ABSTRACT
BACKGROUND: The clinical heterogeneity of myasthenia gravis (MG), an autoimmune disease defined by antibodies (Ab) directed against the postsynaptic membrane, constitutes a challenge for patient stratification and treatment decision making. Novel strategies are needed to classify patients based on their biological phenotypes aiming to improve patient selection and treatment outcomes. METHODS: For this purpose, we assessed the serum proteome of a cohort of 140 patients with anti-acetylcholine receptor-Ab-positive MG and utilised consensus clustering as an unsupervised tool to assign patients to biological profiles. For in-depth analysis, we used immunogenomic sequencing to study the B cell repertoire of a subgroup of patients and an in vitro assay using primary human muscle cells to interrogate serum-induced complement formation. FINDINGS: This strategy identified four distinct patient phenotypes based on their proteomic patterns in their serum. Notably, one patient phenotype, here named PS3, was characterised by high disease severity and complement activation as defining features. Assessing a subgroup of patients, hyperexpanded antibody clones were present in the B cell repertoire of the PS3 group and effectively activated complement as compared to other patients. In line with their disease phenotype, PS3 patients were more likely to benefit from complement-inhibiting therapies. These findings were validated in a prospective cohort of 18 patients using a cell-based assay. INTERPRETATION: Collectively, this study suggests proteomics-based clustering as a gateway to assign patients to a biological signature likely to benefit from complement inhibition and provides a stratification strategy for clinical practice. FUNDING: CN and CBS were supported by the Forschungskommission of the Medical Faculty of the Heinrich Heine University Düsseldorf. CN was supported by the Else Kröner-Fresenius-Stiftung (EKEA.38). CBS was supported by the Deutsche Forschungsgemeinschaft (DFG-German Research Foundation) with a Walter Benjamin fellowship (project 539363086). The project was supported by the Ministry of Culture and Science of North Rhine-Westphalia (MODS, "Profilbildung 2020" [grant no. PROFILNRW-2020-107-A]).
Subject(s)
Autoantibodies , Myasthenia Gravis , Phenotype , Proteomics , Receptors, Cholinergic , Humans , Myasthenia Gravis/blood , Myasthenia Gravis/diagnosis , Myasthenia Gravis/immunology , Myasthenia Gravis/metabolism , Receptors, Cholinergic/immunology , Receptors, Cholinergic/metabolism , Autoantibodies/blood , Autoantibodies/immunology , Proteomics/methods , Female , Male , Middle Aged , Adult , Cluster Analysis , Proteome , Aged , B-Lymphocytes/metabolism , B-Lymphocytes/immunology , Complement ActivationABSTRACT
Kaposi sarcoma associated herpesvirus (KSHV) is associated with around 1% of all human tumors, including the B cell malignancy primary effusion lymphoma (PEL), in which co-infection with the Epstein Barr virus (EBV) can almost always be found in malignant cells. Here, we demonstrate that KSHV/EBV co-infection of mice with reconstituted human immune systems (humanized mice) leads to IgM responses against both latent and lytic KSHV antigens, and expansion of central and effector memory CD4+ and CD8+ T cells. Among these, KSHV/EBV dual-infection allows for the priming of CD8+ T cells that are specific for the lytic KSHV antigen K6 and able to kill KSHV/EBV infected B cells. This suggests that K6 may represent a vaccine antigen for the control of KSHV and its associated pathologies in high seroprevalence regions, such as Sub-Saharan Africa.
