Single-cell transcriptomics of adult gd T-cell lineage commitment highlights its TCR-instructive nature I

Single cell RNA-seq study of T lymphocyte differentiation WT and LAT deficient mice in order to deciphy gd T-cell lineage commitment. Overall design: Single-cell RNA-seq analysis of the T cell stages straddling the developmental bifurcation leading to aß and ?d T cells in adult thymus. Thymocyte subsets encompassing such bifurcation were isolated from 6- to 8-week-old mice using established cell surface markers and subjected to single cell RNA sequencing. Double negative cells (DN, i.e. CD8– and CD4–) cells were sorted into 5 subsets corresponding to DN3a bi-potent aß/?d precursors which rearrange the genes coding for the TCR?, TCRd, and TCRß chains, post-selected aß precursors (DN3b TCR?d– and DN4 TCR?d–; in short DN3b ?d– and DN4 ?d–), post-selected ?d precursors (DN3b TCR?d+; in short DN3b ?d+), and immature ?d T cells in the process of functional diversification (DN4 TCR?d+; in short DN4 ?d+). In addition, to precise the role of ?d TCR signals in the earliest phases of ?d lineage commitment and specification two subsets of DN3 cells expressing signaling-defective ?d TCR from TCRd-H2BEGFP Lat-deficient mice were isolated : DN3 ?dint and DN3 ?d–. Each subset was spike-in using 500 splenic B cells in order to check dataset merging quality.