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

Abstract

One Sentence Summary: Disease relevant B cells during post-rituximab (RTX) relapse emerge from the unsuccessful depletion of pre-existing B cell clones; single-cell transcriptomic phenotyping combined with lineage tracing using paired B cell receptor repertoires identified both persistent antibody-secreting cell as well as memory B cell subsets.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 cellmediated 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.

B cell depletion therapies in autoimmune disease: advances and mechanistic insights

“…In addition to this potential off-target-cell effect, removing B lymphocytes, an entire branch of the immune system, will change the availability of growth and survival factors that were previously consumed by these cells. Following anti-CD20 treatment in patients with MG, the relative proportion of PCs to B cells is increased, and IgA + MBCs outnumber other MBCs 257 . It is unclear why this happens, but it is important to remember that BAFF levels increase after rituximab treatment 102 , 103 .…”

Autoimmune Pathology in Myasthenia Gravis Disease Subtypes Is Governed by Divergent Mechanisms of Immunopathology

“…Long-lived plasma cells populations residing in the thymus produce some of the circulating AChR-specific autoantibodies; plasma cells express low levels of CD20 (32, 34). In contrast, circulating plasmablasts, such as those that secrete MuSK-specific autoantibodies, typically express higher levels of CD20 than their tissue resident plasma cell counterparts (19,184), although some refractory B cell clones were found to emerge during relapse in MuSK MG after treatment with RTX (185). Thus, differences in the efficacy of RTX in AChR and MuSK MG may reflect differences in the tissue localization of disease-causing B cell subsets and/or the susceptibility of different autoantibodyproducing B cell subsets (plasmablasts in MuSK MG and plasma cells in AChR MG, each expressing different levels of CD20) to anti-CD20 depletion.…”

CD20-mediated B cell depletion in acetylcholine receptor autoantibody-positive myasthenia gravis

“…In contrast to AChR MG titers, almost all patients with MuSK MG receiving rituximab show a rapid (within weeks) and marked decline in MuSK autoantibody titer. Interestingly, the intensity of rituximab induction seems to be proportionate to the durability of the response of MuSK MG patients [89], however historic clones (not efficiently depleted by CD20-mediated therapy) can reemerge in many cases, even with intense induction, and cause relapse [31,90]. Finally, in seronegative MG, successful application of rituximab in selected cases points to B cell involvement in pathogenesis and to the fact that seronegative MG may be 'false seronegative' due to the sensitivity threshold of autoantibody detection assays [91,92].…”