RESUMO
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.
Assuntos
Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/metabolismo , Domínios Proteicos , Proteínas Recombinantes de Fusão/metabolismo , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Sítios de Ligação , Sítios de Ligação de Anticorpos , COVID-19/prevenção & controle , Chlorocebus aethiops , Feminino , Células HEK293 , Humanos , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Imunoglobulina G/uso terapêutico , Camundongos Transgênicos , Testes de Neutralização , Ligação Proteica , Proteínas Recombinantes de Fusão/uso terapêutico , SARS-CoV-2/efeitos dos fármacos , Células VeroRESUMO
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/metabolismoRESUMO
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.
Assuntos
Fator de Transcrição E2F1/genética , Endorribonucleases/genética , Antígenos de Histocompatibilidade Classe I/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Proteínas Serina-Treonina Quinases/genética , Resposta a Proteínas não Dobradas/genética , Proteína 1 de Ligação a X-Box/genética , Linhagem Celular Tumoral , Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/genética , Humanos , Ligantes , RNA Mensageiro/genética , Transdução de Sinais/genética , Fatores de Transcrição/genética , Transcrição Gênica/genéticaRESUMO
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.
Assuntos
Regiões 3' não Traduzidas/genética , Coriocarcinoma/patologia , Regulação da Expressão Gênica , Antígenos HLA-G/genética , MicroRNAs/genética , Proteínas de Ligação a RNA/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Coriocarcinoma/genética , Coriocarcinoma/metabolismo , Feminino , Antígenos HLA-G/metabolismo , Humanos , MicroRNAs/metabolismo , Gravidez , Proteínas de Ligação a RNA/genética , Células Tumorais Cultivadas , Neoplasias Uterinas/genética , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologiaRESUMO
NK cells are part of the innate immune system, and are able to identify and kill hazardous cells. The discrimination between normal and hazardous cells is possible due to an array of inhibitory and activating receptors. NKG2D is one of the prominent activating receptors expressed by all human NK cells. This receptor binds stress-induced ligands, including human MICA, MICB, and UL16-binding proteins 1-6. The interaction between NKG2D and its ligands facilitates the elimination of cells under cellular stress, such as tumor transformation. However, the mechanisms regulating the expression of these ligands are still not well understood. Under normal conditions, the NKG2D ligands were shown to be posttranscriptionally regulated by cellular microRNAs and RNA-binding proteins (RBPs). Thus far, only the 3' untranslated regions (UTRs) of MICA, MICB, and UL16-binding protein 2 were shown to be regulated by RBPs and microRNAs, usually resulting in their downregulation. In this study we investigated whether MICB expression is controlled by RBPs through its 5'UTR. We used an RNA pull-down assay followed by mass spectrometry and identified vigilin, a ubiquitously expressed multifunctional RNA-binding protein. We demonstrated that vigilin binds and negatively regulates MICB expression through its 5'UTR. Additionally, vigilin downregulation in target cells led to a significant increase in NK cell activation against said target cells. Taken together, we have discovered a novel mode of MICB regulation.
Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Vigilância Imunológica , Células Matadoras Naturais/imunologia , Proteínas de Ligação a RNA/metabolismo , Estresse Fisiológico/imunologia , Regiões 5' não Traduzidas/genética , Linhagem Celular Tumoral , Regulação da Expressão Gênica/genética , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Ligantes , Ativação Linfocitária , Subfamília K de Receptores Semelhantes a Lectina de Células NK/agonistas , Ligação Proteica , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genéticaRESUMO
MHC class I molecules, in addition to their role in specific activation of the CTL of adaptive immune system, function also as the main ligands for NK cell inhibitory receptors, which prevent NK cells from killing normal, healthy cells. MHC class I proteins are divided into classical and nonclassical proteins. The former group consists of hundreds of HLA-A, B, and C alleles, which are universally expressed, whereas several alleles of the latter group, such as HLA-G, manifest a restricted expression pattern. Despite the important role played by these molecules in innate and adaptive immune responses, their complex expression regulation is not fully known. In our study, we investigated the regulation processes controlling the expression of MHC class I molecules, with a particular focus on their 3' untranslated regions. We identified heterogeneous nuclear ribonucleoprotein R (HNRNPR) as an important positive regulator of classical and nonclassical MHC class I molecules. HNRNPR is a RNA-binding protein belonging to the heterogeneous nuclear ribonucleoprotein family, which has a known role in processing of precursor mRNA. We demonstrated that HNRNPR binds MHC class I mRNAs in their 3' untranslated regions and enhances their stability and consequently their expression. Furthermore, regulation by HNRNPR modulates the cytotoxic activity of NK cells. In conclusion, we show that HNRNPR acts as a general positive regulator of MHC class I expression.
