Antibodies Produced by CLL Phenotype B Cells in Patients With Myasthenia Gravis Are Not Directed Against Neuromuscular Endplates

(A) Representative contour plots of the B-cell compartment of case MBL1-MG and case MBL2-MG during hematologic follow-up flow cytometry screens. CD5⁺ B cells, as gated in the first panel, are highlighted in green to compare the immunophenotype to CD5⁻ B cells of the same patient. The patients met all 5 phenotypic Matutes criteria that are required for a CLL diagnosis. Shown are representative contour plots for 1 patient. (B) Lollipop plot showing the kappa-to-lambda light chain ratio determined by flow cytometry for CD5⁻ B cells (black) and the CD5⁺ B-cell populations (green) of case CLL-MG, case MBL1-MG, and case MBL2. Data are shown in a log scale, with positive values indicating kappa light chain enrichment and negative values indicating lambda chain enrichment. The dashed line indicates equal kappa-to-lambda ratio as present in polyclonal B cells. (C and D) Heatmap displaying light chain (C) and heavy chain (D) sequence similarities obtained from single cells of case CLL-MG, case MBL1-MG, and case MBL2-MG. Variable sequences were aligned and identity matrices computed using Clustal Omega. The heavy chain sequence of case CLL-MG has been obtained from multiple cells. CLL = chronic lymphocytic leukemia; MBL = monoclonal B-cell lymphocytosis; MG = myasthenia gravis.

(A) Representative microscopy images of HEK293T cells transiently transfected with human AChR subunits, cotransfected with rapsyn (to facilitate clustering) and labeled with serum or mAbs derived from case CLL-MG, case MBL1-MG, case MBL2-MG, and the mAb AF122 targeting the α-subunit of the AChR (positive control). Bound antibodies were detected using Alexa Fluor 568–conjugated goat anti-human Abs. Scale bar 15 µm. (B) Bar graph showing the binding of mAbs and patient sera from case CLL-MG, case MBL1-MG, and case MBL2-MG determined by rater-blinded semiquantitative scoring of the cell-based assay as shown in (A). 0 = negative; 1–1.5 = low positive; 2–4 = increasing level of positivity based on fluorescence intensity. AChR = acetylcholine receptor antibody; CLL = chronic lymphocytic leukemia; mAb = monoclonal antibody; MBL = monoclonal B-cell lymphocytosis; MG = myasthenia gravis.

Representative confocal microscopy images of stainings with mAbs (left column) and serum (right column) from case CLL-MG, case MBL1-MG, and case MBL2-MG on murine muscle sections. Bound Abs were detected by Alexa Fluor 488–conjugated goat anti-human Abs. Sections were colabeled with bungarotoxin to identify AChR, anti-neurofilament Abs, and DAPI for cell nuclei. As control, the AF122 mAb targeting the α-subunit of the AChR was used (positive control, left column). Negative control serum (right column) was from a donor without MG. Arrows highlight neuromuscular junctions. Scale bar 20 µm. A zoomed-in view is shown. AChR = acetylcholine receptor antibody; CLL = chronic lymphocytic leukemia; mAb = monoclonal antibody; MBL = monoclonal B-cell lymphocytosis; MG = myasthenia gravis.