Модулятор течения VRAC 1 Потенциальная цель в множественных склерозах.

Introduction

Multiple sclerosis (MS) is a chronic immune-mediated demyelinating disease of the CNS. 1 It is the most common cause of nontraumatic disability in young adults and has a high socioeconomic burden. 2 The heterogeneity in the clinical presentation of the disease and the complexity of its underlying immunopathology have made it difficult to dissect the exact causes of MS and develop effective long-term treatments in patients. 3

Currently available treatment options include either immunosuppressive or immunomodulatory drugs, such as inhibitors of lymphocyte migration into the CNS 4 or monoclonal antibodies (mAbs). One such mAb is alemtuzumab, a humanized anti-CD52 antibody, which not only results in rapid depletion of circulating T cells but also leads to radical and prolonged alterations in the B-cell compartment of patients with MS. 5 , 6

Furthermore, unlike other autoimmune diseases such as myasthenia gravis and rheumatoid arthritis, which are typically characterized by an immune response to a defined set of antigens, 7 , 8 the search for disease-specific autoantigens in MS is still an ongoing endeavor. 9 Some of the potential autoantigens in MS that have been extensively discussed include myelin basic protein, 10 proteolipid protein, 10 myelin oligodendrocyte glycoprotein, 11 and the potassium channel KIR4.1. 12 More recently, the cell adhesion molecule GlialCAM, primarily expressed on astrocytes, was identified as another possible target antigen in MS, with GlialCAM exhibiting high-affinity molecular mimicry with EBV nuclear antigen (EBNA)1. 13 Furthermore, elevated anti-EBNA1 antibodies have been shown to be associated with an increased risk of MS, with Epstein-Barr virus (EBV) being identified as a possible causal agent for MS. 14 However, despite the identification of the different (auto)antigens in MS as those mentioned above, other studies have either not been able to reproduce these findings 15 , 16 or demonstrated that the antibody response in MS is polyspecific and changes over the course of the disease. 17

The aim of this study was to further explore the specificity of the humoral immune response in patients with MS, combining polyclonal stimulation of peripheral blood mononuclear cells (PBMCs) with a human proteome antibody microarray platform followed by a customized software analysis tool.

Methods

Participant Samples and Stratification for Protein Microarray Analysis

Peripheral blood was collected from 20 patients with MS recruited from 4 German centers (the University Hospital Erlangen, the Klinikum St. Marien Amberg, the University Hospital Münster, and Practices for Neurology in Nürnberg and Bamberg) and 9 age-matched and sex-matched healthy controls (HCs) recruited from the Institute of Anatomy and Cell Biology of Friedrich-Alexander-Universität (FAU) Erlangen-Nürnberg. Participants were required to be 18–60 years of age, and all those with MS fulfilled the McDonald criteria for the diagnosis of MS. 18 Of the 20 patients with MS, 19 had relapsing-remitting MS (RRMS) and 1 had primary progressive MS (PPMS). The MS cohort was further divided into 10 patients receiving active therapy with alemtuzumab and 10 patients who were either untreated or receiving disease-modifying treatment other than B-cell–depleting agents. HCs were characterized by the absence of any autoimmune or neurologic disease. The study was approved by the ethics committee of FAU Erlangen-Nürnberg (files 136_18 B and 185_18 B). Written informed consent was obtained from all participants.

ELISPOT Assay

Polyclonally stimulated PBMCs from the protein microarray analysis cohort (N = 10 patients with MS who were either untreated or receiving disease-modifying treatment other than B-cell–depleting agents, N = 10 patients with MS under alemtuzumab therapy, and N = 9 HCs) were tested for reactivity against whole human brain tissue lysate using B-cell enzyme-linked immunospot (ELISPOT) as previously described. 19 Details are provided in eAppendix 1.

Human Proteome-Wide Protein Microarrays

Frozen supernatants from polyclonally stimulated B cells were delivered to Cambridge Protein Arrays Ltd. (Cambridge, United Kingdom). Samples were analyzed using HuProt v4.0 Human Proteome Microarray (CDI Laboratories, Puerto Rico) containing human full-length recombinant proteins, representing more than 16,000 different genes covering approximately 80% of the annotated human protein-coding genome. 20 Although it is not possible to state with certainty whether the proteins are in linear or in the correctly folded conformation, it has been reported that both conformational- as well as linear-epitope recognizing antibodies can be detected for the arrayed proteins. 21 Further information and the tools used for analyzing the protein microarray can be found in eAppendix 1.

Detection of Total IgG in Supernatant, Serum, and CSF

The concentration of total IgG in cell culture supernatants, serum, and CSF was measured using the Human IgG (Total) ELISA Kit (Invitrogen, #BMS2091). Samples were diluted 1:50 for supernatants, 1:1,000 for CSF, and 1:500,000 for serum.

