Gene Expression
Nebulas
Gene Expression NebulasSummary: γδ T lymphocytes represent ~1% of human PBMC and even more cells in most tissues of vertebrates. Although they have important anticancer functions, most current scRNA-Seq studies do not identify γδ T lymphocytes since their transcriptomes at the single cell level are unknown. Here we show that high resolution clustering of large scRNA-Seq data sets and a combination of gene signatures allow the specific detection of human γδ T lymphocytes and identification of their TCRVδ1 and TCRVδ2 subsets in large data sets from complex cell mixtures. In t-SNE plots from blood and tumor samples, the few γδ T lymphocytes appear collectively embedded between cytotoxic CD8 T and NK cells. Their TCRVδ1 and TCRVδ2 subsets form close yet distinct sub-clusters respectively neighbouring NK and CD8 T cells owing to expression of shared and distinct cytotoxic maturation genes. Similar pseudo-time maturation trajectories of TCRVδ1 and TCRVδ2 γδ T lymphocytes were discovered, unveiling in both subsets an unattended pool of TEMRA cells with preserved proliferative capacity, a finding confirmed by in vitro proliferation assays. Overall, the single cell transcriptomes of thousands of individual gd T lymphocytes from different CMV+ and CMV- donors reflect cytotoxic maturation stages driven by the immunological history of donors. This landmark study establishes the rationale for identification, subtyping and deep characterization of human γδ T lymphocytes in further scRNA-Seq studies of complex tissues in physiological and disease conditions.
Overall Design: 6 samples of purified human gd T cells from 3 donors (CMV+ and CMV-). Cell-sorted TCRVdelta1 and TCRVdelta2 T lymphocytes from each donor PBMC.
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| Growth Protocol: | - |
| Treatment Protocol: | - |
| Extract Protocol: | Sequencing library was prepared following the manufacturer’s instructions (10X Genomics), with 14 cycles used for cDNA amplification and 12 cycles for library amplification |
| Library Construction Protocol: | - |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | PAIRED |
| Library Strand: | Forward |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina NextSeq 550 |
| Strand-Specific: | Specific |