Subject(s)
B-Lymphocytes , CD8-Positive T-Lymphocytes , Herpesvirus 8, Human , Animals , Herpesvirus 8, Human/immunology , Humans , B-Lymphocytes/immunology , Mice , CD8-Positive T-Lymphocytes/immunology , Epstein-Barr Virus Infections/immunology , Epstein-Barr Virus Infections/virology , Coinfection/immunology , Coinfection/virology , CD4-Positive T-Lymphocytes/immunology , Herpesvirus 4, Human/immunology , Herpesviridae Infections/immunology , Herpesviridae Infections/virology , Immunoglobulin M/immunology , Antigens, Viral/immunology , Mice, SCID , Lymphoma, Primary Effusion/immunology , Lymphoma, Primary Effusion/virology , Antibodies, Viral/immunologyABSTRACT
Introduction: Given its wide availability and cost-effectiveness, multidimensional flow cytometry (mFC) became a core method in the field of immunology allowing for the analysis of a broad range of individual cells providing insights into cell subset composition, cellular behavior, and cell-to-cell interactions. Formerly, the analysis of mFC data solely relied on manual gating strategies. With the advent of novel computational approaches, (semi-)automated gating strategies and analysis tools complemented manual approaches. Methods: Using Bayesian network analysis, we developed a mathematical model for the dependencies of different obtained mFC markers. The algorithm creates a Bayesian network that is a HC tree when including raw, ungated mFC data of a randomly selected healthy control cohort (HC). The HC tree is used to classify whether the observed marker distribution (either patients with amyotrophic lateral sclerosis (ALS) or HC) is predicted. The relative number of cells where the probability q is equal to zero is calculated reflecting the similarity in the marker distribution between a randomly chosen mFC file (ALS or HC) and the HC tree. Results: Including peripheral blood mFC data from 68 ALS and 35 HC, the algorithm could correctly identify 64/68 ALS cases. Tuning of parameters revealed that the combination of 7 markers, 200 bins, and 20 patients achieved the highest AUC on a significance level of p < 0.0001. The markers CD4 and CD38 showed the highest zero probability. We successfully validated our approach by including a second, independent ALS and HC cohort (55 ALS and 30 HC). In this case, all ALS were correctly identified and side scatter and CD20 yielded the highest zero probability. Finally, both datasets were analyzed by the commercially available algorithm 'Citrus', which indicated superior ability of Bayesian network analysis when including raw, ungated mFC data. Discussion: Bayesian network analysis might present a novel approach for classifying mFC data, which does not rely on reduction techniques, thus, allowing to retain information on the entire dataset. Future studies will have to assess the performance when discriminating clinically relevant differential diagnoses to evaluate the complementary diagnostic benefit of Bayesian network analysis to the clinical routine workup.
Subject(s)
Amyotrophic Lateral Sclerosis , Flow Cytometry , Flow Cytometry/classification , Flow Cytometry/methods , Bayes Theorem , Algorithms , Amyotrophic Lateral Sclerosis/diagnosis , Humans , Models, Theoretical , Male , Female , Adult , Middle Aged , Aged , Aged, 80 and overABSTRACT
Alemtuzumab is a monoclonal antibody that causes rapid depletion of CD52-expressing immune cells. It has proven to be highly efficacious in active relapsing-remitting multiple sclerosis; however, the high risk of secondary autoimmune disorders has greatly complicated its use. Thus, deeper insight into the pathophysiology of secondary autoimmunity and potential biomarkers is urgently needed. The most critical time points in the decision-making process for alemtuzumab therapy are before or at Month 12, where the ability to identify secondary autoimmunity risk would be instrumental. Therefore, we investigated components of blood and CSF of up to 106 multiple sclerosis patients before and after alemtuzumab treatment focusing on those critical time points. Consistent with previous reports, deep flow cytometric immune-cell profiling (n = 30) demonstrated major effects on adaptive rather than innate immunity, which favoured regulatory immune cell subsets within the repopulation. The longitudinally studied CSF compartment (n = 18) mainly mirrored the immunological effects observed in the periphery. Alemtuzumab-induced changes including increased numbers of naïve CD4+ T cells and B cells as well as a clonal renewal of CD4+ T- and B-cell repertoires were partly reminiscent of haematopoietic stem cell transplantation; in contrast, thymopoiesis was reduced and clonal renewal of T-cell repertoires after alemtuzumab was incomplete. Stratification for secondary autoimmunity did not show clear immununological cellular or proteomic traits or signatures associated with secondary autoimmunity. However, a restricted T-cell repertoire with hyperexpanded T-cell clones at baseline, which persisted and demonstrated further expansion at Month 12 by homeostatic proliferation, identified patients developing secondary autoimmune disorders (n = 7 without secondary autoimmunity versus n = 5 with secondary autoimmunity). Those processes were followed by an expansion of memory B-cell clones irrespective of persistence, which we detected shortly after the diagnosis of secondary autoimmune disease. In conclusion, our data demonstrate that (i) peripheral immunological alterations following alemtuzumab are mirrored by longitudinal changes in the CSF; (ii) incomplete T-cell repertoire renewal and reduced thymopoiesis contribute to a proautoimmune state after alemtuzumab; (iii) proteomics and surface immunological phenotyping do not identify patients at risk for secondary autoimmune disorders; (iv) homeostatic proliferation with disparate dynamics of clonal T- and B-cell expansions are associated with secondary autoimmunity; and (v) hyperexpanded T-cell clones at baseline and Month 12 may be used as a biomarker for the risk of alemtuzumab-induced autoimmunity.
