Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 58
Filtrar
Mais filtros

Base de dados
País/Região como assunto
Tipo de documento
Intervalo de ano de publicação
1.
Artigo em Inglês | MEDLINE | ID: mdl-38992472

RESUMO

BACKGROUND: The invariant TCRζ/CD247 homodimer is crucial for TCR/CD3 expression and signaling through its three immunoreceptor tyrosine-based activation motifs (ITAMs). Homozygous null mutations in CD247 lead to immunodeficiency, while carriers exhibit 50% reduced surface CD3. It is unclear whether carriers of other CD247 variants show dominant-negative effects. OBJECTIVE: To analyze and model the potential impact on TCR expression and function of heterozygous nonsense CD247 mutations found in patients with signs of immunodeficiency or autoimmunity. METHODS: Jurkat T cells, either wild-type (WT) or CRISPR/Cas9-edited CD247-deficient (ZKO), were lentivirally transduced with wild-type CD247 or mutations ablating one (Q142X), two (Q101X), or three (Q70X) ITAMs. RESULTS: Three patients from unrelated families were studied. Two heterozygous nonsense CD247 mutations were identified (p.Y152X and p.Q101X), which affected ITAM-3 and ITAM-2+3, respectively. Both mutations were associated with low surface CD3 expression, normal intracellular CD247 levels using a transmembrane-specific antibody but very low intracellular CD247 levels using an ITAM-3-specific one, suggesting the presence of truncated variants in T cells. Transduction of the mutations lacking 1, 2, or 3 ITAMs into ZKO could not restore normal surface CD3 expression (only 60%, 22% and 10%, respectively), whereas in WT they reduced it (to 39%, 19% and 9% of normal levels), and both effects were ITAM number dependent. All six transfectants showed reduced CD69 induction (25-50%), indicating that they were unable to signal downstream properly neither isolated nor associated with wild-type CD247. CONCLUSION: Our results suggest that CD247 variants lacking ITAMs due to nonsense, but not null, mutations are defective for normal TCR assembly and exert a dominant-negative effect on TCR expression and signaling in vitro. This, in turn, may correlate with clinical features in vivo.

2.
Immunology ; 172(3): 440-450, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38514903

RESUMO

Analysis of genetically defined immunodeficient patients allows study of the effect of the absence of specific proteins on human immune function in real-world conditions. Here we have addressed the importance of type I interferon signalling for human NK cell development by studying the phenotype and function of circulating NK cells isolated from patients suffering primary immunodeficiency disease due to mutation of either the human interferon regulatory factor 9 (IRF9) or the signal transducer and activator of transcription 2 (STAT2) genes. IRF9, together with phosphorylated STAT1 and STAT2, form a heterotrimer called interferon stimulated gene factor 3 (ISGF3) which promotes the expression of hundreds of IFN-stimulated genes that mediate antiviral function triggered by exposure to type I interferons. IRF9- and STAT2-deficient patients are unable to respond efficiently to stimulation by type I interferons and so our experiments provide insights into the importance of type I interferon signalling and the consequences of its impairment on human NK cell biology. Surprisingly, the NK cells of these patients display essentially normal phenotype and function.


Assuntos
Interferon Tipo I , Fator Gênico 3 Estimulado por Interferon, Subunidade gama , Células Matadoras Naturais , Fator de Transcrição STAT2 , Transdução de Sinais , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Fator de Transcrição STAT2/metabolismo , Fator de Transcrição STAT2/genética , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Interferon Tipo I/metabolismo , Mutação , Diferenciação Celular , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT1/genética , Células Cultivadas
3.
J Med Virol ; 95(7): e28900, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37403730

