Early B cell tolerance defects in neuromyelitis optica favour anti-AQP4 autoantibody production.

Neuromyelitis optica spectrum disorders (NMOSD) constitute rare autoimmune disorders of the CNS that are primarily characterized by severe inflammation of the spinal cord and optic nerve. Approximately 75% of NMOSD patients harbour circulating pathogenic autoantibodies targeting the aquaporin-4 water channel (AQP4). The source of these autoantibodies remains unclear, but parallels between NMOSD and other autoantibody-mediated diseases posit compromised B cell tolerance checkpoints as common underlying and contributing factors. Using a well established assay, we assessed tolerance fidelity by creating recombinant antibodies from B cell populations directly downstream of each checkpoint and testing them for polyreactivity and autoreactivity. We examined a total of 863 recombinant antibodies. Those derived from three anti-AQP4-IgG seropositive NMOSD patients (n = 130) were compared to 733 antibodies from 15 healthy donors. We found significantly higher frequencies of poly- and autoreactive new emigrant/transitional and mature naïve B cells in NMOSD patients compared to healthy donors (P-values < 0.003), thereby identifying defects in both central and peripheral B cell tolerance checkpoints in these patients. We next explored whether pathogenic NMOSD anti-AQP4 autoantibodies can originate from the pool of poly- and autoreactive clones that populate the naïve B cell compartment of NMOSD patients. Six human anti-AQP4 autoantibodies that acquired somatic mutations were reverted back to their unmutated germline precursors, which were tested for both binding to AQP4 and poly- or autoreactivity. While the affinity of mature autoantibodies against AQP4 ranged from modest to strong (Kd 15.2-559 nM), none of the germline revertants displayed any detectable binding to AQP4, revealing that somatic hypermutation is required for the generation of anti-AQP4 autoantibodies. However, two (33.3%) germline autoantibody revertants were polyreactive and four (66.7%) were autoreactive, suggesting that pathogenic anti-AQP4 autoantibodies can originate from the pool of autoreactive naïve B cells, which develops as a consequence of impaired early B cell tolerance checkpoints in NMOSD patients.

Determining the Spatial Relationship of Membrane-Bound Aquaporin-4 Autoantibodies by STED Nanoscopy.

Determining the spatial relationship of individual proteins in dense assemblies remains a challenge for superresolution nanoscopy. The organization of aquaporin-4 (AQP4) into large plasma membrane assemblies provides an opportunity to image membrane-bound AQP4 antibodies (AQP4-IgG) and evaluate changes in their spatial distribution due to alterations in AQP4 isoform expression and AQP4-IgG epitope specificity. Using stimulated emission depletion nanoscopy, we imaged secondary antibody labeling of monoclonal AQP4-IgGs with differing epitope specificity bound to isolated tetramers (M1-AQP4) and large orthogonal arrays of AQP4 (M23-AQP4). Imaging secondary antibodies bound to M1-AQP4 allowed us to infer the size of individual AQP4-IgG binding events. This information was used to model the assembly of larger AQP4-IgG complexes on M23-AQP4 arrays. A scoring algorithm was generated from these models to characterize the spatial arrangement of bound AQP4-IgG antibodies, yielding multiple epitope-specific patterns of bound antibodies on M23-AQP4 arrays. Our results delineate an approach to infer spatial relationships within protein arrays using stimulated emission depletion nanoscopy, offering insight into how information on single antibody fluorescence events can be used to extract information from dense protein assemblies under a biologic context.

Antibodies produced by clonally expanded plasma cells in multiple sclerosis cerebrospinal fluid cause demyelination of spinal cord explants.

B cells are implicated in the etiology of multiple sclerosis (MS). Intrathecal IgG synthesis, cerebrospinal fluid (CSF) oligoclonal bands and lesional IgG deposition suggest a role for antibody-mediated pathology. We examined the binding of IgG1 monoclonal recombinant antibodies (rAbs) derived from MS patient CSF expanded B cell clones to central nervous system (CNS) tissue. MS rAbs displaying CNS binding to mouse and human CNS tissue were further tested for their ability to induce complement-mediated tissue injury in ex vivo spinal cord explant cultures. The staining of CNS tissue, primary human astrocytes and human neurons revealed a measurable bias in MS rAb binding to antigens preferentially expressed on astrocytes and neurons. MS rAbs that recognize myelin-enriched antigens were rarely detected. Both myelin-specific and some astrocyte/neuronal-specific MS rAbs caused significant myelin loss and astrocyte activation when applied to spinal cord explant cultures in the presence of complement. Overall, the intrathecal B cell response in multiple sclerosis binds to both glial and neuronal targets and produces demyelination in spinal cord explant cultures implicating intrathecal IgG in MS pathogenesis.

The treatment of neuromyelitis optica.

Neuromyelitis optica (NMO) is an autoimmune disorder of the central nervous system directed against astrocytes. Initially diagnosed in individuals with monophasic or relapsing optic neuritis and transverse myelitis, NMO is now recognized as a demyelinating disorder with pleiotropic presentations due to the identification of a specific autoantibody response against the astrocyte water channel aquaporin-4 in the majority of individuals. As visual impairment and neurologic dysfunction in NMO are commonly severe, aggressive treatment of relapses and prophylactic immunomodulatory therapy are the focus of treatment. Although there are no approved treatments for NMO, medications and therapeutic interventions for acute and chronic treatment have been the subject of retrospective study and case reports. The goal of this review is to familiarize the reader with biologic and clinical data supporting current treatments in NMO and highlight future strategies based on advancements in our understanding of NMO pathogenesis.

Magnetic Resonance Imaging Characteristics of LGI1-Antibody and CASPR2-Antibody Encephalitis.

Importance: Rapid and accurate diagnosis of autoimmune encephalitis encourages prompt initiation of immunotherapy toward improved patient outcomes. However, clinical features alone may not sufficiently narrow the differential diagnosis, and awaiting autoantibody results can delay immunotherapy. Objective: To identify simple magnetic resonance imaging (MRI) characteristics that accurately distinguish 2 common forms of autoimmune encephalitis, LGI1- and CASPR2-antibody encephalitis (LGI1/CASPR2-Ab-E), from 2 major differential diagnoses, viral encephalitis (VE) and Creutzfeldt-Jakob disease (CJD). Design, Setting, and Participants: This cross-sectional study involved a retrospective, blinded analysis of the first available brain MRIs (taken 2000-2022) from 192 patients at Oxford University Hospitals in the UK and Mayo Clinic in the US. These patients had LGI1/CASPR2-Ab-E, VE, or CJD as evaluated by 2 neuroradiologists (discovery cohort; n = 87); findings were validated in an independent cohort by 3 neurologists (n = 105). Groups were statistically compared with contingency tables. Data were analyzed in 2023. Main Outcomes and Measures: MRI findings including T2 or fluid-attenuated inversion recovery (FLAIR) hyperintensities, swelling or volume loss, presence of gadolinium contrast enhancement, and diffusion-weighted imaging changes. Correlations with clinical features. Results: Among 192 participants with MRIs reviewed, 71 were female (37%) and 121 were male (63%); the median age was 66 years (range, 19-92 years). By comparison with VE and CJD, in LGI1/CASPR2-Ab-E, T2 and/or FLAIR hyperintensities were less likely to extend outside the temporal lobe (3/42 patients [7%] vs 17/18 patients [94%] with VE; P < .001, and 3/4 patients [75%] with CJD; P = .005), less frequently exhibited swelling (12/55 [22%] with LGI1/CASPR2-Ab-E vs 13/22 [59%] with VE; P = .003), and showed no diffusion restriction (0 patients vs 16/22 [73%] with VE and 8/10 [80%] with CJD; both P < .001) and rare contrast enhancement (1/20 [5%] vs 7/17 [41%] with VE; P = .01). These findings were validated in an independent cohort and generated an area under the curve of 0.97, sensitivity of 90%, and specificity of 95% among cases with T2/FLAIR hyperintensity in the hippocampus and/or amygdala. Conclusions and Relevance: In this study, T2 and/or FLAIR hyperintensities confined to the temporal lobes, without diffusion restriction or contrast enhancement, robustly distinguished LGI1/CASPR2-Ab-E from key differential diagnoses. These observations should assist clinical decision-making toward expediting immunotherapy. Their generalizability to other forms of autoimmune encephalitis and VE should be examined in future studies.

Regulation of neural progenitor cell fate by anandamide.

Exogenous application of neural progenitor cells (NPCs) has successful implications in treating brain disorders, and research is beginning to identify ways to mimic this exogenous application by activating endogenous stem cell compartments. The recent discovery of a functional endocannabinoid system in murine NPCs (mNPCs) represents one potential therapeutic means to influence endogenous stem cell compartments. High levels of the endogenous cannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) persist during CNS inflammation and infection. The goal of this study was to assess the influence of AEA on mNPCs to identify how the endocannabinoid system influences mNPCs in vitro, a potential model to investigate effects of endocannabinoids on endogenous stem cell compartments. Our results show that AEA affects mNPC cell fate determination. Initial glial differentiation was observed, followed by induction of neuronal differentiation with AEA treatment. Cell survival and apoptosis was not affected by AEA. These effects were coupled by an increased phosphorylation of cAMP-responsive element (CRE) binding protein (CREB).

