RESUMO
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.
Assuntos
Memória Imunológica/imunologia , Células Matadoras Naturais/imunologia , Transcriptoma/imunologia , Útero/imunologia , Animais , Linhagem Celular Tumoral , Decídua/imunologia , Decídua/metabolismo , Feminino , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Células Matadoras Naturais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos SCID , Camundongos Transgênicos , Gravidez , Útero/citologia , Fator A de Crescimento do Endotélio Vascular/imunologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Monoclonal antibodies with neutralizing activity against SARS-CoV-2 have demonstrated clinical benefits in cases of mild-to-moderate SARS-CoV-2 infection, substantially reducing the risk for hospitalization and severe disease1-4. Treatment generally requires the administration of high doses of these monoclonal antibodies and has limited efficacy in preventing disease complications or mortality among hospitalized patients with COVID-195. Here we report the development and evaluation of anti-SARS-CoV-2 monoclonal antibodies with optimized Fc domains that show superior potency for prevention or treatment of COVID-19. Using several animal disease models of COVID-196,7, we demonstrate that selective engagement of activating Fcγ receptors results in improved efficacy in both preventing and treating disease-induced weight loss and mortality, significantly reducing the dose required to confer full protection against SARS-CoV-2 challenge and for treatment of pre-infected animals. Our results highlight the importance of Fcγ receptor pathways in driving antibody-mediated antiviral immunity and exclude the possibility of pathogenic or disease-enhancing effects of Fcγ receptor engagement of anti-SARS-CoV-2 antibodies upon infection. These findings have important implications for the development of Fc-engineered monoclonal antibodies with optimal Fc-effector function and improved clinical efficacy against COVID-19 disease.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Tratamento Farmacológico da COVID-19 , COVID-19/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/uso terapêutico , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Neutralizantes/uso terapêutico , Cricetinae , Modelos Animais de Doenças , Feminino , Humanos , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/farmacologia , Imunoglobulina G/química , Imunoglobulina G/imunologia , Masculino , Camundongos , Profilaxia Pré-Exposição , Receptores de IgG/química , Receptores de IgG/imunologia , Resultado do TratamentoRESUMO
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 ProteicaRESUMO
An essential mechanism for severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection begins with the viral spike protein binding to the human receptor protein angiotensin-converting enzyme II (ACE2). Here, we describe a stepwise engineering approach to generate a set of affinity optimized, enzymatically inactivated ACE2 variants that potently block SARS-CoV-2 infection of cells. These optimized receptor traps tightly bind the receptor binding domain (RBD) of the viral spike protein and prevent entry into host cells. We first computationally designed the ACE2-RBD interface using a two-stage flexible protein backbone design process that improved affinity for the RBD by up to 12-fold. These designed receptor variants were affinity matured an additional 14-fold by random mutagenesis and selection using yeast surface display. The highest-affinity variant contained seven amino acid changes and bound to the RBD 170-fold more tightly than wild-type ACE2. With the addition of the natural ACE2 collectrin domain and fusion to a human immunoglobulin crystallizable fragment (Fc) domain for increased stabilization and avidity, the most optimal ACE2 receptor traps neutralized SARS-CoV-2-pseudotyped lentivirus and authentic SARS-CoV-2 virus with half-maximal inhibitory concentrations (IC50s) in the 10- to 100-ng/mL range. Engineered ACE2 receptor traps offer a promising route to fighting infections by SARS-CoV-2 and other ACE2-using coronaviruses, with the key advantage that viral resistance would also likely impair viral entry. Moreover, such traps can be predesigned for viruses with known entry receptors for faster therapeutic response without the need for neutralizing antibodies isolated from convalescent patients.
Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/química , Desenho de Fármacos , Engenharia de Proteínas/métodos , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/genética , Antivirais/metabolismo , Sítios de Ligação , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Mutação , Biblioteca de Peptídeos , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Glicoproteína da Espícula de Coronavírus/químicaRESUMO
Neutrophils play a crucial role in immune defense against and clearance of uropathogenic Escherichia coli (UPEC)-mediated urinary tract infection, the most common bacterial infection in healthy humans. CD300a is an inhibitory receptor that binds phosphatidylserine and phosphatidylethanolamine, presented on the membranes of apoptotic cells. CD300a binding to phosphatidylserine and phosphatidylethanolamine, also known as the "eat me" signal, mediates immune tolerance to dying cells. Here, we demonstrate for the first time that CD300a plays an important role in the neutrophil-mediated immune response to UPEC-induced urinary tract infection. We show that CD300a-deficient neutrophils have impaired phagocytic abilities and despite their increased accumulation at the site of infection, they are unable to reduce bacterial burden in the bladder, which results in significant exacerbation of infection and worse host outcome. Finally, we demonstrate that UPEC's pore forming toxin α-hemolysin induces upregulation of the CD300a ligand on infected bladder epithelial cells, signaling to neutrophils to be cleared.
Assuntos
Infecções por Escherichia coli/prevenção & controle , Neutrófilos/imunologia , Receptores Imunológicos/deficiência , Receptores Imunológicos/imunologia , Infecções Urinárias/imunologia , Escherichia coli Uropatogênica/imunologia , Animais , Apoptose/imunologia , Infecções por Escherichia coli/imunologia , Proteínas de Escherichia coli/metabolismo , Feminino , Técnicas de Inativação de Genes , Proteínas Hemolisinas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fagocitose/genética , Fagocitose/imunologia , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Receptores Imunológicos/genética , Bexiga Urinária/imunologia , Bexiga Urinária/microbiologia , Bexiga Urinária/patologia , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/crescimento & desenvolvimentoRESUMO
INTRODUCTION: The World Health Organization declared that the corona virus disease (COVID 19) is a global pandemic and public health emergency, following the outbreak in Wuhan, China, in December 2019. Fever, dry cough and fatigue were the main manifestations, but the primary concern is cases that deteriorated to severe pneumonia and acute respiratory distress syndrome (ARDS). The outbreak of COVID- 19 in Israel, in March 2020 caused major changes in the routine work in hospitals. Hasharon hospital, a small community hospital in Petah Tikvah was converted to corona patients' admissions only. In accordance with this new reality, the physiotherapy service prepared itself to provide treatments tailored to the clinical needs of the Covid 19 patients and the quarantine conditions of these patients' hospitalization. During a period of 2 months, we built a unique protocol adjusted to isolated patients and staff, including telemedicine and hands-on treatment. This article summarizes our experience of physiotherapy treatment in the acute and rehabilitation period.
Assuntos
COVID-19 , Telemedicina , Humanos , Pandemias , Modalidades de Fisioterapia , SARS-CoV-2RESUMO
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
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.IMPORTANCE 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.
Assuntos
Citomegalovirus/fisiologia , Antígenos de Histocompatibilidade Classe I/genética , Evasão da Resposta Imune , Células Matadoras Naturais/imunologia , Proteínas Virais de Fusão/fisiologia , Linhagem Celular , Citomegalovirus/genética , Citomegalovirus/imunologia , Regulação para Baixo , Humanos , Ativação Linfocitária , Proteínas Virais de Fusão/genéticaRESUMO
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
Natural killer (NK) cells are capable of killing various pathogens upon stimulation of activating receptors. Human metapneumovirus (HMPV) is a respiratory virus, which was discovered in 2001 and is responsible for acute respiratory tract infection in infants and children worldwide. HMPV infection is very common, infecting around 70% of all children under the age of five. Under immune suppressive conditions, HMPV infection can be fatal. Not much is known on how NK cells respond to HMPV. In this study, using reporter assays and NK-cell cytotoxicity assays performed with human and mouse NK cells, we demonstrated that the NKp46-activating receptor and its mouse orthologue Ncr1, both members of the natural cytotoxicity receptor (NCR) family, recognized an unknown ligand expressed by HMPV-infected human cells. We demonstrated that MHC class I is upregulated and MICA is downregulated upon HMPV infection. We also characterized mouse NK-cell phenotype in the blood and the lungs of HMPV-infected mice and found that lung NK cells are more activated and expressing NKG2D, CD43, CD27, KLRG1, and CD69 compared to blood NK cells regardless of HMPV infection. Finally, we demonstrated, using Ncr1-deficient mice, that NCR1 plays a critical role in controlling HMPV infection.
