C57BL/6 wild type mice bred and maintained at ILS animal facility. OT-II and OT-I transgenic mice (gifted by Prof. Hans Acha-orbea, University of Lausanne) and C57BL6
Flt3 transgenic mice (gifted by Ton Rolink) were transported from SWISS. All the animal experiments were performed after getting due approval from the institutional animal ethics committee (ILS/IAEC-164-AH/AUG-19) and (ILS/IAEC-123-AH/AUG-18).
The CD8α+ mutuDC cell line used in this study has been gifted by Prof. Hans Acha-Orbea's group . The cell lines were maintained in culture at 37°C in a humidified incubator with 5% CO2. Cells were cultured in complete IMDM-glutamax medium with all buffered conditions as reported previously. These cells show resemblance in expression of surface markers and mimic splenic ex-vivo immature CD8α+ DCs as shown by extensive characterization done by Prof. Hans Acha-Orbea’s group.
B16F10 cell line obtained from Dr. Shantibhushan Senapati were maintained in DMEM media and were cultured and maintained at 37°C in a humidified incubator with 5% CO2.
For in vitro experiments, the DCs were plated in 12- or 6-well plates at a density of 5×105 or 1×106 cells/ml overnight. The cells were then challenged with different activation media containing TLR9 agonist CpG-B at a concentration of 1ug/ml, TLR3 agonist pIC at 2ug/ml for 2, 6 or 12 h. For performing RT-qPCR analysis the cells were washed in the plate once with PBS followed by addition of RNA-later (LBP) lysis buffer for lysis of cells. The plates were then stored at −80°C until further RNA isolation and processing of samples.
Generation of Stable SMRT KD CD8α+ MutuDCs
For generating stable SMRT knockdown and their comparative control DC, lentiviral vector pLKO.1 (Sigma) containing three different sigma mission shRNA for Ncor2 were picked targeting chromosome 5 on mouse genome against exons 48, 19, and 14 respectively (Key Resources Table). Viral particles packaged with shRNA expressing transfer plasmids were produced in 293T cells using Cal-Phos (CaPO4) mammalian transfection kit according to an optimized protocol. We used a 2nd generation lentiviral system which included PCMVR and PMD2G as packaging and envelope plasmids respectively. Human embryonic kidney (HEK) 293T cells were transfected with transfer plasmids containing three different Ncor2 shRNAs or control shRNAs along with pCMVR8.74 and pMD2G. After 12–14 h the culture medium was replenished and supernatant containing viral particles were collected after 24 h in 50 ml conical tubes. Viral particle-containing culture supernatant was concentrated using ultracentrifugation at 50,000g at 16°C for 2h and preserved at −80°C in small aliquots. For transduction of shRNA containing viruses in CD8α+ cDC1 MutuDC lines, the cells were plated at a density of 1.5 × 105 cells/well of 12 well plate followed by transduction with virus particles containing supernatant. The media was replaced with fresh media after 12h of virus incubation with DCs followed by addition of 1 μg/ml puromycin selection medium after 72 h of media replacement for stable KD cells.
RNA Isolation and RT-qPCR
The extraction of RNA was done using NucleoSpin RNA Plus miniprep kit (Machery Nagel). Briefly, cells were preserved in LBP lysis buffer in −80°C and thawed by placing the plates/tubes on ice. Total RNA was isolated according to the manufacturer's protocol. RNA concentration was estimated by nanodrop (Thermo) and then 1-2 μg of total RNA was used to prepare cDNA using high-capacity cDNA Reverse Transcriptase kit (Applied Biosystems). Quantitative PCR was performed using SYBR Green master (Roche) and PCR amplification was monitored in real-time using LightCycler-480 Instrument. Primer oligonucleotides for qPCR were designed using the universal probe library assay design system and the primer pairs used are listed in Key Resources Table. Primers were optimized for linear and single product amplification by performing standard curve assays.