Assuntos
Regulação da Expressão Gênica , Genes MHC Classe I , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Antígenos de Histocompatibilidade Classe I/genética , Regiões 3' não Traduzidas , Linhagem Celular , Testes Imunológicos de Citotoxicidade , Antígenos HLA-G/imunologia , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Células Matadoras Naturais/imunologia , Ligação Proteica , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Células Matadoras Naturais/imunologiaRESUMO
NK cells kill various cells using activating receptors, such as the natural cytotoxicity receptors (NCRs). NKp46 is a major NCR and is the only NCR expressed in mice (denoted Ncr1). Using Ncr1-deficient mice (Ncr1(gfp/pfp)) we demonstrated that Ncr1 controls various pathologies, and that in its absence Ncr1-related functions are impaired. In 2012, another Ncr1-related mouse was generated, named Noé, in which a random mutation, W32R, in position 32, impaired the Ncr1-Noé cell surface expression. Interestingly, in the Noé mice, Ncr1-dependent deficiencies were not observed. Additionally, the Noé-NK cells were hyperactivated, probably due to increased Helios expression, and the Noé mice demonstrate increased clearance of influenza and murine CMV. In contrast, in the Ncr1(gfp/pfp) mice infection with influenza was lethal and we show in the present study no difference in murine CMV infection between Ncr1(gfp/pfp) and wild-type (WT) mice. Because the foremost difference between the Noé and Ncr1(gfp/gfp) mice is the presence of a mutated Ncr1-Noé protein, we studied its properties. We show that Ncr1-Noé and various other Ncr1 mutants in position 32 can be expressed on the surface, albeit slowly and unstably, and that ligand recognition and function of the various Ncr1-Noé is similar to the WT Ncr1. We further show that the glycosylation pattern of Ncr1-Noé is aberrant, that the Ncr1-Noé proteins accumulate in the endoplasmic reticulum, and that the expression of Ncr1-Noé proteins, but not WT Ncr1, leads to increased Helios expression. Thus, we suggest that the NK hyperactivated phenotype observed in the Noé mice might result from the presence of the Ncr1-Noé protein.
Assuntos
Antígenos Ly/imunologia , Regulação da Expressão Gênica/imunologia , Receptor 1 Desencadeador da Citotoxicidade Natural/imunologia , Animais , Antígenos Ly/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Glicosilação , Humanos , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/imunologia , Camundongos , Camundongos Transgênicos , Mutação , Receptor 1 Desencadeador da Citotoxicidade Natural/genética , Fatores de Transcrição/genética , Fatores de Transcrição/imunologiaRESUMO
Natural killer (NK) cells are currently used in clinical trials to treat tumors. However, such therapies still suffer from problems such as donor variability, reproducibility, and more, which prevent a wider use of NK cells therapeutics. Here we show a potential immunotherapy combining NK cell-mediated tumor eradiation and long non-coding (lnc) RNAs. We overexpressed the interferon (IFN) γ secretion-enhancing lncRNA nettoie Salmonella pas Theiler's (NeST) in the NK cell-like cell line YTS. YTS cells express the co-stimulatory receptor 2B4 whose main ligand is CD48. On YTS cells, 2B4 functions by direct activation. We showed that NeST overexpression in YTS cells resulted in increased IFNγ release upon interaction with CD48 (selectively enhanced (se)YTS cells). Following irradiation, the seYTS cells lost proliferation capacity but were still able to maintain their killing and IFNγ secretion capacities. Finally, we demonstrated that irradiated seYTS inhibit tumor growth in vivo. Thus, we propose seYTS cells as off-the-shelve therapy for CD48-expressing tumors.
RESUMO
F. nucleatum is an anaerobic bacterium that is associated with several tumor entities and promotes tumorigenesis. Recent evidence suggests that F. nucleatum binds the inhibitory receptor carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) via the trimeric autotransporter adhesin CbpF. However, whether this binding is functional or whether other fusobacterial trimeric autotransporter adhesins are involved in CEACAM1 activation is unknown. In this study, using F. nucleatum mutants lacking the type 5c trimeric autotransporter adhesins fvcA (CbpF), fvcB, fvcC, and fvcD, we show that F. nucleatum CbpF binds and activates CEACAM1 and also binds carcinoembryonic antigen (CEA), a tumor-associated protein. We further find that CEACAM antibodies directed against the CEACAM N-terminal domain block the CbpF-CEACAM1 interaction. In functional assays, we demonstrate CbpF-dependent inhibition of CD4+ T cell response. Thus, we characterize an immune evasion mechanism in which F. nucleatum uses its surface protein CbpF to inhibit T cell function by activating CEACAM1.
Assuntos
Molécula 1 de Adesão Celular/imunologia , Infecções por Fusobacterium/imunologia , Evasão da Resposta Imune , Linfócitos T , Fusobacterium nucleatum , Humanos , Linfócitos T/imunologia , Linfócitos T/microbiologiaRESUMO
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.