Assuntos
Antígenos Ly/metabolismo , Células Matadoras Naturais/imunologia , Pulmão/imunologia , Metapneumovirus/imunologia , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Infecções por Paramyxoviridae/imunologia , Animais , Antígenos Ly/genética , Criança , Citotoxicidade Imunológica , Células HEK293 , Humanos , Lactente , Células Matadoras Naturais/virologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptor 1 Desencadeador da Citotoxicidade Natural/genética , Carga ViralRESUMO
PML-RARA and AML1-ETO are important oncogenic fusion proteins that play a central role in transformation to acute myeloid leukemia (AML). Whether these fusion proteins render the tumor cells with immune evasion properties is unknown. Here we show that both oncogenic proteins specifically downregulate the expression of CD48, a ligand of the natural killer (NK) cell activating receptor 2B4, thereby leading to decreased killing by NK cells. We demonstrate that this process is histone deacetylase (HDAC)-dependent, that it is mediated through the downregulation of CD48 messenger RNA, and that treatment with HDAC inhibitors (HDACi) restores the expression of CD48. Furthermore, by using chromatin immunoprecepitation (ChIP) experiments, we show that AML1-ETO directly interacts with CD48. Finally, we show that AML patients who are carrying these specific translocations have low expression of CD48.
Assuntos
Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/imunologia , Evasão Tumoral/genética , Evasão Tumoral/imunologia , Antígenos CD/química , Antígenos CD/genética , Antígenos CD/metabolismo , Sequência de Bases , Antígeno CD48 , Linhagem Celular Tumoral , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/imunologia , Citotoxicidade Imunológica , Expressão Gênica , Regulação da Expressão Gênica , Histona Desacetilases/metabolismo , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Leucemia Mieloide Aguda/metabolismo , Dados de Sequência Molecular , Proteínas de Fusão Oncogênica/metabolismo , Proteína 1 Parceira de Translocação de RUNX1RESUMO
NK cells are innate immune lymphocytes that express a vast repertoire of germ-line encoded receptors for target recognition. These receptors include inhibitory and activating proteins, among the latter of which is CD16, a low affinity binding Fc receptor. Here, we show that human NK cells expand in response to stimulation with various tumor cell lines. We further demonstrate that the tumor-derived expansion of NK cells is accompanied by rapid, cell-dependent, changes in CD16 expression levels. We show that in NK cells expanded in response to the EBV-transformed cell line 721.221, CD16 is shed and therefore approximately half of the expanded 721.221-derived NK-cell population does not express CD16. We also show, in contrast, that in response to 1106mel cells, CD16 expression is maintained on the cell surface of the expanded NK cells due to an antibody-dependent mechanism. Our results may provide a basis for the selective expansion of NK cells that may be used for tumor immunotherapy.
Assuntos
Anticorpos Antineoplásicos/imunologia , Regulação da Expressão Gênica/imunologia , Neoplasias/imunologia , Receptores de IgG/imunologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Técnicas de Cocultura , Feminino , Proteínas Ligadas por GPI/biossíntese , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Regulação da Expressão Gênica/genética , Humanos , Imunoterapia , Células Matadoras Naturais , Masculino , Neoplasias/genética , Neoplasias/metabolismo , Receptores de IgG/biossíntese , Receptores de IgG/genéticaRESUMO
Natural killer (NK) cells are innate immune cells able to rapidly kill virus-infected and tumor cells. Two NK cell populations are found in the blood; the majority (90%) expresses the CD16 receptor and also express the CD56 protein in intermediate levels (CD56(Dim) CD16(Pos)) while the remaining 10% are CD16 negative and express CD56 in high levels (CD56(Bright) CD16(Neg)). NK cells also reside in some tissues and traffic to various infected organs through the usage of different chemokines and chemokine receptors. Kaposi's sarcoma-associated herpesvirus (KSHV) is a human virus that has developed numerous sophisticated and versatile strategies to escape the attack of immune cells such as NK cells. Here, we investigate whether the KSHV derived cytokine (vIL-6) and chemokines (vMIP-I, vMIP-II, vMIP-III) affect NK cell activity. Using transwell migration assays, KSHV infected cells, as well as fusion and recombinant proteins, we show that out of the four cytokine/chemokines encoded by KSHV, vMIP-II is the only one that binds to the majority of NK cells, affecting their migration. We demonstrate that vMIP-II binds to two different receptors, CX3CR1 and CCR5, expressed by naïve CD56(Dim) CD16(Pos) NK cells and activated NK cells, respectively. Furthermore, we show that the binding of vMIP-II to CX3CR1 and CCR5 blocks the binding of the natural ligands of these receptors, Fractalkine (Fck) and RANTES, respectively. Finally, we show that vMIP-II inhibits the migration of naïve and activated NK cells towards Fck and RANTES. Thus, we present here a novel mechanism in which KSHV uses a unique protein that antagonizes the activity of two distinct chemokine receptors to inhibit the migration of naïve and activated NK cells.
