TCR-Vβ Usage in the Thymus and Blood of Myasthenia Gravis Patients

In myasthenia gravis (MG) the muscle acetylcholine receptor (AChR) is the target of an autoimmune response. The anti-AChR response may originate in the thymus, which is abnormal in most MG patients and contains anti-AChR T and B cells. Microbial superantigens (sAg) may trigger autoimmune responses and in this study we sought clues as to whether sAg play a role in the pathogenesis of MG. We investigated the frequency of use of the different TCR Vβ families by the thymus and blood T cells in MG patients and in control subjects, using a multi-primer PCR assay. Identical TCR-Vβ usage was found in the thymi of MG patients and controls, except Vβ2, which showed a small increase in MG patients’ thymi. Blood T cells of MG patients used Vβ4, Vβ6, Vβ15, Vβ16 and Vβ24 significantly more than those of the controls. Vβ4 and Vβ6 are the gene families most frequently used by anti-AChR CD4⁺cells in MG patients. Blood T cells from MG patients used Vβ12, Vβ14, Vβ17 and Vβ18 significantly less than controls. MG patients used Vβ4 and Vβ6 significantly more in the blood than in the thymus, while the opposite occurred for Vβ7, Vβ12 and Vβ14. Controls used Vβ17 more and Vβ24 less in the blood than in the thymus. The preferential expansion of Vβ4 and Vβ6 in MG patients might reflect the immunodominance of certain AChR epitopes, or the action of a sAg outside the thymus. The minimal differences in the TCR-Vβ usage in the blood and thymus of control subjects might be due to expansion of T cell clones specific for common antigens. Identical Vβ usage in the thymi of MG patients and controls does not support an important role of the thymus as the location of anti-AChR sensitization when MG is clinically evident. The differences observed in the Vβ usage in blood and thymi of MG patients are likely to be due to preferential Vβ usage by the anti-AChR T cells in the blood.     

胸腺扩大切除治疗重症肌无力围术期血清IL-17、IL-18、IL-27水平分析

目的探讨胸腺异常合并重症肌无力（MG）患者行胸腺扩大切除术后外周血血清IL—17、IL-18、IL-27等细胞因子的变化情况及其意义。方法选取16例未经免疫抑制治疗的MG患者行胸腔镜下胸腺扩大切除术,采用双抗体夹心ELISA法检测术前及术后第7El外周血血清IL-17、IL．18、IL．27水平,以15例健康献血者的血清IL-17、IL-18、IL-27水平作为正常对照。结果胸腺切除术后MG患者外周血IL-17、IL-18、IL-27水平普遍降低,外周血IL-17、IL-18、IL-27水平在胸腺瘤和胸腺增生两种病理类型中无明显差异。结论手术切除胸腺可降低MG患者外周血IL-17、IL-18、IL-27水平。     

FK506 attenuates thymic output in patients with myasthenia gravis

Introduction

Myasthenia gravis (MG) is an antibody-mediated, T-cell-dependent autoimmune disease. The symptoms are caused by high-affinity IgG against the muscle acetylcholine receptor (AChR) at the neuromuscular junction. The production of these antibodies in B-cells depends on AChR-specific CD4⁺ T-cells and the thymus gland seems to play a significant role in the pathogenesis of MG. Altered thymic T-cell export seems to be associated with a pathological mechanism in myasthenia gravis. Tacrolimus (FK506) has recently been used to treat MG.

Material and methods

We examined the effects of tacrolimus on thymic T-cell export in patients with MG. Sixteen patients with nonthymomatous and/or thymectomized MG were treated with oral administrations of tacrolimus. To assess the effect of tacrolimus on the thymic output, we assayed the levels of T-cell receptor excision circle (TREC), a molecular marker of thymus emigrants.

Results

T-cell receptor excision circle was not significantly different from those in age-matched controls before tacrolimus therapy, but they were partially decreased 4 months after tacrolimus therapy. T-cell receptor excision circle levels were significantly decreased in the thymomatous group (p < 0.05), but not in the nonthymomatous group. Tacrolimus treatment significantly attenuated TREC levels in cultured CD4^–CD8⁺ cells (p < 0.05), but total cell counts were not significantly changed.

Conclusions

These results indicate that TREC levels may become a marker of the curative effect of tacrolimus therapy for thymomatous MG, and that tacrolimus suppresses not only activating T-lymphocytes, but also naïve T-cells.     

