Fig. 4

Characteristics of a persistent MuSK-specific B cell clone. Clonal variants of the MuSK-specific mAb 2E6 were identified in longitudinally collected samples from patient MuSK MG-1. a Clonal lineage containing BCR sequences from bulk RNA sequencing of serial samples collected from patient MuSK MG-1 from whom mAb 2E6 was cloned. A maximum likelihood tree of the 2E6 clone, with clonal variants CVA, CVB, and CVC is shown. Edge lengths represent the expected number of intervening somatic mutations between nodes (see scale bar). Colors correspond to the collection time point (months) at which each clone (sequence) was identified in relation to the first available collection time point. b Binding to MuSK by mAb 2E6 and clonal variants was tested in a CBA over a series of ten two-fold dilutions of each mAb ranging from 10-0.02 µg/ml. Clonal variants, CVA, CVB and CVC were each tested with either the mature (mutated) or an unmutated common ancestor (UCA) of the light chain from mAb 2E6, given that the clonal variants were identified with heavy chain-only sequencing. The MuSK-specific human mAb MuSK1A was used as the positive control and AChR-specific human mAb 637 as the negative control. The ∆MFI was calculated by subtracting the signal acquired with the non-transfected cells from the signal of transfected cells. Each data point represents the mean value from three independent experiments. Bars or symbols represent means and error bars SDs. Values greater than the mean + 4SD of the negative control mAb at 1.25 µg/ml (indicated by the horizontal dotted line) were considered positive. c The x-axis depicts the time in months representing the clinical course of patient MuSK MG-1 from whom the mAb 2E6 was isolated. The bold timepoints indicate longitudinal sample collection and clinical assessment; the italicized timepoints indicate BCDT administration. The left y-axis indicates the MuSK autoantibody titer at each timepoint. The autoantibody titer was measured by CBA using 10 two-fold dilutions ranging from 1:20 to 1:10240. The right y-axis shows the MGC score at each timepoint. Dotted vertical lines indicate the timepoints where the patient received rituximab mediated BCDT. Colored boxes indicate the timepoints at which both clonal variants and the original 2E6 mAb were identified. The blue bars show the MGC score, while the green bars show the autoantibody titer