metabolismo , Infecções por Citomegalovirus/virologia , Proteína DEAD-box 58/metabolismo , DNA Viral/metabolismo , Humanos , Influenza Humana/metabolismo , Influenza Humana/virologia , Fator Regulador 3 de Interferon/metabolismo , Camundongos , Técnicas de Cultura de Órgãos , Biossíntese de Proteínas , Receptores Imunológicos , Serina-Treonina Quinases TOR/metabolismo , Replicação Viral
6.
Dev Cell ; 32(5): 535-45, 2015 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-25662175

RESUMO

Because tissue regeneration deteriorates with age, it is generally assumed that the younger the animal, the better it compensates for tissue damage. We have examined the effect of young age on compensatory proliferation of pancreatic ß cells in vivo. Surprisingly, ß cells in suckling mice fail to enter the cell division cycle in response to a diabetogenic injury or increased glycolysis. The potential of ß cells for compensatory proliferation is acquired following premature weaning to normal chow, but not to a diet mimicking maternal milk. In addition, weaning coincides with enhanced glucose-stimulated oxidative phosphorylation and insulin secretion from islets. Transcriptome analysis reveals that weaning increases the expression of genes involved in replication licensing, suggesting a mechanism for increased responsiveness to the mitogenic activity of high glucose. We propose that weaning triggers a discrete maturation step of ß cells, elevating both the mitogenic and secretory response to glucose.


Assuntos
Biomarcadores/metabolismo , Proliferação de Células , Glucose/farmacologia , Insulina/farmacologia , Ilhotas Pancreáticas/citologia , Desmame , Animais , Apoptose , Western Blotting , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Hipoglicemiantes/farmacologia , Técnicas Imunoenzimáticas , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
7.
Cell Rep ; 10(6): 968-982, 2015 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-25683719

RESUMO

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

8.
Immunity ; 42(2): 344-355, 2015 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-25680274

RESUMO

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.


Assuntos
Adenocarcinoma/imunologia , Adenocarcinoma/microbiologia , Neoplasias do Colo/imunologia , Neoplasias do Colo/microbiologia , Fusobacterium nucleatum/imunologia , Receptores Imunológicos/imunologia , Evasão Tumoral/imunologia , Microambiente Tumoral/imunologia , Animais , Proteínas da Membrana Bacteriana Externa/imunologia , Linhagem Celular , Proliferação de Células , Humanos , Células Matadoras Naturais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Ligação Proteica
9.
Front Immunol ; 5: 4, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24478773

RESUMO

Natural killer (NK) cells are innate immune lymphocytes that function mainly as immune sentinels against viral infection and tumorigenesis. NK cell function is governed by inhibitory and activating signals arising from corresponding receptors. A prominent group of activating NK receptors is the natural cytotoxicity receptors (NCRs), which includes NKp30, NKp44, and NKp46. These receptors bind various diverse ligands of pathogenic, tumor, and even self origin. Type 1 diabetes mellitus (T1D) is a multifactorial autoimmune disease, in which insulin-producing beta (ß) cells are ablated by the immune system. This killing of ß cells is carried out mainly by T cells, but many other immune cells have been implicated in the pathogenesis of this disease. Importantly, NK cells were shown to be key participants in the initial autoimmune attack. It was shown that all ß cells from humans and mice, healthy or sick, express an unknown ligand for the activating NKp46 receptor. In this review, we describe the role played by the NCRs in various pathologies with an emphasis on Type I diabetes.

