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1.
Proc Natl Acad Sci U S A ; 120(50): e2311265120, 2023 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-38055740

RESUMO

Immunoglobulin M (IgM) is an evolutionary conserved key component of humoral immunity, and the first antibody isotype to emerge during an immune response. IgM is a large (1 MDa), multimeric protein, for which both hexameric and pentameric structures have been described, the latter additionally containing a joining (J) chain. Using a combination of single-particle mass spectrometry and mass photometry, proteomics, and immunochemical assays, we here demonstrate that circulatory (serum) IgM exclusively exists as a complex of J-chain-containing pentamers covalently bound to the small (36 kDa) protein CD5 antigen-like (CD5L, also called apoptosis inhibitor of macrophage). In sharp contrast, secretory IgM in saliva and milk is principally devoid of CD5L. Unlike IgM itself, CD5L is not produced by B cells, implying that it associates with IgM in the extracellular space. We demonstrate that CD5L integration has functional implications, i.e., it diminishes IgM binding to two of its receptors, the FcαµR and the polymeric Immunoglobulin receptor. On the other hand, binding to FcµR as well as complement activation via C1q seem unaffected by CD5L integration. Taken together, we redefine the composition of circulatory IgM as a J-chain containing pentamer, always in complex with CD5L.


Assuntos
Linfócitos B , Cadeias J de Imunoglobulina , Imunoglobulina M/metabolismo , Cadeias J de Imunoglobulina/metabolismo , Linfócitos B/metabolismo , Antígenos , Macrófagos/metabolismo
2.
J Immunol ; 211(11): 1725-1735, 2023 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-37843500

RESUMO

Complement activation via the classical pathway is initiated when oligomeric Igs on target surfaces are recognized by C1 of the complement cascade. The strength of this interaction and activation of the complement system are influenced by structural variation of the Ab, including Ab isotype, subclass, and glycosylation profile. Polymorphic variants of IgG have also been described to influence Fc-dependent effector functions. Therefore, we assessed complement binding, deposition, and complement-dependent cytotoxicity (CDC) of 27 known IgG allotypes with anti-trinitrophenyl specificity. Differences between allotypes within subclasses were minor for IgG1, IgG3, and IgG4 allotypes, and more substantial for IgG2. Allelic variant IGHG2*06, containing a unique serine at position 378 in the CH3 domain, showed less efficient complement activation and CDC compared with other IgG2 polymorphisms. We also observed variable cell lysis between IgG1 and IgG3, with IgG3 being superior in lysis of human RBCs and Ramos cells, and IgG1 being superior in lysis of Raji and Wien133 cells, demonstrating that a long-standing conundrum in the literature depends on cellular context. Furthermore, we compared IgG1 and IgG3 under different circumstances, showing that Ag density and Ab hinge length, but not complement regulators, define the context dependency of Ab-mediated CDC activity. Our results point toward a variation in the capacity of IgG subclasses to activate complement due to single amino acid changes and hinge length differences of allotypes to activate complement, which might give new insights on susceptibility to infectious, alloimmune, or autoimmune diseases and aid the design of Ab-based therapeutics.


Assuntos
Ativação do Complemento , Imunoglobulina G , Humanos , Glicosilação
3.
Allergy ; 79(7): 1952-1961, 2024 07.
Artigo em Inglês | MEDLINE | ID: mdl-38439527

RESUMO

BACKGROUND: The noninflammatory immunoglobulin G4 (IgG4) is linked to tolerance and is unique to humans. Although poorly understood, prolonged antigenic stimulation and IL-4-signaling along the T helper 2-axis may be instrumental in IgG4 class switching. Recently, repeated SARS-CoV-2 mRNA vaccination has been linked to IgG4 skewing. Although widely used immunosuppressive drugs have been shown to only moderately affect humoral responses to SARS-CoV-2 mRNA vaccination, the effect on IgG4 switching has not been investigated. METHODS: Here we study the impact of such immunosuppressive drugs, including the IL-4 receptor-blocking antibody dupilumab, on IgG4 skewing upon repeated SARS-CoV-2 mRNA vaccination. Receptor-binding domain (RBD) specific antibody responses were longitudinally measured in 600 individuals, including patients with immune-mediated inflammatory diseases treated with a TNF inhibitor (TNFi) and/or methotrexate (MTX), dupilumab, and healthy/untreated controls, after repeated mRNA vaccination. RESULTS: We observed a substantial increase in the proportion of RBD-specific IgG4 antibodies (median 21%) in healthy/untreated controls after third vaccination. This IgG4 skewing was profoundly reduced in dupilumab-treated patients (<1%). Unexpectedly, an equally strong suppression of IgG4 skewing was observed in TNFi-treated patients (<1%), whereas MTX caused a modest reduction (7%). RBD-specific total IgG levels were hardly affected by these immunosuppressive drugs. Minimal skewing was observed, when primary vaccination was adenoviral vector-based. CONCLUSIONS: Our results imply a critical role for IL-4/IL-13 as well as TNF in vivo IgG4 class switching. These novel findings advance our understanding of IgG4 class switch dynamics, and may benefit humoral tolerance induction strategies, treatment of IgG4 pathologies and mRNA vaccine optimization.


