Patients and controls
In the studies investigating cytokine production, peripheral blood was collected from 13 patients with SLE (12 females and 1 male) with a mean age of 30.6 ± 8.8 years and with a median Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score of 6 (range: 4–15) and 9 HD (8 females and 1 male) with a mean age of 33.4 ± 11.5 years. For the activation analysis and IL-10 production of B cells using flow cytometry (FC), peripheral blood was collected from six female patients with SLE with a mean age of 38.8 ± 12.9 years and a median SLEDAI score of 6 (range: 5–18). Ten HD (8 females and 2 males) with a mean age of 32.9 ± 11.1 years served as controls. The study was approved by the ethics committee at the Charité-Universitätsmedizin Berlin, and written consent was obtained from all donors. All patients met the revised American College of Rheumatology classification criteria for SLE [9]. Disease activity was assessed using the Safety of Estrogens in Lupus Erythematosus National Assessment–SLEDAI score [10].
Peripheral blood mononuclear cells and B cell purification
Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation as previously described [11]. B cells were negatively purified magnetically (B Cell Isolation Kit II; Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. B cells were analyzed regarding their purity to minimize the contamination by other cytokine-producing cells.
B cell purity
A total of 100,000 purified B cells were stained with antibodies against CD14-PacB (M5E2), CD3-PacB (UCHT1) and CD19 PE-Cy7 (SJ25C1) (all from BD Biosciences, San Jose, CA, USA) for 15 minutes at 4 °C. Afterward, 4′,6-diamidino-2-phenylindole (DAPI) was added to the stained cells (dead cell staining) and analyzed by FC using a FACSCanto II flow cytometer (BD Biosciences). Data were evaluated using FlowJo software (version 7; Tree Star, Ashland, OR, USA). The total B cell (CD3−CD14−DAPI−CD19+) purity was 98.5 ± 2.2 % in almost all samples (mean ± standard deviation), with the exception of two cases with 8.2 % and 5.6 % non–B cells (CD3+ cells, CD14+ cells or cell debris). These two samples were outliers but did not show any results substantially different from the remaining samples. Additional analyses also did not show any relationship between cytokine levels in the supernatants of TNF-α, IL-6 or IL-10 and the frequency of non-B cells of these samples, including any relationship of the impact of epratuzumab and the frequency of non-B cells after purification (data not shown).
B cell in vitro stimulation
Cells were resuspended in RPMI 1640 GlutaMAX medium (Life Technologies, Darmstadt, Germany) supplemented with 10 % fetal calf serum (Lonza, Cologne, Germany), 5 % penicillin-streptomycin and 0.05 mM 2-mercaptoethanol (Gibco; Life Technologies) at 1.1 × 106 purified B cells/ml or 1.1 × 107 PBMCs/ml. Cells (90 μl) were seeded, pretreated with 10 μg/ml F(ab′)2 epratuzumab (provided by UCB Celltech, Slough, UK) for 15 minutes and stimulated with 2.5 μg/ml CpG 2006 (TIB MOLBIOL Syntheselabor, Berlin, Germany) and/or 2 μg/ml F(ab′)2 anti-human IgM/IgG (anti-BCR; Jackson ImmunoResearch, Suffolk, UK) for 48 hours at 37 °C in 5 % CO2 [4]. Because the anti-BCR can potentially bind to the crystallizable fragment (Fc) region of the whole anti-CD22 antibody epratuzumab, and because Sieger et al. [6] could show that epratuzumab and F(ab′)2 epratuzumab have the same effect on BCR signaling, the F(ab′)2 fragment of epratuzumab was used to study the effect of the anti-CD22 antibody on the cytokines production after TLR9 and/or BCR activation. For the cytokine analysis, supernatants from the B cell in vitro cultures were harvested and frozen at −70 °C. For the intracellular IL-10 staining, PBMCs stimulated in vitro were used. Unstimulated B cells were used as a negative control.
Interleukin-10 staining for flow cytometry
Intracellular staining for IL-10 was performed on PBMCs after 2 days of in vitro culture. PBMCs were restimulated for the last 4 hours with 10 ng/ml phorbol myristate acetate and 700 ng/ml ionomycin, including 2 hours with 2 μg/ml brefeldin A (all from Sigma-Aldrich, Munich, Germany). PBMCs were stained first on ice for 10 minutes with antibodies against CD14-PacB (M5E2), CD3-PacB (UCHT1), CD27-FITC (L128), CD38-PerCP/Cy5.5 (HIT2) and CD20-PacO (H147) (all from BD Biosciences). After a washing step, PBMCs were incubated with 400 μl of BD FACS Permeabilizing Solution 2 (BD Biosciences) for 10 minutes at room temperature (RT). After another washing step, PBMCs were stained with anti-IL10-APC antibodies (JES3-9D7; Miltenyi Biotec) for 10 minutes at RT. Stained cells were analyzed by FC using a FACSCanto™ II flow cytometer and analyzed using FlowJo software. Unstimulated PBMCs with brefeldin A treatment were used as a control.
Cytokine assay
Frozen supernatants of purified B cells stimulated in vitro were assessed for the cytokine concentrations of IL-1β, IL-4, lL-6, IL-10, IL-17A, IL-17F, IL-22, IL-23, IL-25, IL-31, IL-33, interferon-γ, sCD40L and TNF-α by using the Bio-Plex Pro™ Human Th17 Cytokine Panel (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions. The assay sensitivity depends on the particular cytokines analyzed, ranging from 0.02 pg/ml for IL-1β to 2.13 pg/ml for IL-21. A significant and specific induction after anti-BCR and CpG stimulation above 20 pg/ml was observed only for the cytokines IL-6, TNF-α and IL-10. Therefore, the other cytokines were not considered in further analyses.
Statistical analysis
The statistical analysis was performed with GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA). To compare HD and SLE groups, nonparametric Mann–Whitney U tests were applied. In comparative analyses of data with or without F(ab′)2 epratuzumab incubation, we applied the Wilcoxon signed-rank test test. P values <0.05 were considered to be statistically significant.