The effect of interferon-beta on mouse neural progenitor cell survival and differentiation.

Interferon-beta (IFN-beta) is a mainstay therapy for relapse-remitting multiple sclerosis (MS). However, the direct effects of IFN-beta on the central nervous system (CNS) are not well understood. To determine whether IFN-beta has direct neuroprotective effects on CNS cells, we treated adult mouse neural progenitor cells (NPCs) in vitro with IFN-beta and examined the effects on proliferation, apoptosis, and differentiation. We found that mouse NPCs express high levels of IFNalpha/beta receptor (IFNAR). In response to IFN-beta treatment, no effect was observed on differentiation or proliferation. However, IFN-beta treated mouse NPCs demonstrated decreased apoptosis upon growth factor withdrawal. Pathway-specific polymerase chain reaction (PCR) arrays demonstrated that IFN-beta treatment upregulated the STAT 1 and 2 signaling pathway, as well as GFRA2, NOD1, Caspases 1 and 12, and TNFSF10. These results suggest that IFN-beta can directly affect NPC survival, possibly playing a neuroprotective role in the CNS by modulating neurotrophic factors.

Membrane assembly of aquaporin-4 autoantibodies regulates classical complement activation in neuromyelitis optica.

Neuromyelitis optica (NMO) is an autoimmune CNS disorder mediated by pathogenic aquaporin-4 (AQP4) water channel autoantibodies (AQP4-IgG). Although AQP4-IgG-driven complement-dependent cytotoxicity (CDC) is critical for the formation of NMO lesions, the molecular mechanisms governing optimal classical pathway activation are unknown. We investigated the molecular determinants driving CDC in NMO using recombinant AQP4-specific autoantibodies (AQP4 rAbs) derived from affected patients. We identified a group of AQP4 rAbs targeting a distinct extracellular loop C epitope that demonstrated enhanced CDC on target cells. Targeted mutations of AQP4 rAb Fc domains that enhance or diminish C1q binding or antibody Fc-Fc interactions showed that optimal CDC was driven by the assembly of multimeric rAb platforms that increase multivalent C1q binding and facilitate C1q activation. A peptide that blocks antibody Fc-Fc interaction inhibited CDC induced by AQP4 rAbs and polyclonal NMO patient sera. Super-resolution microscopy revealed that AQP4 rAbs with enhanced CDC preferentially formed organized clusters on supramolecular AQP4 orthogonal arrays, linking epitope-dependent multimeric assembly with enhanced C1q binding and activation. The resulting model of AQP4-IgG CDC provides a framework for understanding classical complement activation in human autoantibody-mediated disorders and identifies a potential new therapeutic avenue for treating NMO.

Mutations of Recombinant Aquaporin-4 Antibody in the Fc Domain Can Impair Complement-Dependent Cellular Cytotoxicity and Transplacental Transport.

Maternal antibodies provide protection for the developing fetus. Transplacental transport of pathogenic autoantibodies might pose a risk for the developing fetus. The transport of antibodies across the placenta to the fetal circulation occurs through the neonatal Fc salvage receptor (FcRn). During gestation, maternal autoantibodies are able to penetrate the embryonic brain before a functional intact blood-brain barrier is established. Brain-reactive antibodies to the water channel protein aquaporin-4 (AQP4) are a hallmark finding in neuromyelitis optica (NMO), a neurological disease that predominantly affects women, many of whom are of childbearing age. AQP4-IgG mediate astrocytic injury in a complement-dependent fashion. Recent studies suggest these antibodies contribute to impaired pregnancy outcome. The aim of the study was to investigate the transplacental transport as well as FcRn binding of a monoclonal AQP4-IgG cloned from an NMO patient (wild-type antibody) compared to five different mutated Fc domain of this antibody containing single amino acid substitutions in the Fc region. All of the Fc-mutated antibodies lack complement-dependent cytotoxicity. Four of the five Fc-mutated antibodies showed limited transplacental transport in vivo. Three mutated Fc with impaired transplacental transport showed persistent binding to rodent FcRn at pH 6 but also at pH 7.2, suggesting that limited transplacental transport could be due to diminished release from FcRn. One mutated Fc with modestly limited transplacental transport showed diminished binding to FcRn at pH 6. This study suggests that mutated Fc with intact transplacental transport may be used to study antibody effector functions and Fc with limited transport may be used as a carrier to deliver therapies to pregnant woman, while sparing the developing fetus.

Optical coherence tomography and T cell gene expression analysis in patients with benign multiple sclerosis.

