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Background and Objectives: Polyneuropathy associated with an immunoglobulin M (IgM) monoclonal gammopathy is characterized by slowly progressive, predominantly distal sensorimotor deficits, sensory ataxia, and electrophysiologic features of demyelination. IgM antibodies against myelin-associated glycoprotein (MAG) are present in serum from most patients. Nerve damage most likely results from the concerted action of binding of anti-MAG antibodies to nerves, followed by complement activation. The interaction of anti-MAG antibodies with complement activation and their relation to clinical characteristics have not been studied in detail. We studied the correlation among anti-MAG antibody titers, complement activation, and IgM-associated polyneuropathy disease severity., Methods: We used serum samples from 101 patients with IgM-associated polyneuropathy to assess IgM anti-MAG titers by ELISA and antibody-mediated complement deposition using both an ELISA-based system and a cell-based system of primate peripheral nerve slides. We studied correlations of complement activation with anti-MAG ELISA titers and clinical characteristics., Results: IgM anti-MAG titers varied from negative to strongly positive. Complement deposition in the ELISA-based system correlated significantly with anti-MAG antibody titer (Spearman rho 0.80; p < 0.0001) despite large variability between serum samples with comparable anti-MAG titers. This variability was even larger in the cell-based assay, which also showed complement deposition in IgM anti-MAG negative patients, indicating the presence of autoantibodies directed against epitopes other than MAG in a subset of patients with IgM-associated polyneuropathy. Clinical characteristics did not correlate with anti-MAG titers or complement activation., Discussion: Anti-MAG antibody titers correlate with the level of complement activation in both ELISA and cell-based systems. However, clinical characteristics of IgM-associated polyneuropathy do not or only weakly correlate with titers or the level of complement deposition. The lack of clear correlations between complement activation and clinical characteristics does, at this stage, not support the use of complement inhibitors in the treatment of IgM-associated polyneuropathy.

Background and Purpose: Complement factor C2 is a potential therapeutic target in immune-mediated neuropathies. However, literature suggests that classical complement pathway activation may proceed to C3 in the absence of C2, a so-called "C2 bypass." Here, we evaluated a C2 bypass mechanism during complement activation by pathogenic human IgM from patients with immune-mediated neuropathies., Methods: IgM autoantibodies from 51 patients with multifocal motor neuropathy (MMN) or anti-myelin-associated glycoprotein (MAG) neuropathy (AMN) were used to activate complement in ex vivo disease models. C2 bypass was evaluated using C2-depleted (C2D) serum and a therapeutic anti-C2 antibody., Results: In two different disease models of MMN, IgM anti-GM1 and IgM anti-GM2 autoantibodies from MMN patients were bound to induced pluripotent stem cell-derived motor neurons and Schwann cells, respectively, and fixed C3 upon incubation with fresh serum. C3 fixation was inhibited by anti-C2 and did not occur with C2D serum. Similarly, in an AMN model, IgM anti-MAG antibodies were incubated with fresh serum fixed C3, which in all cases was abrogated in the absence of C2 or in the presence of anti-C2., Conclusions: In ex vivo disease models of MMN and AMN, complement activation by IgM autoantibodies from 51 patients was in all cases dependent on C2 and was inhibited by an antihuman C2 antibody. No evidence of a C2 bypass mechanism was found., (© 2024 The Author(s). European Journal of Neurology published by John Wiley & Sons Ltd on behalf of European Academy of Neurology.)

Background and Aims: To further substantiate the role of antibody-mediated complement activation in multifocal motor neuropathy (MMN) immunopathology, we investigated the distribution of promotor polymorphisms of genes encoding the membrane-bound complement regulators CD46, CD55, and CD59 in patients with MMN and controls, and evaluated their association with disease course., Methods: We used Sanger sequencing to genotype five common polymorphisms in the promotor regions of CD46, CD55, and CD59 in 133 patients with MMN and 380 controls. We correlated each polymorphism to clinical parameters., Results: The genotype frequencies of rs28371582, a 21-bp deletion in the CD55 promotor region, were altered in patients with MMN as compared to controls (p .009; Del/Del genotype 16.8% vs. 7.7%, p .005, odds ratio: 2.43 [1.27-4.58]), and patients carrying this deletion had a more favorable disease course (mean difference 0.26 Medical Research Council [MRC] points/year; 95% confidence interval [CI]: 0.040-0.490, p .019). The presence of CD59 rs141385724 was associated with less severe pre-diagnostic disease course (mean difference 0.940 MRC point/year; 95% CI: 0.083-1.80, p .032)., Interpretation: MMN susceptibility is associated with a 21-bp deletion in the CD55 promotor region (rs2871582), which is associated with lower CD55 expression. Patients carrying this deletion may have a more favorable long-term disease outcome. Taken together, these results point out the relevance of the pre-C5 level of the complement cascade in the inflammatory processes underlying MMN., (© 2024 The Authors. Journal of the Peripheral Nervous System published by Wiley Periodicals LLC on behalf of Peripheral Nerve Society.)