RESUMO

Antibodies triggering Fc-mediated NK cell activity may contribute to protection against disease caused by SARS-CoV-2 infection in humans. However, how these Fc-mediated humoral responses compare between individuals displaying hybrid immunity (Vac-ex) and those fully vaccinated with no history of SARS-CoV-2 infection (Vac-n) and whether they correlate with neutralizing antibody (NtAb) responses remains largely undetermined. In this retrospective study serum samples from 50 individuals (median age, 44.5 years; range, 11-85; 25 males), 25 Vac-ex and 25 Vac-n were studied. A flow-cytometry-based antibody-mediated NK-cell activation assay was used to quantitate effector NK-cells stimulated to express LAMP1 (lysosomal associated membrane protein 1), MIP1 (Macrophage inflammatory protein 1), and interferon-γ (IFNγ); NK cells isolated from two donors (D1 and D2) were used. NtAb levels targeting the Spike protein of Wuhan-Hu-1 and Omicron BA.1 SARS-CoV-2 variants were quantitated using a SARS-CoV-2 S pseudotyped neutralization assay. Regardless of the SARS-CoV-2 variant S antigen used in the NK-cell activation assay, the frequency of NK cells stimulated to express LAMP-1, MIP1ß, and IFNγ was higher in Vac-ex compared with Vac-n (p values ranging from 0.07 to 0.006) for D1; this was only seen for BA.1 when NK cells from D2 were employed. The frequency of functional NK cells activated by antibody binding to either Wuhan-Hu-1 or Omicron BA.1 S protein was not significantly different for both VAC-ex and VAC-n. In contrast, NtAb titers against BA.1 were around 10-fold lower than that against Wuhan-Hu-1. Vac-ex displayed higher NtAb titers against both (sub)variants than Vac-n. NK-cell responses correlated poorly with NtAb titers (ρ ≤ 0.30). The data demonstrate higher cross-reactivity across variants of concern for antibodies triggering Fc-mediated NK cell than for NtAb. Moreover, Vac-Ex seemed to display more robust functional antibody responses as compared with Vac-n.


Assuntos
Antígenos de Grupos Sanguíneos , COVID-19 , Masculino , Humanos , Adulto , SARS-CoV-2/genética , Anticorpos Neutralizantes , Formação de Anticorpos , Estudos Retrospectivos , Glicoproteína da Espícula de Coronavírus/genética , COVID-19/prevenção & controle , Células Matadoras Naturais , Interferon gama , Anticorpos Antivirais
4.
J Allergy Clin Immunol ; 150(4): 955-964.e16, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35182547

RESUMO

BACKGROUND: Inflammatory phenomena such as hyperinflammation or hemophagocytic lymphohistiocytosis are a frequent yet paradoxical accompaniment to virus susceptibility in patients with impairment of type I interferon (IFN-I) signaling caused by deficiency of signal transducer and activator of transcription 2 (STAT2) or IFN regulatory factor 9 (IRF9). OBJECTIVE: We hypothesized that altered and/or prolonged IFN-I signaling contributes to inflammatory complications in these patients. METHODS: We explored the signaling kinetics and residual transcriptional responses of IFN-stimulated primary cells from individuals with complete loss of one of STAT1, STAT2, or IRF9 as well as gene-edited induced pluripotent stem cell-derived macrophages. RESULTS: Deficiency of any IFN-stimulated gene factor 3 component suppressed but did not abrogate IFN-I receptor signaling, which was abnormally prolonged, in keeping with insufficient induction of negative regulators such as ubiquitin-specific peptidase 18 (USP18). In cells lacking either STAT2 or IRF9, this late transcriptional response to IFN-α2b mimicked the effect of IFN-γ. CONCLUSION: Our data suggest a model wherein the failure of negative feedback of IFN-I signaling in STAT2 and IRF9 deficiency leads to immune dysregulation. Aberrant IFN-α receptor signaling in STAT2- and IRF9-deficient cells switches the transcriptional output to a prolonged, IFN-γ-like response and likely contributes to clinically overt inflammation in these individuals.


Assuntos
Interferon Tipo I , Fator IX , Humanos , Interferon Tipo I/metabolismo , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Interferon-alfa , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT2/genética , Ubiquitina Tiolesterase , Proteases Específicas de Ubiquitina
5.
Eur J Immunol ; 51(11): 2633-2640, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34358329

RESUMO

Here, we describe a new, simple, highly multiplexed serological test that generates a more complete picture of seroconversion than single antigen-based assays. Flow cytometry is used to detect multiple Ig isotypes binding to four SARS-CoV-2 antigens: the Spike glycoprotein, its RBD fragment (the main target for neutralizing antibodies), the nucleocapsid protein, and the main cysteine-like protease in a single reaction. Until now, most diagnostic serological tests measured antibodies to only one antigen and in some laboratory-confirmed patients no SARS-CoV-2-specific antibodies could be detected. Our data reveal that while most patients respond against all the viral antigens tested, others show a marked bias to make antibodies against either proteins exposed on the viral particle or those released after cellular infection. With this assay, it was possible to discriminate between patients and healthy controls with 100% confidence. Analysing the response of multiple Ig isotypes to the four antigens in combination may also help to establish a correlation with the severity degree of disease. A more detailed description of the immune responses of different patients to SARS-CoV-2 virus might provide insight into the wide array of clinical presentations of COVID-19.