Target Antigen Verification by Enzyme-Linked Immunosorbent Assay

To validate antibody reactivity against selected target antigens, enzyme-linked immunosorbent assay (ELISA) was first performed on supernatants collected after polyclonal stimulation of PBMCs (validation cohort 1). A total of 36 patients with MS (RRMS: N = 23; secondary progressive MS with disease activity: N = 6; PPMS: N = 7; all recruited from the Klinikum St. Marien Amberg) and 25 HCs (N = 17 from Cellular Technology Limited's ePBMC library and N = 9 from the Institute of Neuroanatomy, University Hospital Bonn, and the Institute of Anatomy and Cell Biology, FAU Erlangen-Nürnberg) were included. Approval was obtained from the ethics committees of FAU Erlangen-Nürnberg (files 74_18 B and 185_18 B) and the Medical Faculty of the University of Bonn (files 378/21 and 022/22). The total IgG concentration was adjusted to 0.5 µg/mL in each sample. Reactivity was tested against modulator of VRAC current 1 (MLC1), a mixture of different EBV antigens, including viral capsid antigen, EBNA, and early antigen diffuse component, and also against cluster of differentiation 59 (CD59) as an additional antigen to validate the protein microarray results. EBV was included as a positive control antigen to ensure low ELISA plate variability between the different runs, given that >99% of patients with MS have peripheral titers of EBV antibodies. 13

To determine whether MLC1-specific, CD59-specific, and EBV-specific antibodies were also present in the CSF, we performed ELISA on matched serum and CSF samples from 2 independent cohorts (validation cohorts 2 and 3). Cohort 2 comprised 11 patients with MS in remission, 10 patients with MS during relapse, and 20 patients with noninflammatory neurologic diseases (NINDs). Samples were provided by the University Hospital Erlangen (approved by the ethics committee of FAU Erlangen-Nürnberg; files 484_20 B and 24-62-Bp). Cohort 3 comprised 10 patients with MS during relapse, 10 patients with NINDs, and 9 patients with other inflammatory neurologic diseases (OINDs). Samples were provided by the University Hospital of Basel (approved by the ethics committee of the University of Basel; files EKNZ 2021-01858 and EKNZ 332/06). All samples were adjusted to a total IgG concentration of 0.5 µg/mL.

All patients with MS from whom B-cell culture supernatants were collected tested positive for brain lysate in B-cell ELISPOT assays (eAppendix 1). All CSF samples that had been obtained from patients with MS were characterized by the presence of oligoclonal bands. The ELISA protocol is provided in eAppendix 1.

Immunofluorescence Staining and Confocal Imaging

Paraffin-embedded brain tissue sections from patients with MS were obtained from the Department of Pathology, Amsterdam University Medical Center, in cooperation with the Netherlands Brain Bank, with approval of the medical ethics committee of Amsterdam University Medical Center. All participants or next of kin had given informed consent for autopsy and the use of tissues for research purposes.

Immunohistochemistry on human brain tissue was performed as previously described. 22 The protocol and the details of the antibodies used can be found in eAppendix 1.

Images were captured on an A1-HD25 inverted confocal microscope (Nikon, Japan) equipped with a 40× water immersion objective and an A1 large-field-of-view camera (Nikon, Japan). Fluorescence channels of each image were captured sequentially. 3 to 5 Z slices spaced 1.5–2.5 µm apart were captured and Z-projected to generate the final image.

Injection of MLC1-Specific Antibodies Into Mice With Experimental Autoimmune Encephalomyelitis

A total of 14 female wild-type C57BL/6J mice (Charles River, Sulzfeld, Germany) were kept under specific pathogen-free conditions at the Haus für Experimentelle Therapie 4 of the University Hospital Bonn, with a 12-hour light/dark cycle and free access to standard rodent diet and water. All experiments were approved by the Landesamt für Natur-, Umwelt-, und Verbraucherschutz Nordrhein-Westfalen (LANUV; file 81-02.04.2021.A146) and performed according to the criteria of the German Animal Welfare Law. They also complied with the “Principles of Laboratory Animal Care” (NIH publication no. 86-23, revised 1985), the 3R principle, and the Animal Research: Reporting of In Vivo Experiments guidelines. At an age of 13 weeks, 13 mice were immunized with MOG 35-55 (Hooke Laboratories, #EK-2110) and 1 mouse remained nonimmunized as a control. The weight of the mice and clinical signs of experimental autoimmune encephalomyelitis (EAE) were monitored daily using the following scale: 0 = no symptoms; 1 = paralysis of the tail; 2 = complete paralysis of the tail and weakness of the hind limbs; 3 = complete paralysis of the tail and hind limbs; 4 = quadriplegia; 5 = death.

