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| Status |
Public on Jan 01, 2024 |
| Title |
colonTreg_7-3_noGvHD |
| Sample type |
SRA |
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| Source name |
CD4+CD25+ regulatory T cells
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6 Foxp3GFP+
mouse (pool/culture no.): 4209
type: allo
model: BMT control
tissue: colon
treg donor: alloTreg_7
recipient: rec-7-3
pcr2 cy. no.: 12
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| Treatment protocol |
Single cell suspensions from BM (femora and tibiae of both hind legs) and spleen were prepared. T cells were depleted from BM (T cell depleted bone marrow; TCDBM) by using anti-CD90.2 MicroBeads with the MidiMACS® system (Miltenyi Biotec). CD4+CD25- conventional T cells (Tconv) were isolated from splenocytes by depletion of CD25+ cells using anti-CD25-PE (all antibodies used in this study are listed in Supplemental Table 2) and anti-PE UltraPure MicroBeads (Miltenyi Biotec), followed by enrichment of CD4+ cells from the CD25- fraction through labeling with anti-CD4 MicroBeads (Miltenyi Biotec) using the MidiMACS® system. For short-term (d7) FACS and RNA-seq experiments BALB/c recipients were lethally irradiated (8 Gy) on the day of BMT and transplanted i.v. with 2.5 x106 TCDBM cells and 1x106 Tconv from B6-CD45.1 mice with or without in vitro expanded allo or polyTreg from Foxp3gfp mice at a 1:1 ratio. Recipients of the ‘no GvHD’ group received TCDBM and Treg only. In long-term survival experiments lethally irradiated BALB/c recipients were transplanted with 2.5 x106 TCDBM on d0 and 0.25 x106 Tconv were administered on d2 for GvHD induction. Mice of the GvHD prophylaxis groups (allo or poly) received additional 0.25x106 in vitro expanded Treg from B6 WT mice on d0. Recipients were monitored daily, body weight and GvHD symptoms assessed twice weekly by non-blinded investigators applying a standardized scoring system (Cooke et al., 1996). Single cell suspensions from BM, spleen and mesenteric lymph nodes were prepared. Erythrocytes were lysed in splenic and BM cell suspensions. For leukocyte isolation from the liver, the organ was flushed with PBS through the portal vein and the gallbladder was removed before it was excised, transferred to a petri dish and dissected into small pieces using a scalpel. Fragments were suspended in RPMI (Lonza/Biozym)/5% FCS, strained and washed in RPMI w/o FCS before Percoll centrifugation (40%/80%; 800 x g/20 min/20°C, w/o break). Leukocytes were resuspended in RPMI/5% FCS, erythrocytes were lysed, cells were washed in RPMI/10% FCS and kept on ice until further use. For intestinal leukocyte isolation, large intestine was excised, cut into 0.5 cm pieces, incubated twice for 20 min at 37 °C in HBSS/5mM EDTA/1mM DTT (Sigma-Aldrich) followed by vigorous shaking to isolate intraepithelial leukocytes (IEL). For lamina propria leukocytes (LPL), the fragments were transferred to HBSS without phenol red containing calcium and magnesium with 5% FCS together with enzymes of the Lamina propria dissociation kit (Miltenyi Biotec) and incubated for 30 min at 37°C. The fragments were further mechanically dissociated using the GentleMACS system (protocol: m_intestine_01; Miltenyi Biotec), strained, washed and pooled together with IEL before Percoll (Sigma-Aldrich) centrifugation (30%; 512 xg/15 min/20°C). For baseline (no BMT) colon Treg sorts IEL were not included (no Tregs in untreated animals) and colonic LPL were further enriched using anti-CD45 MicroBeads (Miltenyi Biotec) and the AutoMACS® system (Miltenyi Biotec). Splenocytes were enriched for CD25+ cells by MACS® using anti-CD25-PE and anti-PE UltraPure MicroBeads before sorting. Cells were further stained for CD8, CD4 and CD62L (also CD45 for colon-derived cells) and Treg were sorted on a FACSAriaTM IIu or a FACSAriaTM Fusion (Becton Dickinson). From spleen and liver, CD62L+ and CD62L– subpopulations were isolated, from colon, only CD62L–- cells were obtained (sorting strategy is shown in detail in Supplemental Fig.2a). For re-isolation of donor Treg on day 7 after transplantation, single cell suspensions from spleen, liver and colon were prepared as described, stained for H-2Kb, CD45.1, CD45.2, TCRβ and CD4 and sorted on a FACSAria IIuTM or a FACSAriaTM Fusion (for detailed sorting strategy see Supplemental Fig.2b). Staining was performed in PBS/2%FCS with anti-CD16/CD32 antibodies to block FcR-binding and DAPI (Sigma-Aldrich) or Fixable Viability Dye (Thermo Fisher) to exclude dead cells. Detailed information on all antibodies used in this study is listed in Supplemental Table 2. For staining of intracellular markers, the Foxp3/Transcription Factor Staining Buffer Set® (eBioscience) was used. Multicolour FACS staining for surface markers was performed using Brilliant Stain Buffer (BD Biosciences) where applicable. Data was acquired on a FACSymphony® A5 SORP (Becton Dickinson) and analyzed using FlowJo® software (v10.7.1 or v10.8.0; Treestar Inc). For clustering and embedding of multiparameter flow cytometry data (as presented in Supplemental Fig.2a) FlowJo® plugins DownSample_v3.3 and UMAP_v3.1 were used. The UMAP plot in Supplemental Fig. 2a was created on the basis of 27 samples from spleen, liver and colon (all n=3) from 3 experimental groups: alloTreg prophylaxis, polyTreg prophylaxis and GvHD control and 3 independent experiments, with every sample representing a cell pool of 2-3 mice. Cells were stained for CD45.1, CD45.2, H-2kb, TER119, TCRβ, CD8α, CD4, CD19, Nkp46, CD11b, Ly6C, Gr-1 and CD25. Dead cells were excluded with DAPI. Samples were gated on live single CD45.1+TER119- and CD45.2+TER119- cells, downsampled to 15.000 cells each and assembled into one file.