The different roles of the thymus in the pathogenesis of the various myasthenia gravis subtypes

The thymus plays distinct roles in the pathogenesis of the different Myasthenia gravis (MG) subtypes. Inflammatory, neoplastic and age-related alterations of the thymus are of pivotal relevance for the initiation of anti-acetylcholine receptor (AChR) autoimmunity in early onset MG, thymoma-associated MG and, likely, late onset MG, respectively. By contrast, the thymus is presumably not related to MG that is due to autoantibodies to the muscle specific kinase, MuSK. Finally, the role of the thymus is still obscure in MG defined by antibodies against the agrin receptor LRP4 and in MG without all of the above autoantibdies (triple sero-negative MG) since these MG subtypes have been described only recently and thymectomy has not been their standard treatment. This review aims to give an update on intrathymic mechanisms of tolerance breakdown in MG, including abnormal T cell selection and activation, the role of thymic myoid cells, the autoimmune regulator (AIRE) and regulatory T cells.     

Intrathymic expression of neuromuscular acetylcholine receptors and the immunpathogenesis of myasthenia gravis

The thymus has been considered to play an important role in the pathogenesis of myasthenia gravis (MG), an autoimmune disease characterized by skeletal muscle weakness. However, the pathogenic role of the thymus still remains a mystery. The neuromuscular type of acetylcholine receptor (AChR) was the first self-protein associated with a defined autoimmune disease that was found to be expressed by thymic stromal populations. The studies described herein represent our efforts to determine how this “promiscuous” autoantigen expression may be involved in the immunopathogenesis of MG. We review our work, characterizating the expression of the α subunit of AChR (AChRα) in the thymus, and advance a new hypothesis that examines the intrathymic expression of this autoantigen in disease pathogenesis.     

B- and T-cell activation in the thymus of patients with myasthenia gravis

The activation state of thymic T and B lymphocytes was phenotypically and functionally explored in patients with Myasthenia Gravis (MG). We detected no phenotypic signs of activation in fresh total thymic lymphocyte suspensions (CD25 expression) while functional signs of activation were reflected by a significantly higher sensitivity to recombinant IL-2 (rIL-2) without any previous stimulation in MG patients as compared to controls. The response to rIL-2 was time-and dose-dependent, was inhibited by a blocking anti-IL-2 receptor antibody, and was associated to an increase of CD25+ T cells. Thymic B-cell populations purified after T cell and macrophage depletion, expressed at variable levels activation markers such as the transferrin receptor, the CD25, 4F2, CD23 and B8.7 Ag, indicating that a marked proportion of them are activated. Moreover, these B-cell populations were spontaneously sensitive to BCGF-12-kD and to a lesser extent to rIL-2, demonstrating that they also exhibit functional signs of activation. The largest proportion of activated B cells and the most intense response to BCGF-12-kD was found in patients presenting the highest anti-acetylcholine receptor (AChR) titers. Our data confirm the hyperactivity of the thymus gland in MG, reflected by the presence of T and B cells with functional signs of pre-activation. These cells could conceivably be located in lymphoid follicles and may represent autoreactive cells involved in the autoimmune process. Whether they are sensitized to AChR remains to be investigated.     

Antibodies to acetylcholine receptor in parous women with myasthenia: evidence for immunization by fetal antigen

The weakness in myasthenia gravis (MG) is mediated by autoantibodies against adult muscle acetylcholine receptors (AChR) at the neuromuscular junction; most of these antibodies also bind to fetal AChR, which is present in the thymus. In rare cases, babies of mothers with MG, or even of asymptomatic mothers, develop a severe developmental condition, arthrogryposis multiplex congenita, caused by antibodies that inhibit the ion channel function of the fetal AChR while not affecting the adult AChR. Here we show that these fetal AChR inhibitory antibodies are significantly more common in females sampled after pregnancy than in those who present before pregnancy, suggesting that they may be induced by the fetus. Moreover, we were able to clone high-affinity combinatorial Fab antibodies from thymic cells of two mothers with MG who had babies with arthrogryposis multiplex congenita. These Fabs were highly specific for fetal AChR and did not bind the main immunogenic region that is common to fetal and adult AChR. The Fabs show strong biases to VH3 heavy chains and to a single Vkappa1 light chain in one mother. Nevertheless, they each show extensive intraclonal diversification from a highly mutated consensus sequence, consistent with antigen-driven selection in successive steps. Collectively, our results suggest that, in some cases of MG, initial immunization against fetal AChR is followed by diversification and expansion of B cells in the thymus; maternal autoimmunity will result if the immune response spreads to the main immunogenic region and other epitopes common to fetal and adult AChR.     

