Brain antibody sequence evaluation (BASE): an easy-to-use software for complete data analysis in single cell immunoglobulin cloning

Background: Repertoire analysis of patient-derived recombinant monoclonal antibodies is an important tool to study the role of B cells in autoimmune diseases of the human brain and beyond. Current protocols for generation of patient-derived recombinant monoclonal antibody libraries are time-consuming and contain repetitive steps, some of which can be assisted with the help of software automation. Results: We developed BASE, an easy-to-use software for complete data analysis in single cell immunoglobulin cloning. BASE consists of two modules: aBASE for immunological annotations and cloning primer lookup, and cBASE for plasmid sequence identity confirmation before expression. Comparing automated BASE analysis with manual analysis we confirmed the validity of BASE output: identity between manual and automated aBASE analysis was 100% for all outputs, except for immunoglobulin isotype determination. In this case, aBASE yielded correct results in 96% of cases, whereas 4% of cases required manual confirmation. cBASE automatically concluded expression recommendations in 89.8% of cases, 91.8% of which were identical to manually derived results and none of them were false-positive. Conclusions: BASE offers an easy-to-use software solution suitable for complete Ig sequence data analysis and tracking during recombinant mAb cloning from single cells. Plasmid sequence identity confirmation by cBASE offers functionality not provided by existing software solutions in the field and will help to reduce time-consuming steps of the monoclonal antibody generation workflow. BASE can be installed locally or accessed online at Code Ocean

Maternal synapsin autoantibodies are associated with neurodevelopmental delay

Maternal autoantibodies can be transmitted diaplacentally, with potentially deleterious effects on neurodevelopment. Synapsin 1 (SYN1) is a neuronal protein that is important for synaptic communication and neuronal plasticity. While monoallelic loss of function (LoF) variants in the SYN1 gene result in X-linked intellectual disability (ID), learning disabilities, epilepsy, behavioral problems, and macrocephaly, the effect of SYN1 autoantibodies on neurodevelopment remains unclear. We recruited a clinical cohort of 208 mothers and their children with neurologic abnormalities and analyzed the role of maternal SYN1 autoantibodies. We identified seropositivity in 9.6% of mothers, and seropositivity was associated with an increased risk for ID and behavioral problems. Furthermore, children more frequently had epilepsy, macrocephaly, and developmental delay, in line with the SYN1 LoF phenotype. Whether SYN1 autoantibodies have a direct pathogenic effect on neurodevelopment or serve as biomarkers requires functional experiments

Human gestational N‐methyl‐d‐aspartate receptor autoantibodies impair neonatal murine brain function

Objective: Maternal autoantibodies are a risk factor for impaired brain development in offspring. Antibodies (ABs) against the NR1 (GluN1) subunit of the N-methyl-d-aspartate receptor (NMDAR) are among the most frequently diagnosed anti-neuronal surface ABs, yet little is known about effects on fetal development during pregnancy. Methods: We established a murine model of in utero exposure to human recombinant NR1 and isotype-matched nonreactive control ABs. Pregnant C57BL/6J mice were intraperitoneally injected on embryonic days 13 and 17 each with 240μg of human monoclonal ABs. Offspring were investigated for acute and chronic effects on NMDAR function, brain development, and behavior. Results: Transferred NR1 ABs enriched in the fetus and bound to synaptic structures in the fetal brain. Density of NMDAR was considerably reduced (up to -49.2%) and electrophysiological properties were altered, reflected by decreased amplitudes of spontaneous excitatory postsynaptic currents in young neonates (-34.4%). NR1 AB-treated animals displayed increased early postnatal mortality (+27.2%), impaired neurodevelopmental reflexes, altered blood pH, and reduced bodyweight. During adolescence and adulthood, animals showed hyperactivity (+27.8% median activity over 14 days), lower anxiety, and impaired sensorimotor gating. NR1 ABs caused long-lasting neuropathological effects also in aged mice (10 months), such as reduced volumes of cerebellum, midbrain, and brainstem. Interpretation: The data collectively support a model in which asymptomatic mothers can harbor low-level pathogenic human NR1 ABs that are diaplacentally transferred, causing neurotoxic effects on neonatal development. Thus, AB-mediated network changes may represent a potentially treatable neurodevelopmental congenital brain disorder contributing to lifelong neuropsychiatric morbidity in affected children

Multimodal electrophysiological analyses reveal that reduced synaptic excitatory neurotransmission underlies seizures in a model of NMDAR antibody-mediated encephalitis

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

Encephalitis patient-derived monoclonal GABAA receptor antibodies cause epileptic seizures

Autoantibodies targeting the GABAA receptor (GABAAR) hallmark an autoimmune encephalitis presenting with frequent seizures and psychomotor abnormalities. Their pathogenic role is still not well-defined, given the common overlap with further autoantibodies and the lack of patient-derived mAbs. Five GABAAR mAbs from cerebrospinal fluid cells bound to various epitopes involving the α1 and γ2 receptor subunits, with variable binding strength and partial competition. mAbs selectively reduced GABAergic currents in neuronal cultures without causing receptor internalization. Cerebroventricular infusion of GABAAR mAbs and Fab fragments into rodents induced a severe phenotype with seizures and increased mortality, reminiscent of encephalitis patients' symptoms. Our results demonstrate direct pathogenicity of autoantibodies on GABAARs independent of Fc-mediated effector functions and provide an animal model for GABAAR encephalitis. They further provide the scientific rationale for clinical treatments using antibody depletion and can serve as tools for the development of antibody-selective immunotherapies

