Revelations in Thymic Epithelial Cell Biology and Heterogeneity from Single-Cell RNA Sequencing and Lineage Tracing Methodologies.

Thymic epithelial cells (TECs) make up the thymic microenvironments that support the generation of a functionally competent and self-tolerant T-cell repertoire. Cortical (c)TECs, present in the cortex, are essential for early thymocyte development including selection of thymocytes expressing functional TCRs (positive selection). Medullary (m)TECs, located in the medulla, play a key role in late thymocyte development, including depletion of self-reactive T cells (negative selection) and selection of regulatory T cells. In recent years, transcriptomic analysis by single-cell (sc)RNA sequencing (Seq) has revealed TEC heterogeneity previously masked by population-level RNA-Seq or phenotypic studies. We summarize the discoveries made possible by scRNA-Seq, including the identification of novel mTEC subsets, advances in understanding mTEC promiscuous gene expression, and TEC alterations from embryonic to adult stages. Whereas pseudotime analyses of scRNA-Seq data can suggest relationships between TEC subsets, experimental methods such as lineage tracing and reaggregate thymic organ culture (RTOC) are required to test these hypotheses. Lineage tracing - namely, of β5t or Aire expressing cells - has exposed progenitor and parent-daughter cellular relationships within TEC.