Single-cell transcriptomics of adult gd T-cell lineage commitment highlights its TCR-instructive nature I. 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 αβ and γδ 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 αβ/γδ precursors which rearrange the genes coding for the TCRγ, TCRδ, and TCRβ chains, post-selected αβ precursors (DN3b TCRγδ– and DN4 TCRγδ–; in short DN3b γδ– and DN4 γδ–), post-selected γδ precursors (DN3b TCRγδ+; in short DN3b γδ+), and immature γδ T cells in the process of functional diversification (DN4 TCRγδ+; in short DN4 γδ+). In addition, to precise the role of γδ TCR signals in the earliest phases of γδ lineage commitment and specification two subsets of DN3 cells expressing signaling-defective γδ TCR from TCRd-H2BEGFP Lat-deficient mice were isolated : DN3 γδint and DN3 γδ–. Each subset was spike-in using 500 splenic B cells in order to check dataset merging quality.