Mice / cell lines
Animals were housed under specific pathogen-free conditions at the DKFZ, and the governmental committee for animal experimentation approved all animal experiments. In these experiments, we used wild type B/6 mice or Foxp3 GFP-DTR-CD90.1 or Foxp3GFP-
DTR-CD45.1 B/6 mice 171. Rbpjfloxed mice 175 were crossed to Foxp3YFPCre mice to generate
Treg-specific Rbpj knockout mice (Foxp3CreRbpjΔ/Δ). NICDLSL mice were crossed to
Foxp3YFPCre mice to generate Treg-specific NICD knock-in mice (Foxp3CreNICDLSL). NotcheGFP reporter mice were housed at the Weizmann Institute of Science and described in 176.
Isolation of Treg cells from various tissues
We isolated Treg cells from fat, skin, colon, lung, liver, and different lymph nodes as well as spleen and thymus. For lymph nodes and spleen, single-cell suspensions were established and red blood cells were lysed. Cells were stained with antibodies and purified using FACS. For thymus samples, cells were first depleted of double-positive and CD8-single positive thymocytes by staining with CD8 biotinylated mAb and anti- biotin beads followed by AutoMACS-based depletion. Gonadal fat tissue was mechanically dissected following a digestion with buffers containing collagenase II (1 mg/mL), BSA (20 mg/mL) and DNAse (20 µg / mL). Skin and colon tissue was digested using a buffer containing collagenase IV (4 mg/mL), FCS (2% vol/vol), and DNAse (10 µg / mL). Liver tissue cells were isolated with collagenase II (1 mg/mL), BSA (5 mg/mL), and DNAse (20 µg/mL) containing digestion buffer followed by a Ficoll/hypaque gradient centrifugation step. Lung tissue was digested in a buffer containing collagenase IV (2 mg/mL), BSA (0.5% w/vol), and DNAse (20 µg / mL). Digestions were performed at 37°C in a slow-shaking waterbath for 30 minutes to 45 minutes. Afterwards, cells were filtered and stained for FACS-based isolation of target cells.
Flow cytometry, FACS and cell counting (plus Annexin-V and Caspase-3)
For flow cytometric evaluation of surface proteins or cell isolation via FACS, surface staining with monoclonal antibodies was performed at 4°C for 20 minutes followed by
immediate analysis. For intracellular staining, cells were first surface-stained for 20 minutes at 4°C followed by fixation, permeabilization and intracellular staining with the Foxp3 Fix/perm buffer set (eBiosciences) according to manufacturer’s instructions. Annexin-V staining and active Caspase-3 staining was performed according to manufacturer’s instructions (Biolegend, Abcam). Flow cytometric analysis was performed using BD LSRII, LSR Fortessa, or BD Canto II flow cytometers with AccuCheck® counting beads (Life Technologies). Cell sorting was performed using BD FACS ARIA II or III cell sorting machines (BD Biosciences).
Isolation of RNA and reverse transcription followed by qPCR
Sorted cell populations were lysed and RNA was isolated using the RNeasy mini kit (Quiagen). cDNA synthesis was performed with SuperScript reverse transcriptase (Life Technologies) and oligo(dT) primers according to manufacturer’s instructions. qPCR analysis was performed using a ViiA7 instruments (Life Technologies) and either Power SYBR green master mix (Life Technologies) or Taqman master mix (Life Technologies). Gene expression values were normalized to housekeeping genes (Hprt or Gapdh). Both SYBR primer sequences and Taqman order numbers are listed in the appendix section.
Western Blot for Rbpj
We isolated 500,000 CD4posCD25posFoxp3YFP-pos Treg cells and 500,000 CD4posCD25negFoxp3YFPneg Tconv cells from Foxp3CreRbpjΔ/Δ and Foxp3Cre animals. Cells were lysed in RIPA buffer with protease inhibitors and supplemented with loading buffer for SDS-PAGE. 125,000 cells were separated on a gradient acrylamide gel (Biorad). Proteins were blotted onto a PVDF membrane and blocked with 5% milk- PBST for one hour at RT, followed by incubation with primary anti-Rbpj mAb (Cell signaling, clone D10A4) overnight at a 1:3000 dilution. After washing, the membrane was stained with an anti-rabbit HRP conjugated secondary mAb at 1:10000 for one hour at RT. Membrane was washed and specific binding was detected using a chromogenic substrate and a Western Blot detection unit.
In-vitro Treg suppression assay
First, we isolated CD90.1-congenically labeled CD4-positive T-responder cells and MHCII-positive antigen-presenting cells from spleen and liver of Foxp3GFP-DTR-CD90.1 animals. CD90.1pos T-responder cells were labeled with CFSE (final concentration 1 µM, Life Technologies) for 15 minutes at RT followed by stringent washings steps. We incubated 100,000 MHCII-positive APCs with 50,000 CFSE-labeled T-responder cells in a 96-well U-bottom plate. To stimulate T-responder cell division, we added 2µg/mL soluble CD3 mAb to each well for TCR crosslinking through APCs. Next, we isolated CD4posCD25posFoxp3YFPpos Treg cells and CD4posCD25negFoxp3YFPneg Tconv cells from sick Foxp3CreRbpjΔ/Δ and Foxp3Cre mice. Treg and Tconv cells were titrated and added
to each well for a 4:1, 2:1, 1:1, 1:2, 1:4, 1:8, 1:16 and 1:0 Treg or Tconv to T-responder cell ratio. Cells were incubated for 5 days at 37°C (144 hours) followed by re-staining for flow cytometric analysis and measurement on a flow cytometer. T-responder cells were identified by CD90.1 expression, whereas Foxp3 CreRbpjΔ/Δ and Foxp3Cre animals express CD90.2 and MHCIIpos APCs express neither. Cell division was identified by active CFSE dilution of CD90.1pos T-responder cells.