The role of innate immunity in myasthenia gravis

Myasthenia gravis (MG) is a T cell-driven, B cell-mediated and autoantibody-dependent autoimmune disorder against neuromuscular junctions (NMJ). Accumulated evidence has emerged regarding the role of innate immunity in the pathogenesis of MG. In this review, we proposed two hypothesis underlying the pathological mechanism. In the context of gene predisposition, on the one hand, Toll-like receptors (TLRs) pathways were initiated by viral infection in the thymus with MG to generate chemokines and pro-inflammatory cytokines such as Type I interferon (IFN), which facilitate the thymus to function as a tertiary lymphoid organ (TLO). On the another hand, the antibodies against acetylcholine receptors (AChR) generated by thymus then activated the classical pathways on thymus and neuromuscular junction (NMJ). Futher, we also highlight the role of innate immune cells in the pathogenic response. Finally, we provide some future perspectives in developing new therapeutic approaches particularly targeting the innate immunity for MG.     

Myasthenia gravis: A comprehensive review of immune dysregulation and etiological mechanisms

Autoimmune myasthenia gravis (MG) is characterized by muscle weakness caused by antibodies directed against proteins of the neuromuscular junction. The main antigenic target is the acetylcholine receptor (AChR), but the muscle Specific Kinase (MuSK) and the low-density lipoprotein receptor-related protein (LRP4) are also targets. This review summarizes the clinical and biological data available for different subgroups of patients, who are classified according to antigenic target, age of onset, and observed thymic abnormalities, such as follicular hyperplasia or thymoma.Here, we analyze in detail the role of the thymus in the physiopathology of MG and propose an explanation for the development of the thymic follicular hyperplasia that is commonly observed in young female patients with anti-AChR antibodies. The influence of the pro-inflammatory environment is discussed, particularly the role of TNF-α and Th17-related cytokines, which could explain the escape of thymic T cells from regulation and the chronic inflammation in the MG thymus. Together with this immune dysregulation, active angiogenic processes and the upregulation of chemokines could promote thymic follicular hyperplasia.MG is a multifactorial disease, and we review the etiological mechanisms that could lead to its onset. Recent global genetic analyses have highlighted potential susceptibility genes. In addition, miRNAs, which play a crucial role in immune function, have been implicated in MG by recent studies. We also discuss the role of sex hormones and the influence of environmental factors, such as the viral hypothesis. This hypothesis is supported by reports that type I interferon and molecules mimicking viral infection can induce thymic changes similar to those observed in MG patients with anti-AChR antibodies.     

Innate immunity in myasthenia gravis thymus: Pathogenic effects of Toll-like receptor 4 signaling on autoimmunity

The thymus is the main site of immune sensitization to AChR in myasthenia gravis (MG). In our previous studies we demonstrated that Toll-like receptor (TLR) 4 is over-expressed in MG thymuses, suggesting its involvement in altering the thymic microenvironment and favoring autosensitization and autoimmunity maintenance processes, via an effect on local chemokine/cytokine network. Here, we investigated whether TLR4 signaling may favor abnormal cell recruitment in MG thymus via CCL17 and CCL22, two chemokines known to dictate immune cell trafficking in inflamed organs by binding CCR4. We also investigated whether TLR4 activation may contribute to immunodysregulation, via the production of Th17-related cytokines, known to alter effector T cell (Teff)/regulatory T cell (Treg) balance. We found that CCL17, CCL22 and CCR4 were expressed at higher levels in MG compared to normal thymuses. The two chemokines were mainly detected around medullary Hassall's corpuscles (HCs), co-localizing with TLR4⁺ thymic epithelial cells (TECs) and CCR4⁺ dendritic cells (DCs), that were present in higher number in MG thymuses compared to controls. TLR4 stimulation in MG TECs increased CCL17 and CCL22 expression and induced the production of Th17-related cytokines. Then, to study the effect of TLR4-stimulated TECs on immune cell interactions and Teff activation, we generated an in-vitro imaging model by co-culturing CD4⁺ Th1/Th17 AChR-specific T cells, naïve CD4⁺CD25⁺ Tregs, DCs and TECs from Lewis rats. We observed that TLR4 stimulation led to a more pronounced Teff activatory status, suggesting that TLR4 signaling in MG thymic milieu may affect cell-to-cell interactions, favoring autoreactive T-cell activation. Altogether our findings suggest a role for TLR4 signaling in driving DC recruitment in MG thymus via CCL17 and CCL22, and in generating an inflammatory response that might compromise Treg function, favoring autoreactive T-cell pathogenic responses.     