Subject(s)
Autoimmune Diseases , Autoimmunity , Alemtuzumab/adverse effects , Autoimmune Diseases/chemically induced , Humans , Phenotype , ProteomicsABSTRACT
Anti-NMDA receptor (NMDAR) encephalitis is frequently associated with demyelinating disorders (e.g., multiple sclerosis (MS), neuromyelitis optica spectrum disorder (NMOSD), myelin oligodendrocyte glycoprotein-associated disease (MOGAD)) with regard to clinical presentation, neuropathological and cerebrospinal fluid findings. Indeed, autoantibodies (AABs) against the GluN1 (NR1) subunit of the NMDAR diminish glutamatergic transmission in both neurons and oligodendrocytes, leading to a state of NMDAR hypofunction. Considering the vital role of oligodendroglial NMDAR signaling in neuron-glia communication and, in particular, in tightly regulated trophic support to neurons, the influence of GluN1 targeting on the physiology of myelinated axon may be of importance. We applied a myelinating spinal cord cell culture model that contains all major CNS cell types, to evaluate the effects of a patient-derived GluN1-specific monoclonal antibody (SSM5) on neuronal and myelin integrity. A non-brain reactive (12D7) antibody was used as the corresponding isotype control. We show that in cultures at the late stage of myelination, prolonged treatment with SSM5, but not 12D7, leads to neuronal damage. This is characterized by neurite blebbing and fragmentation, and a reduction in the number of myelinated axons. However, this significant toxic effect of SSM5 was not observed in earlier cultures at the beginning of myelination. Anti-GluN1 AABs induce neurodegenerative changes and associated myelin loss in myelinated spinal cord cultures. These findings may point to the higher vulnerability of myelinated neurons towards interference in glutamatergic communication, and may refer to the disturbance of the NMDAR-mediated oligodendrocyte metabolic supply. Our work contributes to the understanding of the emerging association of NMDAR encephalitis with demyelinating disorders.
Subject(s)
Anti-N-Methyl-D-Aspartate Receptor Encephalitis , Neuromyelitis Optica , Humans , Coculture Techniques , Receptors, N-Methyl-D-Aspartate/metabolism , Neuroglia/metabolism , Myelin-Oligodendrocyte Glycoprotein , Autoantibodies , Aquaporin 4ABSTRACT
Seizures are a prominent feature in N-Methyl-D-Aspartate receptor antibody (NMDAR antibody) encephalitis, a distinct neuro-immunological disorder in which specific human autoantibodies bind and crosslink the surface of NMDAR proteins thereby causing internalization and a state of NMDAR hypofunction. To further understand ictogenesis in this disorder, and to test a potential treatment compound, we developed an NMDAR antibody mediated rat seizure model that displays spontaneous epileptiform activity in vivo and in vitro. Using a combination of electrophysiological and dynamic causal modelling techniques we show that, contrary to expectation, reduction of synaptic excitatory, but not inhibitory, neurotransmission underlies the ictal events through alterations in the dynamical behaviour of microcircuits in brain tissue. Moreover, in vitro application of a neurosteroid, pregnenolone sulphate, that upregulates NMDARs, reduced established ictal activity. This proof-of-concept study highlights the complexity of circuit disturbances that may lead to seizures and the potential use of receptor-specific treatments in antibody-mediated seizures and epilepsy.
