Subjects
We recruited 25 SLE patients (22 females and 3 males) from outpatient clinics at the Second Xiangya Hospital of Central South University, fulfilling at least four of the SLE classification criteria of the American College of Rheumatology [46]. Lupus disease activity was assessed using the SLE Disease Activity Index (SLEDAI) [47]. Patient demographic data are shown in Additional file 2: Table S1. 25 Healthy controls (23 females, 2 males) were recruited from the medical staff at the Second Xiangya Hospital, who had no history of cancer, cardiovascular diseases, autoimmune diseases or known infectious diseases. Patients and controls were matched for age and sex. The human sample study followed a protocol approved by the Ethics Committee of Second Xiangya Hospital of Central South University, with written informed consent obtained from the participants.
Cell isolation and transfection
Venous peripheral blood (100 ml) was drawn from patients and control subjects and preserved with heparin. PBMCs were separated by density gradient centrifugation (GE Healthcare, Switzerland). Total CD4+T cells with positive selection and naïve CD4+T cell with negative selection were isolated using Miltenyi beads according to the manufacturer’s instructions (Miltenyi, Germany). A density of 1 × 106 PBMCs/mL cultured in RPMI 1640 culture medium (Gibco, CA, USA) supplemented with 10% foetal bovine serum (FBS) in 24-well plates with 2 μl Leukocyte Activation Cocktail (BD Pharmingen™, USA). Total CD4+ T cells at a density of 5 × 105 cells/mL were cultured in RPMI 1640 culture medium (Gibco, CA, USA) supplemented with 10% FBS and 2 μg/ml of an anti-CD3 antibody (PeproTech, USA) in 24-well plates precoated and 1 μg/ml of an anti-CD28 antibody (PeproTech, USA). Naïve CD4+T cells were transfected with UHRF1-siRNA or Cntl-siRNA (RiboBio Guangzhou, China) using the Human T Nucleofector and Amaxa Nucleofector (Lonza, Switzerland). Then, the cells were seeded and incubated in RPMI 1640 culture medium (Gibco, California, USA) supplemented with 10% FBS and an anti-CD3 antibody (2 μg/ml), anti-CD28 antibody (1 μg/ml) in 24-well plates, recombinant protein IL-6 (20 ng/ml), IL-12 (10 ng/ml), IL-21 (20 ng/ml) and TGF-β (5 ng/ml) for 48 h. The UHRF1 siRNA used in the present study was purchased from RiboBio (China) and had the following sequence: 5′-GGACGAAGTCTTCAAGATT-3’.
Flow cytometry
Cytokines, transcription factors, and surface markers were evaluated using a FACS Canto II instrument (BD Biosciences, USA). Briefly, for cytokine detection, cells were stimulated for 6 h with Leukocyte Activation Cocktail (BD Pharmingen™, USA). Subsequently, the cells were incubated with FcR blocking reagent (Miltenyi, Germany) for 10 min and then incubated with antibodies against surface markers on ice for 30 min in the dark. For intracellular staining, the cells were fixed and permeabilized with a Transcription Factor Buffer Set (BD Pharmingen™, USA) for 40 min and then stained with fluorescent antibodies for an additional 60 min on ice in the dark. Events were collected and analysed with Tree Star FlowJo. The following antibodies were used in the present study: UHRF1 (Abcam, UK), goat anti-Rb IgG Alexa Fluor® 488 (Abcam, UK), PE-cy7 anti-human CD3 (BD Pharmingen™, USA), APC-cy7 anti-human CD4 (BD Pharmingen™, USA), APC anti-human bcl-6 (BD Pharmingen™, USA), FITC anti-human CD4 (BD Pharmingen™, USA), PE-cy7 anti-human PD1 (BD Pharmingen™, USA), PerCP-Cy5.5 anti-human CXCR5 (BD Pharmingen™, USA). Ms CD45R/B220 FITC RA3-6B2 (BD Pharmingen™, USA), Ms T/B-cell antigen Alexa 647 GL7 (BD Pharmingen™, USA), PE/Cy7 anti-human CD95 (Fas) (Biolegend, USA), Ms CXCR5 biotin 2G8 100 μg (BD Pharmingen™, USA), streptavidin-PE 500 μg (BD Pharmingen™, USA), and anti-mouse CD279 (PD-1) (J43) APC-EFLUOR (eBioscience, USA).
ELISA
NP-KLH-specific antibodies were measured with 10 μg/ml of NP-BSA (Bioresearch Technologies) as the coating reagent for ELISA. Diluted serum was incubated in the wells for 1 h at room temperature. Then, NP-KLH-specific antibodies (IgG, IgG1, IgG2a, IgG2b and IgM) were detected using goat polyclonal anti-IgG HRP (Southern Biotech), anti-IgG1 HRP (Southern Biotech), anti-IgG2a HRP (Southern Biotech), anti-IgG2bHRP (Southern Biotech), and anti-IgM HRP (Southern Biotech).
MeDIP-qPCR
UHRF1-siRNA or UHRF1 lentiviral expression vectors (UHRF1-lentivirus) were transfected into naïve CD4+T cells. Genomic DNA was extracted from cells using a Qiagen DNA Extraction kit following the manufacturer’s instructions (QIAGEN, Germany). MeDIP analysis was performed according to the manufacturer’s instructions provided in the MeDIP assay kit (Active Motif, CA, USA). Precipitated DNA was amplified by quantitative PCR using forward and reverse primers specific to the BCL6 promoter sequence (Additional file 3: Table S2).
