Background While extensive studies have been performed to characterize ANCA, little is known about the auto-reactive B cells that produce these autoantibodies. Indirect evidence previously suggested the presence of circulating PR3-specific B cells in patients with PR3-ANCA-associated vasculitis (AAV).
Objectives To develop a method to detect circulating PR3-specific B cells in patients with PR3-AAV, to study their proportion among the different B-cell subsets and to assess their relationship with disease activity.
Methods An enzymatically inactive, conformationally mature, recombinant PR3 (rPR3) was tagged using FITC or biotin. To study the ability of this rPR3 to bind specifically to cells expressing PR3-specific immunoglobulins on their surface, we used two hybridoma cell lines, MCPR3–2 (producing an anti-human PR3 monoclonal antibody) and MCPR3–13 (producing an anti-mouse PR3 monoclonal antibody, with no cross-reactivity with human PR3). We measured the proportion of PR3-FITC positive B cells among PBMCs in 13 patients with PR3-AAV and 14 healthy controls (HCs) by flow cytometry. We then developed a multi-color flow cytometry including CD19, IgD, CD27, CD38, CD24 and biotinylated rPR3 to measure the proportion of PR3-specific B cells among different B-cell subsets in an independent group of 13 patients with PR3-AAV and 11 HCs.
Results rPR3 efficiently bound MCPR3–2 hybridoma cells but not MCPR3–13. Specificity of the staining was confirmed by competition experiments: pre-incubation of MCPR3–2 cells with untagged human rPR3 totally abrogated rPR3-FITC staining, whereas pre-incubation with mouse rPR3 had no effect. Dose-ranging experiments defined the optimal concentration of rPR3 to stain cells expressing anti-PR3 immunoglobulin. The mean (SEM) proportion of rPR3-FITC-stained B cells was higher in patients with PR3-AAV compared to HCs: 2.10% (2.33) vs 0.45% (0.19) respectively, p<0.001. Patients with active disease had numerically higher proportions of PR3-specific B cells than patients in remission: 3.66% (3.28) vs 1.10% (0.52), p=0.09. In HCs, the proportion of PR3-specific B cells was highest among the transitional B-cell subset, and decreased along with the maturation of B cells (figure). Conversely, in patients, the proportion of PR3-specific B cells progressively increased with the maturation of B cells (median 1.9% of naïve B cells, 2.30% of IgD+ memory B cells, 2.37% of IgD- memory B cells, and 3.68% of plasmablasts, p<0.05 for all comparisons with the naïve subset).
Conclusions This study describes an original method to detect and study circulating auto-reactive B cells in patients with PR3-AAV, and suggests that PR3-specific B cells are associated with disease activity and may represent a promising biomarker to predict relapse risk in patients in clinical remission. The progressive enrichment in PR3-specific B cells during the B-cell maturation steps in patients suggest that auto-reactive B cells are actively selected and escape peripheral tolerance checkpoints.
Disclosure of Interest None declared