At the peak of EAE severity, 7 mice received 500 µg of polyclonal rabbit anti-mouse MLC1 antibody (Cusabio, #CSB-PA837739XA01MO) as an intraperitoneal injection and 6 mice received 500 µg of rabbit IgG (Cusabio, #CSB-NP001501Rb-10). Of note, 4 of the 7 mice that were treated with anti-mouse MLC1 antibody died within 24 hours. The remaining mice were killed using CO 2 72 hours later; blood was collected for ELISA, and the mice were perfused transcardially with phosphate-buffered saline for 2 minutes, followed by 4% (w/v) paraformaldehyde for 15 minutes. Brains were removed, postfixed in 4% paraformaldehyde overnight, and embedded in paraffin for histologic analysis. The amount of circulating MLC1-specific antibody was determined by ELISA following the protocol described above. Differences were that Nunc MaxiSorp 96-well plates (Thermo Fisher, #11530627) were used, serum samples were diluted 1:1,000, and biotinylated goat anti-rabbit IgG H&L (Abcam, #ab6720) served as a secondary antibody at 1:50,000 dilution. Plates were read after 30 minutes of development. To investigate in vivo deposition of anti-MLC1 antibody in murine CNS tissue, immunohistochemical staining was performed. The protocol is provided in eAppendix 1.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism version 9.5.1 (GraphPad Software, Inc.). For human protein microarray analysis, a multiple Mann-Whitney U test was used, with a false discovery rate of 0.01 using the Benjamini-Hochberg procedure. For the analysis of ELISA data, the Welch t test was used for normally distributed data points (mouse ELISA) and a Kruskal-Wallis test was applied for measurements that did not follow normal distribution (human ELISA). EAE scores before and after antibody treatment were compared using 2-way analysis of variance while total IgG and protein-specific fluorescence (PSF) values were analyzed using 1-way analysis of variance with the Kruskal-Wallis post hoc test. Nonparametric Spearman correlation was used to compare PSF values from 3 samples that were used in 1:3 dilution with samples from the same patients adjusted to a total IgG concentration of 0.5 µg/mL. For the multiple Mann-Whitney U test, significance was set at q < 0.01; for all other tests, p < 0.05 was considered statistically significant. All tests were 2-sided.

Study Approval

Studies involving human participants were approved by the ethics committees of FAU Erlangen-Nürnberg (files 74_18 B, 136_18 B, 185_18 B, 484_20 B, and 24-62-Bp), the Medical Faculty of the University of Bonn (files 378/21 and 022/22), and the University of Basel (files EKNZ 2021-01858 and EKNZ 332/06). Paraffin-embedded brain tissue sections from patients with MS were obtained from the Department of Pathology, Amsterdam University Medical Center, in co-operation with the Netherlands Brain Bank, with approval of the medical ethics committee of Amsterdam University Medical Center. All participants gave written informed consent before participation. Animal studies were approved by the Landesamt für Natur-, Umwelt-, und Verbraucherschutz Nordrhein-Westfalen (LANUV; file 81-02.04.2021.A146).

Data Availability

The data that support the findings of this study are available from the corresponding author, on reasonable request.

Results

Polyclonal B-Cell Stimulation and Protein Microarray in Combination Reveal Potential MS Target Antigens

PBMCs isolated from 20 patients with MS and 9 age-matched and sex-matched HCs were subjected to polyclonal B-cell stimulation, and cell culture supernatants were collected. Clinical details of all participants are given in the Table . None of the 9 HC donors showed reactivity against whole human brain lysate in a B-cell ELISPOT assay; however, all 10 patients with MS who were not under B-cell depletion therapy showed a positive response ( Figure 1A ), suggesting the presence of CNS-specific memory B cells in these patients. Samples were considered to be positive when the mean spot number was >4.5, as previously described by our group. 19 In addition, 10 patients with MS treated with alemtuzumab, an anti-CD52 mAb depleting both B cells and T cells, 5 were included as controls. None of these 10 patients harbored CNS antigen-specific memory B cells in the blood as determined by ELISPOT, indicating successful depletion of autoreactive B cells by alemtuzumab ( Figure 1A ).

Table Demographic and Clinical Characteristics of the Study Groups at the Time Point of Testing Group/ID Age, y Sex Time since diagnosis, y EDSS score DMT Group characteristics Healthy control (HC) participants  1-1 50 F — — — M:F ratio 0.5:1 Mean age (SD), y: 40.78 (11.22)  1-2 55 F — — —  1-3 43 F — — —  1-4 26 M — — —  1-5 33 M — — —  1-6 24 F — — —  1-7 53 F — — —  1-8 40 F — — —  1-9 43 M — — — Patients with multiple sclerosis (MS)  2-1 28 F 12 2 Daclizumab M:F ratio 0.67:1 Mean age (SD), y: 38.5 (10.16) Mean time since diagnosis (SD), y: 10.55 (6.04) y Mean EDSS score (SD): 2.85 (1.45)  2-2 47 F 14 2 Plasma exchange  2-3 54 M 10 3 Fingolimod  2-4 50 F 18 1.5 —  2–5 a 30 M 0.5 4.5 —  2-6 31 F 8 4.5 Teriflunomide  2-7 24 M 12 2 Fingolimod  2-8 40 F 1 1.5 —  2-9 44 F 12 2 —  2-10 37 M 18 5.5 Dimethyl fumarate Alemtuzumab-treated patients with multiple sclerosis (MS, anti-CD52)  3-1 43 F 17 1 Alemtuzumab M:F ratio: 0.43:1 Mean age (SD): 38.9 (9.36) y Mean time since diagnosis (SD): 10.9 (4.75) y Mean EDSS score (SD): 2.4 (1.58) Mean time since last alemtuzumab dose (SD): 31.3 mo (16.85); range 5–53 mo  3-2 33 F 6 4 Alemtuzumab  3-3 30 F 2 0 Alemtuzumab  3-4 50 F 15 3 Alemtuzumab  3-5 45 F 14 2.5 Alemtuzumab  3-6 44 M 8 4.5 Alemtuzumab  3-7 36 F 8 3.5 Alemtuzumab  3-8 26 F 14 0 Alemtuzumab  3-9 29 M 11 2.5 Alemtuzumab  3-10 53 M 14 3 Alemtuzumab Abbreviations: DMT = disease-modifying therapy; EDSS = Expanded Disability Status Scale; F = female; M = male; MS = multiple sclerosis. a This patient was diagnosed with primary progressive MS.