10.
Cell Host Microbe ; 14(6): 664-74, 2013 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-24331464

RESUMO

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


Assuntos
Proteínas de Escherichia coli/metabolismo , Proteínas Hemolisinas/metabolismo , Interações Hospedeiro-Patógeno , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/fisiologia , Escherichia coli Uropatogênica/imunologia , Escherichia coli Uropatogênica/fisiologia , Fatores de Virulência/metabolismo , Animais , Carga Bacteriana , Sobrevivência Celular , Células Cultivadas , Elementos de DNA Transponíveis , Proteínas de Escherichia coli/genética , Técnicas de Inativação de Genes , Proteínas Hemolisinas/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Insercional , Fator de Necrose Tumoral alfa/imunologia , Escherichia coli Uropatogênica/patogenicidade , Fatores de Virulência/genética
11.
PLoS One ; 8(8): e74033, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24009765

RESUMO

NK cells rapidly kill tumor cells, virus infected cells and even self cells. This is mediated via killer receptors, among which NKp46 (NCR1 in mice) is prominent. We have recently demonstrated that in type 1 diabetes (T1D) NK cells accumulate in the diseased pancreas and that they manifest a hyporesponsive phenotype. In addition, we found that NKp46 recognizes an unknown ligand expressed by beta cells derived from humans and mice and that blocking of NKp46 activity prevented diabetes development. Here we investigated the properties of the unknown NKp46 ligand. We show that the NKp46 ligand is mainly located in insulin granules and that it is constitutively secreted. Following glucose stimulation the NKp46 ligand translocates to the cell membrane and its secretion decreases. We further demonstrate by using several modalities that the unknown NKp46 ligand is not insulin. Finally, we studied the expression of the NKp46 ligand in type 2 diabetes (T2D) using 3 different in vivo models and 2 species; mice and gerbils. We demonstrate that the expression of the NKp46 ligand is decreased in all models of T2D studied, suggesting that NKp46 is not involved in T2D.


Assuntos
Antígenos Ly/metabolismo , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Expressão Gênica , Células Secretoras de Insulina/metabolismo , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Animais , Antígenos Ly/genética , Autoimunidade/genética , Diabetes Mellitus Tipo 2/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/imunologia , Leptina/administração & dosagem , Ligantes , Masculino , Camundongos , Receptor 1 Desencadeador da Citotoxicidade Natural/genética , Ligação Proteica
12.
Eur J Immunol ; 43(8): 2138-50, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23677581

RESUMO

The activity of natural killer (NK) cells is controlled by a balance of signals derived from inhibitory and activating receptors. TIGIT is a novel inhibitory receptor, recently shown in humans to interact with two ligands: PVR and Nectin2 and to inhibit human NK-cell cytotoxicity. Whether mouse TIGIT (mTIGIT) inhibits mouse NK-cell cytotoxicity is unknown. Here we show that mTIGIT is expressed by mouse NK cells and interacts with mouse PVR. Using mouse and human Ig fusion proteins we show that while the human TIGIT (hTIGIT) cross-reacts with mouse PVR (mPVR), the binding of mTIGIT is restricted to mPVR. We further demonstrate using surface plasmon resonance (SPR) and staining with Ig fusion proteins that mTIGIT binds to mPVR with higher affinity than the co-stimulatory PVR-binding receptor mouse DNAM1 (mDNAM1). Functionally, we show that triggering of mTIGIT leads to the inhibition of NK-cell cytotoxicity, that IFN-γ secretion is enhanced when mTIGIT is blocked and that the TIGIT-mediated inhibition is dominant over the signals delivered by the PVR-binding co-stimulatory receptors. Additionally, we identify the inhibitory motif responsible for mTIGIT inhibition. In conclusion, we show that TIGIT is a powerful inhibitory receptor for mouse NK cells.


Assuntos
Moléculas de Adesão Celular/metabolismo , Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Receptores Imunológicos/metabolismo , Receptores Virais/metabolismo , Sequência de Aminoácidos , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Interferon gama/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Nectinas , Receptores Imunológicos/química , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Transdução de Sinais , Ressonância de Plasmônio de Superfície
13.
Oncoimmunology ; 2(1): e22245, 2013 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-23479551

RESUMO

Metastases are responsible for more than 90% of cancer-related deaths. We have recently reported that miR-10b inhibits the expression of MICB, a stress-induced ligand of the activating natural killer (NK)-cell receptor NKG2D. Here, we discuss our findings, which link metastasis formation to immune evasion.

