Thirty-three patients with active disease (SLEDAI ≥ 6) fulfilling the 2012 SLICC classification criteria for SLE [20] were sequentially recruited at the Outpatient Clinic. Twenty-five healthy controls (students and professional staff) comprised the control group. Peripheral blood was collected after informed consent and the study was approved by the institution ethics committee (UNIFESP CEP 1332/09).

A pool of normal human serum [20 normal blood donors] and a pool of SLE serum (33 patients with active SLE—SLEDAI ≥ 6) were used for challenging PBMC cultures. The pools were inactivated for Complement activity, separated into aliquots and kept at -70 °C until use.

PBMC were isolated by standard density gradient centrifugation and 0.5 × 106 viable PBMC/well were cultured for 24 h in RPMI 1640 (Gibco, Grand Island, USA) supplemented with 2 mM L-glutamine (Gibco), 2 mM sodium pyruvate (Gibco), 100 mg/mL penicillin/streptomycin (Gibco) and 100 mM HEPES (Gibco) in 96-well plates (Costar 3799, Corning®, USA) at 37 °C, 5% CO2 and 96% humidity. In addition, individual wells were supplemented with either: (1) 10% fetal calf serum (Gibco) and no human serum; (2) 20% human serum pool (either from SLE patients or from control donors) and no fetal calf serum; and (3) Camptothencin 5uM (Sigma, Missouri, USA) as a positive control for apoptosis induction. After 24 h, cells were washed in phosphate-buffered saline (PBS) and stained with antibodies that characterize each cell phenotype and Annexin-V, as described below (Additional file 1).

Treg cells were identified by staining with APC-Cy3-labeled anti-CD3, V500-labled anti-CD4, PE-Cy7-labeled anti-CD25, and fluorescein isothiocyanate (FITC)-labeled anti-CD127. After 30 min incubation at 4 °C, cells were washed with Pharmingen stain BSA Buffer (Becton Dickinson, San Diego, CA, USA), fixed and permeabilized with FoxP3 fixation/permeabilization buffer (eBioscience, San Diego, CA, USA) and then processed using APC-labeled anti-FoxP3 antibodies (FoxP3 staining kit, eBioscience) according to manufacturer’s instructions. Th17 cells were identified by staining with APC-Cy3-labeled anti-CD3, V500-labeled anti-CD4, PE-Cy7-labeled anti-CCR6, and FITC-labeled anti-CD161. Th2 cells were identified using APC-Cy3-labeled anti-CD3, V500-labeled anti-CD4, and PE-Cy7-labeled anti-CCR4. Th1 cells were identified by staining whit APC-Cy3-labeled anti-CD3, V500-labled anti-CD4, PerCP-Cy5.5-labeled anti-CCR5 and APC-labeled anti-CXCR3. Unless stated otherwise, all monoclonal antibodies were purchased from Becton Dickinson (San Jose, CA, USA) and processed according to the manufacturer’s instructions. Cells were processed in a FACSCanto II™ flow cytometer (Becton Dickinson) and the data obtained were analyzed using the FlowJo software (Tree Star Inc, Ashland, OR, USA) (Additional file 1: Fig. S1). Fluorescence minus one (FMO) was used as gating strategy.

Annexin V was used as a surrogate marker to determine the percentage of cells undergoing apoptosis. PBMC were washed in phosphate-buffered saline (PBS) and 0.5 × 106 cells were incubated with PE-labeled Annexin-V (Annexin V apoptosis detection kit, Becton Dickinson, San Jose, USA) using buffer containing 10 mM HEPES/NaOH (pH 7.4), 0.14 M NaCl, 2.5 mM CaCl2 (Annexin V Binding Buffer—BD).

Parameters were expressed as mean and standard error (SE). The distribution pattern of each quantitative parameter was analyzed by the Kolmogorov-Smirnoff test. One-way analysis of variance (ANOVA) was used for variables with normal distribution. Those with non-parametric distribution were analyzed by the Kruskal–Wallis test followed by the Mann–Whitney test or Dunn´s multiple comparison test. Differences were considered significant if p-values were below 0.05. Data analysis was performed using Prism 5.0 software (GraphPad Software, San Diego, CA, USA).