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Timestamp tracing delineates thymic regulatory T cell ontogeny in native conditions [scRNA-seq]

ABSTRACT: Thymic T-cell selection and regulatory T (Treg) cell development are executed via mechanisms that remain unclear. Here, we develop a timestamp tracing method using Rag1CreER knockin mice in combination with Rosa reporters to reveal the processes governing thymic Treg differentiation in native physiological conditions. It enables tracing of thymic T cells at the double negative and double positive stages upon tamoxifen administration. Following labeled cells over time, we define the processes, kinetics, and gene expression changes during thymic T-cell differentiation; specify thymic Treg maturation with CD73, gene expression changes, and Treg-specific DNA demethylation; characterize precursor cells with 4-1BB and/or CD25 expression that are induced by TCR signaling and differentiate to Treg cells in vitro and in vivo; and determine the features of recirculating or retained mature Treg cells. Thus, Rag1CreER–based timestamp tracing delineates thymic Treg ontogeny, proving the concept for exploring T-cell development and heterogeneity with age-resolved precision.

ORGANISM(S): Mus musculus

PROVIDER: GSE274125 | GEO | 2026/07/21

REPOSITORIES: GEO

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Project description:The mechanism of egress of mature regulatory T cells (Tregs) from the thymus to the periphery remains enigmatic, as does the nature of those factors expressed in the thymic environment. Here, we examined the fate of thymic Tregs in TNFÎ±/RelA double-knockout (TA-KO) mice, because TA-KO mice retain a Treg population in the thymus but have only a small Treg population at the periphery. Transplantation of whole TA-KO thymus to under the kidney capsule of Rag1 null mice failed to induce the production of donor-derived splenic Tregs expressing neuropilin-1 (Nrp1), which was reported to be a marker of naturally occurring Tregs, indicating that TA-KO thymic Tregs either do not leave the thymus or are lost at the periphery. We next transplanted enriched TA-KO thymic Tregs to the peripheries of TA-KO mice and traced mouse survival. Transplantation of TA-KO thymic Tregs rescued the lethality in TA-KO mice, demonstrating that TA-KO thymic Tregs remain functional at the periphery. The TA-KO thymic Treg population had highly demethylated CpG motifs in the foxp3 locus, indicating that the cells were arrested at a late-mature stage. Also, the population included a large subpopulation of Tregs expressing IL-7RÎ±, which is a possible marker of late-mature Tregs. Finally, TA-KO fetal liver chimeric mice developed an Nrp1+ splenic Treg population from TA-KO cells, suggesting that Treg arrest is caused by a lack of RelA in the thymic environment. Together, these results suggest that egress of mature Tregs from the thymus depends on RelA in the thymic environment. For the isolation of thymic Tregs, CD4+CD8Î±-CD25hi thymocytes were isolated from five 1.5- to 2-week-old TNFÎ±-KO or TA-KO mice by using a FACSAria cell sorter. For the isolation of thymic stromal cells, 10 thymi from 1.5- to 2-week-old TNFÎ±-KO or TA-KO mice were minced with scissors and treated with RPMI 1640 supplemented with 2% FCS, 0.2 mg/ml collagenase (Roche, Basel, Switzerland), 0.2 mg/ml dispase I (Roche), and 100 U/ml DNase I (Life Technologies) for 30 min with stirring. Digested thymi were centrifuged in a Percoll (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) gradient (density, 1.115, 1.065, and PBS) at 1400g for 30 min. Cells in the upper layer were collected, and the CD45-EpCAM+ (thymic epithelial cells) and CD45+EpCAM- populations (enriched thymic stromal cells containing macrophages or dendritic cells) were sorted.

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Timestamp tracing delineates thymic regulatory T cell ontogeny in native conditions [scRNA-seq]

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