Assuntos
Anticorpos Antivirais/sangue , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Citometria de Fluxo/métodos , Antígenos Virais/imunologia , COVID-19/imunologia , Ensaios de Triagem em Larga Escala , Humanos , SARS-CoV-2 , Sensibilidade e Especificidade , Testes Sorológicos
6.
Eur J Immunol ; 51(3): 634-647, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33251605

RESUMO

SARS-CoV-2 infection causes an abrupt response by the host immune system, which is largely responsible for the outcome of COVID-19. We investigated whether the specific immune responses in the peripheral blood of 276 patients were associated with the severity and progression of COVID-19. At admission, dramatic lymphopenia of T, B, and NK cells is associated with severity. Conversely, the proportion of B cells, plasmablasts, circulating follicular helper T cells (cTfh) and CD56- CD16+ NK-cells increased. Regarding humoral immunity, levels of IgM, IgA, and IgG were unaffected, but when degrees of severity were considered, IgG was lower in severe patients. Compared to healthy donors, complement C3 and C4 protein levels were higher in mild and moderate, but not in severe patients, while the activation peptide of C5 (C5a) increased from the admission in every patient, regardless of their severity. Moreover, total IgG, the IgG1 and IgG3 isotypes, and C4 decreased from day 0 to day 10 in patients who were hospitalized for more than two weeks, but not in patients who were discharged earlier. Our study provides important clues to understand the immune response observed in COVID-19 patients, associating severity with an imbalanced humoral response, and identifying new targets for therapeutic intervention.


Assuntos
Linfócitos B/imunologia , COVID-19/patologia , Imunoglobulinas/sangue , Células Matadoras Naturais/imunologia , SARS-CoV-2/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Idoso , COVID-19/imunologia , Complemento C3/análise , Complemento C4/análise , Complemento C5/análise , Feminino , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Contagem de Linfócitos , Linfopenia/imunologia , Masculino , Pessoa de Meia-Idade , Síndrome do Desconforto Respiratório/imunologia , Síndrome do Desconforto Respiratório/patologia
7.
J Immunol ; 205(11): 3130-3140, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33148714

RESUMO

Currently, there is a need for reliable tests that allow identification of individuals that have been infected with SARS-CoV-2 even if the infection was asymptomatic. To date, the vast majority of the serological tests for SARS-CoV-2-specific Abs are based on serum detection of Abs to either the viral spike glycoprotein (the major target for neutralizing Abs) or the viral nucleocapsid protein that is known to be highly immunogenic in other coronaviruses. Conceivably, exposure of Ags released from infected cells could stimulate Ab responses that might correlate with tissue damage and, hence, they may have some value as a prognostic indicator. We addressed whether other nonstructural viral proteins, not incorporated into the infectious viral particle, specifically the viral cysteine-like protease, might also be potent immunogens. Using ELISA tests, coating several SARS-CoV-2 proteins produced in vitro, we describe that COVID-19 patients make high titer IgG, IgM, and IgA Ab responses to the Cys-like protease from SARS-CoV-2, also known as 3CLpro or Mpro, and it can be used to identify individuals with positive serology against the coronavirus. Higher Ab titers in these assays associated with more-severe disease, and no cross-reactive Abs against prior betacoronavirus were found. Remarkably, IgG Abs specific for Mpro and other SARS-CoV-2 Ags can also be detected in saliva. In conclusion, Mpro is a potent Ag in infected patients that can be used in serological tests, and its detection in saliva could be the basis for a rapid, noninvasive test for COVID-19 seropositivity.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/metabolismo , Infecções por Coronavirus/sangue , Cisteína Proteases/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Pneumonia Viral/sangue , Saliva/metabolismo , Adulto , Idoso , COVID-19 , Feminino , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , SARS-CoV-2
8.
Proc Natl Acad Sci U S A ; 114(28): E5645-E5654, 2017 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-28652325

