Autoreactive B Cells in ANCA-Associated Vasculitis

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of B-cell driven, autoimmune systemic vasculitides characterized by a relapsing course and the presence of ANCA autoantibodies which are instrumental in their pathogenesis. We aimed to investigate autoreactive proteinase 3 (PR3+) B cells involved in the development of human AAV. We previously developed a customized flow-cytometry method to identify autoreactive B cells among cryopreserved peripheral blood mononuclear cells (PBMC), using labeled PR3, one of the main AAV autoantigens, as a ligand. We therefore used multicolor flow cytometry in combination with bioinformatics and functional in vitro studies on 1) baseline samples of PBMC from 154 well-characterized participants of the RAVE trial (NCT00104299) with severely active PR3-ANCA+ AAV (PR3-AAV) and myeloperoxidase (MPO)-AAV, and 27 healthy controls (HC); 2) samples of matched bone marrow (BM) and peripheral blood from 8 non-vasculitis patients; and 3) 148 longitudinal samples from 23 PR3-AAV patients of the RAVE trial. Clinical data and outcomes from the trial and medical records were correlated with PR3+ B cells (total and subsets). In brief, we identified and phenotypically characterized autoreactive B cells in AAV and healthy controls, reporting their perturbations among the different B cell subsets, and their functional ability to produce PR3-ANCA autoantibodies in vivo and in vitro. We reported their maturation through central and peripheral tolerance checkpoints from BM to peripheral blood, leading to an accumulation of atypical autoreactive PR3+ memory B cells in PR3-AAV patients but not in MPO-AAV and HC. We also described the longitudinal association between autoreactive plasmablast redetection after anti-B cell 13 targeted therapy with the main disease outcome, relapse. Overall, our findings suggest the presence of defective central antigen-independent and peripheral antigen-dependent checkpoints in patients in PR3-AAV, elucidating the selection process of autoreactive B cells, and their association with disease relapse.