Flow Cytometry (FACS)
We performed flow cytometry analysis using the well-established surface and intracellular (IC) staining protocols . 5 x 105 and 1.5 x 106 cells were seeded for surface and IC staining respectively. Cells were either left unstimulated or stimulated with CpG or pIC for 6h. For staining the cells were dissociated and washed with FACS buffer (3% FCS in 1X PBS, 5 mM EDTA). After washing, fluorochrome conjugated antibodies for proteins of interest were added to the cells as a cocktail in the staining buffer. For surface staining cells were stained in FACS buffer for 30 min in dark at 4°C. For IC staining of cytokines the cells were first fixed with 2% paraformaldehyde for 20 min followed by permeabilization using 1x permeabilization buffer (eBiosciences). The fixed and permeabilized cells were then resuspended in IC staining buffer and stained with fluorochrome tagged antibodies for selected cytokines. For optimal staining the cells were incubated with antibodies for 30 min in dark. After incubation the cells were washed twice with FACS wash buffer and then acquired for differential expression analysis using LSRII fortessa flow cytometer (BD Biosciences). The acquired data was analyzed using FlowJo-X software (Treestar). Antibodies used for flow cytometry experiments are listed in the Key Resources Table.
Bio-Plex Assay for Cytokine Quantitation from Cell Culture Supernatants
Bio-Plex assay (multiplex ELISA) was used to estimate the cytokine levels secreted in the cell culture supernatants of SMRT KD and control DC and BMcDC1 after 6 h of CpG stimulation according to previous reports . After culture, the supernatants were stored at −80°C in small aliquots until analysis. Cytokine levels were estimated using 23-plex-mouse cytokine assay kit following the vendor recommended protocol (Biorad).
Generation of Bone Marrow Derived DCs (BMDCs) for ex vivo Studies
Six to eight-week-old female C57BL/6 mice were killed by cervical dislocation and disinfected using 75% ethanol . In short the tibias and femurs were removed under sterile conditions, then soaked in RPMI-1640 medium supplemented with 10% FBS. Cells from both ends of the bone were flushed out with a needle of 1-mL syringe from the bone cavity into a sterile culture dish with RPMI-1640 medium. The cell suspension in the dish was collected and centrifuged at 350g for 5 min, and the supernatant was discarded. The cell pellet was suspended with a 1x RBC lysis buffer (Tonbo) for 5-10 min on ice. Cell clumps were then passed through a 70μm strainer to obtain single cell suspensions. The lysed cells were washed once with RPMI-1640, counted and used for differentiation into DCs.
We followed a well-established protocol for differentiation of BMDCs with slight modifications. The cells, suspended in RPMI-1640 medium supplemented with 10% FBS, were distributed into 6-well plates at a density of 1 × 106 cell/ml/well. Subsequently, 1μl/ml of FLT3L containing sera was added into the medium. The cells were cultured at 37°C in an incubator containing 5% CO2 and left untouched for 5 days. On day 5, the suspended and loosely attached cells were collected.
The cells were plated into 96-well plate for lentiviral transduction using concentrated viruses at a density of 0.4 x 106 cells/well for each Ncor2 shRNA and control shRNA. After 72h the cells were stimulated with CpG for 6h and then immune-profiling was performed using flow cytometry.
Co-culture of DCs with CD4+ T-Cells and CD8+ T-cells for Assessing T-Cell Proliferation and Differentiation
DC-T-cell co-culture experiments were performed according to well established protocol [56, 57]. Naïve CD4+ or CD8+ T-cells were purified from spleen of TCR-transgenic OT-II or OT-I mice using CD4+ or CD8+ T-cell isolation kit. SMRT KD and control CD8α+ cDC1 DCs were seeded at a density of 10,000 cells/well in round bottom 96 well plates followed by pulsing with OVA peptide (323-339) /OT-II at 200nM concentration or OVA peptide (257–264) /OT-I was used at 5nM overnight. Further DCs were stimulated with CpG or pIC for 2h. After 2h, purified OT-II or OT-I T-cells were added at the density of 100,000 cells/well (1:10 ratio). Then T-cell proliferation and differentiation into distinct Th subtypes Th1, Th2, Th17 and Tregs, in case of OT-II, and cytotoxic T-cells, in case of OT-I, were analyzed by FACS. Proliferation was measured using an amine based dye (eFluor 670). The rate of T-cell proliferation was inversely proportional to the median fluorescence intensity (MFI) measured in FACS after 72h of co-culture. For Th and cytotoxic T-cell differentiation profiling after 96h, the co-cultured T-cells were re-stimulated with PMA (10 ng/mL) and ionomycin (500 ng/mL) and followed by Brefeldin-A (10μg/mL) treatment for 5h to block the IC cytokines from being secreted. After 5h, fluorochrome conjugated antibodies specific to different T-cell subtypes were used to profile T-cells into Th1 (T-bet and IFN-γ), Th2 (GATA3, IL-13), Tregs (CD25, FoxP3, IL-10) and Th17 (RORγT, IL-17) or cytotoxic T-cells (perforin, IFN-γ, Granzyme-B). For gating effector T-cells we used CD44 as a marker.
