OP0137 Auto-reactive b cells escape peripheral tolerance checkpoints in patients with pr3-anca associated vasculitis
Authors: D Cornec, A Hummel, T Peikert, J-O Pers, U Specks
Journal: Annals of the Rheumatic Diseases (2017)

<h3>Background</h3> 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). <h3>Objectives</h3> 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. <h3>Methods</h3> 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. <h3>Results</h3> 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&lt;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&lt;0.05 for all comparisons with the naïve subset). <h3>Conclusions</h3> 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. <h3>Disclosure of Interest</h3> None declared