RESEARCH ARTICLE OPEN ACCESS IgG4 Valency Modulates the Pathogenicity of Anti–Neurofascin-155 IgG4 in Autoimmune Nodopathy Alexandre Jentzer, MD, PhD,* Arthur Attal, MD,* Clémence Roué, MS, Julie Raymond, MS, Correspondence Dr. Devaux Cinta Lleixà, MD, PhD, Isabel Illa, MD, PhD, Luis Querol, MD, PhD, Guillaume Taieb, MD, PhD, and jerome.devaux@inserm.fr Jérôme Devaux, PhD, on behalf of the anti–neurofascin-155 study group Neurol Neuroimmunol Neuroinflamm 2022;9:e200014. doi:10.1212/NXI.0000000000200014 Abstract Background and Objectives IgG4 autoantibodies to neurofascin-155 (Nfasc155) are associated with a subgroup of patients with chronic inflammatory demyelinating polyneuropathy (CIDP), currently named autoim- mune nodopathy. We previously demonstrated that those antibodies alter conduction along myelinated axons by inducing Nfasc155 depletion and paranode destruction. In blood, IgG4 have the potency to exchange their moiety with other unrelated IgG4 through a process called Fab-arm exchange (FAE). This process results in functionally monovalent antibodies and may affect the pathogenicity of autoantibodies. Here, we examined this issue and whether FAE is beneficial or detrimental for Nfasc155 autoimmune nodopathy. Methods The bivalency and monospecificity of anti-Nfasc155 were examined by sandwich ELISA in 10 reactive patients, 10 unreactive CIDP patients, and 10 healthy controls. FAE was induced in vitro using reduced glutathione and unreactive IgG4, and the ratio of the κ:λ light chain was monitored. To determine the pathogenic potential of bivalent anti-Nfasc155 IgG4, autoanti- bodies derived from patients were enzymatically cleaved into monovalent Fab and bivalent F(ab’)2 or swapped with unreactive IgG4 and then were injected in neonatal animals. Results Monospecific bivalent IgG4 against Nfasc155 were detected in the serum of all reactive patients, indicating that a fraction of IgG4 have not undergone FAE in situ. These IgG4 were, none- theless, capable of engaging into FAE with unreactive IgG4 in vitro, and this decreased the levels of monospecific antibodies and modulated the ratio of the κ:λ light chain. When injected in animals, monovalent anti-Nfasc155 Fab did not alter the formation of paranodes; by contrast, both native anti-Nfasc155 IgG4 and F(ab’)2 fragments strongly impaired paranode formation. The promotion of FAE with unreactive IgG4 also strongly diminished the pathogenic potential of anti-Nfasc155 IgG4 in animals and decreased IgG4 clustering on Schwann cells. Discussion Our findings demonstrate that monospecific and bivalent anti-Nfasc155 IgG4 are detected in patients and that those autoantibodies are the pathogenic ones. The transformation of anti- Nfasc155 IgG4 into monovalent Fab or functionally monovalent IgG4 through FAE strongly decreases paranodal alterations. Bivalency thus appears crucial for Nfasc155 clustering and paranode destruction. *These authors contributed equally to this work. From the Institute for Neurosciences of Montpellier (A.J., A.A., G.T., J.D.), INSERM U1051, Montpellier University, Hôpital Saint Eloi, France; Department of Immunology (A.J.), CHU Montpellier, France ; Department of Neurology (A.A., G.T.), CHU Montpellier, Hôpital Gui de Chauliac, France; Institut de Génomique Fonctionnelle (C.R., J.R., G.T., J.D.), CNRS UMR5203, France; and Neuromuscular Diseases Unit (C.L., I.I., L.Q.), Hospital de la Santa Creu i Sant Pau, Universitat Autónoma de Barcelona, Spain. Go to Neurology.org/NN for full disclosures. Funding information is provided at the end of the article. Anti–neurofascin-155 study group coinvestigators are listed in appendix 2 at links.lww.com/NXI/A736 the end of the article. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary CASPR1 = contactin-associated protein 1; CIDP = chronic inflammatory demyelinating polyneuropathy; CNTNI = contactin- 1; FAE = Fab-arm exchange; IVIg = IV immunoglobulin; MuSK = muscle-specific kinase; Nfasc155 = neurofascin-155. Cell adhesion molecules at the nodes of Ranvier play a crucial specialization by inhibiting the interaction between CNTN1/ role in the organization of the axonal domains and in the rapid CASPR1 and Nfasc155.6,16,24 FAE may potentially increase the propagation of the nerve impulse along myelinated axons.1 pathogenic effects of these autoantibodies in a similar manner as Particularly, the ternary complex formed by the association of for anti-MuSK IgG4. By contrast, anti-Nfasc155 does not have a neurofascin-155 (Nfasc155), contactin-1 (CNTN1), and function-blocking activity.17 These antibodies induce Nfasc155 contactin-associated protein 1 (CASPR1) is important for the clustering on the Schwann cell surface and lead to its depletion, formation of septate-like junctions at paranodal regions for the thus abolishing paranode formation and maintenance both in clustering/stabilization of voltage-gated sodium channels at patients and animal models.6,17,25-27 This finding suggests that the nodes of Ranvier and for myelin insulation.2-5 Antibodies anti-Nfasc155 IgG4 can aggregate their target and that a fraction of targeting these important cell adhesion molecules have been them may be monospecific in situ. Here, we explored whether discovered in a subset of patients with chronic inflammatory IgG4 valency influences anti-Nfasc155 IgG4 pathogenicity and demyelinating polyneuropathy (CIDP) and have helped im- whether FAE is beneficial for paranode maintenance. For that prove the diagnosis and treatment orientation of this disabling purpose, this study tested the pathogenic potential of monovalent neuromuscular disorder.6-13 Because these patients have distinct and bivalent anti-Nfasc155 IgG4. clinical features, no overt