Autoantibody-producing plasmablasts after B cell depletion identified in muscle-specific kinase myasthenia gravis

Abstract

Myasthenia gravis (MG) is a B cell-mediated autoimmune disorder of neuromuscular transmission. Pathogenic autoantibodies to muscle-specific tyrosine kinase (MuSK) can be found in patients with MG who do not have detectable antibodies to the acetylcholine receptor (AChR). MuSK MG includes immunological and clinical features that are generally distinct from AChR MG, particularly regarding responsiveness to therapy. B cell depletion has been shown to affect a decline in serum autoantibodies and to induce sustained clinical improvement in the majority of MuSK MG patients. However, the duration of this benefit may be limited, as we observed disease relapse in MuSK MG patients who had achieved rituximab-induced remission. We investigated the mechanisms of such relapses by exploring autoantibody production in the reemerging B cell compartment. Autoantibody-expressing CD27+ B cells were observed within the reconstituted repertoire during relapse but not during remission or in controls. Using two complementary approaches, which included production of 108 unique human monoclonal recombinant immunoglobulins, we demonstrated that antibody-secreting CD27hiCD38hi B cells (plasmablasts) contribute to the production of MuSK autoantibodies during relapse. The autoantibodies displayed hallmarks of antigen-driven affinity maturation. These collective findings introduce potential mechanisms for understanding both MuSK autoantibody production and disease relapse following B cell depletion.

Keywords: Autoimmune diseases; Autoimmunity; B cells; Immunology; Immunotherapy.

Figure 1. Representative MuSK cell-based assay (CBA) flow cytometry plots.

Control sera, cell culture supernatants, and monoclonal rIgs were tested for surface binding to MuSK on MuSK-GFP–transfected HEK cells. The x axis represents GFP fluorescence intensity and, consequently, the fraction of HEK cells transfected with MuSK. The y axis represents Alexa Fluor 647 fluorescence intensity, which corresponds to secondary anti–human IgG Fc antibody binding and, consequently, primary antibody binding to MuSK. Hence, transfected cells are located in the right quadrants and cells with MuSK autoantibody binding in the upper quadrants. The upper right quadrant shows cells that are both transfected with MuSK-GFP and that bind MuSK autoantibodies, whereas the upper left quadrant represents nonspecific antibody binding to HEK cell antigens. All results shown were reproduced in duplicate experiments. (A–F) Serum and B cell culture supernatants; (G–L) monoclonal rIg. (A) Post–rituximab relapse (MuSK 2b) serum; (B) post–rituximab remission (MuSK 4) serum; (C) post–rituximab relapse (MuSK 2a) CD27⁺ B cell culture supernatant; (D) post–rituximab remission (MuSK 4) CD27⁺ B cell culture supernatant; (E) post–rituximab relapse (MuSK 2b) plasmablast culture supernatant; (F) post–viral URI (HD 1) plasmablast culture supernatant; (G) 4A3, a humanized murine MuSK–specific monoclonal rIg; (H) 637, a human AChR–specific monoclonal rIg; (I) post–rituximab relapse (MuSK 1) PB–derived rIg 1-1; (J) post–rituximab relapse (MuSK 3) PB–derived rIg 3-29; (K) post–rituximab relapse (MuSK 3) PB–derived rIg 3-33; (L) AChR MG (AChR 7) PB–derived rIg 7-3. AChR, acetylcholine receptor; HD, healthy donor; HEK, human embryonic kidney; MuSK, muscle-specific tyrosine kinase; rIg, recombinant Ig; URI, upper respiratory tract infection.

Figure 2. Summary of MuSK CBA data performed with sera, B cell culture supernatants, and recombinant immunoglobulin (rIg).

Results are presented as Δ% positive cells on the y axis. Δ% positive cells = (%frequency of positive MuSK-GFP–transfected cells/%frequency of MuSK-GFP–transfected cells) – (%frequency of positive GFP-transfected cells/%frequency of GFP-transfected cells). Testing of all samples was performed in duplicate. (A–C) Bars represent means, dots represent individual values, and error bars represent range of values; (D) lines represent means, and dots represent individual rIg values. (A) Sera of MuSK 1–4, AChR 1–8, and HD 1; (B) CD27⁺ B cell culture supernatants of MuSK 1–4, AChR 1–6 and HD 1; (C) Plasmablast culture supernatants from MuSK 1, -2b, -3, -4, AChR 1–3 and HD 1; (D) Plasmablast-derived rIg from MuSK 1 (n = 4), 2b (n = 33), 3 (n = 45) and AChR 7 (n = 15), 8 (n = 11). AChR, acetylcholine receptor; HD, healthy donor; MuSK, muscle-specific tyrosine kinase.

Figure 3. Plasmablast FACS gating strategy and validation.

A representative example of the plasmablast population sorting strategy is shown. PBMCs were initially gated in the SSC/FSC graph (upper left). The SSC/FSC gate was determined based on plasmablast back-gating (not shown). After doublet and dead cell exclusion, CD19⁺CD3^–CD14^– cells were gated as B cells (lower left). Plasmablasts were subsequently gated from the B cell gate as CD27^hiCD38^hi cells (upper right). Naive B cells were defined as IgD⁺CD27^– B cells (lower right). Morphological analysis (400× magnification) is shown for plasmablasts (upper picture) and naive B cells (lower picture). Plasmablasts display an eccentric nucleus, perinuclear hof region, prominent nucleoli, and abundant cytoplasm. In contrast, naive B cells display a large nucleus with a thin rim of cytoplasm. These results were replicated in a repeat experiment. FSC, forward scatter; PBMC, peripheral blood mononuclear cells; SSC, side scatter.

Figure 4. Specificity of positive MuSK MG relapse plasmablast-derived rIg.

Seven monoclonal rIg, MuSK 1-1, 3-1, 3-20, 3-28, 3-29, 3-30, and 3-33, were tested for binding on HEK cells transfected with MuSK (upper panel), AChR (middle panel), and GFP (lower panel). Results are shown as percentage of transfected cells that bound secondary antibody (percent of positive cells). Testing of all samples was performed in duplicate. Bars represent means, dots represent individual values, and error bars represent range of values. AChR, acetylcholine receptor; HD, healthy donor; HEK, human embryonic kidney; MuSK, muscle-specific tyrosine kinase; rIg, recombinant Ig.