Heterogeneity of acetylcholine receptor autoantibody-mediated complement activity in patients with myasthenia gravis

Abstract

Background: Autoantibodies targeting the acetylcholine receptor (AChR) in the serum of myasthenia gravis (MG) patients are broadly polyclonal and heterogeneous in their pathogenic capacity. Specifically, AChR autoantibody-mediated pathology occurs through three mechanisms that include complement-directed tissue damage, blocking of the acetylcholine binding site on the AChR, and modulation (internalization) of the AChR. Clinical assays used for diagnosis and prognosis measure only AChR autoantibody binding and they provide weak association with disease burden, thereby limiting understanding of mechanistic heterogeneity, and monitoring therapeutic response. Objective: To develop an in-vitro cell-based assay that measures AChR autoantibody-mediated complement membrane attack complex (MAC) formation. Methods: A HEK293T cell line, which is commonly used for live cell-based AChR autoantibody binding assays, was modified such that the expression of the complement regulator genes (CD46, CD55 and CD59) were disrupted using CRISPR/Cas9 genome editing. This modified cell line was used to measure serum AChR autoantibody-mediated complement MAC formation via flow cytometry. Results: AChR autoantibody-mediated MAC formation required the use of a modified HEK293T cell line in which the surface expression of three complement regulator genes was absent. Serum samples (n=155) from 97 clinically confirmed AChR patients were tested along with 32 healthy donor (HD) samples; the MG cohort included a wide range of disease burden and AChR autoantibody titer. AChR autoantibodies were detected in 139 of the 155 (89.7%) AChR patient samples via a live cell-based assay. Of the 139 AChR positive samples, autoantibody-mediated MAC formation was detected in 83 (59.7%), while no autoantibodies or MAC formation was detected in samples from the HD group. Autoantibody-mediated MAC formation positively associated with autoantibody binding in most MG patient samples. However, a subset displayed a disassociation between binding and MAC formation. Conclusions: We demonstrate the development of a novel assay for evaluating AChR autoantibody-mediated complement activity. It is anticipated that this assay will afford a deeper understanding of the heterogeneous disease pathology and allow for the identification of MG patients who may benefit from complement inhibitor therapy.

More Efficient Complement Activation by Anti–Aquaporin-4 Compared With Anti–Myelin Oligodendrocyte Glycoprotein Antibodies

“…Although we used HEK293A cells for all our assays, we studied the expression of 2 complement inhibitors (CD46 and CD59) after transfection with MOGα 1 or AQP4-M23. 36 We observed increased CD59 expression of the transfected cells compared with nontransfected cells. However, expression levels were comparable between MOG-and AQP4expressing cells (eFigure 2D).…”

CD59 Expression in Skeletal Muscles and Its Role in Myasthenia Gravis

“…Patients with a higher disease severity of MG presented with a higher MAC formation compared with those with minimal disease burden. 15 During a higher AChR-Ab-mediated complement activity in MG, normal CD59 levels at the muscles could not protect the NMJ against the attack by the formed MAC which was induced by complement activation. 16 The destructive condition of NMJ by complement activation resulted in the decreased expression of both CD59 and AChR.…”

Novel pathophysiological insights in autoimmune myasthenia gravis

“…Two assays designed to measure AChR Ab complement-mediated activity have recently been reported. One is an NMJ bioassay, and the other is based on the live CBA [86 ▪ ,87 ▪ ]. Testing a large cohort of MG patients [87 ▪ ] revealed that a subset of patients lack association between complement membrane attack complex (MAC) formation and Ab binding.…”

“…One is an NMJ bioassay, and the other is based on the live CBA [86 ▪ ,87 ▪ ]. Testing a large cohort of MG patients [87 ▪ ] revealed that a subset of patients lack association between complement membrane attack complex (MAC) formation and Ab binding. Heterogeneous Ab characteristics, among other factors, may play a role in determining MAC formation efficiency, including subunit and epitope specificity, affinity, IgG subclass usage, Fc structure, and post-translation modifications, all of which can alter complement activating capability in addition to structure and stability [88,89].…”