A new genetic model for separating and analyzing Treg cells in unchallenged mice based on their proliferative history

Apart from other cell types, antigen-primed conventional CD4 T cells integrate their functional differentiation into the process of proliferation. However, it is unknown whether the functional maturation of Tregs is also associated with their proliferative history, especially in unchallenged mice. To aid dissection of Treg populations, we developed a reporter system for marking and isolating Tregs undergone proliferative expansion during development or tolerance response towards endogenous antigens. We designed a tandem loxP cassette, named Tlox, to prevent intramolecular recombination. Tlox also acts as a genetic switch placed in front of a fluorescent marker. