Circulating autoreactive proteinase 3+ B cells and tolerance checkpoints in ANCA-associated vasculitis
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 C. Fervenza, Carol A. Langford, Cees G.M. Kallenberg, Peter A. Merkel, Paul A. Monach, Philip Seo, Robert F. Spiera, John H. Stone, Guido Grandi, Jie Sun, Jacques-Olivier Pers, Ulrich Specks, Divi Cornec, for the RAVE-ITN Research Group
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 C. Fervenza, Carol A. Langford, Cees G.M. Kallenberg, Peter A. Merkel, Paul A. Monach, Philip Seo, Robert F. Spiera, John H. Stone, Guido Grandi, Jie Sun, Jacques-Olivier Pers, Ulrich Specks, Divi Cornec, for the RAVE-ITN Research Group
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Clinical Research and Public Health Immunology

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

Abstract

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.

Authors

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 C. Fervenza, Carol A. Langford, Cees G.M. Kallenberg, Peter A. Merkel, Paul A. Monach, Philip Seo, Robert F. Spiera, John H. Stone, Guido Grandi, Jie Sun, Jacques-Olivier Pers, Ulrich Specks, Divi Cornec, for the RAVE-ITN Research Group

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Figure 2

Circulating PR3+ B cells and PR3-ANCA production in patients with PR3-AAV and MPO-AAV and HCs.

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Circulating PR3+ B cells and PR3-ANCA production in patients with PR3-AA...
Representative examples of the gating of PR3+ B cells among total CD19+ cells in a patient with PR3-AAV, a patient with MPO-AAV, and a HC (A). PR3+ B cell frequency and count were increased in patients with PR3-AAV (n = 105) compared with patients with MPO-AAV (n = 49) and HCs (n = 27) (B and C). PBMCs were cultured to promote differentiation into antibody-secreting cells, after which PR3-ANCA secretion was analyzed by means of a Phadia ImmunoCAP 250 analyzer (D). Only patients with PR3-AAV can produce PR3-ANCA IgG in vitro. Correlation of circulating (in vivo) PR3-ANCA IgG with secreted (in vitro) PR3-ANCA IgG in patients with PR3-AAV (E). Data represent median (25%–75% IQR). Multiple comparisons among more than 2 groups were performed with Kruskal-Wallis test. *P < 0.05, ***P < 0.001 after correction for FDR with Benjamini and Hochberg test.