RESUMO

Many activating immunoreceptors associate with signaling adaptor molecules like FcεR1γ or CD247. FcεR1γ and CD247 share high sequence homology and form disulphide-linked homodimers that contain a pair of acidic aspartic acid residues in their transmembrane (TM) domains that mediate assembly, via interaction with an arginine residue at a similar register to these aspartic acids, with the activating immunoreceptors. However, this model cannot hold true for receptors like CD16A, whose TM domains do not contain basic residues. We have carried out an extensive site-directed mutagenesis analysis of the CD16A receptor complex and now report that the association of receptor with the signaling adaptor depends on a network of polar and aromatic residues along the length of the TM domain. Molecular modeling indicates that CD16A TM residues F202, D205, and T206 form the core of the membrane-embedded trimeric interface by establishing highly favorable contacts to the signaling modules through rearrangement of a hydrogen bond network previously identified in the CD247 TM dimer solution NMR structure. Strikingly, the amino acid D205 also regulates the turnover and surface expression of CD16A in the absence of FcεR1γ or CD247. Modeling studies indicate that similar features underlie the association of other activating immune receptors, including CD64 and FcεR1α, with signaling adaptor molecules, and we confirm experimentally that equivalent F, D, and T residues in the TM domain of FcεR1α markedly influence the biology of this receptor and its association with FcεR1γ.


Assuntos
Complexo CD3/metabolismo , Membrana Celular/metabolismo , Receptores de IgG/metabolismo , Motivos de Aminoácidos , Animais , Linhagem Celular , Proteínas Ligadas por GPI/metabolismo , Glicosilação , Células HEK293 , Humanos , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Camundongos , Mutagênese Sítio-Dirigida , Domínios Proteicos , Multimerização Proteica , Receptores de IgE/metabolismo , Receptores Imunológicos/metabolismo , Transdução de Sinais
9.
Blood ; 130(10): 1205-1208, 2017 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-28743717

RESUMO

Mutations in T-cell antigen receptor (TCR) subunit genes cause rare immunodeficiency diseases characterized by impaired expression of the TCR at the cell surface and selective T lymphopenia. Here, detailed analyses of spontaneously arising somatic mutations that recover CD247, and thus TCR expression, in a newly identified CD247-deficient patient are described. The recovery of CD247 expression in some patient T cells was associated with both reversion of the inactivating mutation and a variant with a compensating mutation that could reconstitute TCR expression, but not as efficiently as wild-type CD247. Multiple mutations were found in CD247 complementary DNAs (cDNAs) cloned from the patient as well as in cDNA and genomic DNA from other individuals, suggesting that genetic variation in this gene is frequent. Analyses of other genes mutated in primary immunodeficiency diseases (PIDs) where reversions have been described also revealed a higher rate of mutation than that observed for genes mutated in PIDs where revertants have not been identified or control genes. These data support the hypothesis that the occurrence of somatic mutations that may reconstitute genetic defects in PID is related to an increased propensity of those genes to mutate.


Assuntos
Complexo CD3/genética , Reparo do DNA/genética , Regulação da Expressão Gênica , Síndromes de Imunodeficiência/genética , Humanos , Leucócitos Mononucleares/metabolismo , Mutação/genética , Probabilidade
10.
J Biol Chem ; 292(50): 20472-20480, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-28986447

RESUMO

Self/non-self-discrimination by the innate immune system relies on germline-encoded, non-rearranging receptors expressed by innate immune cells recognizing conserved pathogen-associated molecular patterns. The natural killer group 2D (NKG2D) receptor is a potent immune-activating receptor that binds human genome-encoded ligands, whose expression is negligible in normal tissues, but increased in stress and disease conditions for reasons that are incompletely understood. Here it is not clear how the immune system reconciles receptor binding of self-proteins with self/non-self-discrimination to avoid autoreactivity. We now report that increased expression of NKG2D ligands after virus infection depends on interferon response factors activated by the detection of viral double-stranded RNA by pattern-recognition receptors (RIG-I/MDA-5) and that NKG2D ligand up-regulation can be blocked by the expression of viral dsRNA-binding proteins. Thus, innate immunity-mediated recognition of viral nucleic acids triggers the infected cell to release interferon for NK cell recruitment and to express NKG2D ligands to become more visible to the immune system. Finally, the observation that NKG2D-ligand induction is a consequence of signaling by pattern-recognition receptors that have been selected over evolutionary time to be highly pathogen-specific explains how the risks of autoreactivity in this system are minimized.