14.
J Immunol ; 188(6): 2509-15, 2012 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-22308311

RESUMO

NK cells employ a variety of activating receptors to kill virally infected and tumor cells. Prominent among these receptors are the natural cytotoxicity receptors (NCRs) (NKp30, NKp44, and NKp46), of which only NKp46 has a mouse ortholog (NCR1). The tumor ligand(s) of NKp46/NCR1 is still unknown, but it was shown that the human NKp46 and the mouse NCR1 are involved in tumor eradication both in vitro and in vivo. Whether any of the NK activating receptors is involved in the prevention of tumor metastasis is unknown. To address this question, we studied the activity of the NK cell receptor NKp46/NCR1 in two spontaneous metastasis models, the B16F10.9 melanoma (B16) and the Lewis lung carcinoma (D122) in the NCR1 knockout mouse that was generated by our group, in various in vitro and in vivo assays. We demonstrated that all B16 and D122 tumors, including those generated in vivo, express an unknown ligand(s) for NKp46/NCR1. We have characterized the properties of the NKp46/NCR1 ligand(s) and demonstrated that NKp46/NCR1 is directly involved in the killing of B16 and D122 cells. Importantly, we showed in vivo that NKp46/NCR1 plays an important role in controlling B16 and D122 metastasis. Thus, to our knowledge, in this study we provide the first evidence for the direct involvement of a specific NK killer receptor in preventing tumor metastasis.


Assuntos
Antígenos Ly/imunologia , Receptor 1 Desencadeador da Citotoxicidade Natural/imunologia , Animais , Antígenos Ly/genética , Antígenos Ly/metabolismo , Carcinoma Pulmonar de Lewis , Separação Celular , Citotoxicidade Imunológica/imunologia , Citometria de Fluxo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor 1 Desencadeador da Citotoxicidade Natural/genética , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Invasividade Neoplásica/genética , Metástase Neoplásica/prevenção & controle
15.
J Immunol ; 187(6): 3096-103, 2011 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-21849674

RESUMO

Type 1 diabetes is an incurable disease that is currently treated by insulin injections or in rare cases by islet transplantation. We have recently shown that NKp46, a major killer receptor expressed by NK cells, recognizes an unknown ligand expressed by ß cells and that in the absence of NKp46, or when its activity is blocked, diabetes development is inhibited. In this study, we investigate whether NKp46 is involved in the killing of human ß cells that are intended to be used for transplantation, and we also thoroughly characterize the interaction between NKp46 and its human and mouse ß cell ligands. We show that human ß cells express an unknown ligand for NKp46 and are killed in an NKp46-dependent manner. We further demonstrate that the expression of the NKp46 ligand is detected on human ß cells already at the embryonic stage and that it appears on murine ß cells only following birth. Because the NKp46 ligand is detected on healthy ß cells, we wondered why type 1 diabetes does not develop in all individuals and show that NK cells are absent from the vicinity of islets of healthy mice and are detected in situ in proximity with ß cells in NOD mice. We also investigate the molecular mechanisms controlling NKp46 interactions with its ß cell ligand and demonstrate that the recognition is confined to the membrane proximal domain and stalk region of NKp46 and that two glycosylated residues of NKp46, Thr(125) and Asn(216), are critical for this recognition.


Assuntos
Citotoxicidade Imunológica/imunologia , Células Secretoras de Insulina/imunologia , Células Matadoras Naturais/imunologia , Receptor 1 Desencadeador da Citotoxicidade Natural/química , Receptor 1 Desencadeador da Citotoxicidade Natural/imunologia , Animais , Sítios de Ligação , Separação Celular , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , Imuno-Histoquímica , Células Secretoras de Insulina/metabolismo , Células Matadoras Naturais/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos NOD , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Ligação Proteica
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