Demographic and Clinical Characteristics of the Study Groups at the Time Point of Testing

Abbreviations: DMT = disease-modifying therapy; EDSS = Expanded Disability Status Scale; F = female; M = male; MS = multiple sclerosis.

This patient was diagnosed with primary progressive MS.

Target Antigen Discovery in Patients With MS Based on Human Proteome-Wide Protein Microarrays

(A) Enzyme-linked immmunospot assay results after 6 days of polyclonal stimulation of peripheral blood mononuclear cells. The heatmap displays the memory B-cell response for the whole human brain lysate and reflects the number of antigen-specific spots per well. Three different groups were tested: healthy controls ( N = 9), patients with MS who were untreated or received disease-modifying treatment other than B-cell depletion therapy ( N = 10), and patients with MS who were treated with the monoclonal anti-CD52 antibody alemtuzumab ( N = 10). (B) Stratification strategy used for the analysis of protein microarray data with the numbers in purple being the numbers of proteins analyzed. (C) Heatmap corresponding to the stratification strategy as shown in (B) and comparing healthy controls ( N = 9) and patients with MS who were either untreated or received disease-modifying treatment other than B-cell depletion therapy ( N = 10). Proteins listed in the heatmap are further explained in eTable 1. Anti-CD52 = treatment with the monoclonal anti-CD52 antibody alemtuzumab; FC = fold change; HC = healthy control; MS = multiple sclerosis; PSF = protein-specific fluorescence.

Cell culture supernatants from the 20 patients with MS and 9 HCs were then tested for their response to over 23,000 full-length human protein clones, covered by the HuProt v4.0 proteome microarray platform. Proteins were stratified based on specific criteria displayed in Figure 1B , which identified 23 autoantigens as potentially MS relevant ( Figure 1C ). eTable 1 provides information on each of these proteins. Overall, we noted reactivity against a variety of antigens, most of which were not specific for CNS tissue (eTable 1).

To determine whether the protein microarray results were dependent on the total IgG concentration of a given sample, we tested samples from 3 patients with MS either in 1:3 dilution or adjusted to an IgG concentration of 0.5 µg/mL. The Spearman correlation coefficient ( r ) was >0.75 ( p < 0.0001) for all samples (eFigure 1A), and the top antigen hits were comparable between these 2 strategies (eFigure 1B).

As an alternative approach to analyze the protein microarray data, the reactivity against each protein was assigned a numerical rank in each individual sample based on the PSF values. Because the microarray consisted of 23,156 protein clones, there was the same number of possible ranks per sample. The mean rank values for each protein in the HC group were then subtracted from the corresponding mean rank values in the MS group. Figure 2 illustrates the proteins that were assigned the top 1–20 ranks in the MS group of patients who did not receive B-cell–depleting therapies. As expected, the autoantibody response pattern determined by the protein microarray was comparable between HCs and patients with MS treated with alemtuzumab ( Figure 2 ). eTable 2 provides information on each of these top 20 proteins. While most of the proteins are primarily expressed intracellularly, 2 interesting membrane-bound target antigens that were revealed by this alternative approach were the extracellular surface protein MLC1 23 and CD59, which is a regulator of the membrane attack complex of the complement system. 24 Both antigens are expressed in the CNS and are known to play a role in maintaining astrocyte homeostasis. 24 , 25

Target Antigen Discovery in Patients With MS Based on the Human Proteome-Wide Protein Microarray Platform

Peripheral blood mononuclear cells (PBMCs) were isolated from healthy control participants ( N = 9), patients with MS who did not receive B-cell depletion therapy ( N = 10), or alemtuzumab (anti-CD52)-treated patients with MS ( N = 10). Cells were polyclonally stimulated for 6 days, and culture supernatants were used for human proteome microarray analysis. In contrast to Figure 1 , in each individual patient, all proteins were ranked based on their PSF. For each protein, the mean rank of all patients per group was calculated and the proteins were sorted in descending order, starting with the highest difference between the mean rank of the MS group and the mean rank of the healthy control group. Proteins listed in the heatmap are further explained in eTable 2. Anti-CD52 = treatment with the monoclonal anti-CD52 antibody alemtuzumab; HC = healthy control; MS = multiple sclerosis; PSF = protein-specific fluorescence.

Anti-MLC1 Antibody Titers Are Significantly Elevated in B-Cell Culture Supernatants and Serum Samples From Patients With MS and in the CSF of Patients With Infectious Neuroinflammation

To further explore the role of MLC1 as a potential target antigen in MS, an independent validation cohort of 36 patients with MS and 25 HCs was recruited (validation cohort 1). Details of this cohort are provided in eTable 3.

