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 thymic CD4-CD8αß-TCRαß+NK1.1- IEL precursors (NK1.1- IELPs), we define a developmental trajectory that can be tracked based on the sequential expression of CD122 and T-bet. Moreover, we identify the Id proteins Id2 and Id3 as a novel regulator of IELP development and show that all NK1.1- IELPs progress through a PD-1 stage that precedes the induction of T-bet. The transition from PD-1 to T-bet is regulated by the transcription factor C-Myc, which has far reaching effects on cell cycle, energy metabolism, and the translational machinery during IELP development. In summary, our results provide a high-resolution molecular framework for thymic IEL development of NK1.1- IELPs and deepen our understanding of this still elusive cell type.
Intraepithelial Lymphocytes/immunology , Precursor Cells, T-Lymphoid/immunology , Proto-Oncogene Proteins c-myc/metabolism , T-Box Domain Proteins/metabolism , Thymus Gland/immunology , Animals , Antigens, Ly/metabolism , Cells, Cultured , Gene Expression Regulation , Immunity, Innate , Inhibitor of Differentiation Protein 2/genetics , Inhibitor of Differentiation Protein 2/metabolism , Inhibitor of Differentiation Proteins/genetics , Inhibitor of Differentiation Proteins/metabolism , Interleukin-2 Receptor beta Subunit/genetics , Interleukin-2 Receptor beta Subunit/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , NK Cell Lectin-Like Receptor Subfamily B/metabolism , Sequence Analysis, RNA , Single-Cell Analysis , T-Box Domain Proteins/genetics