Chromatin Immuno-Precipitation (ChIP) for p-STAT3
The ChIP for p-STAT3 was performed according to the methods optimized previously by Raghav and Meyer's lab. For ChIP assays, 40 x 106 CD8a+ cDC1 MutuDCs were seeded in 15 cm2 plates and prepared for four ChIP assays by 10 min cross-linking with 1% formaldehyde (sigma) at room temperature followed by quenching using 2.5 M glycine (sigma) for 10 min. The plates were placed on ice and the cells were scraped and collected in 50 ml conical tubes. The cells were then washed three times using cold 1x PBS at 2,000 rpm for 10 min at 4°C and the cell pellets were stored at −80°C. At the day of the ChIP experiment, the cells were thawed on ice followed by lysis using Farham lysis buffer (5 mM PIPES pH 8.0, 85mM KCl, 0.5% NP-40 supplemented with protease and phosphatase inhibitors (Roche)) made in miliQ. The supernatant was aspirated and the pellet was resuspended in RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS supplemented with Roche protease and phosphatase inhibitor tablet just before use). The chromatin was fragmented using a Bioruptor (Diagenode) sonicator for 30 min using high amplitude and 30s ON & 30s OFF cycles to obtain 200-500 bp size fragments. A cooling unit was used to circulate the cold water during sonication to avoid de-crosslinking because of overheating. After sonication, chromatin length was checked in agarose gel. The fragmented chromatin was centrifuged at 10,000 rpm for 5 min and then clear supernatant was collected in 15 ml conical tubes. The DNA concentration of the chromatin was estimated using a Nano-Drop (Thermo) and the chromatin was diluted with a RIPA buffer to use 150 μg/ml of chromatin for each IP. M-280 sheep anti-rabbit IgG dynabeads 40ul/IP was taken in a 1.5ml MCT tube. 1ml RIPA buffer was added to the beads and placed on a magnetic stand. The MCTs were inverted 5 times and allowed to stand for 3 min. The beads were washed in the same way 3 times. After the 3rd wash the beads were centrifuged shortly and the remaining RIPA buffer was aspirated. To the beads, 5µl of mouse monoclonal anti-p-STAT3 (CST) was added to immunoprecipitated the chromatin complex at 4°C for 8h on rocker shaker. After 8h incubation, the beads were again placed on a magnetic stand and washed with RIPA to get rid of the unbound antibody. Chromatin was added to the beads and placed on a rotating rocker at 4°C overnight. Next day the tubes containing chromatin, antibody, and beads were taken out, placed on a magnetic stand and supernatant was aspirated. The beads were washed 5 times with LiCL IP wash buffer (100mM Tris pH7.5, 500mM LiCl, 1% NP-40, 1% sodium deoxycholate in miliQ) and 2 times with TE buffer (10mM Tris pH7.5, 0.1mM EDTA pH8 in miliQ). After removing the wash buffer completely, protein-bound chromatin complexes were eluted from beads using an elution buffer (1% SDS, 0.1M NaHCO3 in milli-Q water). The chromatin was incubated at room temperature for 30 min in an elution buffer. A short spin was given and the MCT was again placed on a magnetic stand to collect the eluted chromatin. The eluted chromatin was then reverse crosslinked by incubating the eluted supernatant at 65°C overnight on a heat block after adding 8 μl of 5 M NaCl. Next day DNA was purified from the reverse cross-linked chromatin by proteinase-K and RNase digestion followed by purification using PCR purification kit (Qiagen). The purified DNA was eluted in 40μl of elution buffer.
