Single cell RNAseq of human TCRVdelta 1 and TCRVdelta 2 gammadelta T lymphocytes purified from healthy adults blood

?d 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 ?d 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 ?d T lymphocytes and identification of their TCRVd1 and TCRVd2 subsets in large data sets from complex cell mixtures. In t-SNE plots from blood and tumor samples, the few ?d T lymphocytes appear collectively embedded between cytotoxic CD8 T and NK cells. Their TCRVd1 and TCRVd2 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 TCRVd1 and TCRVd2 ?d 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 ?d 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.