Assuntos
Anticorpos Monoclonais Humanizados , Switching de Imunoglobulina , Imunoglobulina G , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/sangue , Feminino , Masculino , Pessoa de Meia-Idade , SARS-CoV-2/imunologia , Inibidores do Fator de Necrose Tumoral/uso terapêutico , COVID-19/prevenção & controle , COVID-19/imunologia , Adulto , Vacinas de mRNA/imunologia , Idoso , Vacinação , Vacinas contra COVID-19/imunologia , Imunossupressores/uso terapêutico , Imunossupressores/farmacologia , Anticorpos Antivirais/imunologia
4.
J Immunol ; 209(1): 16-25, 2022 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-35705253

RESUMO

IgM is secreted as a pentameric polymer containing a peptide called the joining chain (J chain). However, integration of the J chain is not required for IgM assembly and in its absence IgM predominantly forms hexamers. The conformations of pentameric and hexameric IgM are remarkably similar with a hexagonal arrangement in solution. Despite these similarities, hexameric IgM has been reported to be a more potent complement activator than pentameric IgM, but reported relative potencies vary across different studies. Because of these discrepancies, we systematically investigated human IgM-mediated complement activation. We recombinantly generated pentameric and hexameric human IgM (IgM+J and IgM-J, respectively) mAbs and measured their ability to induce complement deposition and complement-dependent cytotoxicity when bound to several Ags at varying densities. At high Ag densities, hexameric and pentameric IgM activate complement to a similar extent as IgG1. However, at low densities, hexameric IgM outcompeted pentameric IgM and even more so IgG1. These differences became progressively more pronounced as antigenic density became critically low. Our findings highlight that the differential potency of hexameric and pentameric IgM for complement activation is profoundly dependent on the nature of its interactions with Ag. Furthermore, it underscores the importance of IgM in immunity because it is a more potent complement activator than IgG1 at low Ag densities.


Assuntos
Imunoglobulina G , Cadeias J de Imunoglobulina , Ativação do Complemento , Proteínas do Sistema Complemento , Humanos , Cadeias J de Imunoglobulina/metabolismo , Imunoglobulina M
5.
J Allergy Clin Immunol ; 151(6): 1646-1654, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36716825

RESUMO

BACKGROUND: Increased prevalence of autoantibody Fab glycosylation has been demonstrated for several autoimmune diseases. OBJECTIVES: To study whether elevated Fab glycosylation is a common feature of autoimmunity, this study investigated Fab glycosylation levels on serum IgG and its subclasses for autoantibodies associated with a range of different B cell-mediated autoimmune diseases, including rheumatoid arthritis, myasthenia gravis subtypes, pemphigus vulgaris, antineutrophil cytoplasmic antibody-associated vasculitis, systemic lupus erythematosus, anti-glomerular basement membrane glomerulonephritis, thrombotic thrombocytopenic purpura, and Guillain-Barré syndrome. METHODS: The level of Fab glycosylated IgG antibodies was assessed by lectin affinity chromatography and autoantigen-specific immunoassays. RESULTS: In 6 of 10 autoantibody responses, in 5 of 8 diseases, the investigators found increased levels of Fab glycosylation on IgG autoantibodies that varied from 86% in rheumatoid arthritis to 26% in systemic lupus erythematosus. Elevated autoantibody Fab glycosylation was not restricted to IgG4, which is known to be prone to Fab glycosylation, but was also present in IgG1. When autoimmune diseases with a chronic disease course were compared with more acute autoimmune illnesses, increased Fab glycosylation was restricted to the chronic diseases. As a proxy for chronic autoantigen exposure, the investigators determined Fab glycosylation levels on antibodies to common latent herpes viruses, as well as to glycoprotein 120 in individuals who are chronically HIV-1-infected. Immunity to these viral antigens was not associated with increased Fab glycosylation levels, indicating that chronic antigen-stimulation as such does not lead to increased Fab glycosylation levels. CONCLUSIONS: These data indicate that in chronic but not acute B cell-mediated autoimmune diseases, disease-specific autoantibodies are enriched for Fab glycans.


