Animal models
Chronic graft-versus-host disease (cGVHD) model. A total of 5×107 CD8+T cells-depleted lymphocytes of Ddx58 CKO and Ddx58f/f female mice were injected into B2D6F1 female mice via tail veins. Urine was collected weekly and the serum samples were collected at the the end of the observation period. By the end of 12 weeks, mice were sacrificed for further experiments.
IMQ-induced lupus model. Epicutaneous Application of imiquimod leads to systemic autoimmunity is a recognized new lupus model59,60. To induce lupus-like disease, Ddx58 CKO and Ddx58f/f mice were treated topically with 5% IMQ cream (Sichuan Med-shine Pharmaceutical, H20030128). Mice were treated with IMQ cream applied to the ear skin three times a week for 8 weeks. Urine samples were collected weekly and serum samples fortnightly. By the end of 8 weeks, mice were sacrificed for further experiments.
The urine protein test kit for urine protein, and the serum anti-double-stranded DNA (dsDNA) IgG and antinuclear antibody (ANA) IgG levels were detected by ELISA kits (CUSABIO, China). Flow cytometry was used to analyze immune cells in the draining lymph nodes (dLNs) and spleens of the models. Analysis of C3 and IgG deposits in kidneys using multi-IHC stains.
EAE disease model. EAE was induced by complete Freund’s adjuvant (CFA)- MOG35-55 peptide immunization (Hooklabs) and scored daily. Briefly, 2×106 naive CD4+ T cells of Ddx58 KO and WT female mice were injected into Rag2-/- female mice via tail veins. Mice were then injected subcutaneously into the neck with 200µl containing 200µg MOG35-55 peptide (Hooklabs) emulsified in complete Freund’s adjuvant (Sigma-Aldrich). Mice were also injected intraperitoneally with 500ng of pertussis toxin (Listlabs) on days 0 and 2 after immunization. Mice were monitored daily for morbidity and scored according to the following scoring criteria: 0, no symptoms, active and mobile; 0.5, partial weakness of the tail; 1, completely paralyzed tails, less active; 2, hind limb weakness, hobbling gait; 2.5, partial paralysis of hind limbs and complete paralysis of a single hind limb; 3, complete hind limb paralysis; 3.5, complete paralysis of both hind limbs, partial paralysis of forelimbs; 4, completely paralyzed of all limbs, losing mobility; 5, moribund or death.
Cell isolation. Density gradient centrifugation (GE Healthcare) was used to isolate the peripheral blood mononuclear cells (PBMCs) from the peripheral blood of healthy controls and SLE patients. Then CD4+ T cells were isolated from the PBMCs by Miltenyi beads according to the manufacturer’s instructions (Miltenyi Biotec).
In vitro human T cell differentiation. Naive CD4+ T cells were purified from PBMCs using the human Naive CD4+ T Cell Isolation Kit (Miltenyi Biotec), and then cells were stimulated with plate-bound anti-CD3 (5µg/ml, Calbiochem, catalog 217570) and anti-CD28 (2µg/ml, Calbiochem, catalog 217669) under the Supplemental Table 3 polarizing conditions. We performed cell culture in 24-well plates with a total volume of 1 ml/well of culture medium with 1×106 naive CD4+ T cells. The medium was refreshed on day 3.
In vitro mouse T cell differentiation. Naive CD4+ T cells from mouse spleen were purified using mouse naive CD4+ T cell isolation kit II (Miltenyi Biotec), and the purity of the enriched subset was validated by flow cytometry and was generally higher than 95%. Purified naive CD4+ T cells were stimulated with plate-bound anti-CD3 (5µg/ml, eBioscience, catalog 16-0031-85) and anti-CD28 (2µg/ml, eBioscience, catalog 16-0281-85) for 3 days under different polarizing conditions (Supplemental Table 4). We performed cell culture in 24-well plates with a total volume of 1 ml/well of culture medium with 1×106 naive CD4+ T cells.
Transfection of siRNA, poly(I:C), and ERV3-16A3_I-int RNA. The Human T Cell Nucleofector Kit and Amaxa Nucleofector System (Lonza) for T cell transfection. Briefly, naive CD4+ T cells were induced differentiation into different T cells for 3 days. The cells were collected and resuspended in 100 µL transfection reagents, and 10µL siRNA (20µM), or 1µL Smart Silence (20µM), or 500ng poly(I:C), or 10µg ERV3-16A3_I-int RNA was added and transfected into the cells by electroporation using the nucleofector program V-024 in the Amaxa Nucleofector apparatus (Lonza). After being cultured under RPMI 1640 complete medium (Gibco) for 6 hours, the cells were transferred to fresh complete medium under different polarizing conditions for 48 to 72 hours and then harvested for subsequent experiments.
Flow cytometry. Briefly, T cells were incubated with fluorescein-labeled surface-labeled antibodies at 4°C for 30 minutes protected from light. For cytokines, cells were stimulated with Leukocyte Activation Cocktail, with BD GolgiPlug™ at 37°C and 5% CO2 for 6 hours. For intracellular staining, cells were fixed and permeabilized using the Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences) or transcription factor buffer set (BD Pharmingen), and then stained with fluorescent antibodies for 30 minutes at 4°C in the dark. Information on antibodies is shown in Supplemental Extended Table 1. The expression of cytokines, surface markers, and transcriptional factors was determined by flow cytometry using FACS Canto II (BD Biosciences) or Cytek® Northern Lights™-CLC (CYTEK Biosciences), and the data were analyzed by the Flowjo software.
