Background Eosinophilic granulomatosis with polyangiitis (EGPA) belongs to the group of ANCA associated vasculitides. While the combination of asthmatic symptoms and vasculitis characterize the disease clinically, eosinophilia and increased serum IgE concentrations are serologic hallmarks. The role of B lymphocytes in EGPA has not been defined so far, but therapeutic response to rituximab in EGPA points towards a role of B-cells in the pathogenesis of EGPA.
Objectives To characterize the peripheral B cell compartment in patients with EGPA, to analyze the in vivo potential of B lymphocytes to class-switch to IgE, and to assess in vitro the differentiation of naïve B cells to IgE-secreting plasmablasts.
Methods Laboratory work-up included ANCA-status, eosinophils, IgE, IgG, IgA, IgM, and peripheral CD19+ B-cell count. B cell subpopulations (naïve, marginal zone, class-switched B cells and plasmablasts) were analyzed by staining PBMCs with fluorescent-labeled monoclonal antibodies against: CD27, CD20, CD38, IgD, IgG, IgA, IgE, CD21, and BAFF-R. For in vitro differentiation assays magnetically isolated B lymphocytes from EGPA patients and aged matched healthy controls were stimulated with CD40L and IL-21 and IL-4 in enriched Iscoves' medium supplemented with 10% FCS, 1 μg/mL insulin, 2.5 μg/mL apo-transferrin, 1% nonessential amino acids, 2 mmol/L glutamine, and 1 μg/mL reduced glutathione. Starting the culture with equal number of B cells, the absolute number of plasmablasts, and IgE class switched cells after 9 days was determined by counting the events in the CD27highCD38high gate or the IgG/A/D-IgE+ gate by flow cytometry. IgE secretion in the supernatant was measured by ELISA.
Results 18 patients (8 females, median age 59 years) with EGPA diagnosed according to ACR and CHC-criteria were included into the study. 22% of patients were ANCA-positive. Immunosuppressive therapy was azathioprine in 11 patients, methotrexate in 3 patients, and leflunomid or mycophenolate in one patient each and two patients received no immunosuppressive treatment. 7 patients had a history of a prior cyclophosphamide therapy. Median lymphocyte count was normal (1.2x103/μl; normal range 1.1 to 3.2x103/μ l) but peripheral B cell numbers were markedly diminished (37/μl, normal range: 100 – 500/μl). While the percentage of naïve B cells (53%) and marginal zone like B cells (10.5%) were within normal range, the percentage of class-switched memory B cells was high (28.2%). To assess the in vitro class switch capacity to IgE the number of IgE class-switched cells after 9 days of culture was determined by counting the events in the IgG/A/D-IgE+ gate. The percentage of IgE-switched cells was 6.8% (range: 0.3%>34%) and not statistically different from healthy controls.
Conclusions In the EGPA-patients we report we observed markedly diminished B-cells despite of normal lymphocyte counts. Within the B cell compartment, there was a shift towards later B cell maturation stages. The in vitro B-cell development to IgE class-switched cells was not increased in EGPA-patients pointing towards a non-B cell intrinsic mechanism.
Disclosure of Interest None declared
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