Assuntos
Fármacos Anti-HIV/farmacologia , Antagonistas dos Receptores CCR5 , Movimento Celular/efeitos dos fármacos , Quimiocinas/farmacologia , Herpesvirus Humano 8/química , Células Matadoras Naturais/efeitos dos fármacos , Receptores de Quimiocinas/antagonistas & inibidores , Receptor 1 de Quimiocina CX3C , Células Cultivadas , Quimiocina CCL5/metabolismo , Quimiocina CX3CL1/metabolismo , Citocinas/genética , Citocinas/metabolismo , Humanos , Immunoblotting , Interleucina-6 , Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Reação em Cadeia da Polimerase , Receptores CCR5/genética , Receptores CCR5/metabolismo , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismoRESUMO
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 SuperfícieRESUMO
Immunoglobulin G (IgG) antibodies contain a single, complex N -glycan on each IgG heavy chain protomer embedded in the hydrophobic pocket between its Cγ2 domains. The presence of this glycan contributes to the structural organization of the Fc domain and determines its specificity for Fcγ receptors, thereby determining distinct cellular responses. On the Fc, the variable construction of this glycan structure leads to a family of highly-related, but non-equivalent glycoproteins known as glycoforms. We previously reported the development of synthetic nanobodies that distinguish IgG glycoforms without cross-reactivity to off-target glycoproteins or free glycans. Here, we present the X-ray crystal structure of one such nanobody, X0, in complex with its specific binding partner, the Fc fragment of afucosylated IgG1. Two X0 nanobodies bind a single afucosylated Fc homodimer at the upper Cγ2 domain, making both protein-protein and protein-carbohydrate contacts and overlapping the binding site for Fcγ receptors. Upon binding, the elongated CDR3 loop of X0 undergoes a conformational shift to access the buried N -glycan and acts as a 'glycan sensor', forming hydrogen bonds with the afucosylated IgG N -glycan that would otherwise be sterically hindered by the presence of a core fucose residue. Based on this structure, we designed X0 fusion constructs that disrupt pathogenic afucosylated IgG1-FcγRIIIa interactions and rescue mice in a model of dengue virus infection.
RESUMO
Immunoglobulin G (IgG) antibodies contain a complex N-glycan embedded in the hydrophobic pocket between its heavy chain protomers. This glycan contributes to the structural organization of the Fc domain and determines its specificity for Fcγ receptors, thereby dictating distinct cellular responses. The variable construction of this glycan structure leads to highly-related, but non-equivalent glycoproteins known as glycoforms. We previously reported synthetic nanobodies that distinguish IgG glycoforms. Here, we present the structure of one such nanobody, X0, in complex with the Fc fragment of afucosylated IgG1. Upon binding, the elongated CDR3 loop of X0 undergoes a conformational shift to access the buried N-glycan and acts as a 'glycan sensor', forming hydrogen bonds with the afucosylated IgG N-glycan that would otherwise be sterically hindered by the presence of a core fucose residue. Based on this structure, we designed X0 fusion constructs that disrupt pathogenic afucosylated IgG1-FcγRIIIa interactions and rescue mice in a model of dengue virus infection.
Assuntos
Imunoglobulina G , Receptores de IgG , Animais , Camundongos , Glicosilação , Receptores de IgG/metabolismo , Fragmentos Fc das Imunoglobulinas/metabolismo , Polissacarídeos/químicaRESUMO
Although dengue virus (DENV) infection typically causes asymptomatic disease, DENV-infected patients can experience severe complications. A risk factor for symptomatic disease is pre-existing anti-DENV IgG antibodies. Cellular assays suggested that these antibodies can enhance viral infection of Fcγ receptor (FcγR)-expressing myeloid cells. Recent studies, however, revealed more complex interactions between anti-DENV antibodies and specific FcγRs by demonstrating that modulation of the IgG Fc glycan correlates with disease severity. To investigate the in vivo mechanisms of antibody-mediated dengue pathogenesis, we developed a mouse model for dengue disease that recapitulates the unique complexity of human FcγRs. In in vivo mouse models of dengue disease, we discovered that the pathogenic activity of anti-DENV antibodies is exclusively mediated through engagement of FcγRIIIa on splenic macrophages, resulting in inflammatory sequelae and mortality. These findings highlight the importance of IgG-FcγRIIIa interactions in dengue, with important implications for the design of safer vaccination approaches and effective therapeutic strategies.
Assuntos
Vírus da Dengue , Dengue , Humanos , Animais , Camundongos , Receptores de IgG , Macrófagos , Imunoglobulina GRESUMO
Monoclonal antibodies (mAbs) with neutralizing activity against SARS-CoV-2 have demonstrated clinical benefit in cases of mild to moderate SARS-CoV-2 infection, substantially reducing the risk for hospitalization and severe disease1-4. Treatment generally requires the administration of high doses of these mAbs with limited efficacy in preventing disease complications or mortality among hospitalized COVID-19 patients5. Here we report the development and evaluation of Fc-optimized anti-SARS-CoV-2 mAbs with superior potency to prevent or treat COVID-19 disease. In several animal models of COVID-19 disease6,7, we demonstrate that selective engagement of activating FcγRs results in improved efficacy in both preventing and treating disease-induced weight loss and mortality, significantly reducing the dose required to confer full protection upon SARS-CoV-2 challenge and treatment of pre-infected animals. Our results highlight the importance of FcγR pathways in driving antibody-mediated antiviral immunity, while excluding any pathogenic or disease-enhancing effects of FcγR engagement of anti-SARS-CoV-2 antibodies upon infection. These findings have important implications for the development of Fc-engineered mAbs with optimal Fc effector function and improved clinical efficacy against COVID-19 disease.
RESUMO
An essential mechanism for SARS-CoV-1 and -2 infection begins with the viral spike protein binding to the human receptor protein angiotensin-converting enzyme II (ACE2). Here we describe a stepwise engineering approach to generate a set of affinity optimized, enzymatically inactivated ACE2 variants that potently block SARS-CoV-2 infection of cells. These optimized receptor traps tightly bind the receptor binding domain (RBD) of the viral spike protein and prevent entry into host cells. We first computationally designed the ACE2-RBD interface using a two-stage flexible protein backbone design process that improved affinity for the RBD by up to 12-fold. These designed receptor variants were affinity matured an additional 14-fold by random mutagenesis and selection using yeast surface display. The highest affinity variant contained seven amino acid changes and bound to the RBD 170-fold more tightly than wild-type ACE2. With the addition of the natural ACE2 collectrin domain and fusion to a human Fc domain for increased stabilization and avidity, the most optimal ACE2 receptor traps neutralized SARS-CoV-2 pseudotyped lentivirus and authentic SARS-CoV-2 virus with half-maximal inhibitory concentrations (IC50) in the 10-100 ng/ml range. Engineered ACE2 receptor traps offer a promising route to fighting infections by SARS-CoV-2 and other ACE2-utilizing coronaviruses, with the key advantage that viral resistance would also likely impair viral entry. Moreover, such traps can be predesigned for viruses with known entry receptors for faster therapeutic response without the need for neutralizing antibodies isolated or generated from convalescent patients.
RESUMO
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.