Single-cell immune repertoire tracing identifies rituximab refractory B cells that emerge during relapse

Abstract Rituximab, an anti-CD20 B cell-depleting therapy, is indicated to treat a growing number of autoimmune disorders. However, disease relapses can occur when the B cell compartment is re-established after treatment. To explore the mechanism of relapse, we studied patients with muscle-specific kinase (MuSK) myasthenia gravis (MG), a paradigm for B cell-mediated autoimmune diseases in which pathology is directly associated with autoantibody production. Whether the failed depletion of specific clones leads to the persistence of autoantibody producing B cell subsets has yet to be elucidated. Here we show that rituximab (RTX) therapy is not fully effective at eliminating disease-associated B cells and provide a mechanistic understanding of relapse. We carried out single-cell transcriptional and B cell receptor (BCR) profiling on longitudinal B cell samples from the peripheral blood of MuSK MG patients who relapsed after rituximab therapy. Computational analysis of these data identified individual B cells that were refractory to rituximab depletion by linking them to clones that were present prior to therapy and continued to persist after therapy. These persistent B cell clones were present at high frequency, and included both antibody-secreting cells and memory B cells. They were disproportionately isotype switched with elevated somatic hypermutation frequencies and a gene expression signature associated with antigen-experience and B cell clonal expansion. We further identified both antibody-secreting cells and memory B cells with specificity for MuSK autoantigen that were clonally expanded during relapse and persistent. Our results provide evidence that post-rituximab relapse in MuSK MG is associated with the failed depletion of autoantigen-specific B cell subsets. This work provides insight into the durability of human B cells during treatment with therapeutic CD20-specific monoclonal antibodies. The transcriptional signatures associated with B cell resistance may represent new therapeutic avenues for treatment and biomarkers of depletion efficacy.