Recent approaches to the development of antigen-specific immunotherapies for myasthenia gravis

Acquired autoimmune myasthenia gravis (MG) is the most common disease that affects the neuromuscular junction (NMJ). MG is associated with autoantibodies (auto-Abs) to components of the NMJ. About 85–90% of MG patients have auto-Abs against the muscle nicotinic acetylcholine receptor (AChR), while about half of the remaining patients have auto-Abs against muscle-specific kinase. Auto-Abs, in combination with local deposition of complement, reduce the number of available post-synaptic nicotinic AChRs and thereby impair neuromuscular transmission. Current medications for MG are non-specific and include acetylcholinesterase inhibitors, immunosuppressants, plasma exchange, intravenous Ig administration and thymectomy. Treatments that selectively target the anti-AChR auto-Abs may prove to be more effective and free of side-effects. We here review two approaches aimed at the development of antigen-specific therapies for MG. The first is specific apheresis of Abs from patients' sera using immobilised recombinant AChR domains as immunoadsorbents. Indeed, we have recently shown that the combined recombinant extracellular domains of all human AChR subunits are capable of specifically immunoadsorbing the majority of pathogenic auto-Abs from several MG sera. The second therapeutic approach is the development of non-pathogenic anti-AChR monoclonal Abs that could potentially be used as protective agents by blocking the binding of patients' auto-Abs to the AChR.     

Do associated auto-antibodies influence the outcome of myasthenia gravis after thymectomy?

Abstract

Myasthenia gravis (MG) is a neuromuscular autoimmune disease, where antibodies against the acetylcholine receptor destroy this receptor. The role of thymectomy in the treatment of MG remains controversial. Because of the frequent association with other autoimmune diseases, we hypothesized that patients with multiple autoantibodies (autoAbs) might have a lower chance of reaching complete stable remission after thymectomy. We analyzed sera of 85 MG patients who underwent a thymectomy between April 2004 and December 2012. We used four different immunodot kits (D-Tek, Mons, Belgium): ANA25 Quantrix, Synthetase 10 Diver, Myositis 7 Diver and Liver 10 profile Diver, all automatized on the BlueDiver Instrument (D-Tek). The Myasthenia Gravis Foundation of America (MGFA) postintervention status was used to determine the outcome after thymectomy. AutoAbs other than anti-acetylcholine receptor (AChR) antibodies were detected in 29.4% of the patients of whom 16.5% clinically had a second autoimmune disease. In none of the seronegative patients other autoAbs were detected. No significant difference was observed in the 3-years remission rate after thymectomy in patients with or without antibodies other than anti-AChR antibodies. Although these autoAbs do not predict outcome in our MG patient cohort, screening for multiple autoAbs in MG patients might be warranted to identify patients with additional autoimmune diseases.     

Circulating CD4+CD25+ and CD4+CD25+ T cells in myasthenia gravis and in relation to thymectomy

Studies in experimental animal models of human autoimmune diseases have revealed that CD4⁺CD25⁺ T regulatory (Tr) cells are of thymic origin and have potentials in preventing auto‐aggressive immunity. Myasthenia gravis (MG) is the best‐characterized autoimmune disease. Changes in the thymus are found in a majority of patients with MG. Thymectomy has beneficial effects on the disease severity and course in a substantial proportion of MG patients. But the occurrence and characteristics of Tr cells have not yet been defined in MG. We determined the frequencies and properties of circulating CD4⁺CD25⁺ versus CD4⁺CD25^– cells in MG patients and healthy controls (HCs), with special focus on the effect of thymectomy on CD4⁺CD25⁺ cells. CD4⁺CD25^high cells comprise only about 2% of blood lymphocytes in both MG patients and HCs. Frequencies of CD4⁺CD25^high cells were similar in MG patients irrespective of treatment with thymectomy. CD4⁺CD25⁺ cells in both MG patients and HCs are mainly memory T cells and are activated to a greater extent than CD4⁺CD25^– cells, as reflected by high levels of CD45RO and human leucocyte antigen (HLA)‐DR‐positive cells. In both MG patients and HCs, CD4⁺CD25⁺ cells also contained a high proportion of CD95‐expressing cells as possible evidence of apoptosis‐proneness. Upon stimulation with anti‐CD3/CD28 monoclonal antibodies, CD4⁺CD25⁺ cells responded more vigorously than CD4⁺CD25^– cells in MG, irrespective of treatment with thymectomy, as well as in HCs. Although CD4⁺CD25^– cells are mainly naïve T cells, in non‐thymectomized MG patients, they are activated to a greater extent as reflected by higher expression of HLA‐DR and CD95 on the surface compared to HCs. The data thus show that there is no deficiency of CD4⁺CD25⁺ cells in MG, nor is the proportion of CD4⁺CD25⁺ cells influenced by thymectomy.     