inflammation or macrophage-mediated demyelination, and a poor response to IV immunoglobulin (IVIg) compared with typical CIDP, this group has recently Methods been named autoimmune nodopathy.14 Nfasc155 is the major Patients’ IgG immune target in autoimmune nodopathy (nearly 70% of the Plasma and sera were obtained from 10 patients with autoimmune cases). The clinical and pathophysiologic distinctiveness of au- nodopathy with anti-Nfasc155 IgG4 antibodies, 10 seronegative toimmune nodopathy relies on the antigen specificity (Nfasc155, patients with CIDP, and 10 healthy donors (Etablissement CNTN1, and CASPR1) and on the fact that these autoanti- Français du Sang, Montpellier, France). All subjects provided bodies predominantly belong to the IgG4 isotype, which are written informed consent. The study was approved by the Ethics unable to activate complement and have low affinity for Fcγ Committee of Montpellier University Hospital (IRB-MTP-2020- receptors.15 Animal models demonstrated that these antibodies 01-20200339). IgG1 and IgG4 were purified using the Captur- can disrupt the paranodal junctions in the absence of comple- eSelect™ affinity matrix (Thermo Fisher Scientific, Waltham, ment activation or macrophage-mediated demyelination.6,16,17 MA), dialyzed to artificial CSF, concentrated at 10 mg/mL, and IgG4 autoantibodies appeared to induce paranodal destruction filter-sterilized. either through a function-blocking activity or by depleting the target antigens. Monoclonal Antibody Production HEK293T cells were transiently transfected with pVITRO1- A growing number of autoimmune diseases implicate IgG4.18 Trastuzumab-IgG4/κ (#61887; Addgene, Watertown, MA) or IgG4 has the unique characteristic of dynamically exchanging pVITRO1-102.1F10-IgG4/λ (#50369; Addgene) for 24 hours; its Fab arms by swapping the heavy chain.19 This process then, the medium was replaced with OptiMEM (Thermo named Fab-arm exchange (FAE) results in bispecific IgG4 Fisher) for antibody production. IgG was purified with a HiTrap that are monovalent to their target. In myasthenia gravis with protein A affinity column (Cytiva, Marlborough, MA) on an antibodies against muscle-specific kinase (MuSK), FAE ap- AKTA Pure (Cytiva). Antibodies were desalted to PBS using a pears to potentiate the IgG4 pathogenic potential.20,21 Anti- HiTrap Desalting column (Cytiva), filter-sterilized, and stored at MuSK IgG4 was shown to block the interaction between −20°C until use. MuSK and low-density lipoprotein receptor–related protein 4.22 Although bivalent anti-MuSK IgG4 can cluster MuSK, the Antibody Cleavage and FAE monovalent form obtained after FAE strongly inhibits the Fab and F(ab’)2 fragments were generated using the Pierce Fab aggregation of acetylcholine receptors at the neuromuscular preparation kit (Thermo Fisher) or FragIT kit (Genovis, Lund, junction and leads to severe muscle weakness in vivo.20,23 Sweden) according to the manufacturer’s protocols. Fab and Insofar, MuSK myasthenia gravis is the sole condition where F(ab’)2 fragments were dialyzed to artificial CSF, concentrated at FAE appears to amplify the pathogenic function of IgG4. 10 mg/mL, and filter-sterilized. FAE was performed as previously described.19,23 IgG4 or IgG1 from Nfasc155-reactive patients In autoimmune nodopathy, the effect of FAE on the pathogenicity were incubated with a 3-fold excess of healthy control IgG4 in the of IgG4 has not yet been evaluated. IgG4 to CNTN1 and presence or absence of 0.5 mM reduced glutathione (GSH) CASPR1 have a function-blocking activity and disrupt paranodal (Merck-Millipore, Burlington, MA) for 24 hours at 37°C. 2 Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 5 | September 2022 Neurology.org/NN Monospecificity ELISA Assay donkey anti-human (709-585-149; 709-545-149). Slides were The extracellular domain of human recombinant Nfasc155 examined using an ApoTome fluorescence microscope (Carl (Met1-Trp1062) with a C-terminal His-tagged was produced in Zeiss MicroImaging GmbH, Oberkochen, Deutschland), and HEK293T cells and purified, then was biotinylated with a 20-fold digital images were manipulated into figures with CorelDraw molar excess of EZ-link sulfo-NHS-biotin (Thermo Fisher) for 30 and Corel Photo-Paint (Corel Corporation, Ottawa, Canada). minutes at 37°C, and desalted to PBS. MaxiSorp microtiter plates were coated with 50 ng of untagged human Nfasc155 overnight at Statistics 4°C. Wells were blocked with 0.5% casein sodium 0.05% Tween Statistical significance was assessed by unpaired and paired 20 in PBS for 1 hour at 37°C and then incubated overnight at 4°C 2-tailed Student t tests, Kolmogorov-Smirnov tests, or one- with 10 μg of IgG4 in blocking solution for regular tests or with way ANOVA followed by Bonferroni post hoc tests using 40 μg of IgG4 for FAE tests (10 μg of patient IgG4 + 30 μg of GraphPad Prism (GraphPad Software, San Diego, CA). Lin- control IgG4). The day after, wells were incubated with 50 ng of ear regression and Spearman correlation were performed biotinylated Nfasc155 for 1 hour at 37°C, and the reactivity was using GraphPad Prism. p values <0.05 were considered revealed using peroxidase-conjugated streptavidin (1:2,000; significant. Merck-Millipore) and SIGMAFAST OPD (Merck-Millipore). As positive controls, IgG1 from Nfasc155-reactive patients were used. Standard Protocol Approvals, Registrations, IgG4 from healthy donors and seronegative patients were used as and Patient Consents negative controls. Dilutions of purified anti-Nfasc155 IgG1 were All animal experiments were in line with the European com- used to obtain a calibration curve, from which the percentage of munity’s guiding principles on the care and use of animals monospecific