Moreover, we have found that CD27+ memory B cells are even further decreased in patients prior to relapse (unpublished data). effector cytokines occurred before relapse, while the percentage of IL10+ B cells significantly decreased. GPA patients in remission had increased serum levels of CCL19 and sCD27, and sCD27 levels increased upon active disease. Conclusions While differences in effector B cell cytokine production were observed between patients and controls, monitoring this in GPA did not clearly distinguish patients about to relapse. Prospective measurements of the regulatory cytokine IL10 may have potential for relapse prediction. Memory B cells appear mainly responsible for effector cytokine production. Increased migration of these cells could explain the decreased presence of TNF+?B cells in the circulation. Electronic supplementary material The online version of this article (doi:10.1186/s13075-016-0978-1) contains supplementary material, which is available to authorized users. anti-neutrophil cytoplasmic antibody, azathioprine, granulomatosis with polyangiitis, healthy controls, mycophenolate mofetil, not applicable, proteinase-3, prednisolone Flow cytometry for analysis of the B cell phenotype Blood was collected in EDTA tubes, and 100?l was incubated with anti-human CD19-eFluor450 (eBioscience, San Diego, CA, USA), CD24-fluorescein isothiocyanate (FITC) (BD biosciences, Franklin Lakes, NJ, USA), CD27-APC-eFluor780 (eBioscience), CD38-PeCy5 (eBioscience), CD5-PerCp-Cy5.5 (Biolegend, San Diego, CA, USA) or the corresponding isotype controls. After 15?minutes cells were treated with fluorescence-activated cell sorting (FACS) Lysing solution (BD Biosciences). Samples were measured using an LSR-II flow cytometer (BD biosciences) and data were analyzed using Kaluza 1.2 flow analysis software Rabbit polyclonal to SORL1 (Beckman Coulter, Brea, CA, USA). B cells were divided into transitional, memory, naive, CD24highCD38high and CD24highCD27+ B cells as described previously [14]. CD5+ B cells were gated on an isotype control. Cell culture and intracellular B cell cytokine pattern upon in vitro stimulation PBMC were isolated and stored in RPMI 1640 (Lonzo, Basel, Switzerland) supplemented with 50?g/mL gentamycin (GIBCO, Life Technologies, Grand Island, NY, USA), 10?% fetal calf serum (FCS) (Lonza) and 10?% dimethyl sulfoxide. The cryopreserved PBMC were thawed, concentrations were adjusted to 106 Dox-Ph-PEG1-Cl cells/mL in RPMI?+?10?% FCS, and cells were seeded in 24-well flat bottom plates (Corning, NY, USA). Cells were left untreated or stimulated using 500?ng/mL CpG-oligodeoxynucleotides (ODN) 2006 (Hycult Biotech, Uden, the Netherlands). Culture plates were incubated for 72?h at 37?C with 5?% CO2. During the last 5?h of incubation 50?ng/mL phorbol myristate acetate (PMA; Sigma-Aldrich, St Louis, MO, USA), 2?mM calcium ionophore (CaI; Sigma-Aldrich) and/or 10?g/mL Brefeldin A (BFA; Sigma-Aldrich) were added to the cell culture. Cells were harvested and stained using anti-human CD19-eFluor450 and CD22-PeCy5 (Biolegend). Subsequently cells were fixed and permeabilized for Dox-Ph-PEG1-Cl intracellular staining using a Fix&Perm kit (Invitrogen, Life Technologies, Grand Island, NY, USA) and incubated with antibodies against human IL10-PE (Biolegend), TNF-Alexa Fluor 488 (BD biosciences), IL6-APC (eBioscience), IL2-PeCy7 (eBioscience) and IFN-Alexa Fluor 700 (BD biosciences). Samples were measured with an LSR-II flow cytometer and data were analyzed using Kaluza 1.2. Samples that had not been stimulated with PMA and CaI were used as negative controls to set the gates during data analysis. Data are presented as the percentage of cytokine-positive B cells within the total CD19+CD22+ population. Enzyme-linked immunosorbent assay (ELISA) for CCL19 and soluble CD27 Serum samples from healthy controls and patients had been collected and stored at ?80?C on the same day as PBMC storage and B cell phenotype analysis. Moreover, serum samples from the relapsing patients were available from the time of active disease. A Human CCL19/MIP-3 beta DuoSet ELISA (R&D Systems, Minneapolis, MN, USA) and a PeliKine Compact? human soluble CD27 ELISA (Sanquin, Amsterdam, the Netherlands) were performed according to the manufacturers instructions. CCL19 levels are expressed as pg/mL and sCD27 levels as units (U)/mL. Statistical analysis Statistical analysis was performed using SPSS v22 (IBM Corporation, Chicago, IL, USA) and GraphPad Prism v5.0 (GraphPad Software, Dox-Ph-PEG1-Cl San Diego, CA, USA). Data are presented as median values with the interquartile range, unless stated otherwise. For comparison between groups the unpaired test was applied for data with a Gaussian distribution and the Mann-Whitney test was used for data with a non-Gaussian distribution. For Dox-Ph-PEG1-Cl intra-individual comparisons the paired test or Wilcoxon matched pairs test was performed for Gaussian- and non-Gaussian-distributed data, respectively. Correlation analysis was performed by calculating the Spearman rank correlation coefficient. values <0.05 were considered statistically significant. Results B cell subset distribution in patients with GPA and healthy controls The B cell subset distribution in patients with GPA in remission differed from healthy controls.