{"doi":"10.4049/jimmunol.2200089","title":"Tracking Regulatory T Cell Development in the Thymus Using Single-Cell RNA Sequencing/TCR Sequencing","abstract":"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.","journal":"The Journal of Immunology","year":2022,"id":260133,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":16,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9462,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":569406,"name":"Rebecca S. La Rue","orcid":null,"position":1,"is_corresponding":false},{"id":267855,"name":"Sarah A. Munro","orcid":"0000-0002-7233-0047","position":2,"is_corresponding":false},{"id":374880,"name":"Michael A. Farrar","orcid":"0000-0002-5569-0366","position":3,"is_corresponding":false},{"id":668447,"name":"David L. Owen","orcid":"0000-0002-1099-0605","position":0,"is_corresponding":true}],"reference_count":47,"raw_metadata":null,"created_at":"2026-07-19T00:26:03.189798Z","pmid":"36038290","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}