ELISA showed that IgG reactivity against MLC1 was significantly elevated in supernatants of polyclonally stimulated B cells from patients with MS compared with HCs ( Figure 3A ). The same applied to CD59, which was used as an additional antigen for validation of the protein microarray results. We also observed significantly increased antibody reactivity against EBV in patients with MS compared with HCs.

Antibody Reactivity Against MLC1, CD59, and EBV Was Significantly Elevated in B-Cell Culture Supernatants and Serum of Patients With MS vs Healthy Control Participants

Enzyme-linked immunosorbent assays were performed to detect immunoglobulin (Ig)G reactivity against MLC1, CD59, and EBV. (A) Supernatant was obtained from peripheral blood mononuclear cells from 36 patients with MS in remission and 25 healthy control participants (validation cohort 1) after 6 days of in vitro polyclonal B-cell stimulation. (B) Matched serum and CSF samples were obtained from a second and third independent cohort (validation cohorts 2 and 3) comprising 11 patients with MS in remission, 20 patients with MS during relapse, 30 patients with noninflammatory neurologic diseases, and 9 patients with other inflammatory neurologic diseases. Patients from validation cohort 2 are displayed in gray and black and patients from validation cohort 3 in white. The total IgG concentration was adjusted to 0.5 µg/mL for each sample. Mean values and standard errors of the mean are shown. * p < 0.05, ** p < 0.01, *** p < 0.001; Kruskal-Wallis test. CD59 = cluster of differentiation 59; EBV = Epstein-Barr virus; HC = healthy control; MLC1 = modulator of VRAC current 1; MS = multiple sclerosis; NIND = noninflammatory neurologic disease; OIND = other inflammatory neurologic disease; Rel = relapse; Rem = remission.

To determine whether MLC1-specific antibodies were also present in the CNS/CSF compartment, we performed ELISA on matched serum/CSF samples from 2 independent validation cohorts (validation cohorts 2 and 3; eTables 4 and 5). As shown in Figure 3B , significantly elevated serum IgG titers to MLC1, CD59, and EBV were observed in patients with MS during relapse compared with patients with NINDs. However, CSF antibody titers to all 3 antigens, i.e., MLC1, EBV, and CD59, were comparable between these 2 cohorts. Patients with MS in remission consistently showed low antibody titers to MLC1 and CD59 in both the peripheral and CSF compartments when compared with the other groups, i.e., patients with MS during relapse, those with NINDs, and patients with OINDs.

Of interest, significantly elevated IgG titers to MLC1 were observed in the serum and CSF of patients with OINDs ( Figure 3B ).

MLC1 Is Expressed by Neurons and Astrocytes in the MS Brain

GlialCAM, a recently described target antigen in MS, 13 exhibits high-affinity molecular mimicry with EBNA1. Of interest, GlialCAM is known to bind MLC1 26 and colocalize in (astrocyte) cell-cell junctions. 27 This link between EBNA1, GlialCAM, and MLC1 made the latter a more promising target antigen over CD59 to follow up. Within the CNS, MLC1 has been described to be primarily expressed by perivascular and subpial astrocytes, Bergmann glia, and ependymal cells while peripheral expression of MLC1 has been detected in leukocytes. 28

In this study, we used a rabbit anti-human MLC1 antibody to stain postmortem MS brain sections ( Figure 4 ). Counterstaining was performed with glial fibrillary acidic protein, SOX10, and NeuN antibodies to detect astrocytes, oligodendrocytes, and neurons, respectively. Negative control staining is shown in eFigure 2. Our results confirm the expression of MLC1 by astrocytes and its absence on oligodendrocytes. Furthermore, in line with previous findings, 28 we observed very strong MLC1 staining on cortical pyramidal neurons.

Immunofluorescence Staining Showed MLC1 Expression by Astrocytes and Neurons in the MS Brain

Postmortem brain tissue sections from a patient with secondary progressive MS were stained for MLC1 using a rabbit polyclonal antibody. Costaining was performed for astrocytes (GFAP + ), oligodendrocytes (SOX10 + ), and neurons (NeuN + ). Scale bars correspond to 50 µm. GFAP = glial fibrillary acidic protein; MLC1 = modulator of VRAC current 1; MS = multiple sclerosis; NeuN = hexaribonucleotide binding protein-3; SOX10 = sex-determining region Y-box transcription factor 10.

MLC1-Specific Antibodies Bind to Cortical Cerebral Neurons When Injected Into Mice With EAE

To study the effect of MLC1-specific antibodies in vivo, mice were immunized with MOG 35-55 and injected with either rabbit anti-mouse MLC1 antibody (N = 7) or nonspecific rabbit IgG (N = 6) at the peak of the disease. Mice were monitored for 72 hours after injection before serum samples were collected for ELISA and CNS tissue was harvested for histologic analysis. As expected, mice that had received MLC1-specific antibody harbored high serum titers of the antibody, whereas nonspecific rabbit IgG-injected mice and the nonimmunized mouse had none to very low MLC1-specific antibody titers ( Figure 5A ).