Benign multiple sclerosis is a retrospective diagnosis based primarily on a lack of motor symptom progression. Recent findings that suggest patients with benign multiple sclerosis experience non-motor symptoms highlight the need for a more prospective means to diagnose benign multiple sclerosis early in order to help direct patient care. In this study, we present optical coherence tomography and T cell neurotrophin gene analysis findings in a small number of patients with benign multiple sclerosis. Our results demonstrated that retinal nerve fiber layer was mildly thinned, and T cells had a distinct gene expression profile that included upregulation of interleukin 10 and leukemia inhibitory factor, downregulation of interleukin 6 and neurotensin high affinity receptor 1 (a novel neurotrophin receptor). These findings add evidence for further investigation into optical coherence tomography and mRNA profiling in larger cohorts as a potential means to diagnose benign multiple sclerosis in a more prospective manner.

IFN-ß alters neurotrophic factor expression in T cells isolated from multiple sclerosis patients - implication of novel neurotensin/NTSR1 pathway in neuroprotection.

Inflammation in relapsing remitting multiple sclerosis (RRMS) is hypothesized to provide neuroprotective effects via altered cytokine/neurotrophin homeostasis. The distinct neurotrophin production from specific cell populations has not been systematically studied and is likely of high yield in understanding the complex regulation of MS pathogenesis. Here, we describe how the mainstream therapy interferon-ß (IFN-ß) modulates neurotrophin expression in T cells isolated from RRMS patients and characterize the neuroprotective capabilities of these factors. We utilize SuperArray gene screen technology to investigate the neurotrophin expression profile of T cells. We demonstrate that IFN-ß induces an anti-inflammatory cytokine expression pattern in T cells. Additionally, IFN-ß upregulates the expression of a novel neurotrophin receptor, the neurotensin high affinity receptor 1 (NTSR1). NTSR1 is expressed in active demyelinating lesions. Furthermore, we demonstrate that the receptor agonist neurotensin is a potent inducer of human neural stem/progenitor cell survival. Our findings highlight the importance of neurotrophin receptors in RRMS and offer insight into disease pathogenesis as well as the mechanisms of action of IFN-ß.

Interferon ß-1b directly modulates human neural stem/progenitor cell fate.

Interferon beta (IFN-ß) is a mainline treatment for multiple sclerosis (MS); however its exact mechanism of action is not completely understood. IFN-ß is known as an immunomodulator; although recent evidence suggests that IFN-ß may also act directly on neural stem/progenitor cells (NPCs) in the central nervous system (CNS). NPCs can differentiate into all neural lineage cells, which could contribute to the remyelination and repair of MS lesions. Understanding how IFN-ß influences NPC physiology is critical to develop more specific therapies that can better assist this repair process. In this study, we investigated the effects of IFN ß-1b (Betaseron®) on human NPCs in vitro (hNPCs). Our data demonstrate a dose-dependent response of hNPCs to IFN ß-1b treatment via sustained proliferation and differentiation. Furthermore, we offer insight into the signaling pathways involved in these mechanisms. Overall, this study shows a direct effect of IFN ß-1b on hNPCs and highlights the need to further understand how current MS treatments can modulate endogenous NPC populations within the CNS.

The cortistatin gene PSS2 rather than the somatostatin gene PSS1 is strongly expressed in developing avian autonomic neurons.

Somatostatin and cortistatin are neuromodulators with divergent expression patterns and biological roles. Whereas expression and function of genes encoding somatostatin (PSS1) and the related peptide cortistatin (PSS2) have been studied in detail for the central nervous system (CNS) and immune system, relatively little is known about their expression patterns in the peripheral nervous system (PNS). We compare the expression patterns of PSS1 and PSS2 in chicken embryos. At E14, PSS1 is higher in the CNS versus PNS, whereas PSS2 is higher in the PNS. During early development, PSS1 is transiently expressed in lumbar sympathetic ganglia and is detectable at low levels throughout the development of dorsal root and ciliary ganglia. In contrast, PSS2 expression increases as development progresses in sympathetic and dorsal root ganglia, whereas levels in ciliary ganglia by E8 are more than 100-fold higher than in sympathetic ganglia. Activin, which induces somatostatin-like immunoreactivity in ciliary ganglion neurons in vivo and in vitro, controls PSS2 expression by stabilizing PSS2 but not PSS1 mRNA. We conclude that much of the somatostatin-like immunoreactivity in the developing avian peripheral nervous system is actually cortistatin, the PSS2 product, as opposed to true somatostatin, which is the PSS1 product. The identification of PSS2 as the predominantly expressed somatostatin gene family member in avian autonomic neurons provides a molecular basis for further functional and pharmacological studies.