Chromatin immunoprecipitation (ChIP) qPCR. Th1 cells transfected with the poly(I:C) were isolated. ChIP with anti-H3K18Ac, anti-H3K18Lac were used to detect IFNG and TBX21 enrichment, which was performed by a ChIP kit (Millipore). The detailed protocols were previously described61. Immunoprecipitated DNA and input DNA were assessed using real-time PCR. The resulting DNA fragments were purified and subjected to PCR with the use of primers encompassing the D-box region of the TBX21 and IFNG gene promoters. The primers used in the present study were shown in Supplemental Table 5.
CUT&Tag. CUT&Tag was performed according to the Hyperactive Universal CUT&Tag Assay Kit for Illumina(Vazyme, TD903) for Th1 cells. In brief, Th1 cells were collected and counted, of which the cell viability was > 85%. Cells were separated into 100,000 cell aliquots in each sample and incubated on ice for 10 min with 100ul of pre-cooled NE buffer for obtaining cell nuclei. Cells were centrifuged at 600×g for 5 min at room temperature and then resuspended in 100µl wash buffer in each sample. ConA Beads were activated in the binding buffer. Transfer 100µl of nuclei to an 8-strip tube containing 10µl activated ConA Beads, invert to mix and incubate at room temperature for 10 min. Beads were separated with a magnetic and supernatant was removed. 1µl of the primary antibody was diluted 1:50 in antibody buffer. The primary antibodies H3K18la (PTM-Bio, PTM1406RM) and H3K18ac (PTM-Bio, PTM-114RM) were used in this study. Cells were incubated overnight at 4°C. The primary antibody was replaced with the secondary antibody diluted to 1:100 in the dig-wash buffer. Samples were incubated for 45min at room temperature on the nutator. The secondary antibody was removed, and samples were washed 3 times in the dig-wash buffer. 2µl pA/G-Tnp was added in 98µl dig-300 buffer for per sample. Samples were incubated for 1h at room temperature on the nutator. Samples were washed 3 times with dig-300 buffer and then resuspended in 50 µl tagmentation buffer. Samples were incubated at 37°C for 1h. DNA was extracted with DNA Extract Beads. Fragmented DNA after purification was amplified by PCR. PCR conditions were set to 72°C for 3min, 95°C for 3min, 11 cycles of 98°C for 10sec, 60°C for 5sec, and 72°C for 1min. VAHTS DNA Clean Beads were used to purify the PCR product. Libraries were indexed using Nextera Indexes, and 150-bp paired-end sequencing was performed on Illumina Novaseq instruments.
RNA extraction and Quantitative reverse transcription PCR (RT-qPCR). Total RNA was extracted from T cells using TRIzol (Invitrogen). RNA quality control was conducted with a NanoDrop spectrophotometer and an Agilent 2100 Bioanalyzer (Thermo Fisher Scientific). 1µg of total RNA was reverse-transcribed using PrimeScript RT reagent Kits With gDNA Eraser (Takara). RT-qPCR was performed on a Fast Real-time PCR system (Roche) with iTaq Universal SYBR Green (BioRad). The relative expression levels of genes were calculated by the 2−ΔCt method, which normalized to the reference gene β-actin. The primers are listed in Supplemental Extended Table 1.
Western Blot. Total protein was isolated from T cells by IP lysis buffer (Beyotime) supplemented with protease inhibitors (Roche) and phosphatase inhibitor (Beyotime). The proteins were quantified by Pierce BCA Protein Assay Kit (Thermo). The primary antibodies and secondary antibodies were used in this study as Supplemental Extended Table 2. The quantification of proteins was normalized to GAPDH or β-actin by densitometry using ImageJ software.
Co-IP. Co-IP assays were performed with the Dynabeads Protein G (Life Technologies) for immunoprecipitation. First, we extracted protein from fresh cells using IP lysis buffer supplemented with protease and phosphatase inhibitors. Rabbit anti-RIG-I Ab (Abcam, ab180675) or Rabbit anti-LDHA Ab (Abcam, ab52488) was added to the lysates, forming a new antibodies-bait-target complex. Then, the antibodies-bait-protein complexes were eluted from the beads and dissociated by boiling in protein loading buffer. Finally, the presence of the target protein was evaluated by Western blot.
Bisulfite sequencing polymerase chain reaction (BSP). Genomic DNA was isolated from CD4+ T cells or Jurkat cells using the QIAamp DNA Mini Kit (Qiagen). DNA was bisulfifite converted using EZ DNA Methylation-Lightning™ Kit (Zymo Research). The bisulfite-treated DNA was amplified via nested PCR amplification reactions with specific primers, which were designed using the online MethPrimer software(http://www.urogene.org/methprimer/). The primer information used to amplify the target fragment are shown in Supplementary Table 6. For each PCR, 0.25mM dNTP mix (Promega), 0.2µM forward and reverse primers, 2.5 U of Taq DNA Polymerase (Promega), and 5×Green GoTaq® Reaction Buffer (Promega) were used in a 20µl total reaction volume. Here 100ng of bisulfite-treated DNA was used as the template of the first PCR, whereas 4µl of PCR1 product was used as the template for the second PCR. Thermal cycling conditions consisted of one cycle of 2 min at 96°C, followed by 40 cycles of 10s at 96°C, 30s at 55°C and 1 min at 72°C, and a final extension at 72°C for 10min. The PCR products were purified by gel extraction (Promega) from a 1.5% agarose gel and ligated into the pMD™18-T Vector (Takara). The ligation products were used to transform competent Escherichia coli cells (strain DH5a) using standard procedures, and blue/white screening was used to select ten independent clones from each specimen were sequenced (Sangon). The final sequence results were analyzed by online QUMA software (http://quma.cdb.riken.jp/).
DsRNA analysis by RNase digestion. According to previous research for dsRNA analysis by RNase A digestion27, 5µg total RNA of CD4+ T cells was dissolved in 32µL H2O and mixed well with 17.5µL NaCl (1 M stock). Then, 0.5µL RNase A (10mg/ml stock, Thermo Fisher Scientific) or H2O was mixed to a total volume of 50µL and incubated at room temperature for 10 min. 1mL TRIzol was added to the mixture. The levels of HERVs were detected by RT-qPCR with ACTB as an internal control. The ratios of (HERV/ACTB)RNaseA/(HERV/ACTB)H2O were calculated as enrichment folds.
DsRNA analysis by J2 pulldown. Purified total RNA from CD4+ T cells was used for the J2 pulldown assay. Purified total RNA from CD4+ T cells was used for the J2 pulldown assay. J2 antibody (Scicons) and mouse IgG control (Merck) (1µg per pulldown) were incubated with Protein G dynabeads (Merck), respectively, for 30min at room temperature. 30µg RNA was mixed with 500µl immunoprecipitation (IP) buffer. Then, the whole mixture was added with washed beads and rotated at 4°C for 2h. Afterward, the beads were incubated in 50µl Proteinase K digestion solution. 1 ml Trizol was directly added to the eluate for RNA purification and RT-qPCR analysis as described above.
RNA-FISH. We used the RNA-FISH to study the subcellular distribution of ERV3-16A3_I-int. Fluoresce-conjugated ERV3-16A3_I-int probes labelled with Cy3 and FISH kits were generated from RiboBio (China). Briefly, 4% paraformaldehyde (supplementing 5% TritonX-100) was used to fix CD4+ T cells of SLE and HCs (30 min). The fixed cells were incubated with ERV3-16A3_I-int probes in hybridization buffer at 37◦C overnight. Nuclei were stained with DAPI.
Immunofluorescent staining. CD4+ T cells were fixed with cold methanol at -20℃ for 15 min. Then the cells were incubated with 0.2% Triton (Sigma) at 4℃ for 5 min. The samples were incubated with primary antibody (Scicons) at 4℃ for overnight and then secondary antibody (Abcam) for 1h at room temperature. Mouse kidney were fixed in formalin and embedded in paraffin. Hematoxylin and eosin (H&E) stains and Periodic Acid-Schiff (PAS) stains were used to assess lymphocytic infiltration and glycogen deposition in the kidney. To assess the immune complex deposition in the kidney, we stained paraffin-embedded renal sections with rabbit anti-C3 antibody (Abcam) and HRP-conjugated anti-mouse IgG. The opal 7-Color Manual IHC Kit (Perkin Elmer) was used for fluorescence labeling. Information on antibodies was provided in Supplemental Extended Table 2.
RNA-Sequencing. RNA-seq dataset was taken from CD4+ T cells of 4 healthy female controls and 4 female SLE patients. The controls ranged in age from 22–51 years of age average age 31.6, and the patients ranged in age from 19–44 years old with an average age of 34.2 years old. The patients without drug therapy had a SLE disease activity index (SLEDAI) ranging from 7 to 23 with an average score of 14.8. RNA library preparation was performed as described in previous researches62. The HERVs were obtained from RepeatMasker for expression analyses by stringtie63,64. The differential analysis was performed using DESeq65.
Whole transcriptome sequencing. Whole transcription sequencing data included 5 healthy female controls and 5 female SLE patients. The controls ranged in age from 21–32 years of age average age 24.2, and the patients ranged in age from 15–32 years old with an average age of 22.2 years old. The patients without drug treatment had SLEDAI ranging from 6 to 13 with an average score of 9.4. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 150bp paired-end reads were generated. The raw data were processed through Fastp66. The data were then mapped to the human reference genome hg38 with Hisat2 v2.0.562. The differential analysis was performed using DESeq65.
Statistical Analysis. SPSS 22.0 was used for statistical analysis and calculation. Comparisons of the means between experimental variables were made via unpaired two-sided Student’s t-test for normally distributed variables or Mann-Whitney for non-normally distributed variables. P < 0.05 was regarded as significant.