Chromatin Immuno-Precipitation (ChIP) for SMRT
The ChIP for SMRT was performed according to the methods optimized previously by Raghav and Deplancke’s lab [25, 58]. In short the cells were lysed in a nuclei extraction buffer for 10 min at 4°C while shaking to isolate the nuclei. The isolated nuclei were then washed using a protein extraction buffer at room temperature for 10 min. Washed nuclei were resuspended in chromatin extraction and incubated for 20 min on ice. The chromatin was fragmented using a Bioruptor (Diagenode) sonicator to obtain 200-500 bp-sized fragments. The fragmented chromatin was centrifuged at 17,000g for 10 min and then clear supernatant was collected in chilled 15ml falcon tubes. The DNA concentration of the chromatin was estimated using a NanoDrop and the sonicated chromatin was diluted with ChIP dilution buffer to get 100 µg/ml of chromatin for each IP. BSA and ssDNA (Salmon Sperm DNA) -preblocked protein-A sepharose (80 µl/IP) beads were added to the samples and incubated for 2h to remove non-specific- binding chromatin. To the supernatant, 5 µl/IP rabbit polyclonal anti-SMRT antibody (Abcam) was added to immuno-precipitate the chromatin complex at 4°C overnight. After the overnight incubation, 50µl blocked beads were added to each sample and incubated for 90 min at 4°C to pull down the respective antibody-chromatin complexes. The beads were then washed four times with a low salt wash buffer followed by two washes with high salt wash buffer, lithium chloride wash buffer and tris-EDTA (TE) buffer. After removing the wash buffer completely, protein-bound chromatin complexes were eluted from beads for 30 min using an elution buffer. The eluted chromatin was then reverse-crosslinked by incubating the eluted supernatant at 65°C overnight on a heat block after adding 8µl of 5M NaCl. The next day, DNA was purified from the reverse crosslinked chromatin by proteinase and RNase digestion followed by purification using Qiagen DNA purification columns. The purified DNA was eluted in 50µl of Qiagen elution buffer.
ChIP/RNA-seq library preparation for sequencing
For RNA-seq library preparation 2ug of total RNA was used to isolate mRNA using magnetic beads with mRNA isolation kit (PolyA mRNA isolation module, NEB). Later mRNA library preparation kit, NEB, was used for RNA-seq library preparation according to manufacturer’s protocol. Concentration of the libraries were estimated by Qubit 2.0 (Invitrogen) and fragment sizes were analysed in Bio-analyzer (Agilent). The libraries were then sequenced on Illumina NextSeq 550 platform.
Similarly for ChIP seq 30 µl ChIP-DNA was processed for library preparation according to ChIP-seq library preparation protocol (NEB) . After library preparation and quality check, the libraries were sent to NGS service provider (Sci Genome, Bangalore, India) for Illumina sequencing using NextSeq-550 instrument.
Cells were collected in RIPA buffer (0.5 M EDTA, 1 M Tris-Cl pH7.5, 1 M NaCl, 200 mM, Roche protease inhibitor) at 0h, 2h and 6h CpG stimulation. Cells were lysed completely by sonication of the samples in Bioruptor (Diagenode) for 10 min using high amplitude and 30s ON & 30s OFF cycles. Protein concentrations were measured in 96 well plates using BCA protein assay kit (BioRad) at 562nM. For western blot of phospho and its respective total protein molecule we first probed the membrane with phospho-antibodies, stripped and re-probed the same membrane with respective total antibodies. For densitometric analysis we first normalized phosphorylated form of STAT3 /mTOR with their respective loading controls. The similar approach was followed for its corresponding total protein. Finally the ratio of normalized values were plotted as relative intensity..
Delayed Type Hypersensitivity (DTH) Assay
DTH was performed using culture grade ovalbumin (OVA) from chicken egg (Sigma) dissolved in 1x PBS at a concentration of 1mg/ml and filtered through 0.2-micron PES syringe filter. 1.5ml alum and 1.5ml OVA was added in a glass beaker and passed through a glass syringe multiple times to make an emulsion. 300µl per mice was injected subcutaneously in the back behind ears in each mice for OVA immunisation. After 14 days control and SMRT KD DCs were pulsed with OVA (100ug/ml) for 4 h. Cells were then stimulated with CpG. After 2h of stimulation the cells were dissociated and injected at 10 x 106 cells/mice. Further after 7 days OVA (20mg/ml) was heated at 80°C for 2h, cooled, and injected in foot pad of mice (25µl/mice). 1x PBS was injected in the alternative footpad. Paw thickness was measured till 72h using Vernier caliper. After 72h the popliteal and inguinal lymph nodes were isolated and checked for T-bet IFN-γ as well as RORγt IL-17.
OVA specific ELISA
To examine OVA specific immune response we performed experiments as described [25, 59]. In brief, we collected sera at day 20 and day 23 after OVA immunization to perform ELISA for OVA specific IgG titer. Elisa plates were coated with 100ug/ml of OVA (Sigma) prepared in a coating buffer (Na2CO3, NaHCO3, Sodium Azide) overnight at 4°C following five washes with washing buffer (PBS with 0.05% tween -20). Blocking was done with PBST containing 0.5% gelatin for 1h at 37°C. After five times washing, 50µl diluted sera were added from mice and kept for 1.5 h at 37°C. IgG1 (dilution 1:10,000) and IgG2a (dilution 1:100) was detected using biotin labelled anti-mouse IgG1 and IgG2a while total IgG (dilution 1:1000) was detected using anti-mouse HRP conjugated IgG followed by anti-mouse streptavidin-HRP (Biolegend). The plates were further incubated at 37°C for 1h and washed 7 times with a washing buffer. Color was developed by TMB (50µl/well) and incubated in dark for 10 min. 2N H2SO4 (50µl/well) was used to stop the reaction. The plates were read using ELISA reader for IgG estimation at 450nm.
Tumor cell lysate preparation
Tumor lysate was prepared from previous reports with some modifications . B16/F10 tumor cells were adjusted to 3 × 106 cells/ml in DMEM medium. Cells were subjected to 3 freeze (− 80°C) / thaw (40°C) cycles of minimum 20 min each. The lysed cells were checked under trypan blue staining and centrifuged at 12,000rpm for 15 min. The supernatant was passed through a 40µm cell strainer before adding to cDC1s seeded at a density of 3 x 106 (DC:tumor cell ratio of 1:1).
B16F10 Tumor Model
Mice were injected subcutaneously (s.c.) with 0.5 x 106 tumor cells into the left flank, and a booster dose was re-injected 3 days later. 7 days after booster dose, 0.1 x 106 cells were injected subcutaneously in mice in the right flank. Tumor growth was measured every day using a vernier caliper till 16 days. Tumors were removed, weighed, and dissociated to make single cell suspensions.
Isolation of tumor and tumor re-stimulation
For IC perforin, granzyme-B, and IFN-γ staining, single cell suspensions were ex vivo re-stimulated with 10ng/ml PMA, 500ng/ml ionomycin, and 5 μg/mL Brefeldin A for 5h. Cells were labelled with indicated surface-staining antibodies, fixed with 2% PFA, permeabilized with permeabilization buffer and stained with IC antibodies.
RNA-seq data processing and analysis
Raw reads of SMRT KD RNA-seq samples and its matched control in unstimulated and 6h CpG stimulation were checked for quality using FASTQC , and aligned to mouse genome (UCSC mm10 ) using hisat2  (with default parameter). Similarly, raw reads of NCoR1 KD and its matched control RNA-seq were processed for quality control and alignment. Raw counts of genes were extracted using featureCount (featureCounts -p -B) . Principal component analysis was performed on variance stabilized transformed (vst) values from DESeq2  using the plotPCA function and plotted using ggplot2 . Further, differential gene expression analysis was performed between NCoR1/SMRT KD compared to its matched control in unstimulated and 6h CpG stimulation condition. Genes were filtered based on log2foldchange (upregulated >= 1 and downregulated <= -1) and adjusted P-value (< 0.05). Total differentially expressed genes were combined from all the comparisons and unsupervised k-means clustering were performed based on log2foldchange values and divided into six clusters. Pathway enrichment analysis for each cluster was performed using Ingenuity pathway analysis.
ChIP-seq data processing and analysis
SMRT ChIP-seq raw reads in unstimulated and 6h CpG stimulation were checked for quality using FASTQC and aligned to mouse genome (RefSeq mm10) using bowtie2 . Reads were filtered using MarkDuplicates function of Picard and mapping quality >=10 using SAMtools [67, 68]. Peak calling was performed using findPeaks (Homer) and factor as style. Peak calling for NCoR1 ChIP-seq data from our previous study were performed with reads down sampled to the level of SMRT i.e. 9M. Distribution analysis of peaks based on distance relative to TSS were performed using ChIPSeeker . Peaks from both NCoR1 and SMRT were merged using bedops merge command and consensus peaks were generated for further downstream analysis . GeneOverlap R package were used to identify differentially expressed genes that are the direct target of SMRT .
ChIP-seq Peak analysis
Differential binding analysis for NCoR1 and SMRT were carried out on the merged peak using the getDifferentialPeaks program of Homer with cut-off of 2-fold enrichment over background . Peaks were filtered out that didn’t show any differential binding in any of the comparisons. Differential peaks were then categorized based on fold change. Peaks from different categories were annotated to nearest genes using the ChIPseeker R package . De novo motif enrichment analysis was performed using findMotifs.pl (size -50, 50 -len 8, 10, 12) using background generated from provided input genomic regions. P-value <=1e-10 were used to filter significantly enriched TF motifs. KEGG pathway enrichment analysis of differentially expressed genes associated with each binding category were performed using clusterProfiler R package .