Rag1-based time-stamp tracing of T-cell developmental origins

Abstract

Abstract The postnatal T‑cell pool comprises cells at diverse developmental stages, complicating efforts to resolve T‑cell differentiation, maturation, and long‑term adaptation. To address this challenge, we developed Rag1‑mediated time-stamp tracing, a genetic strategy that enables time‑resolved tracking of T‑cell developmental origins. We validate the specificity and temporal resolution of this approach across multiple analytical platforms, including flow cytometry, imaging, and single‑cell multiomics. Applying this strategy to thymic regulatory T (Treg) cell development, we show that time-stamp tracing resolves distinct phases of Treg cell induction, maturation, and long‑term adaptation within heterogeneous populations. This analysis identifies precursor populations for thymic Treg cell induction, uncovers a maturation program in nascent Treg cells marked by CD73 upregulation and progressive DNA hypomethylation at Treg cell–specific loci, and distinguishes long‑lived mature Treg cells characterized by activation and clonal expansion. Together, these findings establish Rag1‑based time-stamp tracing as a versatile and unbiased approach for resolving T‑cell developmental origins across immunological contexts.