Assuntos
Artrite Reumatoide , Doenças Autoimunes , Lúpus Eritematoso Sistêmico , Miastenia Gravis , Humanos , Autoanticorpos , Imunoglobulina G , Autoantígenos
6.
J Immunol ; 207(6): 1545-1554, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34408013

RESUMO

Human IgG contains one evolutionarily conserved N-linked glycan in its Fc region at position 297. This glycan is crucial for Fc-mediated functions, including its induction of the classical complement cascade. This is induced after target recognition through the IgG-Fab regions, allowing neighboring IgG-Fc tails to associate through Fc:Fc interaction, ultimately leading to hexamer formation. This hexamerization seems crucial for IgG to enable efficient interaction with the globular heads of the first complement component C1q and subsequent complement activation. In this study, we show that galactose incorporated in the IgG1-Fc enhances C1q binding, C4, C3 deposition, and complement-dependent cellular cytotoxicity in human erythrocytes and Raji cells. IgG1-Fc sialylation slightly enhanced binding of C1q, but had little effect on downstream complement activation. Using various mutations that decrease or increase hexamerization capacity of IgG1, we show that IgG1-Fc galactosylation has no intrinsic effect on C1q binding to IgG1, but enhances IgG1 hexamerization potential and, thereby, complement activation. These data suggest that the therapeutic potential of Abs can be amplified without introducing immunogenic mutations, by relatively simple glycoengineering.


Assuntos
Ativação do Complemento , Imunoglobulina G , Complemento C1q , Humanos , Imunoglobulina G/genética , Mutação
7.
J Immunol ; 205(12): 3491-3499, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33127820

RESUMO

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infections often cause only mild disease that may evoke relatively low Ab titers compared with patients admitted to hospitals. Generally, total Ab bridging assays combine good sensitivity with high specificity. Therefore, we developed sensitive total Ab bridging assays for detection of SARS-CoV-2 Abs to the receptor-binding domain (RBD) and nucleocapsid protein in addition to conventional isotype-specific assays. Ab kinetics was assessed in PCR-confirmed, hospitalized coronavirus disease 2019 (COVID-19) patients (n = 41) and three populations of patients with COVID-19 symptoms not requiring hospital admission: PCR-confirmed convalescent plasmapheresis donors (n = 182), PCR-confirmed hospital care workers (n = 47), and a group of longitudinally sampled symptomatic individuals highly suspect of COVID-19 (n = 14). In nonhospitalized patients, the Ab response to RBD is weaker but follows similar kinetics, as has been observed in hospitalized patients. Across populations, the RBD bridging assay identified most patients correctly as seropositive. In 11/14 of the COVID-19-suspect cases, seroconversion in the RBD bridging assay could be demonstrated before day 12; nucleocapsid protein Abs emerged less consistently. Furthermore, we demonstrated the feasibility of finger-prick sampling for Ab detection against SARS-CoV-2 using these assays. In conclusion, the developed bridging assays reliably detect SARS-CoV-2 Abs in hospitalized and nonhospitalized patients and are therefore well suited to conduct seroprevalence studies.


Assuntos
Anticorpos Antivirais/imunologia , Formação de Anticorpos , COVID-19/imunologia , Proteínas do Nucleocapsídeo/imunologia , SARS-CoV-2/imunologia , Adulto , COVID-19/diagnóstico , Teste de Ácido Nucleico para COVID-19 , Teste Sorológico para COVID-19 , Convalescença , Feminino , Humanos , Testes Imunológicos , Masculino , Pessoa de Meia-Idade
8.
J Immunol ; 202(8): 2220-2228, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30850477