Background: Multifocal motor neuropathy (MMN) is a rare, chronic immune-mediated polyneuropathy characterized by asymmetric distal limb weakness. An important feature of MMN is the presence of IgM antibodies against gangliosides, in particular GM1 and less often GM2. Antibodies against GM1 bind to motor neurons (MNs) and cause damage through complement activation. The involvement of Schwann cells (SCs), expressing GM1 and GM2, in the pathogenesis of MMN is unknown., Methods: Combining the data of our 2007 and 2015 combined cross-sectional and follow-up studies in Dutch patients with MMN, we evaluated the presence of IgM antibodies against GM1 and GM2 in serum from 124 patients with MMN and investigated their binding to SCs and complement-activating properties. We also assessed the relation of IgM binding and complement deposition with clinical characteristics., Results: Thirteen out of 124 patients (10%) had a positive ELISA titer for IgM anti-GM2. Age at onset of symptoms was significantly lower in MMN patients with anti-GM2 IgM. IgM binding to SCs correlated with IgM anti-GM2 titers. We found no correlation between IgM anti-GM2 titers and MN binding or with IgM anti-GM1 titers. IgM binding to SCs decreased upon pre-incubation of serum with soluble GM2, but not with soluble GM1. IgM anti-GM2 binding to SCs correlated with complement activation, as reflected by increased C3 fixation on SCs and C5a formation in the supernatant., Conclusion: Circulating IgM anti-GM2 antibodies define a subgroup of patients with MMN that has an earlier onset of disease. These antibodies probably target SCs specifically and activate complement, similarly as IgM anti-GM1 on MNs. Our data indicate that complement activation by IgM antibodies bound to SCs and MNs underlies MMN pathology., (© 2024. The Author(s).)

Objective: Antibody- and complement-mediated peripheral nerve inflammation are central in the pathogenesis of MMN. Here, we studied innate immune responses to endotoxin in patients with MMN and controls to further our understanding of MMN risk factors and disease modifiers., Methods: We stimulated whole blood of 52 patients with MMN and 24 controls with endotoxin and collected plasma. With a multiplex assay, we determined levels of the immunoregulating proteins IL-1RA, IL-1β, IL-6, IL-10, IL-21, TNF-α, IL-8 and CD40L in unstimulated and LPS-stimulated plasma. We compared baseline and stimulated protein levels between patients and controls and correlated concentrations to clinical parameters., Results: Protein level changes after stimulation were comparable between groups (p > 0.05). IL-1RA, IL-1β, IL-6 and IL-21 baseline concentrations showed a positive correlation with monthly IVIg dosage (all corrected p-values < 0.016). Patients with anti-GM1 IgM antibodies showed a more pronounced IL-21 increase after stimulation (p 0.048)., Conclusions: Altered endotoxin-induced innate immune responses are unlikely to be a susceptibility factor for MMN., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The Fc gamma receptor I (FcγRI or CD64) is the only human Fc receptor with a high affinity for monomeric IgG. It plays a crucial role in immunity, as it mediates cellular effector functions of antibodies including phagocytosis, antigen presentation, and cytokine production. FcγRI is constitutively saturated with monomeric IgG and this feeds the dogma that it has no role in immune responses. However, recent findings have implicated a role for FcγRI in various autoimmune disorders, neuropathies, and antibody therapy in tumor models. By a process known as 'inside-out' signaling, stimulation of myeloid cells with cytokines such as tumor necrosis factor alpha (TNF-α) and interferon-gamma (IFN-γ) enhances FcγRI binding to immune complexes (ICs), including antibody-opsonized pathogens or tumor cells. This review focuses on the current knowledge on interaction of FcγRI with IgG and ICs and the effect of inside-out signaling on FcγRI functioning. Additionally, this review will address potential clinical applications of targeting FcγRI, and the tools that can be used to overcome IC-mediated autoimmune diseases on the one hand, and to enhance antibody-based anti-cancer therapy on the other., (© The Author(s) 2022. Published by Oxford University Press on behalf of the British Society for Immunology.)

