Defects in B-lymphopoiesis and B-cell maturation underlie prolonged B-cell depletion in ANCA-associated vasculitis

Abstract

Objectives: B-cell depletion time after rituximab (RTX) treatment is prolonged in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) compared with other autoimmune diseases. We investigated central and peripheral B-cell development to identify the causes for the defect in B-cell reconstitution after RTX therapy.

Methods: We recruited 91 patients with AAV and performed deep phenotyping of the peripheral and bone marrow B-cell compartment by spectral flow and mass cytometry. B-cell development was studied by in vitro modelling and the role of BAFF receptor by quantitative PCR, western blot analysis and in vitro assays.

Results: Treatment-naïve patients with AAV showed low transitional B-cell numbers, suggesting impaired B-lymphopoiesis. We analysed bone marrow of treatment-naïve and RTX-treated patients with AAV and found reduced B-lymphoid precursors. In vitro modelling of B-lymphopoiesis from AAV haematopoietic stem cells showed intact, but slower and reduced immature B-cell development. In a subgroup of patients, after RTX treatment, the presence of transitional B cells did not translate in replenishment of naïve B cells, suggesting an impairment in peripheral B-cell maturation. We found low BAFF-receptor expression on B cells of RTX-treated patients with AAV, resulting in reduced survival in response to BAFF in vitro.

Conclusions: Prolonged depletion of B cells in patients with AAV after RTX therapy indicates a B-cell defect that is unmasked by RTX treatment. Our data indicate that impaired bone marrow B-lymphopoiesis results in a delayed recovery of peripheral B cells that may be further aggravated by a survival defect of B cells. Our findings contribute to the understanding of AAV pathogenesis and may have clinical implications regarding RTX retreatment schedules and immunomonitoring after RTX therapy.

Keywords: B-lymphocytes; rituximab; vasculitis.

Figure 1. Disturbed peripheral B-cell maturation and prolonged B-cell depletion in patients with antineutrophil cytoplasmic antibody-associated vasculitis (AAV). Peripheral B-cell populations of patients with AAV and healthy donors (HD) were analysed by spectral flow cytometry. (A) Absolute count of B cells/µL blood and frequency (%) of B cells in lymphocytes in HD and AAV treatment groups. (B) Correlation of the frequency (%) of B cells with the duration (months) of B-cell depletion after RTX treatment. (C–H) Absolute count/µL and frequency within total B cells of transitional B cells (IgM⁺⁺CD38⁺⁺) (C), naïve B cells (IgD⁺CD27⁻) (D), marginal zone-like B cells (MZ, IgD⁺CD27⁺) (E), switched memory B cells (IgD⁻CD27⁺) (F), double negative B cells (DN, IgD⁻CD27⁻) (G) and plasmablasts (CD38⁺⁺CD27⁺⁺) (H) in HD and treatment-naïve patients. Data were analysed using Kruskal-Wallis test, corrected for multiple comparisons by Dunn’s multiple comparison tests (A) or Mann-Whitney U test (C–H) and depicted as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Correlation analysis was performed by non-parametric Spearman’s correlation.

Figure 2. RTX treatment unveils a block in B-cell maturation at transitional B-cell stage in patients with antineutrophil cytoplasmic antibody-associated vasculitis (AAV). Spectral flow cytometric analysis of peripheral B-cell populations of patients with AAV and HD. (A–F) Absolute count/µL blood and frequency (%) within total B cells of transitional B cells (IgM⁺⁺CD38⁺⁺) (A), naïve B cells (IgD⁺CD27⁻) (B), marginal zone-like B cells (MZ, IgD⁺CD27⁺) (C), switched memory B cells (IgD⁻CD27⁺) (D), double negative B cells (DN, IgD⁻CD27⁻) (E) and plasmablasts (CD38⁺⁺CD27⁺⁺) (F) in HD and patients with AAV grouped according to treatment. Data were analysed using Kruskal-Wallis test, corrected for multiple comparisons by Dunn’s multiple comparison tests and depicted as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 3. Impaired B-lymphopoiesis in bone marrow (BM) of patients with antineutrophil cytoplasmic antibody-associated vasculitis (AAV). Ex vivo BM aspirates of AAV and healthy donors (HD) were analysed by mass cytometry by time of flight. (A) Vaevictis dimensionality reduction projections of one HD and five patients with AAV with outlined regions of subpopulations. Defined regions show 95% probability contouring from indicated subpopulations. (B) Distribution of subpopulations as frequency of live (cPARP^-/cleaved caspase 3⁻) cells of analysed BM samples. (C) Frequency of B lineage^- (B lin^-) early progenitor cells and CD10⁺CD38⁺ lymphoid precursor cells in live (cPARP⁻cleaved caspase 3⁻) cells. (D) Frequency of common lymphoid progenitors (CLP)/pro-B cells, pre-BI, pre-BII and immature B cells within CD10⁺CD38⁺ lymphoid precursor cells. (C, D) Filled circles: treatment-naïve except for short-term glucocorticoids; empty circle: RTX-induction therapy+LEF maintenance therapy; empty triangle: CYC+high-dose prednisolone. Data were analysed using Mann-Whitney U test and depicted as *p<0.05.

