BACKGROUND. Little is known about the autoreactive B cells in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). We aimed to investigate tolerance checkpoints of circulating antigen-specific proteinase 3 (PR3+) B cells. METHODS. Multicolor flow cytometry in combination with bioinformatics and functional in vitro studies were performed on baseline samples of peripheral blood mononuclear cells from 154 well-characterized participants of the RAVE trial (NCT00104299) with severely active PR3-AAV and myeloperoxidase (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 PR3-AAV (4.77% [3.98%-6.01%]), than in MPO-AAV (3.16% [2.51%-5.22%]), and in AAV compared to HCs (1.67% [1.27%-2.16%], p<0.001 for all comparisons), implying a defective central tolerance checkpoint in patients. Only PBMC from PR3-AAV contained PR3+ B cells capable of secreting PR3-ANCA IgG in vitro, proving to be functionally distinct from those of MPO-AAV and HCs. Unsupervised clustering identified subtle subsets of atypical autoreactive PR3+ memory B cells accumulating through the maturation process in PR3-AAV patients. PR3+ B cells were enriched in the memory B cell compartment of 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). CONCLUSIONS. This study suggests 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.
Alvise Berti, Sophie Hillion, Amber M. Hummel, Young Min Son, Nedra Chriti, Tobias Peikert, Eva M. Carmona, Wayel H. Abdulahad, Peter Heeringa, Kristina M. Harris, E. William St. Clair, Paul Brunetta, Fernando Fervenza, Carol Langford, Cees Kallenberg, Peter Merkel, Paul A. Monach, Philip Seo, Robert F. Spiera, John H. Stone, Guido Grandi, Jie Sun, Jacques-Olivier Pers, Ulrich Specks, Divi Cornec