Reemergence of pathogenic, autoantibody-producing B cell clones in myasthenia gravis following B cell depletion therapy

Abstract

Myasthenia gravis (MG) is an autoantibody-mediated autoimmune disorder of the neuromuscular junction. A small subset of patients (<10%) with MG, have autoantibodies targeting muscle-specific tyrosine kinase (MuSK). MuSK MG patients respond well to CD20-mediated B cell depletion therapy (BCDT); most achieve complete stable remission. However, relapse often occurs. To further understand the immunomechanisms underlying relapse, we studied autoantibody-producing B cells over the course of BCDT. We developed a fluorescently labeled antigen to enrich for MuSK-specific B cells, which was validated with a novel Nalm6 cell line engineered to express a human MuSK-specific B cell receptor. B cells (≅ 2.6 million) from 12 different samples collected from nine MuSK MG patients were screened for MuSK specificity. We successfully isolated two MuSK-specific IgG4 subclass-expressing plasmablasts from two of these patients, who were experiencing a relapse after a BCDT-induced remission. Human recombinant MuSK mAbs were then generated to validate binding specificity and characterize their molecular properties. Both mAbs were strong MuSK binders, they recognized the Ig1-like domain of MuSK, and showed pathogenic capacity when tested in an acetylcholine receptor (AChR) clustering assay. The presence of persistent clonal relatives of these MuSK-specific B cell clones was investigated through B cell receptor repertoire tracing of 63,977 unique clones derived from longitudinal samples collected from these two patients. Clonal variants were detected at multiple timepoints spanning more than five years and reemerged after BCDT-mediated remission, predating disease relapse by several months. These findings demonstrate that a reservoir of rare pathogenic MuSK autoantibody-expressing B cell clones survive BCDT and reemerge into circulation prior to manifestation of clinical relapse. Overall, this study provides both a mechanistic understanding of MuSK MG relapse and a valuable candidate biomarker for relapse prediction.

The mechanisms of immunopathology underlying B cell depletion therapy-mediated remission and relapse in patients with MuSK MG

“…The B cell subsets that secrete autoantibodies (31) are short-lived plasmablasts and plasma cells. Some plasmablasts may express low levels of CD20, while plasma cells do not express CD20 (18,32,33). The response to RTX observed in MuSK MG patients often includes a rapid and near-complete reduction of autoantibody titer and subsequent disease remission.…”

“…To perform a more rigorous experimental demonstration, we next produced recombinant human MuSK monoclonal autoantibodies (mAbs) from these plasmablasts (33)(34)(35). We also included experienced (memory) B cells in our cell isolation approach; the result of which was that most of our MuSK mAbs originated from plasmablasts, while the rest were derived from memory B cells (33)(34)(35).…”

“…To perform a more rigorous experimental demonstration, we next produced recombinant human MuSK monoclonal autoantibodies (mAbs) from these plasmablasts (33)(34)(35). We also included experienced (memory) B cells in our cell isolation approach; the result of which was that most of our MuSK mAbs originated from plasmablasts, while the rest were derived from memory B cells (33)(34)(35). Recombinant production of human mAbs allowed for an unlimited source of human autoantibodies for study, given that those secreted in the culture media by stimulated B cells are limited in quantity.…”

“…The MuSK mAbs that we (33)(34)(35) and others (42) generated contain multiple mutations in the sequences of their variable region, which is the characteristic hallmark of the affinity maturation process. The reversion of these sequences to their corresponding germline-encoded form, which would be found in the naïve B cell precursors, is a common approach that is used to investigate the development or origin of autoantibodies (43,44).…”

Addressing technical pitfalls in pursuit of molecular factors that mediate immunoglobulin gene regulation

“…The adaptive immune system employs B cell receptors (BCR) to mount effective responses to antigens. Variation in the BCR/antibody (Ab) repertoire has been linked to differential immune responses in a variety of disease contexts including infection, cancer, and autoimmunity [1][2][3][4][5][6]. This has prompted interest to better understand the development of the BCR repertoire and factors that contribute to variation in the circulating Ab repertoire in both healthy and disease states [1,[7][8][9][10][11][12].…”