Figure 4. Delayed but intact B-cell development in vitro of antineutrophil cytoplasmic antibody-associated vasculitis (AAV) bone marrow (BM). (A) Experimental setup of in vitro development of BM-derived CD34⁺ cells to immature B cells. Isolated CD34⁺cells from the BM of HD (n=8) and patients with AAV (n=6) were cultured as described and analysed by flow cytometry every 7 days. (B) Representative plots show the development of CD10⁺CD38⁺ lymphoid precursors to IgM⁺CD19⁺ immature B cells over 49 days in HD (upper row) and AAV (lower row). (C–E) Development of subpopulations over time. Absolute counts of CD10⁺CD38⁺ lymphoid precursors (C), common lymphoid progenitors (CLP)/pro-B cells, pre-B cells and immature B cells (D) as well as frequency of CLP/pro-B cells, pre-B cells and immature B cells in CD10⁺CD38⁺ (E). (C–E) Filled circles: treatment-naïve except for short-term glucocorticoids; empty triangle: CYC+high-dose prednisolone. Mean of 3–5 technical replicates per donor±SEM is shown.

Figure 5. Expression of BAFF-receptor (BAFF-R) and TACI is reduced in recirculating B cells after rituximab (RTX) treatment. (A) Representative flow cytometry plot of BAFF-R expression (left) and bar graph of median fluorescence intensity (MFI) of BAFF-R (right) in non-plasmablast B cells. (B) BAFF concentration (pg/mL) was measured in the serum of healthy donors (HD) and patients with antineutrophil cytoplasmic antibody-associated vasculitis (AAV) using LEGENDplex assay. (C) Correlation of BAFF-R MFI in non-plasmablast B cells with absolute count of B cells/µL blood (left) and BAFF concentration in serum (right). (D) Representative flow cytometry plot of TACI expression (left) and bar graph of MFI of TACI (right) in marginal zone-like (MZ-like) and switched memory B cells. (E) Correlation of BAFF-R MFI with TACI MFI. (F) Soluble TACI (sTACI) concentration (pg/mL) was measured in the serum of HD and patients with AAV by ELISA. Data were analysed using Kruskal-Wallis test, corrected for multiple comparisons by Dunn’s multiple comparison tests and depicted as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Correlation analysis was performed by non-parametric Spearman’s correlation.

Figure 6. Low BAFF-receptor (BAFF-R) expression by enhanced BAFF-R processing contributes to impaired B-cell survival in antineutrophil cytoplasmic antibody-associated vasculitis (AAV). (A) TNFRSF13C mRNA expression relative to RPLPO was determined by quantitative PCR. (B) ADAM10 and ADAM17 expression was analysed as median fluorescence intensity (MFI) in B cells. (C) Western blot analysis of ex vivo isolated B cells of BAFF-R c-terminal fragment (BAFF-R ct) in patients with AAV compared with HD. Representative western blot analysis (left) and quantification of BAFF-R ct in relation to β-actin (right). (D) Isolated B cells were cultured in medium+3% fetal calf serum (FCS) for 5 days. BAFF-R expression in B cells was analysed as MFI. (E) Experimental setup of performed survival assay. Isolated B cells were cultured as indicated in medium+3% FCS in absence or presence of 50 ng/mL BAFF-60mer. Cells were analysed by flow cytometry. (F) Survival benefit of BAFF addition to the culture was analysed. Counts of B cells on day 5 of both culture conditions were compared (left). Ratio of BAFF/medium treated B cells (counts) on day 5 (right). Data were analysed using Kruskal-Wallis test, corrected for multiple comparisons by Dunn’s multiple comparison tests or Mann-Whitney U test and depicted as *p<0.05.