RESUMO

Abs can acquire N-linked glycans in their V regions during Ag-specific B cell responses. Among others, these N-linked glycans can affect Ag binding and Ab stability. Elevated N-linked glycosylation has furthermore been associated with several B cell-associated pathologies. Basic knowledge about patterns of V region glycosylation at different stages of B cell development is scarce. The aim of the current study is to establish patterns of N-glycosylation sites in Ab V regions of naive and memory B cell subsets. We analyzed the distribution and acquisition of N-glycosylation sites within Ab V regions of peripheral blood and bone marrow B cells of 12 healthy individuals, eight myasthenia gravis patients, and six systemic lupus erythematosus patients, obtained by next-generation sequencing. N-glycosylation sites are clustered around CDRs and the DE loop for both H and L chains, with similar frequencies for healthy donors and patients. No evidence was found for an overall selection bias against acquiring an N-glycosylation site, except for the CDR3 of the H chain. Interestingly, both IgE and IgG4 subsets have a 2-fold higher propensity to acquire Fab glycans compared with IgG1 or IgA. When expressed as rmAb, 35 out of 38 (92%) nongermline N-glycosylation sites became occupied. These results point toward a differential selection pressure of N-glycosylation site acquisition during affinity maturation of B cells, which depends on the location within the V region and is isotype and subclass dependent. Elevated Fab glycosylation represents an additional hallmark of TH2-like IgG4/IgE responses.


Assuntos
Linfócitos B/imunologia , Imunoglobulina G , Região Variável de Imunoglobulina , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Ativação Linfocitária/genética , Linfócitos B/patologia , Glicosilação , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Lúpus Eritematoso Sistêmico/patologia
9.
Proc Natl Acad Sci U S A ; 115(8): 1901-1906, 2018 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-29432186

RESUMO

A hallmark of B-cell immunity is the generation of a diverse repertoire of antibodies from a limited set of germline V(D)J genes. This repertoire is usually defined in terms of amino acid composition. However, variable domains may also acquire N-linked glycans, a process conditional on the introduction of consensus amino acid motifs (N-glycosylation sites) during somatic hypermutation. High levels of variable domain glycans have been associated with autoantibodies in rheumatoid arthritis, as well as certain follicular lymphomas. However, the role of these glycans in the humoral immune response remains poorly understood. Interestingly, studies have reported both positive and negative effects on antibody affinity. Our aim was to elucidate the role of variable domain glycans during antigen-specific antibody responses. By analyzing B-cell repertoires by next-generation sequencing, we demonstrate that N-glycosylation sites are introduced at positions in which glycans can affect antigen binding as a result of a specific clustering of progenitor glycosylation sites in the germline sequences of variable domain genes. By analyzing multiple human monoclonal and polyclonal (auto)antibody responses, we subsequently show that this process is subject to selection during antigen-specific antibody responses, skewed toward IgG4, and positively contributes to antigen binding. Together, these results highlight a physiological role for variable domain glycosylation as an additional layer of antibody diversification that modulates antigen binding.


Assuntos
Região Variável de Imunoglobulina/genética , Anticorpos , Anticorpos Monoclonais , Afinidade de Anticorpos , Artrite Reumatoide/imunologia , Autoanticorpos , Linfócitos B/metabolismo , Glicosilação , Humanos , Imunoglobulina G/genética
10.
Clin Exp Allergy ; 50(3): 401-405, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31880850

RESUMO

BACKGROUND: Small, basic peanut proteins are often poorly extracted in pH-neutral buffers that are optimal for the extraction of peanut storage proteins such as Ara h 1. As a result, such proteins are easily missed as potential allergens. OBJECTIVE: To analyse the allergenic composition of the basic peanut protein (BPP) fraction. METHODS: A peanut extract prepared at pH 4 was fractionated by physicochemical procedures. Chemical analysis was performed by SDS-PAGE and mass spectrometry. Because immunoblotting was found to be inefficient for most of these small basic proteins, IgE-binding activity was measured by coupling the fractions to CNBr-activated Sepharose, followed by incubation with sera from 55 Dutch peanut-allergic children and 125 I-labelled anti-IgE. RESULTS: Most IgE reactivity of the BPP fraction was due to the 5-7 kDa amino-terminal fragment of Ara h 1. This finding was confirmed by the use of the fragment in recombinant form, to which 25/55 of the sera was IgE-positive. CONCLUSION: The amino-terminal fragment of Ara h 1, a member of a family of small anti-microbial proteins, is an allergen independent of the carboxy-terminal fragment of Ara h 1.


Assuntos
Sequência de Aminoácidos , Antígenos de Plantas/imunologia , Imunoglobulina E/imunologia , Proteínas de Membrana/imunologia , Proteínas de Plantas/imunologia , Proteínas Citotóxicas Formadoras de Poros/imunologia , Antígenos de Plantas/genética , Feminino , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Plantas/genética , Proteínas Citotóxicas Formadoras de Poros/genética
11.
Clin Exp Rheumatol ; 38(2): 306-313, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31498069

RESUMO

OBJECTIVES: Tumour necrosis factor (TNF) inhibitors like certolizumab, elicit an immunogenic response leading to the formation of anti-drug antibodies (ADAs). We sought to mechanistically investigate the relationship between certolizumab concentrations, ADAs, and the effective TNF neutralising capacity in sera of rheumatoid arthritis (RA) patients. TNF neutralising capacity of certolizumab was compared to the neutralising capacity of adalimumab. METHODS: Serum samples were collected from 40 consecutive certolizumab-treated RA patients at baseline and 4, 16, 28 and 52 weeks after treatment initiation [Dutch Trial Register NTR (Nederlands Trial Register) Trial NL2824 no. 2965]. Certolizumab concentration and ADA titre were measured with a certolizumab bridging enzyme-linked immunosorbent assay (ELISA) and a drug-tolerant radioimmunoassay (RIA), respectively. TNF neutralisation by certolizumab and adalimumab, in presence or absence of ADAs, was analysed with the TNF-sensitive WEHI bioassay. RESULTS: Despite a high incidence of ADAs during one year of follow-up (65%; 26/40 patients), certolizumab levels of >10 µg/ml were measured in most patients. The capacity for TNF neutralisation highly correlated with certolizumab serum concentration, whereas no association with ADAs was observed. Similar results were obtained for adalimumab. The relative in vitro neutralising potency was higher for certolizumab compared to adalimumab. CONCLUSIONS: Anti-certolizumab antibodies were detected in a large proportion of patients, but in most cases where ADAs were detected, certolizumab was also present in high concentrations, directly correlating with in vitro neutralising capacity. These results indicate that measurement of certolizumab drug levels, rather than ADAs, have direct clinical significance.


Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Antirreumáticos , Fragmentos Fab das Imunoglobulinas/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adalimumab , Anticorpos , Anticorpos Neutralizantes/imunologia , Antirreumáticos/imunologia , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/imunologia , Certolizumab Pegol , Humanos , Infliximab
12.
J Gastroenterol Hepatol ; 34(10): 1878-1886, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30345709

RESUMO

BACKGROUND AND AIM: Immunoglobulin subclass G4-related disease (IgG4-RD) is characterized by an abundance of IgG4 antibodies in the serum and tissue. Glycosylation status of antibodies can impact on immune effector functions and disease pathophysiology. We sought to establish glycosylation patterns in a prospective cohort of patients with IgG4-RD and the relationship with disease activity and response to treatment. METHODS: We assessed IgG Fc-tail and Fab-arm glycosylation status in patients with IgG4-RD (n = 22), disease controls with primary sclerosing cholangitis (PSC) (n = 22), and healthy controls (n = 22). Serum IgG and subclasses were quantified using ELISA. Fc and Fab glycosylation were analyzed by mass spectrometry and lectin affinity chromatography, respectively. Disease activity, organ damage, and response to treatment were assessed using the IgG4 Responder Index. RESULTS: Immunoglobulin G Fab sialylation was increased in IgG4-RD compared with PSC and healthy control (P = 0.01), with a preferential increase in IgG4-specific Fab sialylation, which was independent of IgG4 Fab-arm exchange. There was a reduction in IgG1-specific Fc bisection and hybrid structures in IgG4-RD (P < 0.01), which recovered upon steroid treatment and correlated with disease activity. Overall, IgG Fc galactosylation was reduced in both IgG4-RD and PSC (P < 0.01), with a preferential reduction in IgG1-specific sialylation and enhancement of IgG4-specific bisection in PSC. IgG4 fucosylation and IgG1/2/3 hybrid structures negatively correlated with complement C3 and C4 levels in IgG4-RD (P < 0.01), but not PSC. CONCLUSION: We report the first study showing unique antibody glycosylation status in a prospective cohort of IgG4-RD and PSC patients, which may determine modulation of the immune system and contribute to disease pathophysiology.


Assuntos
Colangite Esclerosante/sangue , Fragmentos Fab das Imunoglobulinas/sangue , Fragmentos Fc das Imunoglobulinas/sangue , Doença Relacionada a Imunoglobulina G4/sangue , Imunoglobulina G/metabolismo , Processamento de Proteína Pós-Traducional , Corticosteroides/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Colangite Esclerosante/diagnóstico , Colangite Esclerosante/imunologia , Feminino , Glicosilação , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Doença Relacionada a Imunoglobulina G4/diagnóstico , Doença Relacionada a Imunoglobulina G4/tratamento farmacológico , Doença Relacionada a Imunoglobulina G4/imunologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Fatores de Tempo , Resultado do Tratamento
13.
Ann Rheum Dis ; 77(10): 1471-1479, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29945923

RESUMO

OBJECTIVES: Therapeutic antibodies can provoke an antidrug antibody (ADA) response, which can form soluble immune complexes with the drug in potentially high amounts. Nevertheless, ADA-associated adverse events are usually rare, although with notable exceptions including infliximab. The immune activating effects and the eventual fate of these 'anti-idiotype' complexes are poorly studied, hampering assessment of ADA-associated risk of adverse events. We investigated the in vitro formation and biological activities of ADA-drug anti-idiotype immune complexes using patient-derived monoclonal anti-infliximab antibodies. METHODS: Size distribution and conformation of ADA-drug complexes were characterised by size-exclusion chromatography and electron microscopy. Internalisation of and immune activation by complexes of defined size was visualised with flow imaging, whole blood cell assay and C4b/c ELISA. RESULTS: Size and conformation of immune complexes depended on the concentrations and ratio of drug and ADA; large complexes (>6 IgGs) formed only with high ADA titres. Macrophages efficiently internalised tetrameric and bigger complexes in vitro, but not dimers. Corroborating these results, ex vivo analysis of patient sera demonstrated only dimeric complexes in circulation.No activation of immune cells by anti-idiotype complexes was observed, and only very large complexes activated complement. Unlike Fc-linked hexamers, anti-idiotype hexamers did not activate complement, demonstrating that besides size, conformation governs immune complex potential for triggering effector functions. CONCLUSIONS: Anti-idiotype ADA-drug complexes generally have restricted immune activation capacity. Large, irregularly shaped complexes only form at high concentrations of both drug and ADA, as may be achieved during intravenous infusion of infliximab, explaining the rarity of serious ADA-associated adverse events.


Assuntos
Anticorpos/imunologia , Formação de Anticorpos/efeitos dos fármacos , Complexo Antígeno-Anticorpo/imunologia , Antirreumáticos/imunologia , Infliximab/imunologia , Cromatografia em Gel , Ensaio de Imunoadsorção Enzimática , Humanos , Soro/imunologia
14.
Anal Biochem ; 437(2): 118-22, 2013 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-23499975

RESUMO

Although several techniques exist for the measurement of high-affinity interactions, it is still challenging to determine dissociation constants around or even below 1pM. During the analysis of several human-derived monoclonal antibodies to adalimumab, we found a clone with a very high affinity that could not be measured using conventional surface plasmon resonance assays. We developed a straightforward and robust method to measure affinities in the nanomolar to sub-picomolar range. The assay is based on separation of bound and free fluorescently labeled antigen using size exclusion chromatography and quantification by in-line fluorescence detection. We describe optimal conditions and procedures that result in a very sensitive assay that can be used to reliably determine ultra-high affinities. Using the method described in this article, a dissociation constant of 0.78pM could be determined for the anti-adalimumab antibody.


Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Anticorpos/fisiologia , Afinidade de Anticorpos/fisiologia , Cromatografia Líquida de Alta Pressão/métodos , Adalimumab , Anticorpos/química , Anticorpos Monoclonais Humanizados/uso terapêutico , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Linfócitos B/metabolismo , Fluorescência , Humanos , Interleucina-6/imunologia , Sensibilidade e Especificidade , Toxoide Tetânico/imunologia
15.
Sci Rep ; 12(1): 62, 2022 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-34996950