Repertoire analysis of monoclonal antibodies in the cerebrospinal fluid from patients with anti-NMDA-Receptor-Encephalitis

Die Anti-N-Methyl-D-Aspartat-Rezeptor(NMDAR)-Enzephalitis ist die häufigste Form autoimmun vermittelter Hirnentzündungen. Die PatientInnen entwickeln typischerweise zunächst psychotische Veränderungen wie inhaltliche Denkstörungen und Halluzinationen und in der Folge neurologische Symptome, wie Bewusstseinsstörungen, Dyskinesien, epileptische Anfälle, autonome Dysregulationen und zentrale Hypoventilation. Der Nachweis von Antikörpern gegen die NR1-Untereinheit des NMDAR im Liquor gilt als wichtigstes Diagnosekriterium. Bisherige Arbeiten mit extrahiertem Gesamt-Immunglobulin aus Liquor und Serum von PatientInnen zeigten Störungen der synaptischen Funktion in vitro und in vivo. Der Einfluss weiterer möglicher Autoantikörper konnte dabei nicht ausgeschlossen werden und der Beweis für die Pathogenität NR1-spezifischer Antikörper war bisher ausstehend. Dazu generierten wir monoklonale rekombinante humane Antikörper aus Liquorproben von acht Patientinnen mit NMDAR-Enzephalitis. Wir isolierten einzelne Gedächtnis-B-Zellen sowie Plasmazellen mittels Durchflusszytometrie. Die variablen Regionen der für die schweren und leichten Immunglobulinketten kodierenden Genabschnitte amplifizierten mittels Polymerase-Kettenreaktion. Diese klonierten wir in Vektoren, die den konstanten Genabschnitt humaner Antikörper enthalten. Mit dem jeweiligen Vektorenpaar aus schwerer und leichter Immunglobulinkette co-transfizierten wir menschliche embryonale Nierenzellen (HEK-Zellen) und gewannen aus dem Kulturmedium die monoklonalen Antikörper. Sechs Prozent der generierten rekombinanten humanen Antikörper zeigten eine Reaktivität auf NR1-transfizierten HEK-Zellen, ebenso das für NR1-spezifische Antikörper typische Färbemuster auf hippocampalen Neuronen und auf Mäusehirnschnitten. Alle NR1-spezifischen Antikörper waren vom IgG-Isotyp, sie ließen sich aus Plasmazellen sowie Gedächtnis-B-Zellen isolieren. Letztere könnten als Ausgangspunkt möglicher späterer Rezidive klinisch relevant sein. Einige der NR1-spezifischen Antikörper zeigten eine klonale Expansion mit 100 Prozent identischen Klonen und wiesen wenige somatische Hypermutationen auf, was auf eine kürzlich stattgefundene periphere Immunreaktion hindeutet. Wir fanden zudem drei völlig unmutierte NR1-spezifische Antikörper („naturally occuring antibodies“), was bei diesen Patientinnen eine unvollständige Immuntoleranz gegenüber NR1 nahelegt. Auf hippocampalen Neuronen führte die Inkubation mit monoklonalen NR1-spezifischen Antikörpern zu einer Senkung der NMDAR-Dichte sowie zu einer Reduktion der NMDAR-spezifischen Ströme. Nach intravenösen Injektionen in Mäuse ließ sich eine Anreicherung der Antikörper im Hippocampus und Kleinhirn nachweisen. Für über 95 Prozent der nicht NR1-bindenden Antikörper zeigte sich eine spezifische Reaktivität auf Mäusehirnschnitten, unter anderem an neuronalen Oberflächen, an Gliazellen und an Endothel. Mit diesen Daten konnten wir nachweisen, dass monoklonale NR1-spezifische Antikörper allein neurotoxisch sind. Damit ist jeder Nachweis dieser Autoantikörper als ein Risikofaktor für neuropsychiatrische Symptome anzusehen. In zukünftigen Arbeiten gilt es zu klären, ob das Vorhandensein verschiedener NR1-spezifischer Antikörper prognostisch relevant ist und ob weitere Autoantikörper zur Pathophysiologie beitragen.The anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is the most common form of autoimmune mediated brain inflammation. Typically patients initially develop psychotic changes such as substantive thought disorders and hallucinations, followed by neurological symptoms like impaired consciousness, dyskinesia, seizures, autonomic dysregulations and central hypoventilation. The detection of antibodies against the NR1 subunit of the NMDAR in the cerebrospinal fluid (CSF) is the most important diagnostic criterion. Previous works with extracted whole immunoglobulins from patients‘ CSF and serum have shown synaptic dysfunction in vitro and in vivo. Thereby an influence of further possible autoantibodies could not be eliminated and the proof of pathogenicity of NR1-specific antibodies was pending. Therefore we generated monoclonal recombinant human antibodies from CSF samples from eight patients with NMDAR encephalitis. We isolated single memory B cells and plasma cells via flow cytometry. The variable gene regions coding the heavy and light immunoglobulin chains were amplified by polymerase chain reaction. These were cloned into vectors, containing the constant gene sequence of human antibodies. We co-transfected human embryonic kidney (HEK) cells with the respective pair of heavy and light immunoglobulin chains and harvested monoclonal antibodies from the culture medium. Six percent of the generated monoclonal recombinant human antibodies showed reactivity to NR1-transfected HEK cells, likewise the typical NR1-specific staining pattern on hippocampal neurons and mouse brain sections. All NR1-specific antibodies were from the IgG isotype and have been isolated from plasma cells as well as memory B cells. Last-mentioned ones might be clinically relevant as possible origin of future relapses. Several of the NR1-specific antibodies showed clonal expansion with 100 percent identical clones and few somatic hypermutations, indicating a recent peripheral immune reaction. Furthermore we found three completely unmutated NR1-specific antibodies („naturally occurring antibodies“), suggesting incomplete immune tolerance against NR1 in these patients. Incubating hippocampal neurons with monoclonal NR1-specific antibodies caused a reduction of the NMDAR density and NMDAR-specific currents. After intravenous injections into mice, antibody enrichment in the hippocampus and cerebellum was detected. More than 95 percent of the non- NR1-binding antibodies showed specific reactivity on mouse brain sections, amongst others to neuronal surfaces, glia cells and endothelium. With these data we proved that monoclonal NR1-specific antibodies alone are neurotoxic. Therewith, any detection of these autoantibodies needs to be considered as a risk factor for neuro-psychiatric symptoms. Future work will clarify whether the presence of different NR1-specific antibodies is of prognostic relevance and whether further autoantibodies contribute to the pathophysiology