Assuntos
Regulação da Expressão Gênica , Imunidade Inata , Células Matadoras Naturais/metabolismo , Lentivirus/fisiologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/agonistas , RNA Viral/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular , Células Cultivadas , Cricetinae , Proteína DEAD-box 58/química , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismo , Regulação Viral da Expressão Gênica , Genes Reporter , Humanos , Helicase IFIH1 Induzida por Interferon/química , Helicase IFIH1 Induzida por Interferon/genética , Helicase IFIH1 Induzida por Interferon/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/virologia , Lentivirus/imunologia , Ligantes , Mutação , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptores Imunológicos , Proteínas Recombinantes/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
11.
J Immunol ; 195(4): 1676-84, 2015 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-26179905

RESUMO

HSV-1 latently infects most humans, causing a variable clinical picture that depends, in part, on host genetic factors. Both IgG and its cellular FcRs, CD16A and CD32A-C (encoded by FCGR3A and FCGR2A-C, respectively, on chromosome 1), display polymorphisms that could affect their defensive function. Of potential relevance are a FCGR3A dimorphism resulting in CD16A-valine/phenylalanine-158 allotypes with different IgG affinity, variations conditioning NK cell expression of CD32B or CD32C, and IgG1 H chain (IGHG1) and kappa-chain (IGKC) polymorphisms determining allotypes designated G1m and Km. In this study, we assessed the contribution of Ig genetic variations and their interaction with FcR polymorphism to HSV-1 susceptibility, as well as their impact on NK cell-mediated Ab-dependent cellular cytotoxicity (ADCC). Our results show an epistatic interaction between IGHG1 and FCGR3A such that the higher affinity CD16A-158V/V genotype associates with an asymptomatic course of HSV-1 infection only in homozygotes for G1m3. Furthermore, CD16A-158V and G1m3 allotypes enhanced ADCC against opsonized HSV-1-infected fibroblasts. Conversely, Km allotypes and CD32B or CD32C expression on NK cells did not significantly influence HSV-1 susceptibility or ADCC. NK cells degranulating against immune serum-opsonized HSV-1-infected fibroblasts had heterogeneous phenotypes. Yet, enhanced ADCC was observed among NK cells showing a differentiated, memory-like phenotype (NKG2C(bright)NKG2A(-)CD57(+)FcRγ(-)), which expand in response to human CMV. These results extend our knowledge on the importance of immunogenetic polymorphisms and NK cell-Ab interplay in the host response against HSV-1 and point to the relevance of interactions between immune responses elicited during chronic coinfection by multiple herpesviruses.


Assuntos
Herpes Simples/imunologia , Herpesvirus Humano 1/imunologia , Imunoglobulina G/imunologia , Células Matadoras Naturais/imunologia , Animais , Citotoxicidade Celular Dependente de Anticorpos/genética , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Degranulação Celular/genética , Degranulação Celular/imunologia , Linhagem Celular , Suscetibilidade a Doenças , Epistasia Genética , Expressão Gênica , Variação Genética , Genótipo , Herpes Simples/genética , Humanos , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias kappa de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/imunologia , Imunofenotipagem , Fenótipo , Polimorfismo Genético , Receptores de IgG/genética , Receptores de IgG/metabolismo , Ativação Viral/imunologia
12.
Immunol Cell Biol ; 94(5): 479-85, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26732147

RESUMO

The expression of NKG2D ligands (NKG2D-L) flag stressed cells for immune recognition and destruction. A precise control of the cell surface expression of these proteins is therefore required to ensure an appropriate immune response and it is becoming clear that NKG2D ligand expression is regulated at multiple levels. We now report that the surface stability of the human glycosyl-phosphatidyl-inositol (GPI)-anchored ligand ULBP1 (UL16-binding protein) at the plasma membrane is lower than other ULBP molecules. This difference in stability is due neither to shedding nor to a higher internalization rate of ULBP1 but rather occurs because of a rapid degradation of ULBP1 protein after internalization from the cell surface that is blocked by proteasome inhibition. These data indicate that, in addition to the known transcriptional and post-translational mechanisms, surface expression of human NKG2D-L is also regulated by protein turnover and that the brief residence of ULBP1 could contribute to the fine tuning of immune responses.


