Session Type: Poster Session (Monday)
Session Time: 9:00AM-11:00AM
Background/Purpose: We previously developed a method to recognize proteinase 3 (PR3)-specific B cells by flow cytometry. We reported that, in a small number of subjects, the proportion of PR3-specific B cells was higher in patients with PR3-ANCA-associated vasculitis (AAV) compared to healthy controls (HCs) (Cornec et al, J Autoimmunity 2017). The objectives of this study were to replicate our previous findings on a larger number of subjects, including anti-myeloperoxidase (MPO)-AAV patients as disease controls, and to study the association of PR3-specific B cell proportions with clinical and biological features in patients with PR3-AAV.
Methods: We analyzed samples from 161 patients who participated in the RAVE trial, including 110 patients with PR3-AAV and 51patients with MPO-AAV, as well as 27 HCs. We measured the proportion of PR3-specific B cells and subsets among cryopreserved PBMCs using a multi-color flow cytometry panel including CD19, IgD, CD27, CD38, CD24, and biotinylated PR3. SPADE (Spanning-tree Progression Analysis of Density-normalized Events) algorithm was used for the unsupervised clustering of flow cytometry data, based on the level of expression of each of the 6 markers on each cell.
Results: The proportion of PR3-specific B cells was higher in patients with PR3-AAV and MPO-AAV (median [25-75% interquartile range]: 2.82% [2.35-3.74] and 2.87 [2.15-3.84], respectively) compared to HCs (1.51 [1.25-1.73], both p< 0.001), while they did not differ substantially between PR3-AAV and MPO-AAV (p=0.517). We observed a significant shift towards a more mature phenotype of these PR3-specific B cells in patients with PR3-AAV compared to MPO-AAV and HCs, as represented by a higher proportion of PR3-specific B cells among the unswitched memory B-cell subsets (IgD+CD27+; 6.14% [4.27-8.74] versus 4.47% [3.11-6.87] versus 2.89% [2.19-4.23], p< 0.01 for all comparisons), switched memory B-cell subsets (IgD-CD27+; 2.98% [1.67-5.00] versus 2.1% [1.38-3.09] versus 1.19% [0.96-1.60], p< 0.001 for all comparisons), and double negative B-cell subsets (IgD-CD27-; 2.23% [1.54-3.76] versus 1.44% [0.83-2.53] versus 0.80% [0.00-1.34], p< 0.01 for all comparisons). In patients with PR3-AAV, no associations between PR3-specific B cell proportion and age, sex, new diagnosis vs relapsing disease, or disease activity (BVAS/WG) was observed. The SPADE clustering resulted in the definition of 200 distinct subsets detectable in each sample with variable proportions, and showed a strong segregation of patients and HCs, and PR3-AAV and MPO-AAV patients. A cluster belonging to the switched memory subset was PR3-reactive only in PR3-AAV (figure).
Conclusion: Circulating PR3-specific B cells can be detected in the peripheral blood. The proportion of PR3-specific B cells was higher in patients than in HCs. Interestingly, PR3-specific B cell repartition is shifted towards a memory phenotype in PR3-AAV patients. Further studies are ongoing to characterize the dynamics of the PR3-specific B cells after therapy, and to understand the differences of these autoreactive B cells between patients with PR3-AAV and HCs.
To cite this abstract in AMA style:Berti A, Hillion S, Hummel A, Carmona E, Peikert T, Langford C, Merkel P, Monach P, Seo P, Spiera R, St. Clair E, Fervenza F, Harris K, Stone J, Pers J, Heeringa P, Abdulahad W, Specks U, Cornec D. Circulating PR3-Specific B Cells in Patients with Active ANCA-Associated Vasculitis [abstract]. Arthritis Rheumatol. 2019; 71 (suppl 10). https://acrabstracts.org/abstract/circulating-pr3-specific-b-cells-in-patients-with-active-anca-associated-vasculitis/. Accessed December 5, 2020.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/circulating-pr3-specific-b-cells-in-patients-with-active-anca-associated-vasculitis/