IgG4 was calculated. (2010/63/EU) and were approved by the local ethical com- mittee and by the “ministére de l’éducation nationale de Quantification of κ/λ Ratios l’enseignement supérieur et de la recherche” (APAFIS#3847- The amounts of λ and κ light chains in anti-Nfasc155 IgG4 were 2016012610089856v5). determined by ELISA against Nfasc155 as indicated above, and the plates were revealed using the peroxidase-conjugated goat Data Availability anti-human λ or κ light chain (1:2000; Bio-Rad, Hercules, CA). Data are available from the corresponding author on request. Purified recombinant IgG4/κ and IgG4/λ were used to obtain a calibration curve. For sandwich ELISA, MaxiSorp microtiter plates were coated with 1 μg of donkey anti-human IgG (Jackson Results ImmunoResearch, West Grove, PA) in 50 μL of PBS overnight A Fraction of IgG4 to Nfasc155 Are at 4°C. Wells were blocked as indicated above and then in- Monospecific and Bivalent in Reactive Patients cubated overnight at 4°C with 1 ng of IgG4 from healthy donors Our previous finding indicated that IgG4 autoantibodies or patients. The day after, the reactivity was revealed using the target and cluster Nfasc155 on the Schwann cell surface. We peroxidase-conjugated goat anti-human λ or κ light chain. The thus examined whether patients’ IgG4 were able to cross-link quantity of λ or κ light chains was expressed as a percentage of Nfasc155 in vitro. For that purpose, we selected 10 patients the total amount of λ + κ light chains. reactive to Nfasc155 from whom a large volume of serum or Animals plasma was available. The clinical history and antibody titers In vitro nerve incubation experiments and neonatal injection of these patients are indicated in Table 1. IgG1 and IgG4 were performed as previously described.17 New-born Wistar were purified from these patients, as well as 10 healthy rats received a single intraperitoneal injection of 250 μg of controls and 10 seronegative patients with CIDP. To de- control IgG4, native Nfasc155-reactive IgG4, Fab or F(ab’)2 termine the percentage of monospecific antibodies, IgG4 fragments, or 1 mg of swapped antibodies in artificial CSF. were tested with a monospecificity ELISA assay based on Animals were then killed 2 days after the injection. Teased the ability of IgG4 to link soluble biotinylated Nfasc155 to fibers were analyzed from 4 animals for each group. immobilized untagged Nfasc155 (Figure 1A). IgG4 from seronegative patients with CIDP and healthy controls did Immunolabeling not couple Nfasc155. By contrast, IgG4 from Nfasc155- Immunolabeling was performed as previously described.17 reactive patients significantly cross-linked Nfasc155 with The following primary antibodies were used: rabbit antisera biotinylated Nfasc155 with variable intensities, indicating against CASPR1 (1/2000); mouse monoclonal antibodies that patients have different levels of monospecific antibodies against Nav channels (K58/35; 1:500; Merck-Millipore); (Figure 1B). Dilutions of anti-Nfasc155 IgG1 were used to mouse monoclonal antibodies against Myc (11667149001; 1/ calculate the percentage of monospecific IgG4 in each pa- 200; Roche, Basel, Switzerland); and/or goat antibody against tient (Table 1). The potency to cross-link Nfasc155 CNTN1 (1/2000; R&D Systems, Minneapolis, MN). The (Figure 1B) and the percentage of monospecific IgG4 sig- following secondary antibodies from Jackson ImmunoR- nificantly correlated with the titers of anti-Nfasc155 IgG4 esearch were used at a 1:500 dilution: donkey anti-mouse in the patients (Spearman p = 0.018 and p = 0.031, re- (715-545-151; 715-585-151), donkey anti-goat (705-545- spectively). However, no correlation was found between 147; 705-585-147), donkey anti-rabbit (711-585-152), and Nfasc155 cross-linking by monospecific antibodies and the Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 5 | September 2022 3 Table 1 Antibody Titers and Clinical Features of Nfasc155-Reactive Patients Monospecific Clinical score Titers anti-Nfasc155 IgG4 (%) Sex Age (mRS score) Response to treatment AN1 2,600 10 M 46 3 IVIg−, Cx partial, Plex+, Ritux+ AN2 19,700 20 M 25 3 IVIg−, Cx+, Plex+ AN3 1,600 19 M 22 3 IVIg−, Cx−, Plex+, Ritux+ AN4 20,500 78 M 53 4 IVIg−, Cx−, Ritux−, Plex+ AN5 6,000 17 M 33 2 IVIg−, Cx+ AN6 3,000 8 M 50 4 IVIg−, Cx+, Plex+, Ritux+ AN7 10,000 21 M 16 3 IVIg−, Cx−, Ritux+ AN8 1,800 7 M 37 3 IVIg partial, Cx partial, Plex+ AN9 6,000 17 M 68 4 IVIg+, Ritux+, Plex+ AN10 6,000 29 M 46 1 IVIg+, Ritux+ Abbreviations: Cx = corticosteroids; IVIg = IV Immunoglobulin; mRS = modified Rankin Scale; Nfasc155 = neurofascin-155; NT = not tested; Plex = plasma exchange; Ritux = rituximab. clinical severity or the age of the patients (eFigure 1, links. whether the κ/λ ratios of anti-Nfasc155 IgG4 differ from those lww.com/NXI/A736). Similarly, no correlation was found of the total IgG4 fraction not only in each patient but also in between antibody titers and clinical severity. controls, the κ/λ ratios were quantified by sandwich ELISA in the purified IgG4 fractions from all patients and controls IgG4 Reactive to Nfasc155 Can Undergo FAE (Figure 2). Although the κ/λ ratios of anti-Nfasc155 IgG4 were In Vitro slightly more dispersed that those found in the total IgG4 To determine whether anti-Nfasc155 IgG4 can undergo FAE, fractions and presented different SDs (p < 0.05 with a Bartlett IgG4 from Nfasc155-positive patients were incubated in vitro test), the mean values were not significantly different (p > 0.05 with a reducing agent, reduced glutathione (GSH), in the by one-way ANOVA followed by Bonferroni post hoc tests). presence or absence of 3-fold excess of IgG4 from a healthy Also, no significant differences were found between