B Cell Immunophenotyping and Transcriptional Profiles of Memory B Cells in Patients with Myasthenia Gravis

Abstract

Myasthenia gravis (MG) is an autoantibody-mediated postsynaptic neuromuscular junction disorder. Anti-acetylcholine receptor antibodies are found in about 80% of patients with MG [1], and numerous auto-antibodies have been recently discovered; muscle-specific tyrosine kinase (MuSK) [2], low-density lipoprotein receptor-related protein 4 (LRP4) [3], agrin [4], voltage-gated K+ channel Kv1.4 [5], ryanodine receptor [6] and cortactin [7]. Chronic immunosuppressive treatment (ISTX), such as tacrolimus, azathioprine, mycophenolate mofetil and prednisolone, is required in most patients at some point in their courses, in order to maintain disease stability. Nevertheless, appropriate biomarkers that reflect disease activity or toxicity of ISTX lack [8]. Herein, we immunophenotyped peripheral blood mononuclear cells (PBMC) from participants and tested if B cell subsets were altered by disease activity or ISTX. Furthermore, given that MG is an apparently antibody-specific autoimmune disease, we analyzed transcriptional profiles of memory B cells (CD19+ CD27+) in order to develop appropriate biomarkers to monitor disease status. For transcriptome study, we used Nanostring analysis which is a digital multiplexed mRNA assay that provides highly reproducible data even in small-sized samples, by detecting native RNAs directly, without reverse transcription or amplification [9]. MATERIALS AND METHODS Study subjects A total of 21 patients with MG and 10 healthy controls were recruited from December 2015 to August 2019 in Seoul Metropolitan Government Boramae Medical Center. Written informed consent was obtained from all participants. This study was approved by the local institutional review board (IRB no. 20151016/16-2015-147/111).

Double-negative (DN) B cells: an under-recognized effector memory B cell subset in autoimmunity

“…In healthy individuals, DN B cells mainly exist in peripheral blood and tonsil (6,33). The mean frequency of DN B cells is approximately 5% (3.6-5.95%) of all peripheral blood B cells (6,7,(11)(12)(13)26,34,35), and is relatively higher in elderly people (8.79-15.8%) (Table 1) (11,21,34). The frequencies of DN B cells are increased in many diseases, as discussed later.…”

“…Other autoimmune diseases DN B cells are also increased in myasthenia gravis (MG) (13) and multiple sclerosis (MS) (11,12). After ex-vivo CD40L stimulation, DN B cells of MS patients could produce comparable or higher levels of proinflammatory cytokines such as TNF-α, lymphotoxin-α and granzyme B than CD27 + switched-memory B cells (11).…”

“…These two ostensibly contradictory models raise questions about the actual origin and function of DN B cells. However, for a long time this unique B cell subset has been poorly characterized, until recent studies indicated their potential roles in diseases (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16).…”

“…The expansion of DN B cells has been detected in various diseases, especially in autoimmune diseases (6)(7)(8)(9)(10)(11)(12)(13). Thus, studies on these B cells have been increasing in the last 15 years, and emerging evidence has further improved our understanding of their roles in immune responses.…”

Roles of cytokines and T cells in the pathogenesis of myasthenia gravis

“…The proportion of memory B cells is elevated in MG (generalized MG > ocular MG) [31]. B cell profiles in patients with MG differ according to disease status, and may guide treatment decisions [57]. An increase in the number of CD19 + BAFF-R + B cells in MG patients (including thymoma-associated MG and seronegative MG patients) [58] and the efficacy of treatment with rituximab (an anti-B cell agent) have been reported in MG [59,60].…”