(B) H-CDR3 mean length in amino acid residues, (C) GRAVY index, and (D) mean net charge at pH 7
(B) H-CDR3 mean length in amino acid residues, (C) GRAVY index, and (D) mean net charge at pH 7.4 for each subject is shown as a single point for both the naive (Na?ve-IgM) and memory (Memory-IgM, Memory-IgG and Memory-IgA) compartments. positive selection of somatic mutations in the H-CDR regions and altered H-CDR3 physicochemical properties. The VL repertoire of MuSK-MG was specifically characterized by reduced V/J segment distance in recombined sequences, suggesting diminished VL receptor editing during B cell development. Our results identify large-scale abnormalities in both the na?ve and memory B cell repertoires. Particular abnormalities were unique to either AChR-MG or MuSK-MG indicating that the repertoires reflect the distinct properties of the subtypes. These repertoire abnormalities are consistent with previously observed defects in B AFX1 cell tolerance checkpoints in MG, thereby offering additional insight regarding the impact of tolerance defects on peripheral autoimmune repertoires. These collective findings point toward a deformed B cell repertoire as a fundamental component of MG. Introduction Myasthenia gravis (MG) is a chronic autoimmune disorder of neuromuscular signal transmission characterized by muscle weakness and fatigability (1). The estimated annual incidence is 1C2 per 100,000 and the prevalence is as high as 20C50 per 100,000 (2, 3). Recent epidemiological studies indicate that, like other autoimmune diseases, its incidence is rising considerably (4). Approximately 80C85% of MG Doripenem Hydrate patients have circulating autoantibodies that target the acetylcholine receptor (AChR)(1) while up to 10% harbor autoantibodies that target muscle specific kinase (MuSK) (5). Autoantibodies to lipoprotein-related protein 4 (LRP4) are detected in a fraction (18%) of AChR and MuSK autoantibody double-negative MG patients, but also in a small fraction ( 6%) that are AChR or MuSK autoantibody positive (6, 7). A minor fraction ( 10%) of MG patients do not have detectable autoantibodies directed toward any of these recognized antigens. The majority of MG patients lacking thymomas have been commonly classified into two major distinct subtypes based on whether they test positive for anti-AChR (AChR-MG) or anti-MuSK (MuSK-MG) antibodies. While both AChR-MG and MuSK-MG demonstrate neuromuscular junction pathologies, these two subtypes differ in the degree of gender bias, clinical presentation, response to immunotherapies, and association with specific HLA alleles (1, 8C10). Thus, implying that non-thymoma AChR-MG and MuSK-MG may have different underlying disease mechanisms and genetics, despite broadly similar presentations. AChR-MG and MuSK-MG are among the few autoimmune diseases for which the molecular immunopathology is well understood. The autoantibodies in AChR-MG, which are primarily IgG1, lead to the loss of AChR through internalization and localized complement-mediated postsynaptic tissue damage (11, 12). The majority of autoantibodies that recognize MuSK are from the IgG4 subclass. They inhibit the agrin-LRP4-MuSK pathway by preventing agrin-activated LRP4 binding to MuSK, leading to dispersal of the AChRs (13, 14). Transfer (both active and passive) of these autoantibodies from humans to animal models affect the disease, demonstrating the direct role these molecules play in its pathology (15C18). While the role of autoantibodies in both AChR-MG and MuSK-MG pathologies are clear, the immune dysfunction that precedes their production is less well understood. The role of the thymus in the immunopathology of many AChR MG patients is conspicuous (19), this is further highlighted by a recent clinical trial demonstrating the clinical benefit following thymectomy (20). The thymus in a subset of MG patients includes (19) AChR Doripenem Hydrate expression by thymic epithelial cells and myoid cells, the presence of proinflammatory cytokines, and defective regulatory T cells. B cells often organize in the hyperplastic thymus within tertiary lymphoid organs, frequently exemplifying many characteristics of germinal centers. To better define the Doripenem Hydrate role of B cells in MG, a number of studies have focused on those which characteristically populate the thymus. B cells populating the hyperplastic thymus express markers of activation and display functional signs of activation (21). Limited-throughput Sanger-based sequencing and B cell immortalization studies have shown that the B cells resident in these MG thymi are broadly clonally heterogeneous; lacking a dominant clone(s) among the infiltrate (22, 23). They harbor the characteristics of antigen experience, including somatic hypermutation (24C26) and biased variable region gene segment usage, which include over-representation of the VH3 family at the expense of VH4 genes (22). Importantly, among these B cells are plasmablasts and plasma cells that produce autoantibodies directed toward AChR. However, they represent a minor fraction of the total B cell infiltrate, thus do not appear to be highly enriched (27, 28). While these studies.