the κ/λ donor. The samples were then tested for their ability to cate- ratios of Nfasc155-reactive patients and those of healthy donors nate immobilized Nfasc155 with soluble biotinylated Nfasc155. or seronegative patients. In vitro FAE with a monoclonal IgG4/ In the absence of GSH, anti-Nfasc155 IgG4 potently inter- λ antibody increased mainly the proportion of the λ light chain connected immobilized Nfasc155 and biotinylated Nfasc155 in Nfasc155-reactive IgG4 and resulted in a significant decrease (Figure 1C). The addition of reducing agents potently di- of the ratio κ:λ (Figure 2C). In vitro FAE with a monoclonal minished the ability of IgG4 to cross-connect Nfasc155 in most IgG4/κ antibody confirmed this observation and resulted in a patients (Figure 1C). This effect was further potentiated by the significant increase in the proportion of the κ light chain in addition of a 3-fold excess of IgG4 from healthy donors, which Nfasc155-reactive IgG4 and in an increase of the ratio κ:λ led to a 43% reduction of the levels of monospecific antibodies. (Figure 2D). By contrast, reducing conditions did not affect the mono- specificity of anti-Nfasc155 IgG1 (Figure 1D), which is in The correlations between laboratory finding and clinical history keeping with previous observations.19,23 indicated that FAE efficiency with recombinant IgG4/λ appeared to slightly follow clinical severity, and anti-Nfasc155 IgG4 from Ratio of κ and λ Light Chains in Nfasc155- patients with a higher score showed a lower potency to undergo Reactive Patients FAE (eFigure 1B, links.lww.com/NXI/A736). However, patients The κ light chain is generally slightly more prevalent than the λ with a high clinical score also showed a higher proportion of λ light chain in mature B cells.28 We exploited this characteristic light chains; it is thus possible that FAE potency with IgG4/λ is to confirm our finding and monitored the changes in the κ and underestimated in those patients because antibodies are already λ light chain proportion of anti-Nfasc155 IgG4 after in vitro predominantly IgG4/λ before FAE (eTable 2). induction of FAE with monoclonal IgG4/λ or IgG4/κ anti- bodies. In a first manner, the amount of κ and λ light chains was Bivalency Is Crucial for Pathogenicity, Whereas measured in anti-Nfasc155 IgG4 by ELISA. The amount of κ Monovalency Decreases the Pathogenicity of light chains was found to strongly correlated with that of λ light Nfasc155-Reactive IgG4 chains (p = 0.0016; Figure 2, A–C), and the mean κ/λ ratio was To determine whether the valency of IgG4 is important for the 1,4 ± 1,0 (eTable 1, links.lww.com/NXI/A736). To determine pathogenic function, we have performed enzymatic cleavage of 4 Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 5 | September 2022 Neurology.org/NN Figure 1 Anti-Nfasc155 IgG4 Cross-Link Nfasc155 and Undergo Fab-Arm Exchange In Vitro (A) Principle of Fab-arm exchange (FAE) and monospecificity assay. Reduced glutathione (GSH) favors FAE of monospecific IgG4 to generate bispecific IgG4. Monospecific antibodies cross-link untagged Nfasc155 with biotinylated Nfasc155. Bispecific antibodies are unable to cross-link these proteins. (B) Purified IgG4 from healthy controls (HC; n = 10), seronegative CIDP (CIDP−; n = 10) and Nfasc155+ autoimmune nodopathy (AN+; n = 10) were tested by ELISA against Nfasc155 and then incubated with a biotinylated Nfasc155 and revealed with streptavidin-HRP. IgG4 from HCs or seronegative patients did not cross-link Nfasc155. By contrast, IgG4 from Nfasc155+ patients cross-linked Nfasc155 (***p < 0.001 by one-way ANOVA followed by Bonferroni post hoc tests). The percentage of monospecific Nfasc155 antibodies correlated with the titers of anti-Nfasc155 IgG4. p Value, Spearman correlation coefficient (r), R square (R2), and 95% confidence band (dotted lines) are indicated on the graph. (C) To determine whether anti-Nfasc155 IgG4 can undergo FAE, IgG4 from Nfasc155+ patients (n = 9) were incubated with GSH in the absence (1:0) or presence of a 3-fold excess of HC IgG4 (1:3). The addition of GSH decreased IgG4 ability to cross-link Nfasc155. This effect was exacerbated by the addition of 3-fold excess of HC IgG4. The decrease in monospecific antibodies is shown on the right as ratio (**p < 0.005 compared with control condition in the absence of GSH or HC IgG4 using paired Student t tests). (D) As controls, IgG1 from Nfasc155+ patients (n = 7) were tested against Nfas155 with the monospecificity ELISA assay. Purified IgG1 cross-linked Nfasc155. GSH and 3-fold IgG4 excess did not affect IgG1-mediated cross-linking (p > 0.005 using paired Student t tests). Gray shades delineate positivity limit values. Bars represent mean and SEM, CIDP = chronic inflammatory demyelinating polyneuropathy; ns = nonsignificant; Nfasc155 = neurofascin-155. purified IgG4 from 2 Nfasc155-reactive patients (AN1 and confirmed by Western blot, and the ability of Fab and F(ab’)2 AN2) with immobilized papain or IdeS to generate monova- to bind Nfasc155 and paranodes was demonstrated not only by lent Fab or bivalent F(ab’)2 fragments. IgG4 cleavage was immunohistology on transfected cells and on teased nerve Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 5 | September 2022 5 Figure 2 Fab-Arm Exchange Modulates the Ratio of κ:λ Light Chain in Nfasc155-Reactive IgG4 (A and B) The levels of λ and κ light chains of anti-Nfasc155 IgG4 were measured by ELISA against Nfasc155 in 10 reactive patients (A). The levels of λ (green dots) and κ (blue dots) light chains were also quantified by sandwich ELISA in the purified IgG4 fractions from healthy controls (n = 10), unreactive CIDP patients (CIDP−; n = 10), and reactive autoimmune nodopathy