Single cell RNA sequencing identifies the developmental origin of C-Myc dependent T-bet+thymic intraepithelial lymphocyte precursor

Natural intraepithelial lymphocytes (IELs) are thymus-derived adaptive immune cells, which are important contributors to intestinal immune homeostasis. Similar to other innate-like T cells they are induced in the thymus through high-avidity interaction that would otherwise lead to clonal deletion in conventional CD4 and CD8 T cells. By applying single-cell RNA-sequencing (scRNA-seq) on a heterogeneous population of IEL precursors (IELPs) we define a developmental trajectory that identifies Id3 as a novel regulator of IELP development and shows that all natural TCRaß+ IELPs progress through a PD-1 stage as a result of strong agonist selection. Furthermore, with the use of newly developed PD-1 fate map and conditional T-bet knockout mice we demonstrate that this PD-1 stage precedes the induction of T-bet. The transition from PD-1 to T-bet is regulated by the transcription factor C-Myc, as T cell-specific conditional C-myc knockout mice lack PD-1-T-bet+ IELPs, while PD-1+T-bet- IELPs are still present. In summary, our results provide a high-resolution molecular framework for thymic IEL development and deepen our understanding of this still elusive cell type. Overall design: Single cell RNA sequencing was performed on thymic intraepithelial lymphocytes by sorting CD4-CD8-TCRb+NK1.1+ fresh isolated thymocytes from one TBGR mouse. A replicating experiment was executed by comparing fresh isolated IELPs from one TBGR and TGBR C-mycD/DCd4 mouse. Please note that some of the readme_*.coutt.txt files are identical, because the same cell barcodes have been used but different illumina barcodes.