Media and reagents
RPMI-1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 ng/ml streptomycin, and heat-inactivated 10% FBS (Biosource International) was used for cell culturing throughout the experiments. For human cell stimulation, we used 5 mM CpG-ODN 2216 (Invivogen), 1 mg/ml R848 (InvivoGen), and different doses of MPA (Sigma) dissolved in methanol; as a vehicle control, methanol was diluted in parallel.
Cell isolation and culture
Human mDCs and pDCs were isolated from peripheral blood mononuclear cells (PBMCs) from healthy adult donors, as described previously [3,27]. CD11c+/BDCA4-/lineage-/CD4+ cells (defined as mDCs) and CD11c-/BDCA4+/lineage-/CD4+ cells (defined as pDCs) were sorted by FACS Aria® (BD Biosciences) (Suppl. Fig. 1) to reach greater than 99% purity according to re-staining with anti-BDCA1 and anti-BDCA2 antibodies. The purified mDCs and pDCs were incubated for 24 h with R848 and CpG-ODN, respectively, in flat-bottomed 96-well plates at a concentration of 2×104 cells in a final volume of 200 ml of medium per well, in the presence of MPA or the vehicle control.
Stimulation of lupus serum
Experiments using lupus serum to stimulate pDCs were conducted as described previously (21). Lupus sera were obtained from five active SLE patients with low complement levels prior to steroid therapy who satisfied 2019 European League Against Rheumatism (EULAR)/American College of Rheumatology (ACR) classification criteria for SLE. All patients had anti-double-stranded DNA antibody. Written informed consent was obtained from all SLE patients. In these experiments, pDCs were stimulated with 20% lupus serum in flat-bottomed 96-well plates at a concentration of 2´104 cells in 200 ml of medium per well.
Analyses of cells and supernatants
Human DC subsets were stained with PE-labeled CD86 (2331) and FITC-labeled CD80 (L307.4) monoclonal antibodies (all from BD Biosciences) and then analyzed by FACScalibur® (BD Biosciences). The production of cytokines in the culture supernatants after 24 h was determined by an enzyme-linked immunosorbent assay (ELISA). The ELISA kits for detecting human IL-12p40 and TNF were purchased from R&D Systems. The ELISA kit for human IFN-a was purchased from PBL Biomedical Laboratories.
For the viability assay, cells were washed with PBS containing 2 mM EDTA, and viable cells were counted in triplicate, using trypan-blue exclusion to identify the dead cells. Viable cells were also evaluated as the annexin V-negative fraction using an Annexin V–FITC Kit (Sigma).
For intracellular staining, pDCs were stimulated with CpG-ODN 2216 for 90 min or 3 h, and the cells were immediately fixed and stained with Alexa Fluor-647 anti-pAKT (pS473; BD Biosciences) (after 90 min-culture) and Alexa Fluor-anti-human IRF-7 (G-8; sc-74472 AF488, Santa Cruz Biotechnology) (after 3 h-culture) using a BD Cytofix/Cytoperm™ Fixation and Permeabilization Solution (BD Biosciences) according to the manufacturer’s instructions. Then the cells were analyzed by flow cytometry.
Purified DC subsets were stimulated with R848 or CpG-ODN in the presence of 10 mM MPA for 4 or 8 h. The cells were then collected for cDNA synthesis using the QuantiTect. Reverse Transcription (RT) kit (QIAGEN). The DNA resulting from each RT reaction was subjected to real-time PCR using the SYBR® Green Master Mix (QIAGEN) for the detection of CD80, CD86, IFNA1, IL12B, TNF, IRF7, and STAT4. Primers for CD80 (QT00000497), CD86 (QT00033915), IL12B (QT00000364), IRF7 (QT00210595), and STAT4 (QT00014133) were purchased from QIAGEN. Primers for IFNA1 and TNF were purchased from Bio-rad and Sino biological, respectively. We selected glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the reference gene. Quantification of the mRNA transcription level was determined using the comparative cycle threshold (Ct) method. All samples were examined in duplicate, and the mean Ct value was calculated by Rotor Gene Q software version 2.2.3. The fold change in gene transcription was calculated as the ratio of D/DCt in the treated samples to untreated samples.
Purified pDCs were stimulated with CpG-ODN in the presence or absence of 10 mM MPA for 3 h. The cells were then seeded on glass slides by a cytospin, mounted, and fixed using a BD Transcription Factor Buffer set (Cat. No. 562725).
Samples were labeled with Alexa Fluor-anti-human IRF-7 and 4',6'-diamidino-2- phenylindole (DAPI). Images were acquired using a fluorescence microscope (BZ-9000, Keyence). Cells were regarded as negative when the expression level of IRF7 in the cytoplasm was higher and distinguishable from that in the nucleus, as shown by DAPI staining. The ratio of nuclear IRF7-negative cells was calculated based on 50 cells from each donor.
Data were analyzed using paired t-tests. A value of p < 0.05 was considered statistically significant. Data analysis was carried out using Prism (GraphPad Software).