Assuntos
Membrana Celular/metabolismo , Endocitose , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Animais , Células CHO , Membrana Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Endocitose/efeitos dos fármacos , Glicosilfosfatidilinositóis/metabolismo , Meia-Vida , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Inibidores de Proteassoma/farmacologia , Estabilidade Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos
13.
J Immunol ; 193(3): 1344-52, 2014 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-24973455

RESUMO

Proteolytic shedding of ligands for the NK group 2D (NKG2D) receptor is a strategy used by tumors to modulate immune recognition by NK cells and cytotoxic T cells. A number of metalloproteases, especially those of the A disintegrin and metalloprotease (ADAM) family, can mediate NKG2D ligand cleavage and this process can be modulated by expression of the thiol isomerase ERp5. In this article, we describe that an increased shedding of the NKG2D ligand MICA is observed postinfection with several strains of human CMV due to an enhanced activity of ADAM17 (TNF-α converting enzyme) and matrix metalloprotease 14 caused by a reduction in the expression of the endogenous inhibitor of metalloproteases tissue inhibitors of metalloproteinase 3 (TIMP3). This decrease in TIMP3 expression correlates with increased expression of a cellular miRNA known to target TIMP3, and we also identify a human CMV-encoded microRNA able to modulate TIMP3 expression. These observations characterize a novel viral strategy to influence the shedding of cell-surface molecules involved in immune response modulation. They also provide an explanation for previous reports of increased levels of various ADAM17 substrates in the serum from patients with CMV disease. Consistent with this hypothesis, we detected soluble MICA in serum of transplant recipients with CMV disease. Finally, these data suggest that it might be worthwhile to prospectively study ADAM17 activity in a larger group of patients to assay whether this might be a useful biomarker to identify patients at risk for development of CMV disease.


Assuntos
Infecções por Citomegalovirus/imunologia , Regulação para Baixo/imunologia , Regulação Viral da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , MicroRNAs/genética , Inibidor Tecidual de Metaloproteinase-3/antagonistas & inibidores , Inibidor Tecidual de Metaloproteinase-3/biossíntese , Linhagem Celular Tumoral , Citomegalovirus/genética , Citomegalovirus/imunologia , Infecções por Citomegalovirus/enzimologia , Infecções por Citomegalovirus/genética , Regulação para Baixo/genética , Antígenos de Histocompatibilidade Classe I/sangue , Humanos , Metaloproteinase 14 da Matriz/sangue , Metaloproteinase 14 da Matriz/metabolismo , MicroRNAs/biossíntese , Cultura Primária de Células , Especificidade por Substrato/genética , Especificidade por Substrato/imunologia , Inibidor Tecidual de Metaloproteinase-3/sangue , Regulação para Cima/genética , Regulação para Cima/imunologia
15.
Immunology ; 146(1): 70-80, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25980678

RESUMO

After immune interactions, membrane fragments can be transferred between cells. This fast transfer of molecules is transient and shows selectivity for certain proteins; however, the constraints underlying acquisition of a protein are unknown. To characterize the mechanism and functional consequences of this process in natural killer (NK) cells, we have compared the transfer of different NKG2D ligands. We show that human NKG2D ligands can be acquired by NK cells with different efficiencies. The main findings are that NKG2D ligand transfer is related to immune activation and receptor-ligand interaction and that NK cells acquire these proteins during interactions with target cells that lead to degranulation. Our results further demonstrate that NK cells that have acquired NKG2D ligands can stimulate activation of autologous NK cells. Surprisingly, NK cells can also re-transfer the acquired molecule to autologous effector cells during this immune recognition that leads to their death. These data demonstrate that transfer of molecules occurs as a consequence of immune recognition and imply that this process might play a role in homeostatic tuning-down of the immune response or be used as marker of interaction.


Assuntos
Degranulação Celular/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células Matadoras Naturais/imunologia , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Citotoxicidade Imunológica/imunologia , Proteínas Ligadas por GPI/metabolismo , Glicosilfosfatidilinositóis , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Transporte Proteico , Receptores de Células Matadoras Naturais/imunologia
17.
Biochem J ; 454(2): 295-302, 2013 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-23772752

