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

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.