Pathogenesis of myasthenia gravis. Acetylcholine receptor-related antigenic determinants in tumor-free thymuses and thymic epithelial tumors.

The authors describe an immunohistologic study of acetylcholine receptor (AChR)-related antigenic determinants in tumor-free thymuses of myasthenia gravis (MG) patients (13 cases) and nonmyasthenic controls (10 cases) and in thymic epithelial tumors of patients with MG (8 cases) and without MG (6 cases). Monoclonal antibodies (MAbs) to the cytoplasmic part and to the extracellular main immunogenic region (MIR) of the alpha subunit of AChRs were used. Their intrathymic binding sites were defined by double-immunostaining, and compared with alpha-bungarotoxin (alpha-Bgt) labeling demonstrated by fluorescence microscopy. Tumor-free thymuses of MG patients and control patients contained cytoplasmic AChR epitopes and alpha-Bgt binding sites on myoid cells and some epithelial cells. Only myoid cells also expressed extracellular MIR epitopes, suggesting that they bear complete AChRs, and are important targets for the autoimmune attack in tumor-free MG thymus. Evidence that AChR-related antigenic determinants of epithelial cells are also significant for MG is provided by our findings in thymic epithelial tumors. All eight tumors with MG but only two out of six tumors without MG showed cytoplasmic AChR epitopes and alpha-Bgt binding sites on neoplastic epithelial cells. Myoid cells and MIR epitopes did not occur in the neoplasms, but in some tumor-free thymic remnants beside thymomas. It is assumed that nonneoplastic and neoplastic thymic epithelial cells contain only incomplete AChRs or AChR-like molecules. The different expression of AChR epitopes in thymic epithelial tumors and tumor-free thymuses might explain some of the heterogeneous region specificities of anti-AChR antibodies in sera of MG patients with and without thymoma.     

Cellular aspects of myasthenia gravis

Several cellular aspects were investigated in a large series of patients with MG. First, non-Ag-specific proliferation was tested by measuring the response to r-IL2. Thymocytes from most MG patients showed hyperactivity to r-IL2. Peripheral blood lymphocytes (PBL) from some patients also showed a high response to r-IL2. These responding patients were generally those tested before thymectomy, presenting a high anti-AChR Ab titer and a severe form of the disease. Second, Ag-specific proliferation of MG PBL was assayed using 8 synthetic peptides corresponding to selected domains of torpedo or human AChR. Only 2 peptides gave a positive response in a significant number of patients, essentially in those presenting high anti-AChR Ab titer. The first is located near the alpha-bungarotoxin binding site and the second is in a cytoplasmic domain, according to models predicting the AChR transmembrane orientation. The positive results were essentially obtained with the human peptides; the corresponding torpedo peptides were positive in very few patients. Both human and torpedo peptides which include a part of the alpha-bungarotoxin binding site were negative. Finally, although morphological abnormalities were clearly visible in thymic hyperplasia, no correlation could be established between the thymus type and the cellular proliferation either to r-IL2, or to the peptides. Overall, our data indicate that cell-dependent mechanisms participate in the pathogenesis of MG, but the level of their involvement deserves further investigation.     