In Vivo Binding of MLC1-Specific Antibodies to Cerebral Cortical Neurons Was Demonstrated in Mice With Experimental Autoimmune Encephalomyelitis (EAE)

MOG 35-55 -immunized C57BL/6J mice received either a single dose of polyclonal rabbit anti-mouse MLC1 antibody ( N = 7) or nonspecific rabbit IgG ( N = 6). Mice that survived >24 hours, i.e., 3 for the anti-MLC1 antibody vs 6 for the isotype control-treated group, were sacrificed 72 hours later. (A) ELISA measurement of MLC1-specific serum antibodies. *** p < 0.001; Welch t test. (B) Development and course of clinical EAE. Mean EAE scores ± standard errors of the mean (SEM) are shown. (C) Mean percentage of mouse initial weight (±SEM). One nonimmunized control mouse is shown for comparison. (D) Upper panels: sections of the cerebral cortex of MOG 35-55 -immunized mice treated either with rabbit anti-mouse MLC1 antibody (left) or rabbit IgG (right). Lower panels: sections from the cerebral cortex of a healthy nonimmunized mouse, incubated with serum samples from MOG 35-55 -immunized mice that had received either rabbit anti-mouse MLC1 (left) or rabbit IgG (right). The scale bars correspond to 50 µm. IgG = immunoglobulin G; MLC1 = modulator of VRAC current 1; MOG = myelin oligodendrocyte glycoprotein; OD = optical density.

Of the 7 mice that were treated with anti-mouse MLC1 antibody, 4 mice died within 24 hours after antibody injection. In the remaining mice, there were no significant differences in EAE score development or weight between anti-MLC1 antibody-specific and nonspecific rabbit IgG-injected mice ( Figure 5 , B and C ).

Strong binding of MLC1-specific antibodies to cerebral cortical neurons was observed in anti-MLC1 antibody-injected mice ( Figure 5D ) while staining was absent in nonspecific rabbit IgG-injected mice. Similar to the staining of in vivo antibody deposition, serum from anti-MLC1 antibody-treated mice stained cerebral cortical neurons, whereas no staining was observed when brain sections were incubated with serum from the control group.

Discussion

Despite the development of highly sensitive assays and advances in proteomics, the search for an MS-specific antibody signature has been ongoing for several decades, with conflicting findings between different research groups. 15 , 16

In this study, we used a novel approach to identify relevant target antigens in patients with MS that could be of diagnostic relevance. To this end, we compared the presence or absence of CNS antigen-specific memory B cells in the MS group vs age-matched and sex-matched HCs and a cohort of alemtuzumab-treated patients with MS, respectively. As expected, the immunologic fingerprint was skewed toward autoimmune B-cell responses to CNS antigens, particularly in the patients with MS compared with the HCs or patients who had received anti-CD52 therapy. Treatment with alemtuzumab not only results in prolonged T-cell lymphopenia, but differentiation of naïve B cells to memory B cells is also impaired, 29 which can be attributed to the strong reduction in the number of follicular regulatory T cells and follicular T helper (Tfh) 17 cells. 30

Furthermore, in line with the literature, 17 , 31 our first set of analyses confirmed that the antibody response is heterogeneous between individual patients and targets a diverse set of ubiquitously expressed self-antigens that are not CNS-specific. For example, the mean PSF for FAM84A (family with sequence similarity 84, member A), a protein that may play a role in cell motility, 32 was significantly higher in patients with MS vs HCs. FAM84A has previously been reported as an autoantigen, not only in the context of MS 31 but also in patients with inflammatory bowel disease. 33 Similarly, the antigen UBE3A (ubiquitin protein ligase E3A) has also been associated with MS. 34 The intracellular localization of many of these targets suggests a secondary reaction to cellular debris that may not be MS specific. 31 In other words, such a reactivity may be an epiphenomenon of general tissue damage due to (neuro)inflammation. It is conceivable that intracellular or otherwise “cryptic” antigens from injured neuronal or glial cells may activate autoreactive T and B cells, eliciting strong secondary inflammation. 35 A similar mechanism has been proposed in patients with ischemic stroke, where intracellular epitopes are encountered by the immune system, thereby triggering autoimmune responses to CNS tissue. 36

As an alternative strategy for the analysis of the protein microarray data, every protein was assigned a rank based on the PSF difference between patients with MS, those who were not under B-cell depletion therapy, and HCs. This rank mirrored the degree to which a protein was targeted by autoantibodies in an individual patient sample. In this case, an antigen that triggered our interest was MLC1.

