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. 2025 Mar;12(2):e200374.
doi: 10.1212/NXI.0000000000200374. Epub 2025 Feb 11.

Modulator of VRAC Current 1 Is a Potential Target Antigen in Multiple Sclerosis

Johannes Raffael Dahl  1   2 , Alicia Weier  2   3 , Christopher Winter  4 , Maik Hintze  1   2 , Veit Rothhammer  5 , Thanos Tsaktanis  5 , Anne-Katrin Proebstel  6   7   8 , Tradite Neziraj  6   7   8 , Elisabeth Poessnecker  6   7   8 , Johanna Oechtering  6   7   8 , Jens Kuhle  6   7   8 , Boris-Alexander Kallmann  9 , Gabriele Luber  10 , Thorsten Heider  11 , Luisa Klotz  12 , Rittika Chunder  2 , Stefanie Kuerten  2
Affiliations

Modulator of VRAC Current 1 Is a Potential Target Antigen in Multiple Sclerosis

Johannes Raffael Dahl et al. Neurol Neuroimmunol Neuroinflamm. 2025 Mar.

Abstract

Background and objectives: Multiple sclerosis (MS) is a chronic immune-mediated demyelinating disease of the CNS. Highlighted by the success of B-cell-depleting therapies such as the monoclonal anti-CD20 antibodies rituximab, ocrelizumab, and ofatumumab, B cells have been shown to play a central role in the immunopathology of the disease. Yet, the target antigens of the pathogenic B-cell response in MS remain unclear.

Methods: We combined polyclonal B-cell stimulation of peripheral blood mononuclear cells with a human proteome-wide protein microarray to identify target antigens of MS by comparing samples from 20 patients with MS with 9 age-matched and sex-matched healthy controls. Results were verified by enzyme-linked immunosorbent assay (ELISA) in 3 independent validation cohorts (N = 47 patients with MS in remission; N = 20 patients with MS during relapse; N = 25 HCs; N = 30 patients with other noninflammatory neurologic diseases; N = 9 patients with other inflammatory neurologic diseases). Experimental autoimmune encephalomyelitis (EAE) was used as an animal model to evaluate the pathogenicity of the antibodies of choice.

Results: Our results corroborate the existing concept of a highly diverse autoimmune response in MS. Yet, a significantly elevated antibody response against the membrane protein modulator of VRAC current 1 (MLC1) was noted in B-cell culture supernatants and serum samples of patients with MS. Furthermore, significantly elevated titers to MLC1 were observed in the CSF of patients with neuroinflammatory diseases other than MS. Neurons and astrocytes were identified as the main cell types expressing MLC1 in the brain of a patient with MS. Injection of anti-MLC1 antibodies into mice with EAE led to strong in vivo binding to cerebral cortical neurons and to the death of 4 of the 7 injected mice.

Discussion: Future studies will have to address the diagnostic and prognostic value of MLC1-specific antibodies in neuroinflammatory disorders such as MS and characterize the functional role of MLC1 expression in neurons and astrocytes.

Trial registration information: The study has been registered in the German Clinical Trials Register (study number DRKS00015528).

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Conflict of interest statement

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 “ImmunoSensation2” (EXC2151–390873048). Go to Neurology.org/N for full disclosures.

Figures

Figure 1
Figure 1. 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.
Figure 2
Figure 2. 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.
Figure 3
Figure 3. 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.
Figure 4
Figure 4. 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.
Figure 5
Figure 5. In Vivo Binding of MLC1-Specific Antibodies to Cerebral Cortical Neurons Was Demonstrated in Mice With Experimental Autoimmune Encephalomyelitis (EAE)
MOG35-55-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 MOG35-55-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 MOG35-55-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.
See this image and copyright information in PMC

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