Subject(s)
Autoantibodies/adverse effects , Synaptic Transmission , Animals , Anti-N-Methyl-D-Aspartate Receptor Encephalitis/chemically induced , Disease Models, Animal , Male , Rats , Rats, WistarABSTRACT
OBJECTIVE: Antibodies against neuronal N-methyl-D-aspartate receptors (NMDARs) in patients with anti-NMDAR encephalitis alter neuronal synaptic function and plasticity, but the effects on other cells of the nervous system are unknown. METHODS: Cerebrospinal fluid (CSF) of patients with anti-NMDAR encephalitis (preabsorbed or not with GluN1) and a human NMDAR-specific monoclonal antibody (SSM5) derived from plasma cells of a patient, along the corresponding controls, were used in the studies. To evaluate the activity of oligodendrocyte NMDARs and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in vitro after exposure to patients' CSF antibodies or SSM5, we used a functional assay based on cytosolic Ca2+ imaging. Expression of the glucose transporter (GLUT1) in oligodendrocytes was assessed by immunocytochemistry. RESULTS: NMDAR agonist responses were robustly reduced after preincubation of oligodendrocytes with patients' CSF or SSM5 but remained largely unaltered with the corresponding controls. These effects were NMDAR specific, as patients' CSF did not alter responses to AMPA receptor agonists and was abrogated by preabsorption of CSF with HEK cells expressing GluN1 subunit. Patients' CSF also reduced oligodendrocyte expression of glucose transporter GLUT1 induced by NMDAR activity. INTERPRETATION: Antibodies from patients with anti-NMDAR encephalitis specifically alter the function of NMDARs in oligodendrocytes, causing a decrease of expression of GLUT1. Considering that normal GLUT1 expression in oligodendrocytes and myelin is needed to metabolically support axonal function, the findings suggest a link between antibody-mediated dysfunction of NMDARs in oligodendrocytes and the white matter alterations reported in patients with this disorder. ANN NEUROL 2020;87:670-676.
Subject(s)
Anti-N-Methyl-D-Aspartate Receptor Encephalitis/metabolism , Autoantibodies/immunology , Oligodendroglia/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Adolescent , Adult , Animals , Anti-N-Methyl-D-Aspartate Receptor Encephalitis/immunology , Autoantibodies/cerebrospinal fluid , Autoantibodies/pharmacology , Autoantigens/immunology , Cells, Cultured , Child , Female , Glucose Transporter Type 1/biosynthesis , Humans , Male , Oligodendroglia/drug effects , Rats , Rats, Sprague-Dawley , Receptors, N-Methyl-D-Aspartate/immunology , Young AdultABSTRACT
N-Methyl-D-aspartate (NMDA) receptors (NMDARs) are among the most important excitatory neurotransmitter receptors in the human brain. Autoantibodies to the human NMDAR cause the most frequent form of autoimmune encephalitis involving autoantibody-mediated receptor cross-linking and subsequent internalization of the antibody-receptor complex. This has been deemed to represent the predominant antibody effector mechanism depleting the NMDAR from the synaptic and extra-synaptic neuronal cell membrane. To assess in detail the molecular mechanisms of autoantibody-induced NMDAR endocytosis, vesicular trafficking, and exocytosis we transiently co-expressed rat GluN1-1a-EGFP and GluN2B-ECFP alone or together with scaffolding postsynaptic density protein 95 (PSD-95), wild-type (WT), or dominant-negative (DN) mutant Ras-related in brain (RAB) proteins (RAB5WT, RAB5DN, RAB11WT, RAB11DN) in HEK 293T cells. The cells were incubated with a pH-rhodamine-labeled human recombinant monoclonal GluN1 IgG1 autoantibody (GluN1-aAbpH-rhod) genetically engineered from clonally expanded intrathecal plasma cells from a patient with anti-NMDAR encephalitis, and the pH-rhodamine fluorescence was tracked over time. We show that due to the acidic luminal pH, internalization of the NMDAR-autoantibody complex into endosomes and lysosomes increases the pH-rhodamine fluorescence. The increase in fluorescence allows for mechanistic assessment of endocytosis, vesicular trafficking in these vesicular compartments, and exocytosis of the NMDAR-autoantibody complex under steady state conditions. Using this method, we demonstrate a role for PSD-95 in stabilization of NMDARs in the cell membrane in the presence of GluN1-aAbpH-rhod, while RAB proteins did not exert a significant effect on vertical trafficking of the internalized NMDAR autoantibody complex in this heterologous expression system. This novel assay allows to unravel molecular mechanisms of autoantibody-induced receptor internalization and to study novel small-scale specific molecular-based therapies for autoimmune encephalitis syndromes.
