Combined transient ablation and single-cell RNA-sequencing reveals the development of medullary thymic epithelial cells

Abstract

Medullary thymic epithelial cells (mTECs) play a critical role in central immune tolerance by mediating negative selection of autoreactive T cells through the collective expression of the peripheral self-antigen compartment, including tissue-specific antigens (TSAs). Recent work has shown that gene expression patterns within the mTEC compartment are remarkably heterogenous and include multiple differentiated cell states. To further define mTEC development and medullary epithelial lineage relationships, we combined lineage tracing and recovery from transient in vivo mTEC ablation with single cell RNA-sequencing in Mus musculus. The combination of bioinformatic and experimental approaches revealed a non-stem transit-amplifying population of cycling mTECs that preceded Aire expression. Based on our findings, we propose a branching model of mTEC development wherein a heterogeneous pool of transit-amplifying cells gives rise to Aire- and Ccl21a-expressing mTEC subsets. We further use experimental techniques to show that within the Aire-expressing developmental branch, TSA expression peaked as Aire expression decreased, implying Aire expression must be established before TSA expression can occur. Collectively, these data provide a higher order roadmap of mTEC development and demonstrate the power of combinatorial approaches leveraging both in vivo models and high-dimensional datasets.

Sirt6 Regulates the Development of Medullary Thymic Epithelial Cells and Contributes to the Establishment of Central Immune Tolerance

“…Due to the overall reduction of mTECs, the absolute number of CD80 + mTECs, MHC II high mTECs and Aire + mTECs all decreased dramatically ( Figure 3B ). Specifically, Aire expressed on CD80 + MHC II high mTECs (mTEC high ) and then regulated the expression of thousands of TRAs ( Heino et al, 2000 ; Gray et al, 2007 ; Wells et al, 2020 ). Sirt6 deletion accelerated the maturation of CD80 – Aire – mTECs to the differentiation of CD80 + Aire – mTEC, but did not affect its further differentiation into CD80 + Aire + mTECs ( Figures 3C,D ).…”

Post-Aire Medullary Thymic Epithelial Cells and Hassall’s Corpuscles as Inducers of Tonic Pro-Inflammatory Microenvironment

“…It is now widely accepted that at least during fetal development both the cortical as well as the medullary epithelial cells are derived from a single bipotent progenitor ( 20 , 21 ), while the existence of the bipotent progenitor in the adult thymus has remained controversial. After receiving a signal yet to be identified, some of the progenitors are directed toward the mTEC lineage and upregulate the proliferation marker Ki67 ( 22 ) to become more populous and can still give rise to different mature mTEC lineages. Recent advances in single-cell transcriptomics in mice ( 22 – 26 ) and humans ( 27 ) have highlighted the heterogeneity of these functionally diverse thymic cells and although minor differences exist between the results, most of the respective studies agree that in mice the mature mTECs can be divided into four subpopulations: 1) mTEC I, characterized by the dependency of lymphotoxin (LT)β signaling, high expression of CCL21 and lack of MHCII expression ( 28 , 29 ); 2) mTEC II, characterized by RANK-dependency and high expression of Aire, MHCII and thousands of tissue-restricted genes ( 30 , 31 ); 3) mTEC III, known as post-Aire cells or corneocyte-like mTECs, which express low/mid MHCII and whose gene expression profile resembles late-stage keratinocytes/corneocytes (see below); and 4) mTEC IV, known as thymic tuft cells, which express IL-25 and whose gene expression profile resembles intestinal tuft cells ( 23 , 24 ).…”

The Early Postnatal Life: A Dynamic Period in Thymic Epithelial Cell Differentiation

“…CCl21 + cells emerge during embryogenesis and their numbers also undergo a marked increase during the first weeks of life (57). Recent single cell RNA sequencing analysis suggests that Aire-and Ccl21aexpressing mTEC subsets do not share a direct lineage relationship (58). Moreover, the discoveries that Aire + mTECs differentiate into Post-Aire cells (59, 60) further extended our view on the heterogeneity within thymus medulla.…”