MLC1 is expressed in the CNS 28 and is known to play a crucial role in regulating blood-brain barrier (BBB) integrity. 23 , 30 , 37 Variations in the MLC1 gene cause megalencephalic leukoencephalopathy with subcortical cysts, which is a rare form of leukodystrophy. 25 Of interest, the same disease can also result from GLIALCAM gene variation. 38 GlialCAM functions as a β-subunit of MLC1, with histologic studies demonstrating colocalization of GlialCAM with its binding partner MLC1. 39 While GlialCAM has recently been identified as a possible target antigen in MS, 13 the expression of MLC1 is also said to be increased in the MS brain, particularly in areas of immunologically active demyelinated lesions of the white matter. 25

Given the close relationship and spatial proximity of GlialCAM and MLC1, 39 it is tempting to speculate that patients with MS develop antibodies to MLC1 as a result of epitope spreading 40 after an immune response to GlialCAM. Although GlialCAM did not fulfil the requirements of our stratification strategy and was hence not investigated further, it should be noted that the patients with MS recruited for the protein microarray analysis had a mean disease duration of 10.55 years (range 0.5–18 years) and were in remission. By contrast, samples that were used by in a previous study 13 to demonstrate molecular mimicry between the EBV transcription factor EBNA1 and GlialCAM were collected from patients with MS at the onset of their disease or during an acute episode. It is possible that GlialCAM-specific antibodies are disease stage-dependent and more prevalent in newly diagnosed MS patients.

In validation experiments, supernatants from polyclonally stimulated peripheral B cells from an independent cohort of patients with MS showed elevated IgG titers to MLC1, CD59, and EBV compared with HCs ( Figure 3A ), hence confirming our proteome microarray results. Similarly, serum antibody titers to all 3 antigens were significantly elevated in patients with MS during relapse, compared with patients with NINDs ( Figure 3B ). On the contrary, there was no increase in IgG titers to MLC1 in patients with MS in the CSF, neither in remission nor during relapse ( Figure 3B ).

The phenotypic differences of B cells in the periphery vs CSF result in only specific autoantigens driving B-cell survival and proliferation in the CNS/CSF compartment, as previously discussed. 41 This could explain why we did not observe elevated anti-MLC1 antibody titers in the CSF in either of our 2 MS validation cohorts in this study. A proteome microarray on cultured B cells isolated from the CSF of patients with MS would most likely reveal another assortment of potential target antigens.

One of the more interesting findings of this study was the significant increase in IgG titers to MLC1 in the CSF of patients with OINDs compared with the other groups ( Figure 3B ). Of note, 5 of the 9 patients in the OIND group were diagnosed with different forms of infection-associated/related neuropathy (eTable 5). We propose that these abnormally elevated IgG titers to MLC1 in the CSF speak for possible postviral/infectious humoral autoimmunity, 42 where (auto)antibodies against MLC1 may serve as biomarkers of an overall activated immune system rather than directly mediating disease pathogenesis. 43 , 44 In this case, regardless of the initiating event, activation and recruitment of autoreactive T and B cells result from (chronic) tissue damage, culminating in an immune response to otherwise “disease-irrelevant” antigens such as MLC1 as a result of bystander activation. 45 Along these lines, the presence of anti-GAD (glutamic acid decarboxylase) antibodies has been associated with several neurologic symptoms, yet there is no concrete evidence that these antibodies are pathogenic. 46 Similarly, the presence of autoantibodies in different types of cancer is believed to reflect an overactive immune system rather than directly contributing to cancer pathology. 47

Although the number of patients in our current OIND cohort is too small to draw a meaningful conclusion, we believe that addressing the diagnostic and prognostic value of an antibody response against MLC1, using larger cohorts of patients, especially those with viral/bacterial neuroinflammation, may be interesting.

Finally, our initial experiment performed in the MOG 35-55 EAE model demonstrated strong in vivo binding of MLC1-specific antibodies to cerebral cortical neurons, confirming the results obtained when staining MS brain tissue ( Figure 4 ). As shown in Figure 5B , we observed no worsening of the clinical score in mice that had received MLC1-specific vs control antibodies. However, one must take into account that of the 7 mice that were treated with anti-MLC1 antibody, 4 mice died within the first 24 hours after antibody injection. All mice in the EAE cohort that were treated with the isotype control antibody survived the duration of the experiment. This leaves room for speculation that anti-MLC1 antibodies played a role in aggravating disease severity. Unfortunately, it was not possible to perform CNS histopathologic analysis of the tissue from the dead mice, given the poor quality of tissue architecture. Our theory why the injection of anti-MLC1 antibody led to death in some animals while not worsening disease in others is that the outcome depended on the time point of antibody injection. In animals that had died, disease was just developing at the time point of injection (EAE scores between 1.0 and 2.0), whereas the mice that survived had full-blown EAE with scores between 2.5 and 3.0. We suggest that the integrity of the BBB played an important role in anti-MLC1 antibody-mediated immunopathology. Previous studies have shown that a breach in the brain barriers typically occurs before immune cell infiltration and subsequent demyelination. 48 Furthermore, it was demonstrated that the BBB is dramatically but transiently compromised in myelin antigen-immunized mice at least one week before the development of the paralytic phase of EAE. 49 , 50 Hence, we assume that the BBB in mice with developing EAE was more compromised than in those with established EAE. Further experiments with a larger sample size will have to be performed to verify the findings regarding the pathogenicity of MLC1-specific antibodies.

Acknowledgment

The authors thank Ji-Young Chang and Ursula Lebherz for administrative assistance and Birgit Blanck, Marion Michels, and Angelika Zoons for providing technical expertise. The authors are also grateful to Cambridge Protein Arrays for performing the microarray screens and to Sandra Amor for providing human brain tissue samples. This work was performed in (partial) fulfillment of the requirements for obtaining the degree “Dr. med.”

