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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jan 17, 2023 |
| Title |
Deep phenotypin heart-specific Tregs |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Myocardial infarction (MI) is a sterile inflammatory condition that results in the activation of T- cells targeting cardiac antigens. T-cells have been shown to contribute to reparative and maladaptive remodeling post-MI. However, the differentiation trajectories and in situ activity of heart-specific CD4+T cells and the effect of distinct T-cell phenotypes on MI repair remains poorly understood. Herein, we combined T-cell receptor transgenic models and tetramer staining targeting myocardial protein with single-cell transcriptomics (scRNAseq) and functional phenotyping to elucidate how the myosin-specific CD4+ T cells (TCR-M) differentiate in the murine infarcted myocardium and influence tissue repair. Furthermore, we transferred pro-inflammatory versus regulatory pre-differentiated heart-specific T-cells to dissect how they differentially regulate post-myocardial infarction (MI) inflammation.
Flow cytometry and scRNAseq findings reveled that transferred TCR-M cells acquired an induced regulatory phenotype (iTreg) in the infarcted myocardium and blunt local inflammation. Myocardial TCR-M cells differentiated into two main lineages enriched with cell activation and pro-fibrotic transcripts (e.g. Tgfb1) or with suppressor immune checkpoints (e.g. Pdcd1), which we also found in human myocardial tissue. These cells produced high levels of latency-associated peptide (LAP) and inhibited interleukine-17 (IL-17) responses. Tetramer staining further identified endogenous myosin-specific T-cells that acquired a regulatory phenotype in the heart post-MI. Notably, TCR-M cells that were pre-differentiated in vitro towards a regulatory phenotype maintained a stable in vivo FOXP3 expression and anti-inflammatory activity whereas TH17 partially converted towards a regulatory phenotype in the injured myocardium, which was associated to blunted myocardial inflammation. Moreover, myocardial scar of Treg TCR-M transferred mice showed blunted inflammatory transcripts and enrichment for growth factors supporting fibroblasts and endothelial cells.
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| Overall design |
Digested hearts and MedLNs were obtained from pooled TCR-M transferred mice at 5 and 7 days after sham or MI operation (n: 5-7 mice per group). Cell suspension was obtained as previously described and following surface staining, heart CD4+ T-cells were enriched by magnetic cell sorting, using anti-PE microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany). Before sorting, samples were separately labeled with an anti-CD45/H2-K hashtag antibody (Biolegend TotalSeqC antibodies, San Diego, CA, USA, 1:150 dilution). Additionally, all samples were labeled with anti-Thy1.1 (clone OX-7) and anti-CD25 (clone PC61) Cite-Seq antibodies (Biolegend TotalSeqC antibodies, San Diego, CA, USA, 1:100). Sodium azide was omitted in the buffers used for sorting purposes. After staining, heart and MedLN samples were separately pooled and CD4+ T-cells were sorted using a FACS Aria III instrument (BD, Heidelberg, Germany). Following a washing step, all samples were pooled and suspended in 65 µL of PBS/0.04% BSA and loaded in the 10x Genomics Chromium system as one multiplexed sample. A total of two libraries from the same experiment were generated and merged for analysis.
Myosin-specific T-cells (TCR-M cells) were transferred to WT Balb/C mice one day prior to the induction of experimental myocardial infarction (MI). CD4+ T-cells and transferred TCR-M cells were isolated from heart and mediastinal lymph nodes at 5 and 7 days post sham or MI operation by fluorescence activated cell sorting according to the congenical marker Thy1.1 expressed on TCR-M cells. In addition, CD4+ T-cells from sham and MI operated animals which have not received TCR-M cells were obtained at 5 days post-surgery. Pooled CD4+ T-cells and TCR-M cells stained with different hashtag and CITE-seq antibodies were analyzed using scRNAseq. Comparative gene expression profiling analysis of RNA-seq data from infarcted myocardial tissue of control non-T-cell transferred mice versus myocardial tissue from mice receiving adoptive transfer of Treg-polarized myosin-specific T-cells. Samples were collected at 5 days post-surgery and a total of 8 control samples and 6 Treg-transferred samples were analyzed.
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| Contributor(s) |
Delgobo M, Weiß E, Ashour DE, Richter L, Popiolkowski L, Arampatzi P, Stangl V, Arias-Loza P, Mariotti-Ferrandiz E, Reiner PP, Saliba A, Ludewig B, Hofmann U, Frantz S, Ramos GC |
| Citation(s) |
36744467 |
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| Submission date |
Mar 07, 2022 |
| Last update date |
Mar 10, 2023 |
| Contact name |
Antoine-Emmanuel Saliba |
| E-mail(s) |
emmanuel.saliba@helmholtz-hzi.de
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| Phone |
+49-931-31-81341
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| Organization name |
Helmholtz Institute for RNA-based Infection Research
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| Street address |
Josef-Schneider-Straße 2 / D15
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| City |
Würzburg |
| ZIP/Postal code |
97080 |
| Country |
Germany |
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| Platforms (2) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
| GPL30172 |
NextSeq 2000 (Mus musculus) |
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| Samples (23)
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| Relations |
| BioProject |
PRJNA813551 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE198059_RAW.tar |
183.6 Mb |
(http)(custom) |
TAR (of CSV, FASTA, MTX, TAR, TSV) |
| GSE198059_README_demux.txt |
445 b |
(ftp)(http) |
TXT |
| GSE198059_Reads_per_gene.csv.gz |
2.1 Mb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
| Processed data provided as supplementary file |
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