Objectives To develop a method to detect and phenotype circulating proteinase 3 (PR3)-specific B-cells in patients with PR3-ANCA associated vasculitis (AAV). Methods Recombinant human PR3 (rPR3) was tagged with FITC or biotin, and its binding characteristics were studied by flow cytometry using three hybridoma cell lines secreting antibodies (Ab) against human PR3, mouse PR3 (no cross-reactivity with human PR3), and human neutrophil elastase. We measured the proportion of PR3-specific B-cells and studied their surface phenotype in patients with PR3-AAV and healthy controls (HC). Results Labeled rPR3 efficiently and specifically bound to hybridoma cells producing anti-human-PR3-Ab but not anti-mouse-PR3-Ab or anti-human-elastase-Ab. The proportion of rPR3-stained B cells was higher in patients with PR3-AAV compared to HCs: median (IQR) 1.11% (0.81–2.43) vs 0.45% (0.26–0.62) respectively, p < 0.001. There was a trend towards a higher proportion of PR3-specific B cells among patients with active disease compared to patients in remission: 2.91% (1.18–6.52) vs 0.99% (0.72–1.58), p = 0.09. In HCs, the proportion of PR3-specific B cells was highest among the transitional B-cell subset, and decreased with the maturation of B cells. Conversely, in patients, the proportion of PR3-specific B cells progressively increased with the maturation of B cells (median 1.90% of naïve B cells, 2.30% of unswitched memory B cells, 2.37% of switched memory B cells, and 3.68% of plasmablasts). Conclusions Circulating PR3-specific B cells can be detected in HC and patients with PR3-AAV. Their progressive enrichment during B-cell maturation suggests that they are actively selected and escape peripheral tolerance checkpoints in patients.

Identification and phenotyping of circulating autoreactive proteinase 3-specific B cells in patients with PR3-ANCA associated vasculitis and healthy controls / Cornec, D; Berti, A; Hummel, A; Peikert, T; Pers, Jo; Specks, U. - In: JOURNAL OF AUTOIMMUNITY. - ISSN 0896-8411. - 2017, 84:(2017), pp. 122-131. [10.1016/j.jaut.2017.08.006]

Identification and phenotyping of circulating autoreactive proteinase 3-specific B cells in patients with PR3-ANCA associated vasculitis and healthy controls

Berti A
Secondo
;
2017-01-01

Abstract

Objectives To develop a method to detect and phenotype circulating proteinase 3 (PR3)-specific B-cells in patients with PR3-ANCA associated vasculitis (AAV). Methods Recombinant human PR3 (rPR3) was tagged with FITC or biotin, and its binding characteristics were studied by flow cytometry using three hybridoma cell lines secreting antibodies (Ab) against human PR3, mouse PR3 (no cross-reactivity with human PR3), and human neutrophil elastase. We measured the proportion of PR3-specific B-cells and studied their surface phenotype in patients with PR3-AAV and healthy controls (HC). Results Labeled rPR3 efficiently and specifically bound to hybridoma cells producing anti-human-PR3-Ab but not anti-mouse-PR3-Ab or anti-human-elastase-Ab. The proportion of rPR3-stained B cells was higher in patients with PR3-AAV compared to HCs: median (IQR) 1.11% (0.81–2.43) vs 0.45% (0.26–0.62) respectively, p < 0.001. There was a trend towards a higher proportion of PR3-specific B cells among patients with active disease compared to patients in remission: 2.91% (1.18–6.52) vs 0.99% (0.72–1.58), p = 0.09. In HCs, the proportion of PR3-specific B cells was highest among the transitional B-cell subset, and decreased with the maturation of B cells. Conversely, in patients, the proportion of PR3-specific B cells progressively increased with the maturation of B cells (median 1.90% of naïve B cells, 2.30% of unswitched memory B cells, 2.37% of switched memory B cells, and 3.68% of plasmablasts). Conclusions Circulating PR3-specific B cells can be detected in HC and patients with PR3-AAV. Their progressive enrichment during B-cell maturation suggests that they are actively selected and escape peripheral tolerance checkpoints in patients.
2017
Cornec, D; Berti, A; Hummel, A; Peikert, T; Pers, Jo; Specks, U
Identification and phenotyping of circulating autoreactive proteinase 3-specific B cells in patients with PR3-ANCA associated vasculitis and healthy controls / Cornec, D; Berti, A; Hummel, A; Peikert, T; Pers, Jo; Specks, U. - In: JOURNAL OF AUTOIMMUNITY. - ISSN 0896-8411. - 2017, 84:(2017), pp. 122-131. [10.1016/j.jaut.2017.08.006]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11572/376471
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