Hodgkin Lymphoma Cell Lines and Tissues Express mGluR5: A Potential Link to Ophelia Syndrome and Paraneoplastic Neurological Disease

Ophelia syndrome is characterized by the coincidence of severe neuropsychiatric symptoms, classical Hodgkin lymphoma, and the presence of antibodies to the metabotropic glutamate 5 receptor (mGluR5). Little is known about the pathogenetic link between these symptoms and the role that anti-mGluR5-antibodies play. We investigated lymphoma tissue from patients with Ophelia syndrome and with isolated classical Hodgkin lymphoma by quantitative immunocytochemistry for mGluR5-expression. Further, we studied the L-1236, L-428, L-540, SUP-HD1, KM-H2, and HDLM-2 classical Hodgkin lymphoma cell lines by FACS and Western blot for mGluR5-expression, and by transcriptome analysis. mGluR5 surface expression differed significantly in terms of receptor density, distribution pattern, and percentage of positive cells. The highest expression levels were found in the L-1236 line. RNA-sequencing revealed more than 800 genes that were higher expressed in the L-1236 line in comparison to the other classical Hodgkin lymphoma cell lines. High mGluR5-expression was associated with upregulation of PI3K/AKT and MAPK pathways and of downstream targets (e.g., EGR1) known to be involved in classical Hodgkin lymphoma progression. Finally, mGluR5 expression was increased in the classical Hodgkin lymphoma-tissue of our Ophelia syndrome patient in contrast to five classical Hodgkin lymphoma-patients without autoimmune encephalitis. Given the association of encephalitis and classical Hodgkin lymphoma in Ophelia syndrome, it is possible that mGluR5-expression in classical Hodgkin lymphoma cells not only drives tumor progression but also triggers anti-mGluR5 encephalitis even before classical Hodgkin lymphoma becomes manifest

Human NMDAR autoantibodies disrupt excitatory-inhibitory balance, leading to hippocampal network hypersynchrony

Anti-NMDA receptor autoantibodies (NMDAR-Abs) in patients with NMDAR encephalitis cause severe dis-ease symptoms resembling psychosis and cause cognitive dysfunction. After passive transfer of patients' cerebrospinal fluid or human monoclonal anti-GluN1-autoantibodies in mice, we find a disrupted excit-atory-inhibitory balance resulting from CA1 neuronal hypoexcitability, reduced AMPA receptor (AMPAR) signaling, and faster synaptic inhibition in acute hippocampal slices. Functional alterations are also reflected in widespread remodeling of the hippocampal proteome, including changes in glutamatergic and GABAergic neurotransmission. NMDAR-Abs amplify network g oscillations and disrupt q -g coupling. A data-informed network model reveals that lower AMPAR strength and faster GABAA receptor current kinetics chiefly ac-count for these abnormal oscillations. As predicted in silico and evidenced ex vivo, positive allosteric mod-ulation of AMPARs alleviates aberrant g activity, reinforcing the causative effects of the excitatory-inhibitory imbalance. Collectively, NMDAR-Ab-induced aberrant synaptic, cellular, and network dynamics provide con-ceptual insights into NMDAR-Ab-mediated pathomechanisms and reveal promising therapeutic targets that merit future in vivo validation