Gene Expression
Nebulas
Gene Expression NebulasSummary: The fetus is thought to be protected from exposure to foreign antigens, yet CD45RO+ T-cells reside in the fetal intestine. We combined functional assays with mass cytometry, single-cell RNA-sequencing, and high-throughput TCR-sequencing to characterize the fetal intestinal CD4+ T-cell compartment. We identified 22 CD4+ T-cell clusters, including naive-like, regulatory-like, and memory-like subpopulations, which were confirmed and further characterized at the transcriptional level. Memory-like cells expressed high levels of Ki-67, indicating cell division, and CD5, a surrogate marker of TCR-avidity, and produced IFN-γ and IL-2. Pathway analysis revealed a differentiation trajectory associated with cellular activation and proinflammatory effector functions, and TCR-repertoire analysis demonstrated clonal expansions, distinct repertoire characteristics, and interconnections between subpopulations of memory-like CD4+ T-cells. Imaging-mass cytometry further showed that memory-like CD4+ T-cells colocalized with antigen-presenting cells. Collectively, these results provided evidence for the generation of memory-like CD4+ T-cells in the human fetal intestine, consistent with exposure to foreign antigens.
Overall Design: Single, live, CD3+CD8a–TCRγδ–CD4+/– T-cells from one human fetal intestines were processed on the Chromium 3' single cell platform (10x Genomics) and sequenced on an Illumina HiSeq 4000.
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| Growth Protocol: | - |
| Treatment Protocol: | Single, live, CD3+CD8a–TCRγδ–CD4+/–T-cells from the intestines of fetus #6 were sorted using a FACSAria III flow cytometer (BD Biosciences). Post-sort purity was 96.5%. |
| Extract Protocol: | A single-cell suspension was prepared following the 10X Genomics single cell sample preparation protocol. |
| Library Construction Protocol: | Droplet-based single-cell partitioning and single-cell RNA-Seq libraries were generated using the Chromium Single-Cell 3'Reagent v2 Kit (10X Genomics, Pleasanton, CA) as per the manufacturer' protocol. |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | PAIRED |
| Library Strand: | Forward |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 4000 |
| Strand-Specific: | Specific |