patients (AN+; n = 10) and were compared with those found in anti-Nfasc155 IgG4 (B). The levels of κ light chain strongly correlated with those of λ light chain in Nfasc155+ patients. No significant differences in the levels of λ and κ light chains were found between the groups (p > 0.01 by one-way ANOVA followed by Bonferroni post hoc tests). p Value, Spearman correlation coefficient (r), R square (R2), and 95% confidence band (dotted lines) are indicated on the graph. (C and D) IgG4 from Nfasc155+ patients (n = 10) were incubated with of a 3-fold excess of monoclonal IgG4/λ (C) or IgG4/κ (D) in the presence or absence of reduced glutathione (GSH). The levels of κ and λ light chains were then measured by ELISA against Nfasc155, and the ratio κ/λ (red dots) was calculated. Fab-arm exchange (FAE) with IgG4/λ significantly increased the levels of λ light chains and decreased the levels of κ light chains in anti-Nfasc155 IgG4 and resulted in a decrease of the κ/λ ratio. Reversely, FAE with FAE with IgG4/κ significantly increased the levels of κ light chains and decreased those of λ light chains in anti-Nfasc155 IgG4 and resulted in an increase of the κ/λ ratio (*p < 0.05; **p < 0.005; ***p < 0.001 compared with control condition in the absence of GSH using paired Student t tests). The percentage of increase or decrease of the κ/λ ratio following FAE (gray dots) with IgG4/λ or IgG4/κ was quantified for each patient. Changes in the κ/λ ratio were significantly different following FAE with IgG4/λ or IgG4/κ (***p < 0.001 using paired Student t tests). CIPD = chronic inflammatory demyelinating polyneuropathy; Nfasc155 = neurofascin-155. fibers (eFigure 2, links.lww.com/NXI/A736) but also by nerve barrier is permeable, and IgG4 from Nfasc155-reactive ELISA. The enzymatic cleavage did not alter the affinity of anti- patients can inhibit paranode formation.17 Animals were then Nfasc155 Fab and F(ab’)2 for Nfasc155. killed 2 days after injection, and the percentages of voltage- gated sodium (Nav) channel clusters with 1, 2, or no The pathogenic potential of those fractions was then tested by CASPR1-positive paranodes were quantified. As controls, performing intraperitoneal antibody injection in new-born rat animals received a single dose of control IgG4 from healthy pups (n = 4 animals in each group). At these ages, the blood- donors. The native IgG4 from both Nfasc155-reactive 6 Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 5 | September 2022 Neurology.org/NN Figure 3 Bivalent F(ab’)2 From Nfasc155-Reactive IgG4 Abrogate Paranode Formation but Not Monovalent Fab (A and B) New-born rat pups received an intraperitoneal injection of 250 μg of anti-Nfasc155 IgG4, Fab, or F(ab’)2 from patient AN1 (n = 4 animals for each condition and age). As controls, animals received 250 μg of IgG4 from healthy donors. Two days after injection, animals were killed, and sciatic nerve fibers were fixed. Teased sciatic nerve fibers were then immunolabeled for voltage-gated sodium channels (Nav; green) to label nodes and heminodes and for CASPR1 (red) to label paranodes. (C and D) The percentage of Nav clusters lacking CASPR1-positive paranodes (arrowheads) or with 1 or 2 flanking CASPR1 positive paranodes (double arrowheads) was quantified, as well as the paranodal length (D) (n = 100–200 nodes or paranodes for each condition). The injection of native IgG4 or F(ab’)2 fragments from patient AN1 strongly abrogated the formation of CASPR1-positive paranodes and resulted in a higher percentage of heminodes lacking paranodes (****p < 0.0001, ***p < 0.001, **p < 0.005, and *p < 0.05 by one-way ANOVA followed by Bonferroni post hoc tests). The mean length of paranodes was also shorter after treatment with native IgG4 or F(ab’)2 fragments reactive to Nfasc155 (****p < 0.0001 and ***p < 0.001 by one-way ANOVA followed by Bonferroni post hoc tests). By contrast, the injection of the monovalent Fab fragment of IgG4 did not affect the formation or the length of paranodes (G and H). Scale bar: 10 μm. Bars represent mean and SEM CASPR1 = contactin-associated protein 1; ns = nonsignificant; Nfasc155 = neurofascin-155. patients strongly impaired paranode formation (Figure 3 and effects on the formation of paranodes, and the percentages of eFigure 3, links.lww.com/NXI/A736), and nearly 60% of the Nav clusters with 1, 2, or no CASPR1-positive paranodes were Nav channel clusters lacked paranodes by P2 vs 21%–25% in similar to controls (Figure 3 and eFigure 3). Also, monovalent controls (more than 100 nodes were analyzed in 4 animals per Nfasc155-reactive Fab did not affect paranodal length groups). The remaining CASPR1-positive paranodes were (Figure 3), indicating that monovalency abolishes the path- shorter compared with control animals (Figure 3; the mean of ogenicity of anti-Nfasc155 IgG4. length of more than 100 paranodes was calculated in 4 animals per condition and averaged), whereas the mean node length Bivalent F(ab’)2 IgG4 Antibodies Can Cluster was not affected (the mean of length of more than 100 nodes Nfasc155 and Abrogate Paranode Formation was calculated in 4 animals per condition and averaged). To investigate whether enzymatic cleavage also alters the F(ab’)2 fragments from patients AN1 and AN2 had similar clustering of Nfasc155 on the Schwann cell surface, sciatic effects and resulted in a significant increase of the percentage nerves were incubated in vitro for 3 hours in the presence of of Nav clusters lacking paranodes (Figure 3 and eFigure 3). native IgG4 or Fab and F(ab’)2 fragments (Figure 4). An The remaining paranodes were significantly shorter compared important clustering of IgG4 was found at the vicinity of the with controls but not to native