Circulating autoreactive proteinase 3(+) B cells and tolerance checkpoints in ANCA-associated vasculitis

BACKGROUND: Little is known about the autoreactive B cells in antineutrophil cytoplasmic antibody–associated (ANCA-associated) vasculitis (AAV). We aimed to investigate tolerance checkpoints of circulating antigen-specific proteinase 3–reactive (PR3(+)) B cells. METHODS: Multicolor flow cytometry in combination with bioinformatics and functional in vitro studies were performed on baseline samples of PBMCs from 154 well-characterized participants of the RAVE trial (NCT00104299) with severely active PR3-AAV and myeloperoxidase-AAV (MPO-AAV) and 27 healthy controls (HCs). Clinical data and outcomes from the trial were correlated with PR3(+) B cells (total and subsets). RESULTS: The frequency of PR3(+) B cells among circulating B cells was higher in participants with PR3-AAV (4.77% median [IQR, 3.98%–6.01%]) than in participants with MPO-AAV (3.16% median [IQR, 2.51%–5.22%]) and participants with AAV compared with HCs (1.67% median [IQR, 1.27%–2.16%], P < 0.001 for all comparisons), implying a defective central tolerance checkpoint in patients with AAV. Only PBMCs from participants with PR3-AAV contained PR3(+) B cells capable of secreting PR3-ANCA IgG in vitro, proving they were functionally distinct from those of participants with MPO-AAV and HCs. Unsupervised clustering identified subtle subsets of atypical autoreactive PR3(+) memory B cells accumulating through the maturation process in patients with PR3-AAV. PR3(+) B cells were enriched in the memory B cell compartment of participants with PR3-AAV and were associated with higher serum CXCL13 levels, suggesting an increased germinal center activity. PR3(+) B cells correlated with systemic inflammation (C-reactive protein and erythrocyte sedimentation rate, P < 0.05) and complete remission (P < 0.001). CONCLUSION: This study suggests the presence of defective central antigen-independent and peripheral antigen-dependent checkpoints in patients with PR3-AAV, elucidating the selection process of autoreactive B cells. TRIAL REGISTRATION: ClinicalTrials.gov NCT00104299. FUNDING: The Vasculitis Foundation, the National Institute of Allergy and Infectious Diseases of the NIH, and the Mayo Foundation for Education and Research.