Background and Objectives: To determine the role of complement in the disease pathology of multifocal motor neuropathy (MMN), we investigated complement activation, and inhibition, on binding of MMN patient-derived immunoglobulin M (IgM) antibodies in an induced pluripotent stem cell (iPSC)-derived motor neuron (MN) model for MMN., Methods: iPSC-derived MNs were characterized for the expression of complement receptors and membrane-bound regulators, for the binding of circulating IgM anti-GM1 from patients with MMN, and for subsequent fixation of C4 and C3 on incubation with fresh serum. The potency of ARGX-117, a novel inhibitory monoclonal antibody targeting C2, to inhibit fixation of complement was assessed., Results: iPSC-derived MNs moderately express the complement regulatory proteins CD46 and CD55 and strongly expressed CD59. Furthermore, MNs express C3aR, C5aR, and complement receptor 1. IgM anti-GM1 antibodies in serum from patients with MMN bind to MNs and induce C3 and C4 fixation on incubation with fresh serum. ARGX-117 inhibits complement activation downstream of C4 induced by patient-derived anti-GM1 antibodies bound to MNs., Discussion: Binding of IgM antibodies from patients with MMN to iPSC-derived MNs induces complement activation. By expressing complement regulatory proteins, particularly CD59, MNs are protected against complement-mediated lysis. Yet, because of expressing C3aR, the function of these cells may be affected by complement activation upstream of membrane attack complex formation. ARGX-117 inhibits complement activation upstream of C3 in this disease model for MMN and therefore represents an intervention strategy to prevent harmful effects of complement in MMN., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Background: The addition of monoclonal antibody therapy against GD2 to the treatment of high-risk neuroblastoma led to improved responses in patients. Nevertheless, administration of GD2 antibodies against neuroblastoma is associated with therapy-limiting neuropathic pain. This severe pain is evoked at least partially through complement activation on GD2-expressing sensory neurons., Methods: To reduce pain while maintaining antitumor activity, we have reformatted the approved GD2 antibody ch14.18 into the IgA1 isotype. This novel reformatted IgA is unable to activate the complement system but efficiently activates leukocytes through the FcαRI (CD89)., Results: IgA GD2 did not activate the complement system in vitro nor induced pain in mice. Importantly, neutrophil-mediated killing of neuroblastoma cells is enhanced with IgA in comparison to IgG, resulting in efficient tumoricidal capacity of the antibody in vitro and in vivo., Conclusions: Our results indicate that employing IgA GD2 as a novel isotype has two major benefits: it halts antibody-induced excruciating pain and improves neutrophil-mediated lysis of neuroblastoma. Thus, we postulate that patients with high-risk neuroblastoma would strongly benefit from IgA GD2 therapy., Competing Interests: Competing interests: JL is co-founder of TigaTx and ME is employed by TigaTx., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Activation of the classical and lectin pathway of complement may contribute to tissue damage and organ dysfunction of antibody-mediated diseases and ischemia-reperfusion conditions. Complement factors are being considered as targets for therapeutic intervention., Objective: We sought to characterize ARGX-117, a humanized inhibitory monoclonal antibody against complement C2., Methods: The mode-of-action and binding characteristics of ARGX-117 were investigated in detail. Furthermore, its efficacy was analyzed in in vitro complement cytotoxicity assays. Finally, a pharmacokinetic/pharmacodynamic study was conducted in cynomolgus monkeys., Results: Through binding to the Sushi-2 domain of C2, ARGX-117 prevents the formation of the C3 proconvertase and inhibits classical and lectin pathway activation upstream of C3 activation. As ARGX-117 does not inhibit the alternative pathway, it is expected not to affect the antimicrobial activity of this complement pathway. ARGX-117 prevents complement-mediated cytotoxicity in in vitro models for autoimmune hemolytic anemia and antibody-mediated rejection of organ transplants. ARGX-117 exhibits pH- and calcium-dependent target binding and is Fc-engineered to increase affinity at acidic pH to the neonatal Fc receptor, and to reduce effector functions. In cynomolgus monkeys, ARGX-117 dose-dependently reduces free C2 levels and classical pathway activity. A 2-dose regimen of 80 and 20 mg/kg separated by a week, resulted in profound reduction of classical pathway activity lasting for at least 7 weeks., Conclusions: ARGX-117 is a promising new complement inhibitor that is uniquely positioned to target both the classical and lectin pathways while leaving the alternative pathway intact., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Development of chronic rejection is still a severe problem and causes high mortality rates after lung transplantation (LTx). Complement activation is important in the development of acute rejection (AR) and bronchiolitis obliterans syndrome, with C3 as a key complement factor. Methods: We investigated a single nucleotide polymorphism (SNP) in the C3 gene (rs2230199) in relation to long-term outcome after LTx in 144 patient-donor pairs. In addition, we looked at local production of donor C3 by analyzing bronchoalveolar lavage fluid (BALF) of 6 LTx patients using isoelectric focusing (IEF). Results: We demonstrated the presence of C3 in BALF and showed that this is produced by the donor lung based on the genotype of SNP rs2230199. We also analyzed donor and patient SNP configurations and observed a significant association between the SNP configuration in patients and episodes of AR during 4-years follow-up. Survival analysis showed a lower AR-free survival in homozygous C3 slow patients ( p = 0.005). Furthermore, we found a significant association between the SNP configuration in donors and BOS development. Patients receiving a graft from a donor with at least one C3 fast variant for rs2230199 had an inferior BOS-free survival ( p = 0.044). Conclusions: In conclusion, our data indicate local C3 production by donor lung cells. In addition, a single C3 SNP present in recipients affects short-term outcome after LTx, while this SNP in donors has an opposite effect on long-term outcome after LTx. These results could contribute to an improved risk stratification after transplantation., (Copyright © 2019 Kardol-Hoefnagel, Budding, van de Graaf, van Setten, van Rossum, Oudijk and Otten.)