anti-Nfasc155 IgG4 (Figure 3 nodes of Ranvier and at the mesaxon in myelinated fibers and eFigure 3), indicating that both native IgG4 and F(ab’)2 incubated with native Nfasc155-reactive IgG4 or F(ab’)2 fragments were similarly pathogenic. Reversely, the mono- fragments. By contrast, no clustering was observed on the valent Fab fragment of IgG4 from both AN1 and AN2 had no surface of Schwann cells incubated with Fab fragments or Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 5 | September 2022 7 Figure 4 Bivalent Antibodies Cluster on the Schwann Cell Surface of Myelinated Fibers (A–D) Rat sciatic nerve were incubated in vitro for 3 hours with control IgG4 (A) or with Nfasc155-reactive IgG4 (B), Fab fragment (C), or F(ab’)2 fragment (D) from patient AN1. Fibers were then immunostained for IgG (green) and contactin-1 (CNTN1; red) to label paranodes. By contrast to control IgG4, Nfasc155- reactive IgG4 accumulated at paranode vicinity (double arrowheads) and along the outer mesaxon (arrows). The cleavage into the Fab fragment inhibited IgG4 accumulation at the mesaxon and around paranodes (C). By contrast, the cleavage into the F(ab’)2 fragment did not alter the ability to aggregate at the mesaxon and paranode vicinity despite the absence of the Fc region (D). Scale bars: 10 μm. Nfasc155 = neurofascin-155. healthy control IgG4. Altogether, this further indicated that native anti-Nfasc155 IgG4 readily clustered along the bivalency is crucial for Nfasc155 clustering and paranodal Schwann and near Nav channels (eFigure 4). This clustering alterations. This also indicated that the Fc portion of IgG4 was strongly decreased in animals treated with swapped is dispensable and is not involved in the pathogenic IgG4 but not abolished (eFigure 4). mechanisms. FAE Modulates the Pathogenicity of Anti- Nfasc155 IgG4 Discussion The above finding indicates that in vitro FAE can decrease In this study, we demonstrate that a proportion of mono- the monospecificity of anti-Nfasc155 IgG4 and results in specific anti-Nfasc155 IgG4 can be detected in patients’ serum functionally monovalent antibodies. We thus tested whether and can cross-link Nfasc155 both in vitro and in vivo on promoting FAE may diminish the pathogenic action of these Schwann cells. The enzymatic cleavage into monovalent Fab autoantibodies. For that purpose, IgG4 from 2 Nfasc155- or bivalent F(ab’)2 enabled us to demonstrate that IgG4 reactive patients AN1 and AN2 were incubated in vitro with bivalency is crucial for the pathogenic action of anti-Nfasc155 a 3-fold excess of healthy control IgG4 and GSH to promote IgG4 and that monovalent antibodies are less pathogenic. Our FAE and were then injected into newborn pups. By contrast data demonstrate that these antibodies can undergo FAE in to native antibodies, the swapped antibodies had no effects vitro and in situ and that FAE modulates the pathogenic on the formation of paranodes in newborn pups, and the potential of autoreactive IgG4. The presence of monospecific percentages of Nav clusters with 1, 2, or no CASPR1- IgG4 antibodies was not due to an inherent inability of anti- positive paranodes were similar to those of control animals Nfasc155 IgG4 to undergo interchain exchange because the (Figure 5 and eFigure 4, links.lww.com/NXI/A736). How- simple addition of GSH was sufficient to diminish the ever, paranodal length appeared shorter in animals treated amounts of monospecific antibodies. Perhaps anti-Nfasc155 with swapped antibodies compared with control animals. IgG4 have not all undergone FAE in situ with other circu- The quantification of paranodal length confirmed this ob- lating IgG4. In keeping with this, the κ:λ ratios of anti-Nfasc155 servation, and paranodes were found to be significantly IgG4 did not perfectly match with those of circulating IgG4 in shorter in animals treated with swapped IgG4 compared each patient. Nonetheless, patients with Nfasc155 autoimmune with control animals but still significantly longer than those nodopathy did not carry exclusively monospecific bivalent, and of animals treated with native Nfasc155-reactive IgG4 the percentage of monospecific bivalent anti-Nfasc155 IgG4 (Figure 5 and eFigure 4). This indicated that FAE can po- ranged between 7% and 78% in patients. tently decrease the pathogenic effect of anti-Nfasc155 IgG4. In our previous study, we found that anti-Nfasc155 IgG4 FAE enables the formation of functionally monovalent IgG4 aggregate near the nodes in injected animals.17 Here also, that are anti-inflammatory and participate to immune 8 Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 5 | September 2022 Neurology.org/NN Figure 5 Fab-Arm Exchange Decreases the Pathogenicity of Nfasc155-Reactive IgG4 (A) Fab-arm exchange (FAE) was induced in vitro between IgG4 from patient AN1 and healthy donor IgG4. New-born rat pups were injected at birth with control IgG4 (250 μg per animal), IgG4 from patient AN1 (250 μg per animal), or swapped IgG4 from patient AN1 (FAE; 1 mg per animal) and killed after 2 days. Teased fibers were immunostained for voltage-gated sodium channels (Nav; green) and CASPR1 (red) (n = 4 animals for each condition and age). (B and C) The percentage of Nav clusters lacking CASPR1-positive paranodes (arrows) and Nav clusters with 1 or 2 flanking paranodes (double arrowheads) was counted in each group. The injection of native Nfasc155-reactive IgG4 reduced the formation of CASPR1-positive paranodes and significantly decreased the mean length of paranodes. By contrast, swapped reactive Nfasc155 IgG4 did not significantly affect paranode formation. Nonetheless, paranodal length was decreased in animals treated with swapped IgG4 (****p < 0.0001, ***p < 0.001, **p < 0.005, and *p < 0.05 by one-way ANOVA followed by Bonferroni post hoc tests) (n = 100–200 nodes or paranodes for each condition). Scale bars: 10 μm. Bars represent mean and SEM. CASPR1 = contactin-associated protein 1; ns = nonsignificant; Nfasc155 = neurofascin-155. tolerance through the blocking of the autoantigen epitope and unreactive IgG4. Also, we cannot exclude the fact that a fraction by competing with other subclass antibodies.29 Class switch to of the swapped antibodies rearranged in vivo into monospecific IgG4 following chronic exposure to antigens is thus suspected anti-Nfasc155 IgG4. Thus, the limited effects of swapped anti- to dampen the immune reaction. Nonetheless, IgG4 anti- Nfasc155 IgG4 are likely mediated by residual monospecific bodies are associated with numerous autoimmune patholo- antibodies. To avoid this issue, a mutant form of IgG4 (S228P, gies,30 and recently, FAE has been shown to promote the F405L, and R409K) and of anti-MuSK IgG4 (S228P) were pathogenicity of anti-MuSK antibodies in myasthenia gravis recently used to create under controlled reducing conditions a through function-blocking activity.20 In a previous study, we stable bispecific antibody.21 We did not use this technique here showed that anti-Nfasc155 IgG4 does not have a function- because anti-Nfasc155 IgG4 has not been cloned. blocking activity.17 Instead, anti-Nfasc155 antibodies appeared to cross-link Nfasc155, promote Nfasc155 degra- Using IgG1 against Nfasc155, we have been able to esti- dation, and thus abolish the association with the CNTN1/ mate the percentage of monospecific anti-Nfasc155 IgG4. CASPR1 complex at the paranode. In neonatal animals, the These levels were generally low with a median of 18% and injection of native anti-Nfasc155 IgG4 thereby inhibited para- were proportional to antibody titers, suggesting that node formation and resulted in either shorter or no paranode. monospecific antibodies may either be newly synthesized Proteolytic cleavage into monovalent Fab abrogated the path- or may simply persist because of their prevalence. However, ogenic capacity of anti-Nfasc155 IgG4. Promoting FAE in vitro these levels did not correlate with clinical severity or re- also strongly decreased the effect of anti-Nfasc155 IgG4 but did sponse to treatment. Most patients were unresponsive to not abolish paranode shortening. FAE is a dynamic process, IVIg but responded positively to either plasmapheresis or and our data demonstrate that a proportion of IgG4 still remain rituximab. This further highlighted that low levels of monospecific even in the presence of a three-fold excess of monospecific anti-Nfasc155 IgG4 are sufficient to be Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 5 | September 2022 9 detrimental and that antibody-depleting strategies are the levels of GSH have been reported to be decreased not only in most efficient in those patients. some inflammatory diseases but also during aging.42 FAE may thus be less efficient in some patients because of GSH de- The absence of clinical correlate with the percentage of ficiency. A correlation was previously reported between the monospecific anti-Nfasc155 IgG4 is the main limitation of our severity of CIDP and the age of patients with antibodies to the study. From this small cohort study, the quantification of nodes of Ranvier.43 However, we did not find such correlation monospecific IgG4 does not seem to have clinical value for here, and generally, patients with anti-Nfasc155 IgG4 were prognosis or treatment choice. However, a larger cohort study younger.6,35,37,44 Thus, patients’ age may not be the critical recently demonstrated that anti-Nfasc155 IgG4 titers follow factor influencing IgG4 monospecificity. Nonetheless, we clinical severity scores and are useful to monitor disease sta- cannot rule out that low GSH blood levels may participate in tus.31 Anyhow, this study demonstrates that IgG4 valency the persistence of monospecific antibodies. We did not mea- dictates the pathogenicity of anti-Nfasc155 and that FAE can sure GSH blood levels in our cohort because blood samples potently decrease the pathogenic potential of these autoan- were not available and plasma levels of GSH are marginally low tibodies. Of course, one question comes to mind: why are compared with blood levels. those patients unresponsive to IVIg6,12,32-35 if IgG4 effectively decreases autoantibody pathogenicity? IgG4 represent less Altogether, our data indicate that bivalent IgG4 are patho- than 3% of IVIg. Thus, the amount of IgG4 present in a usual genic in Nfasc155 autoimmune nodopathy and that pro- dose of IVIg (2 g/kg) may be insufficient to mask patients’ moting FAE using healthy IgG4 and GSH can decrease the pathogenic antibodies. In addition, IVIg are known to act pathogenic potential of these autoantibodies. It has to be kept through several modes of action involving the Fc portion of in mind that promoting FAE may be detrimental in anti- IgG including the activation of immunomodulatory FcγRIIb, MuSK myasthenia gravis.20,21 Future works should examine the saturation of FcRn, or the inhibition of complement.36 whether FAE is beneficial or detrimental in anti-CNTN1 and IgG4 does not activate complement, FcγRIIb, and our data anti-CASPR1 autoimmune nodopathy. further demonstrated that the Fc domain of anti-Nfasc155 IgG4 is dispensable. Thus, by contrast to other isotypes, IgG4 Acknowledgment are not acting through Fc-mediated effector function, and On behalf of all her trainees and colleagues, this paper is the mode of actions of IVIg appears irrelevant for Nfasc155 dedicated to the memory of Isabel Illa. The authors thank Drs autoimmune nodopathy. Laurence Goutebroze for generous