Single-cell RNA-sequencing identifies the developmental trajectory of C-Myc-dependent NK1.1- T-bet+ intraepithelial lymphocyte precursors

Mucosal Immunol. 2020 Mar;13(2):257-270. doi: 10.1038/s41385-019-0220-y. Epub 2019 Nov 11.


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-CD8αβ-TCRαβ+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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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
  • Intraepithelial Lymphocytes / immunology*
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • NK Cell Lectin-Like Receptor Subfamily B / metabolism
  • Precursor Cells, T-Lymphoid / immunology*
  • Proto-Oncogene Proteins c-myc / metabolism*
  • Sequence Analysis, RNA
  • Single-Cell Analysis
  • T-Box Domain Proteins / genetics
  • T-Box Domain Proteins / metabolism*
  • Thymus Gland / immunology*


  • Antigens, Ly
  • Idb2 protein, mouse
  • Inhibitor of Differentiation Protein 2
  • Inhibitor of Differentiation Proteins
  • Interleukin-2 Receptor beta Subunit
  • Klrb1c protein, mouse
  • Myc protein, mouse
  • NK Cell Lectin-Like Receptor Subfamily B
  • Proto-Oncogene Proteins c-myc
  • T-Box Domain Proteins
  • T-box transcription factor TBX21
  • Idb3 protein, mouse