Glossary

blood-brain barrier

cluster of differentiation 59

experimental autoimmune encephalomyelitis

Epstein-Barr virus

EBV nuclear antigen

enzyme-linked immunosorbent assay

enzyme-linked immunospot

Ethikkommission Nordwest- und Zentralschweiz

Friedrich-Alexander-Universität

noninflammatory neurologic disease

other inflammatory disease

primary progressive MS

protein-specific fluorescence

relapsing-remitting MS

Footnotes

Editorial, page e200383

Author Contributions

J.R. Dahl: major role in the acquisition of data; analysis or interpretation of data. A. Weier: major role in the acquisition of data; analysis or interpretation of data. C. Winter: analysis or interpretation of data. M. Hintze: analysis or interpretation of data. V. Rothhammer: major role in the acquisition of data. T. Tsaktanis: major role in the acquisition of data. A-K. Proebstel: major role in the acquisition of data; analysis or interpretation of data. T. Neziraj: major role in the acquisition of data; analysis or interpretation of data. E. Poessnecker: major role in the acquisition of data. J. Oechtering: major role in the acquisition of data. J. Kuhle: major role in the acquisition of data. B-A. Kallmann: major role in the acquisition of data. G. Luber: major role in the acquisition of data. T. Heider: major role in the acquisition of data. L. Klotz: major role in the acquisition of data. R. Chunder: drafting/revision of the manuscript for content, including medical writing for content; major role in the acquisition of data; analysis or interpretation of data. S. Kuerten: drafting/revision of the manuscript for content, including medical writing for content; major role in the acquisition of data; study concept or design; analysis or interpretation of data.

Study Funding

This study was funded by Sanofi (grant number GZ-2016-11659) and by the German Research Foundation (DFG) project 460333672 CRC1540 EBM.

Disclosure

A. Weier, J.R. Dahl, M. Hintze, C. Winter, J. Oechtering, J. Kuhle, G. Luber, and T. Heider have declared that no conflict of interest exists. V. Rothhammer was funded by an ERC Starting Grant from the European Research Council (HICI 851693), a Heisenberg Fellowship, and a research grant provided by the German Research Foundation (Deutsche Forschungsgemeinschaft [DFG] RO4866-3/1, RO4866-4/1; project ID 401772351). He was also supported by transregional and collaborative research centers provided by the DFG (project ID 408885537–TRR 274; project ID 261193037–CRC 1181; project ID 505539112–KFO 5024). T. Tsaktanis was funded by the Kommission für Klinische Forschung, Klinikum rechts der Isar, as well as by the Clinician Scientist Program of the Medical Faculty of Friedrich-Alexander-Universität Erlangen-Nürnberg. A-K. Proebstel has received financial compensation for participation in advisory boards and/or consultations from Biogen, Novartis, Roche, and UCB, and research support from Biogen. She has received funding from the Swiss National Science Foundation (Eccellenza Professorship 194609; Starting Grant 211318), the National Multiple Sclerosis Society Kathleen C. Moore Fellowship: FG-1708-28871, the European Union Horizon Europe research and innovation program and Swiss State Secretariat for Education, Research and Innovation (SERI) (101136582), the Propatient Foundation, the Fondation Pierre Mercier pour la science, and the Gottfried & Julia Bangerter-Rhyner-Foundation. T. Neziraj is funded by a Department of Defense Multiple Sclerosis Research Program Early Investigator Research Award (MS220186). B-A. Kallmann has received speaker fees and consultancy honoraria from Hexal, Alexion, Biogen, BMS, Viatris, Novartis, Merck, Janssen, Roche, and Sanofi. L. Klotz has received compensation for serving on scientific advisory boards for Alexion, Genzyme, Janssen, Merck, Novartis, and Roche; speaker honoraria and travel support from Bayer, Biogen, Genzyme, Grifols, Merck, Novartis, Roche, Santhera, and Teva; and research support from the DFG, the German Ministry for Education and Research (BMBF), the Interdisziplinäres Zentrum für Klinische Forschung (IZKF) Münster, the research program Innovative Medizinische Forschung (IMF) Münster, Biogen, Novartis, and Merck. R. Chunder has received funding from the DFG under Germany's Excellence Strategy (EXC2151–390873048) and from Novartis (Oppenheim-Förderpreis für Multiple Sklerose 2023). S. Kuerten reports funding from Novartis, F. Hoffmann-La Roche, and Sanofi, and speaker fees and consultancy honoraria from Novartis, F. Hoffmann-La Roche, Sanofi, and Teva. S. Kuerten receives funding from the German Research Foundation (DFG) IRTG 2168 (grant no. 272482170) and from DFG project 460333672 CRC1540 EBM. She is a member of the Excellence Cluster “ImmunoSensation 2 ” (EXC2151–390873048). Go to Neurology.org/N for full disclosures.

References

Associated Data

Data Availability Statement

The data that support the findings of this study are available from the corresponding author, on reasonable request.