gift of antibodies. The authors thank Mallory Poncelet for technical assistance. The fact that bivalent antibodies are pathogenic points out that IgG1 or IgG3 could also be pathogenic. IgG1 or IgG3 to Study Funding Nfasc155 are infrequently found in autoimmune nodopathy. Supported by the Agence Nationale pour la Recherche Only a few patients here presented with IgG1 to Nfasc155, and (NECCIN; GT and JJD) and from the Association Française lower titers of IgG1 have previously been reported compared contre les Myopathies (grant no. 23593; GT and JJD). with IgG4.37 Nonetheless, we found here that IgG1 antibodies were able to cross-link Nfasc155 and may thus be pathogenic. Disclosure Several recent studies described the presence of IgG1 or IgG3 to C. Roué, J. Raymond, A. Attal, A. Jentzer, C. Lleixà, I. Illa, and pan-neurofascin in patients with CIDP or Guillain-Barré G. Taieb have no relevant disclosures. L. Querol received syndrome.9,38,39 In addition to complement activation, these research funds from Novartis Spain, Sanofi-Genzyme, and antibodies may alter paranodal regions by favoring Nfasc155 Grifols. J. Devaux received a research grant from CSL Behring. aggregation and degradation. Recently, IgG3 to CNTN1 have Go to Neurology.org/NN for full disclosures. also been shown to affect the surface expression of CNTN1 in neurons.40 The cleavage of anti-CNTN1 IgG into Fab, but not Publication History the F(ab’)2 fragment, abolished these effects. This further sug- Received by Neurology: Neuroimmunology & Neuroinflammation gests that the monospecificity of IgG1 and IgG3 to CNTN1 may February 10, 2022. Accepted in final form May 18, 2022. Submitted and externally peer reviewed. The handling editor was Marinos C. participate in their pathogenic actions. It still remains to be Dalakas, MD, FAAN. demonstrated whether this is also true for anti-CNTN1 IgG4. In any cases, affinity maturation of IgG4 appears to be required for the development of the pathogenic activity, as demonstrated in MusK myasthenia gravis.41 Thus, antibody affinity and titers are Appendix 1 Authors likely to further influence antibody pathogenicity. Name Location Contribution The mechanisms responsible for IgG4 swapping in situ are not Alexandre Institute for Neurosciences of Drafting and revision of Jentzer, Montpellier, INSERM U1051, the manuscript, yet well defined. GSH was originally suspected to enable FAE MD, PhD Montpellier University, Hôpital acquisition of data, and in cells because a low-molecular-weight compound extracted Saint Eloi, France analysis and from erythrocyte lysate could promote FAE.19 Here, GSH also Department of Immunology, CHU Montpellier, France interpretation of data promoted the FAE of anti-Nfasc155 IgG4 in vitro. The blood 10 Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 5 | September 2022 Neurology.org/NN Appendix 1 (continued) Appendix 2 (continued) Name Location Contribution Name Location Role Contribution Arthur Institute for Neurosciences of Drafting and revision of Thierry Hôpital de la Pitié Coinvestigator Provided Attal, MD Montpellier, INSERM U1051, the manuscript, Maisonobe Salpêtrière, Paris, France samples and Montpellier University, Hôpital acquisition of data, and clinical data Saint Eloi, France analysis and Department of Neurology, CHU interpretation of data Philippe CHU Amiens Picardie, Coinvestigator Provided Montpellier, Hôpital Gui de Merle Amiens, France samples and Chauliac, France clinical data Clémence Institut de Génomique Acquisition of data and Chokri Habib Bourguiba Coinvestigator Provided Roué, MS Fonctionnelle, CNRS UMR5203, analysis and Mhiri University hospital, 3029 samples and 141 rue de la Cardonille, France interpretation of data Sfax, Tunisia clinical data Julie Institut de Génomique Acquisition of data Salma Habib Bourguiba Coinvestigator Provided Raymond, Fonctionnelle, CNRS UMR5203, Sakka University hospital, 3029 samples and MS 141 rue de la Cardonille, France Sfax, Tunisia clinical data Cinta Neuromuscular Diseases Unit, Drafting and revision of Karine Hôpital de la Pitié Coinvestigator Provided Lleixà, MD, Hospital de la Santa Creu i Sant the manuscript, Viala Salpêtrière, Paris, France samples and PhD Pau, Universitat Autónoma de acquisition of data, and clinical data Barcelona, Spain analysis and interpretation of data Adrien Hôpital Foch, Suresnes, Coinvestigator Provided Wang France samples and Isabel Illa, Neuromuscular Diseases Unit, Drafting and revision of clinical data MD, PhD Hospital de la Santa Creu i Sant the manuscript, Pau, Universitat Autónoma de acquisition of data, and Barcelona, Spain analysis and interpretation of data References 1. 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Neurol Neuroimmunol Neuroinflamm 2022;9; DOI 10.1212/NXI.0000000000200014 This information is current as of August 10, 2022 Updated Information & including high resolution figures, can be found at: Services http://nn.neurology.org/content/9/5/e200014.full.html References This article cites 44 articles, 8 of which you can access for free at: http://nn.neurology.org/content/9/5/e200014.full.html##ref-list-1 Citations This article has been cited by 1 HighWire-hosted articles: http://nn.neurology.org/content/9/5/e200014.full.html##otherarticles Permissions & Licensing Information about reproducing this article in parts (figures,tables) or in its entirety can be found online at: http://nn.neurology.org/misc/about.xhtml#permissions Reprints Information about ordering reprints can be found online: http://nn.neurology.org/misc/addir.xhtml#reprintsus Neurol Neuroimmunol Neuroinflamm is an official journal of the American Academy of Neurology. 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