Assuntos
Interações Hospedeiro-Patógeno , Vírus da Influenza B/fisiologia , Influenza Humana/metabolismo , Influenza Humana/virologia , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Animais , Citotoxicidade Imunológica , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Humanos , Influenza Humana/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Camundongos , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/virologia , Ligação Proteica , Treonina/metabolismoRESUMO
Rheumatoid Arthritis (RA) causes chronic inflammation of joints. The cytokines TNFα and IFNγ are central players in RA, however their source has not been fully elucidated. Natural Killer (NK) cells are best known for their role in elimination of viral-infected and transformed cells, and they secrete pro-inflammatory cytokines. NK cells are present in the synovial fluids (SFs) of RA patients and are considered to be important in bone destruction. However, the phenotype and function of NK cells in the SFs of patients with erosive deformative RA (DRA) versus non-deformative RA (NDRA) is poorly characterized. Here we characterize the NK cell populations present in the blood and SFs of DRA and NDRA patients. We demonstrate that a distinct population of activated synovial fluid NK (sfNK) cells constitutes a large proportion of immune cells found in the SFs of DRA patients. We discovered that although sfNK cells in both DRA and NDRA patients have similar phenotypes, they function differently. The DRA sfNK secrete more TNFα and IFNγ upon exposure to IL-2 and IL-15. Consequently, we suggest that sfNK cells may be a marker for more severely destructive RA disease.
Assuntos
Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Células Matadoras Naturais/imunologia , Líquido Sinovial/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Artrite Reumatoide/sangue , Estudos de Casos e Controles , Feminino , Humanos , Interferon gama/metabolismo , Masculino , Pessoa de Meia-Idade , Fenótipo , Receptores de Células Matadoras Naturais/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Fusobacterium nucleatum (F. nucleatum) is an oral anaerobe found to be enriched in colorectal cancer (CRC). Presence of F. nucleatum in CRC has been correlated with resistance to chemotherapy and poor prognosis. We previously demonstrated that the Fap2 outer-surface protein of F. nucleatum 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 F. nucleatum 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.
RESUMO
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.
RESUMO
Expression of the stress-induced ligands MICA, MICB and ULBP 1-6 are up-regulated as a cellular response to DNA damage, excessive proliferation or viral infection; thereby, they enable recognition and annihilation by immune cells that express the powerful activating receptor NKG2D. This receptor is present not exclusively, but primarily on NK cells. Knowledge about the regulatory mechanisms controlling ULBP expression is still vague. In this study, we report a direct interaction of the oncogenic RNA binding protein (RBP) IMP3 with ULBP2 mRNA, leading to ULBP2 transcript destabilization and reduced ULBP2 surface expression in several human cell lines. We also discovered that IMP3 indirectly targets MICB with a mechanism functionally distinct from that of ULBP2. Importantly, IMP3-mediated regulation of stress-ligands leads to impaired NK cell recognition of transformed cells. Our findings shed new light on the regulation of NKG2D ligands and on the mechanism of action of a powerful oncogenic RBP, IMP3.
Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Proteínas de Ligação a RNA/metabolismo , Evasão Tumoral , Linhagem Celular Tumoral , Proteínas Ligadas por GPI/antagonistas & inibidores , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Células Matadoras Naturais/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismoRESUMO
Natural Killer (NK) cells are critical in the defense against viruses in general and against influenza in particular. We previously demonstrated that the activating NK cell receptor NKp46 is involved in the killing of influenza-virus infected cells through its interaction with viral hemagglutinin (HA). Furthermore, the recognition by NKp46 and consequent elimination of influenza infected cells were determined to be sialic-acid dependent. Here, we show that the human co-activating receptors 2B4 and NTB-A directly recognize the viral HA protein and co-stimulate killing by NK cells. We demonstrate that the 2B4/NTB-A-HA interactions require the sialylation of these receptors, and we identified the binding sites mediating these interactions. We also show that the virus counters these interactions through its neuraminidase (NA) protein. These results emphasize the critical role played by NK cells in eliminating influenza, a significant cause of worldwide morbidity and mortality.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Influenza Humana/imunologia , Células Matadoras Naturais/imunologia , Família de Moléculas de Sinalização da Ativação Linfocitária/imunologia , Linhagem Celular Tumoral , Citotoxicidade Imunológica/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1RESUMO
Natural killer (NK) cells form an important arm of the innate immune system and function to combat a wide range of invading pathogens, ranging from viruses to bacteria. However, the means by which NK cells accomplish recognition of pathogens with a limited repertoire of receptors remain largely unknown. In the current study, we describe the recognition of an emerging fungal pathogen, Candida glabrata, by the human NK cytotoxic receptor NKp46 and its mouse ortholog, NCR1. Using NCR1 knockout mice, we observed that this receptor-mediated recognition was crucial for controlling C. glabrata infection in vitro and in vivo. Finally, we delineated the fungal ligands to be the C. glabrata adhesins Epa1, Epa6, and Epa7 and demonstrated that clearance of systemic C. glabrata infections in vivo depends on their recognition by NCR1. As NKp46 and NCR1 have been previously shown to bind viral adhesion receptors, we speculate that NKp46/NCR1 may be a novel type of pattern recognition receptor.