ABSTRACT
Objective: Autoimmune encephalitis is most frequently associated with anti-NMDAR autoantibodies. Their pathogenic relevance has been suggested by passive transfer of patients' cerebrospinal fluid (CSF) in mice in vivo. We aimed to analyze the intrathecal plasma cell repertoire, identify autoantibody-producing clones, and characterize their antibody signatures in recombinant form. Methods: Patients with recent onset typical anti-NMDAR encephalitis were subjected to flow cytometry analysis of the peripheral and intrathecal immune response before, during, and after immunotherapy. Recombinant human monoclonal antibodies (rhuMab) were cloned and expressed from matching immunoglobulin heavy- (IgH) and light-chain (IgL) amplicons of clonally expanded intrathecal plasma cells (cePc) and tested for their pathogenic relevance. Results: Intrathecal accumulation of B and plasma cells corresponded to the clinical course. The presence of cePc with hypermutated antigen receptors indicated an antigen-driven intrathecal immune response. Consistently, a single recombinant human GluN1-specific monoclonal antibody, rebuilt from intrathecal cePc, was sufficient to reproduce NMDAR epitope specificity in vitro. After intraventricular infusion in mice, it accumulated in the hippocampus, decreased synaptic NMDAR density, and caused severe reversible memory impairment, a key pathogenic feature of the human disease, in vivo. Interpretation: A CNS-specific humoral immune response is present in anti-NMDAR encephalitis specifically targeting the GluN1 subunit of the NMDAR. Using reverse genetics, we recovered the typical intrathecal antibody signature in recombinant form, and proved its pathogenic relevance by passive transfer of disease symptoms from man to mouse, providing the critical link between intrathecal immune response and the pathogenesis of anti-NMDAR encephalitis as a humorally mediated autoimmune disease.
ABSTRACT
OBJECTIVE: Intravenous immunoglobulin (IVIG) is an established treatment for numerous autoimmune conditions. Clinical trials of IVIG for multiple sclerosis, using diverse dose regimens, yielded controversial results. The aim of this study is to dissect IVIG effector mechanisms on demyelination in an ex vivo model of the central nervous system (CNS)-immune interface. METHODS: Using organotypic cerebellar slice cultures (OSC) from transgenic mice expressing green fluorescent protein (GFP) in oligodendrocytes/myelin, we induced extensive immune-mediated demyelination and oligodendrocyte loss with an antibody specific for myelin oligodendrocyte glycoprotein (MOG) and complement. Protective IVIG effects were assessed by live imaging of GFP expression, confocal microscopy, immunohistochemistry, gene expression analysis and flow cytometry. RESULTS: IVIG protected OSC from demyelination in a dose-dependent manner, which was at least partly attributed to interference with complement-mediated oligodendroglia damage, while binding of the anti-MOG antibody was not prevented. Staining with anti-CD68 antibodies and flow cytometry confirmed that IVIG prevented microglia activation and oligodendrocyte death, respectively. Equimolar IVIG-derived Fab fragments or monoclonal IgG did not protect OSC, while Fc fragments derived from a polyclonal mixture of human IgG were at least as potent as intact IVIG. INTERPRETATION: Both intact IVIG and Fc fragments exert a dose-dependent protective effect on antibody-mediated CNS demyelination and microglia activation by interfering with the complement cascade and, presumably, interacting with local immune cells. Although this experimental model lacks blood-brain barrier and peripheral immune components, our findings warrant further studies on optimal dose finding and alternative modes of application to enhance local IVIG concentrations at the site of tissue damage.