Representative flow cytometry dot plots from a healthy individual

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Representative flow cytometry dot plots from a healthy individual Representative flow cytometry dot plots from a healthy individual. Lymphocytes were gated based on the forward-side scatter profile. (A) Within the lymphocyte population, B cells were distinguished by expression of CD19. (B) Cell surface markers CD27 and CD38 were used to further characterize B cell subpopulations. Transitional B cells were CD19<sup>+</sup>CD27<sup>−</sup>CD38<sup>high</sup>, naive B cells were defined as CD19<sup>+</sup>CD27<sup>−</sup>CD38<sup>−/low</sup> and memory B cells were CD19<sup>+</sup>CD27<sup>+</sup>CD38<sup>−/low</sup>. The two putative regulatory B cell subsets were defined as (C) CD19<sup>+</sup>CD24<sup>high</sup>CD38<sup>high</sup> or (D) CD19<sup>+</sup>CD24<sup>high</sup>CD27<sup>+</sup>. From: Altered B cell balance, but unaffected B cell capacity to limit monocyte activation in anti-neutrophil cytoplasmic antibody-associated vasculitis in remission Rheumatology (Oxford). 2014;53(9):1683-1692. doi:10.1093/rheumatology/keu149 Rheumatology (Oxford) | © The Author 2014. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Distribution of B cell subsets in AAV patients and HCs Distribution of B cell subsets in AAV patients and HCs. (A–F) Graphs represent data for 41 HCs, 36 patients in clinical remission and 12 patients with active disease. Horizontal lines indicate median values. A P-value <0.05 was considered statistically significant. *P < 0.05, **P < 0.001, ***P < 0.0001. AAV: ANCA-associated vasculitis; HCs: healthy controls. From: Altered B cell balance, but unaffected B cell capacity to limit monocyte activation in anti-neutrophil cytoplasmic antibody-associated vasculitis in remission Rheumatology (Oxford). 2014;53(9):1683-1692. doi:10.1093/rheumatology/keu149 Rheumatology (Oxford) | © The Author 2014. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

(A and B) For detection of B10 cells, whole blood samples were stimulated with PMA and calcium ionophore in the presence of BFA for 4 h. (C and D) For induction of B10pro cells, PBMCs were stimulated with CpG-ODN for 3 days. PMA, calcium ionophore and BFA were added in the final 5 h of the culture. (A and C) Representative flow cytometry dot plots from a healthy individual, demonstrating the gating strategy for (A) B10 cells and (C) B10pro cells. Samples incubated with BFA only were used to set the gate. (B) Frequency of B10 cells from HCs (n = 10) and AAV patients (n = 10) in remission. (D) Percentages of B10pro cells in HCs (n = 18), AAV patients in remission (n = 15) and AAV patients with active disease at the time of inclusion (n = 6). PMA: phorbol myristate acetate; BFA: brefeldin A; HCs: healthy controls; AAV: ANCA-associated vasculitis. From: Altered B cell balance, but unaffected B cell capacity to limit monocyte activation in anti-neutrophil cytoplasmic antibody-associated vasculitis in remission Rheumatology (Oxford). 2014;53(9):1683-1692. doi:10.1093/rheumatology/keu149 Rheumatology (Oxford) | © The Author 2014. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

In five HCs and five AAV patients in remission, B cell suppressive capacity was tested using LPS-activated monocytes as a responder population. Intracellular TNF-α expression in monocytes was measured by flow cytometry. (A) Representative histograms showing TNF-α expression in monocytes. Monocytes cultured alone without adding LPS were used as a negative control. Monocytes activated with LPS served as a positive control sample. Monocytes were co-cultured with either unstimulated B cells or with B cells that were preactivated with CpG-ODN and anti-CD40. (B) Data from five HCs and five GPA patients. For each donor the median FI of TNF-α expression in the positive control condition was used as a reference sample and was normalized to 100%. Median FI values measured in other samples are expressed relative to the reference sample. LPS: lipopolysaccharide; HCs: healthy controls; GPA: granulomatosis with polyangiitis; FI: fluorescence intensity. From: Altered B cell balance, but unaffected B cell capacity to limit monocyte activation in anti-neutrophil cytoplasmic antibody-associated vasculitis in remission Rheumatology (Oxford). 2014;53(9):1683-1692. doi:10.1093/rheumatology/keu149 Rheumatology (Oxford) | © The Author 2014. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com