Recent studies have demonstrated that regulatory T cells (Tregs) develop in the thymus via two pathways involving distinct Treg progenitors (TregP): CD25+FOXP32 (CD25+ TregP) and CD252FOXP3lo (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+FOXP32CD732 TregP, CD252FOXP3loCD732 TregP, newly generated mature CD25+FOXP3+CD732 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.
Bibliographical noteFunding Information:
This work was supported by National Institutes of Health Grants R01 AI124512 and 2T32AI007313.
This work was supported by National Institutes of Health Grants R01 AI124512 and 2T32AI007313. We thank A. Rost, N. Keller, G Hubbard, and L. Heltemes-Harris for technical assistance with mouse breeding and genotyping; the University of Minnesota's Supercomputing Institute for providing computing and bioinformatic resources; Emma Stanley, Jerry Daniel, and Kenneth Beckman for assistance with 10× Genomics single-cell capture, library preparation, and sequencing; Elyse Froehling for bulk RNA-Seq library preparation at the University of Minnesota Genomics Center; and Terese Martin, Jason Motl, and Paul Champoux for cell sorting and maintenance of the Flow Cytometry Core Facility at the University of Minnesota.
© 2022 by The American Association of Immunologists, Inc.
- Forkhead Transcription Factors/genetics
- Interleukin-2 Receptor alpha Subunit/metabolism
- Receptors, Antigen, T-Cell/metabolism
- Sequence Analysis, RNA
- T-Lymphocytes, Regulatory/metabolism
- Thymus Gland/metabolism
PubMed: MeSH publication types
- Journal Article
- Research Support, N.I.H., Extramural