Key Points

Tregs develop via distinct agonist and postagonist stages.

CD25+ TregP agonist cells are a main source of IL2 needed for Treg development.

Thymic recirculating Tregs have highly diverse transcriptomes and TCRs.

Abstract

Recent studies have demonstrated that regulatory T cells (Tregs) develop in the thymus via two pathways involving distinct Treg progenitors (TregP): CD25+FOXP3− (CD25+ TregP) and CD25−FOXP3lo (FOXP3lo TregP) Treg progenitors. To examine this process in more detail, we carried out single-cell RNA sequencing (scRNA-Seq) and TCR-Seq on sorted murine CD4+CD8+ double-positive (DP) thymocytes, CD4+ single-positive (CD4SP) thymocytes, CD25+FOXP3−CD73− TregP, CD25−FOXP3loCD73− TregP, newly generated mature CD25+FOXP3+CD73− Tregs, and FOXP3+CD73+ recirculating/long-term resident Tregs (RT-Tregs). Sorted populations were individually hashtagged and then combined into one scRNA-Seq/TCR-Seq library before sequencing and subsequent analysis. We found that both CD25+ TregP and FOXP3lo TregP arise via an initial agonist-activated state that gives rise to a second transitional stage before differentiating into mature Tregs. Using both scRNA-Seq and bulk RNA-Seq on sorted thymocyte subsets, we demonstrate that CD25+ TregP are significantly enriched for Il2 production, suggesting that they are the major source of IL-2 needed to convert TregP into mature Tregs. Using TCR-Seq, we found that several TCRs were clearly biased in favor of the conventional or Treg lineages, but that a large fraction of TCRs were found in both these lineages. Finally, we found that RT-Tregs in the thymus are not monomorphic but are composed of multiple distinct subsets and that these RT-Tregs express the most diverse TCR repertoire of all CD4SP thymocytes. Thus, our studies define multiple stages of Treg differentiation within the murine thymus and serve as a resource for future studies on CD4+ thymocyte development and Treg differentiation.

Footnotes

This work was supported by National Institutes of Health Grants R01 AI124512 and 2T32AI007313.

Raw and processed data are available through the Gene Expression Omnibus accession numbers GSE195820 (single-cell RNA-sequencing data) and GSE202871 (bulk RNA sequencing); the single-cell RNA-sequencing data in Fig. 5C were previously deposited under Gene Expression Omnibus accession number GSE123067 (https://www.ncbi.nlm.nih.gov/geo/).

The online version of this article contains supplemental material.

Abbreviations used in this article:

DNdouble-negativeDPdouble-positiveeTregeffector regulatory T cellHTOhashtag oligoRT-Tregrecirculating/long-term resident regulatory T cellscRNA-Seqsingle-cell RNA sequencingTregregulatory T cellSPsingle positiveTregPTreg progenitorUMAPUniform Manifold Approximation and ProjectionReceived January 25, 2022.Accepted July 26, 2022.Copyright © 2022 by The American Association of Immunologists, Inc.