RESUMO

The human MICA (MHC I-related chain A) gene, encoding a ligand for the NKG2D (NKG2-D type II integral membrane protein) receptor, is highly polymorphic. A group of MICA alleles, named MICA 5.1 (prototype, MICA*008), produce a truncated protein due to a nucleotide insertion in the transmembrane domain. These alleles are very frequent in all of the human populations studied and they have different biological properties, compared with full-length alleles, e.g. recruitment into exosomes, which makes them very potent for down-modulating the NKG2D receptor in effector immune cells. Moreover, MICA*008 is not affected by viral immune evasion mechanisms that target other MICA alleles. In the present study, we demonstrate that MICA*008 acquires a GPI (glycosylphosphatidylinositol) anchor and that this modification is responsible for many of the distinct biological features of the truncated MICA alleles, including recruitment of the protein to exosomes. MICA*008 processing is also unusual as it is observed in the endoplasmic reticulum as a Triton™ X-114 soluble protein, partially undergoing GPI modification while the rest is exocytosed, suggesting a new model for MICA*008 release. This is the first report of a GPI-anchored MICA allele. The finding that this modification occurs in both families of human NKG2D ligands, as well as in the murine system, suggests positive pressure to maintain this biochemical feature.


Assuntos
Proteínas Ligadas por GPI/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Polimorfismo Genético , Alelos , Animais , Células CHO , Cricetinae , Cricetulus , Retículo Endoplasmático/metabolismo , Exossomos/metabolismo , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Glicosilfosfatidilinositóis/análise , Células HEK293 , Células HeLa , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Ligantes , Mutagênese Insercional , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidade
19.
Front Immunol ; 15: 1273942, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38410511

RESUMO

Introduction: It is now clear that coronavirus disease 19 (COVID-19) severity is associated with a dysregulated immune response, but the relative contributions of different immune cells is still not fully understood. SARS CoV-2 infection triggers marked changes in NK cell populations, but there are contradictory reports as to whether these effector lymphocytes play a protective or pathogenic role in immunity to SARS-CoV-2. Methods: To address this question we have analysed differences in the phenotype and function of NK cells in SARS-CoV-2 infected individuals who developed either very mild, or life-threatening COVID-19 disease. Results: Although NK cells from patients with severe disease appeared more activated and the frequency of adaptive NK cells was increased, they were less potent mediators of ADCC than NK cells from patients with mild disease. Further analysis of peripheral blood NK cells in these patients revealed that a population of NK cells that had lost expression of the activating receptor NKG2D were a feature of patients with severe disease and this correlated with elevated levels of cell free NKG2D ligands, especially ULBP2 and ULBP3 in the plasma of critically ill patients. In vitro, culture in NKG2DL containing patient sera reduced the ADCC function of healthy donor NK cells and this could be blocked by NKG2DL-specific antibodies. Discussion: These observations of reduced NK function in severe disease are consistent with the hypothesis that defects in immune surveillance by NK cells permit higher levels of viral replication, rather than that aberrant NK cell function contributes to immune system dysregulation and immunopathogenicity.


Assuntos
COVID-19 , Citotoxicidade Imunológica , Humanos , COVID-19/patologia , Células Matadoras Naturais , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , SARS-CoV-2/metabolismo
20.
J Leukoc Biol ; 115(5): 985-991, 2024 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-38245016

RESUMO

The membrane (M) glycoprotein of SARS-CoV-2 is one of the key viral proteins regulating virion assembly and morphogenesis. Immunologically, the M protein is a major source of peptide antigens driving T cell responses, and most individuals who have been infected with SARS-CoV-2 make antibodies to the N-terminal, surface-exposed peptide of the M protein. We now report that although the M protein is abundant in the viral particle, antibodies to the surface-exposed N-terminal epitope of M do not appear to neutralize the virus. M protein-specific antibodies do, however, activate antibody-dependent cell-mediated cytotoxicity and cytokine secretion by primary human natural killer cells. Interestingly, while patients with severe or mild disease make comparable levels of M antigen-binding antibodies, M-specific antibodies from the serum of critically ill patients are significantly more potent activators of antibody-dependent cell-mediated cytotoxicity than antibodies found in individuals with mild or asymptomatic infection.


Assuntos
Anticorpos Antivirais , Citotoxicidade Celular Dependente de Anticorpos , COVID-19 , Estado Terminal , Células Matadoras Naturais , SARS-CoV-2 , Humanos , COVID-19/imunologia , SARS-CoV-2/imunologia , Anticorpos Antivirais/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Receptores Fc/imunologia , Receptores Fc/metabolismo , Anticorpos Neutralizantes/imunologia , Proteínas M de Coronavírus/imunologia , Feminino , Pessoa de Meia-Idade , Masculino
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA