RESUMO
Autoantibodies against myelin oligodendrocyte glycoprotein (MOG) have recently been established to define a new disease entity, MOG-antibody-associated disease (MOGAD), which is clinically overlapping with multiple sclerosis. MOG-specific antibodies (Abs) from patients are pathogenic, but the precise effector mechanisms are currently still unknown and no therapy is approved for MOGAD. Here, we determined the contributions of complement and Fc-receptor (FcR)-mediated effects in the pathogenicity of MOG-Abs. Starting from a recombinant anti-MOG (mAb) with human IgG1 Fc, we established MOG-specific mutant mAbs with differential FcR and C1q binding. We then applied selected mutants of this MOG-mAb in two animal models of experimental autoimmune encephalomyelitis. First, we found MOG-mAb-induced demyelination was mediated by both complement and FcRs about equally. Second, we found that MOG-Abs enhanced activation of cognate MOG-specific T cells in the central nervous system (CNS), which was dependent on FcR-, but not C1q-binding. The identification of complement-dependent and -independent pathomechanisms of MOG-Abs has implications for therapeutic strategies in MOGAD.
Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Animais , Humanos , Glicoproteína Mielina-Oligodendrócito , Autoanticorpos , Receptores Fc , Proteínas do Sistema Complemento , Anticorpos MonoclonaisRESUMO
Antibodies to myelin oligodendrocyte glycoprotein (MOG-Abs) define a distinct disease entity. Here we aimed to understand essential structural features of MOG required for recognition by autoantibodies from patients. We produced the N-terminal part of MOG in a conformationally correct form; this domain was insufficient to identify patients with MOG-Abs by ELISA even after site-directed binding. This was neither due to a lack of lipid embedding nor to a missing putative epitope at the C-terminus, which we confirmed to be an intracellular domain. When MOG was displayed on transfected cells, patients with MOG-Abs recognized full-length MOG much better than its N-terminal part with the first hydrophobic domain (P < 0.0001). Even antibodies affinity-purified with the extracellular part of MOG recognized full-length MOG better than the extracellular part of MOG after transfection. The second hydrophobic domain of MOG enhanced the recognition of the extracellular part of MOG by antibodies from patients as seen with truncated variants of MOG. We confirmed the pivotal role of the second hydrophobic domain by fusing the intracellular part of MOG from the evolutionary distant opossum to the human extracellular part; the chimeric construct restored the antibody binding completely. Further, we found that in contrast to 8-18C5, MOG-Abs from patients bound preferentially as F(ab')2 rather than Fab. It was previously found that bivalent binding of human IgG1, the prominent isotype of MOG-Abs, requires that its target antigen is displayed at a distance of 13-16 nm. We found that, upon transfection, molecules of MOG did not interact so closely to induce a Förster resonance energy transfer signal, indicating that they are more than 6 nm apart. We propose that the intracellular part of MOG holds the monomers apart at a suitable distance for bivalent binding; this could explain why a cell-based assay is needed to identify MOG-Abs. Our finding that MOG-Abs from most patients require bivalent binding has implications for understanding the pathogenesis of MOG-Ab associated disorders. Since bivalently bound antibodies have been reported to only poorly bind C1q, we speculate that the pathogenicity of MOG-Abs is mostly mediated by other mechanisms than complement activation. Therefore, therapeutic inhibition of complement activation should be less efficient in MOG-Ab associated disorders than in patients with antibodies to aquaporin-4 .
Assuntos
Autoanticorpos/imunologia , Epitopos/imunologia , Glicoproteína Mielina-Oligodendrócito/imunologia , Adulto , Feminino , Humanos , MasculinoRESUMO
High levels of antibodies against glutamic acid decarboxylase (GAD) are observed in patients with different neurological disorders, but cells producing these autoantibodies are largely unexplored. We detect circulating GAD-reactive B cells in peripheral blood that readily differentiate into antibody-producing cells. These cells are highly elevated in most patients with GAD-antibody-associated disorders (n = 15) compared to controls (n = 19). They mainly produce GAD65 antibodies of the IgG1 and IgG4 subclasses and are as abundant as B cells reactive for common recall antigens. Bone marrow cells represent an additional source of GAD antibodies. The identification of GAD-antibody-producing cells has implications for the selection of cell-specific biologics. ANN NEUROL 2019;85:448-454.
Assuntos
Autoanticorpos/imunologia , Linfócitos B/imunologia , Ataxia Cerebelar/imunologia , Glutamato Descarboxilase/imunologia , Encefalite Límbica/imunologia , Plasmócitos/imunologia , Rigidez Muscular Espasmódica/imunologia , Adolescente , Adulto , Idoso , Células da Medula Óssea/imunologia , Estudos de Casos e Controles , Feminino , Humanos , Imunoglobulina G/imunologia , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
OBJECTIVE: Autoantibodies against myelin oligodendrocyte glycoprotein (MOG) occur in a proportion of patients with inflammatory demyelinating diseases of the central nervous system (CNS). We analyzed their pathogenic activity by affinity-purifying these antibodies (Abs) from patients and transferring them to experimental animals. METHODS: Patients with Abs to MOG were identified by cell-based assay. We determined the cross-reactivity to rodent MOG and the recognized MOG epitopes. We produced the correctly folded extracellular domain of MOG and affinity-purified MOG-specific Abs from the blood of patients. These purified Abs were used to stain CNS tissue and transferred in 2 models of experimental autoimmune encephalomyelitis. Animals were analyzed histopathologically. RESULTS: We identified 17 patients with MOG Abs from our outpatient clinic and selected 2 with a cross-reactivity to rodent MOG; both had recurrent optic neuritis. Affinity-purified Abs recognized MOG on transfected cells and stained myelin in tissue sections. The Abs from the 2 patients recognized different epitopes on MOG, the CC' and the FG loop. In both patients, these Abs persisted during our observation period of 2 to 3 years. The anti-MOG Abs from both patients were pathogenic upon intrathecal injection in 2 different rat models. Together with cognate MOG-specific T cells, these Abs enhanced T-cell infiltration; together with myelin basic protein-specific T cells, they induced demyelination associated with deposition of C9neo, resembling a multiple sclerosis type II pathology. INTERPRETATION: MOG-specific Abs affinity purified from patients with inflammatory demyelinating disease induce pathological changes in vivo upon cotransfer with myelin-reactive T cells, suggesting that these Abs are similarly pathogenic in patients. Ann Neurol 2018;84:315-328.
Assuntos
Autoanticorpos/sangue , Encéfalo/metabolismo , Encéfalo/patologia , Glicoproteína Mielina-Oligodendrócito/sangue , Adulto , Idoso , Animais , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Feminino , Cobaias , Humanos , Inflamação/sangue , Inflamação/diagnóstico , Masculino , Pessoa de Meia-Idade , Ratos , Ratos Endogâmicos Lew , Adulto JovemRESUMO
OBJECTIVES: Neurodegeneration is considered a relevant pathophysiologic feature in neurologic disorders associated with antibodies against glutamic acid decarboxylase 65 (GAD65). In this study, we investigate surrogates of neuroaxonal damage in relation to disease duration and clinical presentation. METHODS: In a multicentric cohort of 50 patients, we measured serum neurofilament light chain (sNfL) in relation to disease duration and disease phenotypes, applied automated MRI volumetry, and analyzed clinical characteristics. RESULTS: In patients with neurologic disorders associated with GAD65 antibodies, we detected elevated sNfL levels early in the disease course. By contrast, this elevation of sNfL levels was less pronounced in patients with long-standing disease. Increased sNfL levels were observed in patients presenting with cerebellar ataxia and limbic encephalitis, but not in those with stiff person syndrome. Using MRI volumetry, we identified atrophy predominantly of the cerebellar cortex, cerebellar superior posterior lobe, and cerebral cortex with similar atrophy patterns throughout all clinical phenotypes. DISCUSSION: Together, our data provide evidence for early neuroaxonal damage and support the need for timely therapeutic interventions in GAD65 antibody-associated neurologic disorders.
Assuntos
Ataxia Cerebelar , Doenças do Sistema Nervoso , Rigidez Muscular Espasmódica , Humanos , Atrofia , AutoanticorposRESUMO
The formation of neuronal circuits is a key process of development, laying foundations for behavior. The cellular mechanisms regulating circuit development are not fully understood. Here, we reveal Psidin as an intracellular regulator of Drosophila olfactory system formation. We show that Psidin is required in several classes of olfactory receptor neurons (ORNs) for survival and subsequently for axon guidance. During axon guidance, Psidin functions as an actin regulator and antagonist of Tropomyosin. Accordingly, Psidin-deficient primary neurons in culture display growth cones with significantly smaller lamellipodia. This lamellipodial phenotype, as well as the mistargeting defects in vivo, is suppressed by parallel removal of Tropomyosin. In contrast, Psidin functions as the noncatalytic subunit of the N-acetyltransferase complex B (NatB) to maintain the number of ORNs. Psidin physically binds the catalytic NatB subunit CG14222 (dNAA20) and functionally interacts with it in vivo. We define the dNAA20 interaction domain within Psidin and identify a conserved serine as a candidate for phosphorylation-mediated regulation of NatB complex formation. A phosphomimetic mutation of this serine showed severely reduced binding to dNAA20 in vitro. In vivo, it fully rescued the targeting defect but not the reduction in neuron numbers. In addition, we show that a different amino acid point mutation shows exactly the opposite effect by rescuing only the cell number but not the axon targeting defect. Together, our data suggest that Psidin plays two independent developmental roles via the acquisition of separate signaling pathways, both of which contribute to the formation of olfactory circuits.
Assuntos
Axônios/fisiologia , Proteínas Sanguíneas/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila/fisiologia , Neurônios/fisiologia , Condutos Olfatórios/fisiologia , Acetiltransferases/metabolismo , Animais , Western Blotting , Contagem de Células , Células Cultivadas , Genótipo , Cones de Crescimento/fisiologia , Imunoprecipitação , Hibridização In Situ , Rede Nervosa/citologia , Rede Nervosa/crescimento & desenvolvimento , Rede Nervosa/fisiologia , Condutos Olfatórios/citologia , Condutos Olfatórios/crescimento & desenvolvimento , Fenótipo , Fosforilação/fisiologia , Pseudópodes/fisiologia , Interferência de RNA , Saccharomyces cerevisiae/metabolismo , Tropomiosina/farmacologiaRESUMO
Although some COVID-19 patients maintain SARS-CoV-2-specific serum immunoglobulin G (IgG) for more than 6 months postinfection, others eventually lose IgG levels. We assessed the persistence of SARS-CoV-2-specific B cells in 17 patients, 5 of whom had lost specific IgGs after 5-8 months. Differentiation of blood-derived B cells in vitro revealed persistent SARS-CoV-2-specific IgG B cells in all patients, whereas IgA B cells were maintained in 11. Antibodies derived from cultured B cells blocked binding of viral receptor-binding domain (RBD) to the cellular receptor ACE-2, had neutralizing activity to authentic virus, and recognized the RBD of the variant of concern Alpha similarly to the wild type, whereas reactivity to Beta and Gamma were decreased. Thus, differentiation of memory B cells could be more sensitive for detecting previous infection than measuring serum antibodies. Understanding the persistence of SARS-CoV-2-specific B cells even in the absence of specific serum IgG will help to promote long-term immunity.
RESUMO
Autoimmune disorders of the central nervous system (CNS) comprise a broad spectrum of clinical entities. The stratification of patients based on the recognized autoantigen is of great importance for therapy optimization and for concepts of pathogenicity, but for most of these patients, the actual target of their autoimmune response is unknown. Here we investigated oligodendrocyte myelin glycoprotein (OMGP) as autoimmune target, because OMGP is expressed specifically in the CNS and there on oligodendrocytes and neurons. Using a stringent cell-based assay, we detected autoantibodies to OMGP in serum of 8/352 patients with multiple sclerosis, 1/28 children with acute disseminated encephalomyelitis and unexpectedly, also in one patient with psychosis, but in none of 114 healthy controls. Since OMGP is GPI-anchored, we validated its recognition also in GPI-anchored form. The autoantibodies to OMGP were largely IgG1 with a contribution of IgG4, indicating cognate T cell help. We found high levels of soluble OMGP in human spinal fluid, presumably due to shedding of the GPI-linked OMGP. Analyzing the pathogenic relevance of autoimmunity to OMGP in an animal model, we found that OMGP-specific T cells induce a novel type of experimental autoimmune encephalomyelitis dominated by meningitis above the cortical convexities. This unusual localization may be directed by intrathecal uptake and presentation of OMGP by meningeal phagocytes. Together, OMGP-directed autoimmunity provides a new element of heterogeneity, helping to improve the stratification of patients for diagnostic and therapeutic purposes.
Assuntos
Autoanticorpos/imunologia , Autoimunidade/imunologia , Encefalomielite Aguda Disseminada/imunologia , Esclerose Múltipla/imunologia , Glicoproteína Oligodendrócito-Mielina/imunologia , Adulto , Animais , Estudos de Casos e Controles , Criança , Pré-Escolar , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/imunologia , Feminino , Humanos , Imunoglobulina G/imunologia , Masculino , Camundongos , Pessoa de Meia-Idade , Transtornos Psicóticos/imunologia , Ratos , Linfócitos T/imunologia , Adulto JovemRESUMO
OBJECTIVE: To identify circulating myelin oligodendrocyte glycoprotein (MOG)-specific B cells in the blood of patients with MOG antibodies (Abs) and to determine whether circulating MOG-specific B cells are linked to levels and epitope specificity of serum anti-MOG-Abs. METHODS: We compared peripheral blood from 21 patients with MOG-Abs and 26 controls for the presence of MOG-specific B cells. We differentiated blood-derived B cells in vitro in separate culture wells to Ab-producing cells via engagement of Toll-like receptors 7 and 8. We quantified the anti-MOG reactivity with a live cell-based assay by flow cytometry. We determined the recognition of MOG epitopes with a panel of mutated variants of MOG. RESULTS: MOG-Ab-positive patients had a higher frequency of MOG-specific B cells in blood than controls, but MOG-specific B cells were only detected in about 60% of these patients. MOG-specific B cells in blood showed no correlation with anti-MOG Ab levels in serum, neither in the whole group nor in the untreated patients. Epitope analysis of MOG-Abs secreted from MOG-specific B cells cultured in different wells revealed an intraindividual heterogeneity of the anti-MOG autoimmunity. CONCLUSIONS: This study shows that patients with MOG-Abs greatly differ in the abundance of circulating MOG-specific B cells, which are not linked to levels of MOG-Abs in serum suggesting different sources of MOG-Abs. Identification of MOG-specific B cells in blood could be of future relevance for selecting patients with MOG-Abs for B cell-directed therapy.