Epigenetic analysis of the TSDR
To analyze specific demethylation of Treg-specific demethylated region, we isolated Treg and Tconv cells from sick Foxp3 CreRbpjΔ/Δ and Foxp3Cre control mice via FACS. We purified genomic DNA with the DNA Blood and Tissue Kit (Quiagen) followed by sodium-bisulfite conversion (Epitect BS conversion kit) according to manufacturer’s protocol. Barcode-labeled amplicons from BS-DNA were generated with TSDR- specific BS primers (see appendix). Equimolar amounts of amplicons were combined and processed on a GS junior sequencer (Roche). Sequence reads were aligned to a BS- converted mouse genome and methylation levels were visualized in heat maps.
TCR sequencing
We isolated spleen as well as inguinal, axial, brachial, and cervical LN from sick Foxp3CreRbpjΔ/Δ and Foxp3Cre animals. We pre-purified Treg cells via CD25-positive
selection via magnetic bead enrichment (Miltenyi Biotec) and isolated CD4posCD25posFoxp3pos Treg cells from via FACS. Genomic DNA was isolated with
adjustment. 500 ng of gDNA were submitted for immunoSEQ® TCR-β Survey Sequencing (Adaptive Biotechnologies) followed by bioinformatic analysis.
Intracellular cytokine secretion assay
Whole spleen was isolated from sick Foxp3CreRbpjΔ/Δ and Foxp3Cre animals and red blood cells were lysed. Splenocytes were resuspended in fresh complete medium and treated with 1X PMA/Ionomycin stimulation cocktail plus transport inhibitors or just transport inhibitors (eBiosciences). Cells were incubated for eight hours at 37°C, followed by surface antibody staining for 20 minutes at 4°C. Cells were then fixed, permeabilized, and stained intracellularly with the Foxp3 Fix/perm buffer set (eBiosciences) and anti-cytokine antibodies. Cells were analyzed on a LSR II flow cytometer.
Ig subtype ELISA
Peripheral blood serum was isolated from sick Foxp3CreRbpjΔ/Δ and Foxp3Cre control animals via intracardial puncture. Blood was allowed to clot for 30 minutes at RT, followed by centrifugation (20,000xg for 15 minutes) and removal of blood serum. Blood serum was titrated for Ig subtypes with an ELISA against mouse IgG1, IgG2a, IgG2b, IgG3, IgE and IgM.
Autoantibody screening with RAG2 KO organ protein via Western Blot
We isolated organs of the gastro-intestinal tract (stomach, small intestine, large intestine), lymphatic system (lymph nodes), endocrine system (pancreas), brain, eye, spleen, salivary gland, liver, heart, lung, and testis from a RAG2 -/- animal. Protein was extracted using ProteoJet lysis buffer with protease inhibitors for 10 minutes at RT and orbital shaking (1000 rpm). Lysate was cleared for 15 minutes at 16,000xg and supernatant was measured for protein content with BCA (Pierce). 20 µg of protein were loaded onto a gradient acrylamide gel (Biorad) and separated using SDS-PAGE. Proteins were transferred onto a PVDF membrane and blocked with 5% Milk-PBS- Tween. Membranes were cut into strips and incubated individually with peripheral blood serum in 5% Milk overnight (1:500 serum concentration). Membrane strips were washed and incubated with an HRP-conjugated donkey-anti mouse IgG mAb (1:3000 in
5% milk-PBST) for one hour at RT. Membrane strips were washed, re-assembled and measured with a chromogenic detection kit and a Western Blot detection unit.
Gene expression microarray
We isolated 50,000 CD4posCD25posFoxp3YFPpos Treg and 50,000 CD4posCD25negFoxp3YFPneg Tconv cells from sick Foxp3CreRbpjΔ/Δ and Foxp3Cre animals using FACS ARIA II or III cell sorters. Furthermore, we isolated CD4posCD25posFoxp3-
NICDYFPposFoxp3GFPpos Treg and 50,000 CD4posCD25posFoxp3-NICDYFPnegFoxp3GFPpos
Treg cells from Foxp3 CreNotchLSL mice using FACS ARIA II or III cell sorters. RNA was extracted using the RNEasy Plus Micro Kit (Qiagen) followed by amplification and hybridization of material to the MouseWG-6 v2.0 Expression BeadChip (Illumina) through the DKFZ Genomics and Proteomics Core Facility. Microarray data will be deposited at the Gene Expression Omnibus (GEO).
Chromatin-IP with Rbpj
Chromatin-IP experiments were performed with an anti-Rbpj monoclonal antibody (Cell Signaling, clone D10A4, concentration 25 µg / mL) at a 1:50 dilution with the Magnify ChIP system (Lie Technologies). We used three to four biological replicates of CD4posCD25posFoxp3YFPpos Treg cells from Foxp3Cre animals, CD4posCD25posFoxp3YFPpos Treg cells from Foxp3CreRbpjΔ/Δ animals, and CD4posCD25negFoxp3YFPneg Tconv cells from Foxp3 Cre animals. Chromatin IP was performed according to manufacturers recommendations. Bead-bound DNA was eluted in 100µL elution buffer and used for Real-time PCR based measurements with Sybr primers specific for Hes1, Dtx1, and IL7R putative RBPJ binding sites (see appendix). Real-time PCR measurements were performed using the Sybr Master Mix and a Viia 7 RT-PCR system (Life Technologies). Results were normalized to input controls, and Rbpj-specific binding at different loci was identified by calculation of binding in Rbpj- proficient Treg cells vs. binding in Rpbj-deficient Treg cells.