RESUMO

The neonatal Fc receptor (FcRn) is known to mediate placental transfer of IgG from mother to unborn. IgE is widely known for triggering immune responses to environmental antigens. Recent evidence suggests FcRn-mediated transplacental passage of IgE during pregnancy. However, direct interaction of FcRn and IgE was not investigated. Here, we compared binding of human IgE and IgG variants to recombinant soluble human FcRn with ß2-microglobulin (sFcRn) in surface plasmon resonance (SPR) at pH 7.4 and pH 6.0. No interaction was found between human IgE and human sFcRn. These results imply that FcRn can only transport IgE indirectly, and thereby possibly transfer allergenic sensitivity from mother to fetus.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoglobulina E/metabolismo , Placenta/metabolismo , Receptores Fc/metabolismo , Transporte Biológico , Feminino , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina G/metabolismo , Troca Materno-Fetal , Gravidez , Ligação Proteica , Ressonância de Plasmônio de Superfície , Microglobulina beta-2/metabolismo
16.
Front Immunol ; 13: 1082154, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36591315

RESUMO

The generation of high-affinity antibodies requires an efficient germinal center (GC) response. As differentiating B cells cycle between GC dark and light zones they encounter different oxygen pressures (pO2). However, it is essentially unknown if and how variations in pO2 affect B cell differentiation, in particular for humans. Using optimized in vitro cultures together with in-depth assessment of B cell phenotype and signaling pathways, we show that oxygen is a critical regulator of human naive B cell differentiation and class switch recombination. Normoxia promotes differentiation into functional antibody secreting cells, while a population of CD27++ B cells was uniquely generated under hypoxia. Moreover, time-dependent transitions between hypoxic and normoxic pO2 during culture - reminiscent of in vivo GC cyclic re-entry - steer different human B cell differentiation trajectories and IgG class switch recombination. Taken together, we identified multiple mechanisms trough which oxygen pressure governs human B cell differentiation.


Assuntos
Linfócitos B , Oxigênio , Humanos , Oxigênio/metabolismo , Centro Germinativo , Hipóxia/metabolismo , Diferenciação Celular , Imunoglobulina G
17.
Front Immunol ; 13: 891316, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35572548

RESUMO

Glycosylation of CD45RB (RB+) has recently been identified to mark antigen-experienced B cells, independent of their CD27 expression. By using a novel combination of markers including CD45RB glycosylation, CD27 and IgM/IgD isotype expression we segregated human peripheral blood B cell subsets and investigated their IGHV repertoire and in vitro functionality. We observed distinct maturation stages for CD27-RB+ cells, defined by differential expression of non-switched Ig isotypes. CD27-RB+ cells, which only express IgM, were more matured in terms of Ig gene mutation levels and function as compared to CD27-RB+ cells that express both IgM and IgD or cells that were CD27-RB-. Moreover, CD27-RB+IgM+ cells already showed remarkable rigidity in IgM isotype commitment, different from CD27-RB+IgMD+ and CD27-RB- cells that still demonstrated great plasticity in B cell fate decision. Thus, glycosylation of CD45RB is indicative for antigen-primed B cells, which are, dependent on the Ig isotype, functionally distinct.


Assuntos
Subpopulações de Linfócitos B , Antígenos Comuns de Leucócito/imunologia , Subpopulações de Linfócitos B/metabolismo , Glicosilação , Humanos , Imunoglobulina D/metabolismo , Isotipos de Imunoglobulinas/metabolismo , Imunoglobulina M/metabolismo , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo
18.
Nat Commun ; 13(1): 6073, 2022 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-36241613

RESUMO

Binding to the neonatal Fc receptor (FcRn) extends serum half-life of IgG, and antagonizing this interaction is a promising therapeutic approach in IgG-mediated autoimmune diseases. Fc-MST-HN, designed for enhanced FcRn binding capacity, has not been evaluated in the context of a full-length antibody, and the structural properties of the attached Fab regions might affect the FcRn-mediated intracellular trafficking pathway. Here we present a comprehensive comparative analysis of the IgG salvage pathway between two full-size IgG1 variants, containing wild type and MST-HN Fc fragments, and their Fc-only counterparts. We find no evidence of Fab-regions affecting FcRn binding in cell-free assays, however, cellular assays show impaired binding of full-size IgG to FcRn, which translates into improved intracellular FcRn occupancy and intracellular accumulation of Fc-MST-HN compared to full size IgG1-MST-HN. The crystal structure of Fc-MST-HN in complex with FcRn provides a plausible explanation why the Fab disrupts the interaction only in the context of membrane-associated FcRn. Importantly, we find that Fc-MST-HN outperforms full-size IgG1-MST-HN in reducing IgG levels in cynomolgus monkeys. Collectively, our findings identify the cellular membrane context as a critical factor in FcRn biology and therapeutic targeting.