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| Growth protocol |
Splenocytes were stained with anti-CD25-PE and anti-PE UltraPure MicroBeads and enriched for CD25+ cells by MACS® (Miltenyi Biotec). The CD25+ fraction was further stained for CD8, CD4 and CD62L and Treg were sorted on a FACSAria IIuTM or a FACSAriaTM Fusion (BD Biosciences) as CD4+CD25highCD62L+ cells; purity of sorted cells was >98%. Polyclonal in vitro expansion of Treg was performed as recently described {Riegel, 2020 #61}. In brief, sorted Treg were cultured in DMEM (high glucose, Gibco/Invitrogen) with 10% FCS, 2mM L-glutamine, 10mM HEPES, 1% NEAA (PAN Biotech), 50U/ml penicillin, 50µg/ml streptomycin and 5x10-5 M 2-mercaptoethanol (Gibco/Invitrogen) (cDMEM) in 96-U-well plates (1x104/well) and stimulated with CD3/CD28-beads (Treg-Expansion Kit, Miltenyi Biotec, 4 beads/cell) and rhIL-2 (2000 U/ml; Proleukin®, Chiron). Cultures received 100 µl/well fresh medium on d4, were restimulated on d7 with 1 bead/cell after transfer into 24-well-plates (1x106 cells/ml/well), fed and split on d10 as needed and harvested on d12. For allospecific in vitro expansion sorted Treg were seeded at 5x104/well in cDMEM together with 2,5x104/well (d0) or 1x104/well (restimulation on d7) anti-CD11c MicroBead-enriched (Miltenyi Biotec) and irradiated (30 Gy) CD11c+ DC from BALB/c mice. Cells received fresh medium on d4 and d10 and were harvested on d12.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total cellular RNA was isolated from Treg (freshly isolated, in vitro expanded, or re-isolated after BMT) using the RNeasy Mini Kit or the RNeasy Micro Kit (Qiagen). Following the manufacturer’s recommendations, samples originating from 500 to 500k cells were processed with the Micro Kit whereas the Mini Kit was applied to all samples exceeding 500k cells. RNA concentration and quality were simultaneously assessed using RNA Screentape Analysis or High Sensitivity RNA Screentape Analysis (Agilent), depending on the expected yield.
Preparation of dsDNA libraries for Illumina sequencing was facilitated using the SMART-Seq® Stranded Kit (Takara) according to the manufacturer’s instructions. Depending on sample quality and abundance, fragmentation times and second PCR cycle numbers were adjusted, respectively. For some samples, first PCR products were pooled to minimize carry-over losses. The concentration of dsDNA libraries was measured with the Qubit™ dsDNA HS Assay Kit (Thermo Fisher Scientific). DNA fragment size distribution was assessed using the High Sensitivity D1000 ScreenTape Assay (Agilent). Libraries were stored at -20°C until sequencing was performed using the Illumina NextSeq550 sequencer and libraries are summarized in Supplemental Tables 4-7.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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| Description |
re-isolated
244-4209_C_ra
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| Data processing |
base calling: Base-calling and demultiplexing of sequencing reads was carried out using the bcl2fastq Conversion Software (v1.8.4) provided by Illumina (Illumina Inc., San Diego, California, USA).
mapping: Sequencing reads were mapped to the mouse genome (GRCm38.p5) using STAR v2.5.3a. The genome index incorporated gene annotation from GENCODE Mouse release M16 to aid in spliced alignment.
generation of unique raw reads per geneTables of raw uniquely mapped read counts per gene were generated during mapping using the built-in --quantMode GeneCounts option in STAR.
Differential expression analysis was carried out on raw gene counts using edgeR (v3.34.0) in R (v4.1.0)
Assembly: Gencode Release M16 (GRCm38.p5); https://www.gencodegenes.org/mouse/release_M16.html
Supplementary files format and content: raw reads:column 1: gene ID (Ensemble); other columns: counts for the 2nd read strand aligned with RNA (column 4 of STAR output) for individual samples
Supplementary files format and content: normalized reads: column 1-5: Ensembl ID, Gene Symbol,Transcript Length, Gene Type; other columns: log2 transformed, batch-corrected and normalized read counts for individual samples (edgeR output).
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| Submission date |
Jan 26, 2023 |
| Last update date |
Jan 01, 2024 |
| Contact name |
Michael Rehli |
| E-mail(s) |
michael.rehli@klinik.uni-r.de
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| Organization name |
University Hospital Regensburg
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| Department |
Internal Med III
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| Street address |
F.-J.-Strauss-Allee 11
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| City |
Regensburg |
| ZIP/Postal code |
93042 |
| Country |
Germany |
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| Platform ID |
GPL21626 |
| Series (2) |
| GSE223796 |
Donor regulatory T cells rapidly adapt to recipient tissues to control acute graft-versus-host disease [bulk RNA-seq] |
| GSE223800 |
Donor regulatory T cells rapidly adapt to recipient tissues to control acute graft-versus-host disease |
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| Relations |
| BioSample |
SAMN32932068 |
| SRA |
SRX19189060 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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