Immunofluorescence
For tissue samples, mesenteric lymph nodes (mLNs) were fixed with formalin and embedded with paraffin. The following antibodies were used for immunofluorescence analysis: anti-peanut agglutinin (anti-PNA, 20 μg/ml, Vector Laboratories), an anti-goat HRP-linked antibody (Abcam, UK), and an anti-CD3 antibody (Abcam, UK). Images were obtained using a laser scanning microscope (Olympus, Japan).
RNA isolation and real-time quantitative polymerase chain reaction
Naïve CD4+T cells, transfected with UHRF1-siRNA or Cntl-siRNA for 48 h and UHRF1-lentivirus or Cntl-lentivirus, were lysed with TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed using a LightCycler 96 (Roche, Basel, Switzerland), and the mRNA or DNA levels were quantified using a SYBR Prime Script RT-qPCR kit (Takara, Dalian, China). β-Actin was amplified and used as a loading control. The relative mRNA or DNA levels were calculated using the 2 −ΔCt (ΔCt = Ct target gene—Ct β-actin) method. The sequences of the primers are shown in Additional file 3: Table S2.
Western blot analysis
Cells were lysed with whole cell lysis buffer and denatured at 100 °C for 5 min. Subsequently, the cellular proteins were separated by SDS polyacrylamide gel electrophoresis and transferred to PVDF membranes. The membranes were blocked in TBST buffer that contained 5% non-fat dry milk and then incubated overnight at 4 ℃ with a rabbit anti-UHRF1 Ab (1:1000, Abcam, UK) and a rabbit anti-β-actin Ab (1:1000, Abcam, UK).
Chromatin immunoprecipitation (ChIP)-qPCR
ChIP was performed according to the instructions provided with ChIP assay kit (Millipore, Billerica, MA, USA). In brief, Naïve CD4+ T cells transfected with UHRF1-siRNA or Cntl-siRNA, UHRF1-lentivirus or Cntl-lentivirus were harvested and fixed for 10 min at RT with 1% formaldehyde. Glycine was subsequently added to a final concentration of 0.125 M to quench the formaldehyde. Cells were pelleted, washed once with ice-cold PBS, and lysed with SDS buffer. Lysates were pelleted, resuspended, and sonicated to reduce DNA to fragments of 200 to 1000 base pairs. Chromatin was precipitated with protein A agarose beads for 1 h and then incubated with 5 μl anti-acetyl histone H3 (Active Motif), or 5 μl anti-H3K27me3 (Active Motif) or normal IgG (Millipore) overnight. The immunocomplexes were further precipitated with protein A agarose beads, washed, and eluted in 100 ml of TE with 0.5% SDS and 200 mg/ml proteinase K. Precipitated DNA was further purified with phenol/chloroform extraction and ethanol. The relative enrichment level was quantified using qPCR and calculated relative to the respective input DNA. The primers are shown in Additional file 3: Table S2.
Lentivirus transfection
UHRF1-lentivirus and Cntl-lentivirus were packaged and synthesized by Genechem Biotechnology (Genechem, Shanghai, China). We infected 0.5 × 106 naïve CD4+T cells at an appropriate multiplicity of infection (MOI = 30) for 12 h, after which the cells were seeded and incubated in RPMI 1640 culture medium (Gibco, CA, USA) supplemented with 10% FBS and anti-CD3 antibody (2 μg/ml) and anti-CD28 antibody (1 μg/ml) in 24-well plates and then treated with recombinant IL-6 (20 ng/ml), IL-12 (10 ng/ml), IL-21 (20 ng/ml) and TGF-β (5 ng/ml) for 72 h.
NP-KLH immunization
Age-matched mice were immunized with NP-KLH (Bioresearch Technologies, USA) emulsified in Imject™ Alum Adjuvant (Thermo Fisher, USA) that was administered to each mouse by subcutaneous injection. After immunization for one week, we injected the same amount of NP-LKH to boost mouse immunity for another week. Then, we collected the spleen, lymph nodes and serum for subsequent experiments.
Cell cycle analysis
Naïve CD4+ T cells transfected with UHRF1-siRNA or Cntl-siRNA for 96 h were harvested. Cells were fixed in 75% ethanol under −20 ℃ overnight and washed by PBS, then cells were added with 500 μl PI staining solution and incubated for 15 min in the dark at room temperature. All cells were evaluated using a FACS Canto II instrument (BD Biosciences, USA) and the dates were analyzed with FlowJo software (Tree Star FlowJo).
Apoptosis assays
Naïve CD4+T cells transfected with UHRF1-siRNA and Cntl-siRNA for 96 h were harvested. Apoptotic cell populations were detected by a FACS Canto II instrument (BD Biosciences, USA) using the Annexin V-FITC Apoptosis Kit (Roche, Basel, Switzerland) according to the manufacturer’s instruction.
Proliferation assay
Briefly, 1 × 10 4 UHRF1-siRNA or Cntl-siRNA naïve CD4+ T cells were transferred into a 96-well cell culture plate with 100 μl the RPMI 1640 medium and cultured for 96 h. Later on, 10 μl CCK-8 was added to each well, and then the plates were incubated for 2 h. Eventually, absorbance was measured at 450 nm with a microplate reader (BioRad Laboratories, CA, USA).
Statistical analysis
Data are presented as the means ± standard deviation (S.D.). We used an unpaired two-tailed t-test to compare the difference between two groups. All analyses were performed with SPSS 19.0 (SPSS, Inc., Chicago, IL). P < 0.05 was considered to indicate a significant difference.