Obstructive chronic lung allograft dysfunction (BOS) is the major limiting factor for lung transplantation (LTx) outcome. PTPN22 is described as the hallmark autoimmunity gene, and one specific single nucleotide polymorphism (SNP), rs2476601, is associated with multiple autoimmune diseases, impaired T cell regulation, and autoantibody formation. Taking into consideration the contribution of autoimmunity to LTx outcome, we hypothesized that polymorphisms in the PTPN22 gene could be associated with BOS incidence. We selected six SNPs within PTPN22 and analyzed both patient and donor genotypes on BOS development post-LTx. A total of 144 patients and matched donors were included, and individual SNPs and haplotype configurations were analyzed. We found a significant association between patients carrying the heterozygous configuration of rs2476601 and a higher risk for BOS development ( p = 0.005, OR: 4.400, 95%CI: 1.563-12.390). Kaplan-Meier analysis showed that heterozygous patients exhibit a lower BOS-free survival compared to patients homozygous for rs2476601 ( p = 0.0047). One haplotype, which solely contained the heterozygous risk variant, was associated with BOS development ( p = 0.015, OR: 7.029, 95%CI: 1.352-36.543). Our results show that LTx patients heterozygous for rs2476601 are more susceptible for BOS development and indicate a deleterious effect of the autoimmune-related risk factor of PTPN22 in patients on LTx outcome.

Cellular protection against undesired effects of complement activation is provided by expression of membrane-bound complement regulatory proteins including CD59. This protein prevents membrane attack complex formation and is considered to be involved in graft accommodation. Also, CD59 downregulates CD4+ and CD8+ T-cell activation and proliferation. It is unknown whether CD59 expression is affected by transplantation. The aim of this study was to evaluate the quantitative CD59 antigen expression on distinct leukocyte subsets following lung transplantation ( n  = 26) and to investigate whether this differs from pretransplantation ( n  = 9). The results show that CD59 expression on leukocytes is significantly lower posttransplantation compared with healthy controls ( p  = 0.002) and pretransplantation ( p  < 0.0001). Moreover, the CD59 expression diminishes posttransplantation on all distinct lymphocyte subsets ( p  < 0.02). This effect appeared to be specific for CD59 since the expression of other surface markers remained stable or inclined following transplantation. The highest antigen expression posttransplantation was observed on CD4+ T cells and monocytes ( p  ≤ 0.002). These findings show that CD59 expression on leukocytes diminishes posttransplantation, which could result in decreased resistance against complement and enhanced T-cell activation. If such reduction in CD59 expression also occurs on endothelial cells from the transplanted organ, this could lead to a change into a prothrombotic and proinflammatory phenotype.

