Single-cell repertoire tracing identifies rituximab-resistant B cells during myasthenia gravis relapses
Ruoyi Jiang, Miriam L. Fichtner, Kenneth B. Hoehn, Minh C. Pham, Panos Stathopoulos, Richard J. Nowak, Steven H. Kleinstein, Kevin C. O’Connor
Ruoyi Jiang, Miriam L. Fichtner, Kenneth B. Hoehn, Minh C. Pham, Panos Stathopoulos, Richard J. Nowak, Steven H. Kleinstein, Kevin C. O’Connor
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Research Article Immunology

Single-cell repertoire tracing identifies rituximab-resistant B cells during myasthenia gravis relapses

Abstract

Rituximab, a B cell–depleting therapy, is indicated for treating a growing number of autoantibody-mediated autoimmune disorders. However, relapses can occur after treatment, and autoantibody-producing B cell subsets may be found during relapses. It is not understood whether these autoantibody-producing B cell subsets emerge from the failed depletion of preexisting B cells or are generated de novo. To further define the mechanisms that cause postrituximab relapse, we studied patients with autoantibody-mediated muscle-specific kinase (MuSK) myasthenia gravis (MG) who relapsed after treatment. We carried out single-cell transcriptional and B cell receptor profiling on longitudinal B cell samples. We identified clones present before therapy that persisted during relapse. Persistent B cell clones included both antibody-secreting cells and memory B cells characterized by gene expression signatures associated with B cell survival. A subset of persistent antibody-secreting cells and memory B cells were specific for the MuSK autoantigen. These results demonstrate that rituximab is not fully effective at eliminating autoantibody-producing B cells and provide a mechanistic understanding of postrituximab relapse in MuSK MG.

Authors

Ruoyi Jiang, Miriam L. Fichtner, Kenneth B. Hoehn, Minh C. Pham, Panos Stathopoulos, Richard J. Nowak, Steven H. Kleinstein, Kevin C. O’Connor

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Figure 5

Unique characteristics of persistent ASCs.

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Unique characteristics of persistent ASCs. (A) Expression of log-normali...
(A) Expression of log-normalized CD20 expression (Z score) visualized by intensity over t-SNE plot. (B) The expression of CD20 (the receptor target of RTX, gene symbol MS4A1) and BAFF or APRIL receptors (TACI or TNFRSF13B, BAFF-R or TNFRSF13C, BCMA or TNFRSF17) for persistent and nonpersistent members of the ASC cluster. Normalized gene expression values are computed from counts of gene expression transcripts. Paired 2-tailed t tests were used to test for the significant differential expression of each gene. Horizontal bars show the average expression of ASC cluster members for each patient for cells of a given status. (C) Clonal expansion expressed as Simpson’s diversity for ASC and memory B cell clusters. A 1-tailed t test was used to assess the significance of the null hypothesis that ASCs would not be less diverse than memory B cell clones. Horizontal bars show the average diversity of cluster members for each patient of memory B cells or ASCs. (D) Simpson’s diversity for persistent and nonpersistent members of the ASC cluster. A 1-tailed t test was used to assess the significance of the null hypothesis that persistent clones would not be less diverse than nonpersistent clones. Horizontal bars show the average diversity of ASC cluster members for each patient for cells of a given status. (E) The frequency of ASCs in post-RTX MuSK MG patients and a control AChR MG patient. The fraction of ASC cluster members is quantified as a ratio of the number of cells in the ASC cluster divided by the number of total B cells for each patient. A 1-sample t test was used to assess the significance of the null hypothesis that ASCs would not be more abundant in post-RTX samples compared with the asymptomatic AChR MG patient sample. Horizontal bars show the average frequency of ASC cluster members for each patient of a given status. Values belonging to the same patient are paired with a gray line for all graphs. Data for the same n = 3 patients are shown for all panels except panel E, which includes an asymptomatic patient sample (n = 4). Violin plots are used in place of error bars to show the full range of values. Statistical differences are shown only when significant (***P < 0.001; **P < 0.01; *P < 0.05).