Assuntos
Anticorpos Monoclonais , Doenças Autoimunes , Animais , Doenças Autoimunes/tratamento farmacológico , Antígenos de Histocompatibilidade Classe I , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G , Macaca fascicularis/metabolismo , Ligação Proteica , Receptores Fc
19.
EBioMedicine ; 81: 104109, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35752106

RESUMO

BACKGROUND: Immunoglobulin G (IgG) antibodies serve a crucial immuno-protective function mediated by IgG Fc receptors (FcγR). Absence of fucose on the highly conserved N-linked glycan in the IgG Fc domain strongly enhances IgG binding and activation of myeloid and natural killer (NK) cell FcγRs. Although afucosylated IgG can provide increased protection (malaria and HIV), it also boosts immunopathologies in alloimmune diseases, COVID-19 and dengue fever. Quantifying IgG fucosylation currently requires sophisticated methods such as liquid chromatography-mass spectrometry (LC-MS) and extensive analytical skills reserved to highly specialized laboratories. METHODS: Here, we introduce the Fucose-sensitive Enzyme-linked immunosorbent assay (ELISA) for Antigen-Specific IgG (FEASI), an immunoassay capable of simultaneously quantitating and qualitatively determining IgG responses. FEASI is a two-tier immunoassay; the first assay is used to quantify antigen-specific IgG (IgG ELISA), while the second gives FcγRIIIa binding-dependent readout which is highly sensitive to both the IgG quantity and the IgG Fc fucosylation (FcγR-IgG ELISA). FINDINGS: IgG Fc fucosylation levels, independently determined by LC-MS and FEASI, in COVID-19 responses to the spike (S) antigen, correlated very strongly by simple linear regression (R2=0.93, p < 0.0001). The FEASI method was then used to quantify IgG levels and fucosylation in COVID-19 convalescent plasma which was independently validated by LC-MS. INTERPRETATION: FEASI can be reliably implemented to measure relative and absolute IgG Fc fucosylation and quantify binding of antigen-specific IgG to FcγR in a high-throughput manner accessible to all diagnostic and research laboratories. FUNDING: This work was funded by the Stichting Sanquin Bloedvoorziening (PPOC 19-08 and SQI00041) and ZonMW 10430 01 201 0021.


Assuntos
Fucose , Imunoglobulina G , Receptores de IgG , COVID-19/diagnóstico , COVID-19/terapia , Ensaio de Imunoadsorção Enzimática/métodos , Fucose/química , Fucose/metabolismo , Humanos , Imunização Passiva , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Receptores de IgG/química , Soroterapia para COVID-19
20.
Science ; 371(6532)2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33361116

RESUMO

Immunoglobulin G (IgG) antibodies are crucial for protection against invading pathogens. A highly conserved N-linked glycan within the IgG-Fc tail, which is essential for IgG function, shows variable composition in humans. Afucosylated IgG variants are already used in anticancer therapeutic antibodies for their increased activity through Fc receptors (FcγRIIIa). Here, we report that afucosylated IgG (approximately 6% of total IgG in humans) are specifically formed against enveloped viruses but generally not against other antigens. This mediates stronger FcγRIIIa responses but also amplifies brewing cytokine storms and immune-mediated pathologies. Critically ill COVID-19 patients, but not those with mild symptoms, had high concentrations of afucosylated IgG antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), amplifying proinflammatory cytokine release and acute phase responses. Thus, antibody glycosylation plays a critical role in immune responses to enveloped viruses, including COVID-19.


Assuntos
Anticorpos Antivirais/imunologia , COVID-19/imunologia , Imunoglobulina G/imunologia , SARS-CoV-2/imunologia , Adulto , Idoso , Anticorpos Antivirais/sangue , Anticorpos Antivirais/química , COVID-19/fisiopatologia , Células Cultivadas , Estado Terminal , Citomegalovirus/imunologia , Feminino , Fucose/análise , Glicosilação , HIV/imunologia , Vacinas contra Hepatite B/imunologia , Humanos , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/química , Inflamação , Interleucina-6/biossíntese , Interleucina-6/imunologia , Macrófagos/imunologia , Masculino , Pessoa de Meia-Idade , Parvovirus B19 Humano/imunologia , Índice de Gravidade de Doença , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Adulto Jovem
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