Lung transplantation (LTx) outcome is hampered by development of chronic rejection, often manifested as the bronchiolitis obliterans syndrome (BOS). Low serum levels of thymus and activation-regulated chemokine (TARC/CCL17), a chemoattractant, measured during the first month post-LTx are predictive for BOS development. Since TARC/CCL17 promotor polymorphisms correlate with serum TARC/CCL17 levels, we investigated seven single-nucleotide polymorphisms (SNPs) within this region and their potential association with LTx outcome. We analyzed donor and patient SNP configurations and haplotypes and observed a trend between a donor SNP (rs223899) configuration and patient TARC/CCL17 serum levels post-LTx ( p  = 0.066). Interestingly, this SNP configuration in patients did not show any correlation with pre-LTx TARC/CCL17 serum levels ( p  = 0.776). Survival analysis showed that receiving a graft from a donor heterozygous for rs223899 has a disadvantageous impact on transplantation outcome. When stratified per donor SNP genotype, patients receiving a transplant from a heterozygous donor showed a lower BOS-free survival ( p  = 0.023) and survival rate ( p  = 0.0079). Since rs223899 is located within a NFκB binding site, heterozygosity at this position could result in a reduced TARC/CCL17 expression. Our data indicate that a single TARC/CCL17 promotor SNP in the donor correlates with lower serum TARC/CCL17 levels measured 1 month after LTx and affects clinical outcome after LTx.

Antibodies against HLA and non-HLA are associated with transplantation outcome. Recently, pretransplant serum IgG antibody levels against apoptotic cells were found to correlate with kidney allograft loss. We investigated the presence of these antibodies in lung transplantation (LTx) patients and evaluated the correlation of pre-LTx serum levels of IgG antibodies against apoptotic cells with LTx outcome. These cells included donor lung endothelial cells (ECs) obtained from lung perfusion fluid collected during LTx procedure. Cells were isolated, expanded in vitro , and analyzed as targets for antiapoptotic cell reactivity. Cultured cells exhibited EC morphology and were CD31+, CD13+, and vWF+. End-stage lung disease patients showed elevated serum IgG levels against apoptotic lung EC ( p  = 0.0018) compared to healthy controls. Interestingly, the levels of circulating antibodies directed against either apoptotic Jurkat cells or apoptotic lung ECs did not correlate, suggesting a target cell specificity. We observed no correlation between chronic or acute rejection and pre-LTx serum levels of antiapoptotic antibodies. Also, these levels did not differ between matched patients developing chronic rejection or not during follow-up or at the time of diagnosis, as they remained as high as prior to transplantation. Thus, circulating levels of antiapoptotic cell antibodies are elevated in end-stage lung disease patients, but our data do not correlate with outcome after LTx.

CD59 is a complement regulatory protein that inhibits membrane attack complex formation. A soluble form of CD59 (sCD59) is present in various body fluids and is associated with cellular damage after acute myocardial infarction. Lung transplantation (LTx) is the final treatment for end-stage lung diseases, however overall survival is hampered by chronic lung allograft dysfunction development, which presents itself obstructively as the bronchiolitis obliterans syndrome (BOS). We hypothesized that, due to cellular damage and activation during chronic inflammation, sCD59 serum levels can be used as biomarker preceding BOS development. We analyzed sCD59 serum concentrations in 90 LTx patients, of whom 20 developed BOS. We observed that BOS patients exhibited higher sCD59 serum concentrations at the time of diagnosis compared to clinically matched non-BOS patients (p = 0.018). Furthermore, sCD59 titers were elevated at 6 months post-LTx (p = 0.0020), when patients had no BOS-related symptoms. Survival-analysis showed that LTx patients with sCD59 titers ≥400 pg/ml 6 months post-LTx have a significant (p < 0.0001) lower chance of BOS-free survival than patients with titers ≤400 pg/ml, 32% vs. 80% respectively, which was confirmed by multivariate analysis (hazard ratio 6.2, p < 0.0001). We propose that circulating sCD59 levels constitute a novel biomarker to identify patients at risk for BOS following LTx.

After lung transplantation (LTx), circulating mononuclear cell composition and their subsets may be predictive for the bronchiolitis obliterans syndrome (BOS). We investigated the cellular composition in patients developing BOS, or not, by analyzing peripheral blood taken at multiple time points after transplantation. PBMCs of 11 BOS and 39 non-BOS patients were analyzed by FACS for monocytes, dendritic cells, NK-, NKT-, B- and T cells as well as B- and T cell subsets. Analysis of blood samples taken monthly during the first year post-LTx showed that circulating NK, NKT and dendritic cell percentages were not indicative of BOS development, whereas increases in T cells, monocytes and lowered fractions of B cells were related to BOS development. B- and T cell subset analysis at month 5 post-LTx indicated that IgM+IgD- memory B cells and central memory CD8+ T cells were decreased, whereas NKT cells were increased in BOS patients compared to non-BOS patients. Prior to BOS diagnosis, the composition of specific mononuclear cells on a group level differs from patients remaining BOS free. However, given the overlap in percentages of cellular frequencies between the patient groups investigated, this analysis does not allow prediction or risk stratification for development of BOS in individual patients., (Copyright © 2015 Elsevier B.V. All rights reserved.)