Diverse Fab specific for acetylcholine receptor epitopes from a myasthenia gravis thymus combinatorial library

The muscle weakness in myasthenia gravis (MG) is caused by heterogeneous high-affinity IgG autoantibodies to the nicotinic acetylcholine receptor (AChR), a complex ion channel glycoprotein. These antibodies are clearly responsible for reducing AChR numbers at the neuromuscular junction in myasthenia; however, the origins, diversity, specificity and pathogenicity of individual antibodies have not yet been established. We have cloned and characterized four different AChR-specific Fab from an MG patient's thymus by screening an IgG1/kappa gene combinatorial lambda phage library with soluble human AChR labeled with [125I] alpha-bungarotoxin. Unlike most previously cloned human antibodies, all four Fab immunoprecipitated soluble human muscle AChR. Two Fab strongly inhibited binding of mAb to the main immunogenic region on the alpha subunits and one Fab bound to an epitope on the fetal-specific gamma subunit. In sensitivity and fine specificity, these Fab resembled the anti-AChR antibodies found in many MG patients, including the donor. The closest germline counterparts for their heavy chains were in VH families 1, 3 and 4; however, there were many differences consistent with an antigen-driven response of diverse B cell clones. The combinatorial approach holds promise for further analysis of human autoantibodies.      

Pathogenesis of myasthenia gravis

 Various studies over the last 25 years in Man and animal models have revealed many steps in the pathogenesis of myasthenia gravis (MG) which is now considered the classical organ specific, autoantibody mediated and T cell dependent human autoimmune disease. Though not a disease entity, MG is associated with pathological alterations of the thymus in about 80% of cases. These are described here with reference to distinct models of autoimmunization against the acetylcholine receptor (AChR). In MG with thymitis, intrathymic production of AChR-specific autoantibodies is the result of a classical antigen-driven immune reaction that occurs completely inside the thymus and probably involves AChR on myoid cells as the triggering (myasthenogenic) antigen. Genetic factors contribute essentially to the pathogenesis of this form of MG. In thymoma-associated MG genetic factors are probably of marginal significance. Neither intratumour autoantibody production nor T cell activation seem to occur and the AChR is not the myasthenogenic antigen. Instead, abnormal neurofilaments that share epitopes with the AChR and other autoantigen targets in paraneoplastic MG are expressed in thymomas and may trigger autoantigen-specific, non-tolerogenic T cell selection by molecular mimicry. These data support the hypothesis that initial steps in the pathogenesis of most MG cases take place within abnormal thymic microenvironments, be they inflammatory or neoplastic. Where these initial steps occur in MG cases without thymic pathology is not known. Likewise, the factors involved in the initial triggering of MG remain enigmatic in all MG subtypes. Received: 16 September 1996 / Accepted: 28 October 1996     

A comprehensive analysis of the epidemiology and clinical characteristics of anti-LRP4 in myasthenia gravis

Double-seronegative myasthenia gravis (dSN-MG, without detectable AChR and MuSK antibodies) presents a serious gap in MG diagnosis and understanding. Recently, autoantibodies against the low-density lipoprotein receptor-related protein 4 (LRP4) have been identified in several dSN-MG sera, but with dramatic frequency variation (∼2–50%). We have developed a cell based assay (CBA) based on human LRP4 expressing HEK293 cells, for the reliable and efficient detection of LRP4 antibodies. We have screened about 800 MG patient sera from 10 countries for LRP4 antibodies. The overall frequency of LRP4-MG in the dSN-MG group (635 patients) was 18.7% but with variations among different populations (range 7–32.7%). Interestingly, we also identified double positive sera: 8/107 anti-AChR positive and 10/67 anti-MuSK positive sera also had detectable LRP4 antibodies, predominantly originating from only two of the participating groups. No LRP4 antibodies were identified in sera from 56 healthy controls tested, while 4/110 from patients with other neuroimmune diseases were positive. The clinical data, when available, for the LRP4-MG patients were then studied. At disease onset symptoms were mild (81% had MGFA grade I or II), with some identified thymic changes (32% hyperplasia, none with thymoma). On the other hand, double positive patients (AChR/LRP4-MG and MuSK/LRP4-MG) had more severe symptoms at onset compared with any single positive MG subgroup. Contrary to MuSK-MG, 27% of ocular dSN-MG patients were LRP4 antibody positive. Similarly, contrary to MuSK antibodies, which are predominantly of the IgG4 subtype, LRP4 antibodies were predominantly of the IgG1 and IgG2 subtypes. The prevalence was higher in women than in men (female/male ratio 2.5/1), with an average disease onset at ages 33.4 for females and 41.9 for males. Overall, the response of LRP4-MG patients to treatment was similar to published responses of AChR-MG rather than to MuSK-MG patients.