wilfordii (Guizhou Han Prescription Pharmacy, Guizhou, China). One month after the beginning of the treatment, their blood samples were BMN 673 mw collected again for subsequently laboratory examination. The full blood counts and erythrocyte sedimentation rates (ESR) of individual subjects were examined. The levels of serum C-reactive protein (CRP), rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) were determined by scatter turbidimetry using a Siemens special protein analyser (Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany). Peripheral blood mononuclear cells (PBMCs) were isolated from individual patients by density-gradient centrifugation using Ficoll-Paque Plus (Amersham Biosciences,

Little Chalfont, UK). PBMCs at 5 × 105/tube were stained in duplicate with APC-cyanin 7 (Cy7)-anti-CD3 (BD Bioscience, San Diego, CA, USA), peridinin chlorophyll (PerCP)-anti-CD19, phycoerythrin (PE)-anti-CD38, APC-anti-CD86 or APC-Cy7-anti-CD3, PerCP-anti-CD19, fluorescein isothiocyanate (FITC)-anti-IgD, PE-anti-CD27 and APC-anti-CD95 (BD PharMingen, San Diego, CA, USA) for 30 min, and APC-Cy7-anti-IgG (BD Bioscience), PerCP-anti-IgG1, PE-anti-IgG1 APC-anti-IgG1 and FITC-anti-IgG (BD PharMingen) as the isotype controls. Furthermore, PBMCs (5 × 105/tube) were stained in duplicate with PerCP-anti-CXCR5 (Biolegend, San Diego, CA, USA), APC-anti-CD4, PE-anti-ICOS, FITC-anti-PD-1, APC-Cy7-anti-CD3 or isotype-matched

controls (BD Bioscience) for 30 min. After being washed with phosphate-buffered saline (PBS), the cells were characterized on a BD fluorescence activated cell sorter (FACS)Aria

II. PBMCs at 4 × 106/ml were stimulated find more in duplicate with or without 3 μg/ml of CpGB (cytosine-phosphate-guanine class B) (R&D Systems, PAK6 Minneapolis, MN, USA) in the presence of 10 ng/ml of recombinant IL-2 (R&D Systems) in RPMI-1640 supplemented with 10% fetal calf serum (FCS) (Hyclone, Logan, UT, USA) in 5% CO2 at 37°C for 3 days [22]. The cells were harvested and then stained in duplicate with PerCP-anti-CD19 and APC-Cy7-anti-CD3 for 30 min, fixed, permeabilized with permeabilization solution (BD Bioscience) and stained with APC-anti-Toll-like receptor (TLR)-9 or the isotype control, followed by flow cytometry analysis of TLR-9 expression. The concentrations of serum IL-21 in individual patients and HC were determined by ELISA using the human IL-21 ELISA kit, according to the manufacturer’s instructions (R&D Systems). Briefly, individual sera at 1:4 dilutions were subjected to ELISA analysis, and the concentrations of serum IL-21 in individual samples were calculated according to the standard curve established by using the recombinant IL-21 provided. The limitation of detection for the level of IL-21 was 10 ng/l. Data are expressed as median and range or individual mean values. The difference between the groups was analysed by Mann–Whitney U non-parametric test using spss version 19·0 software.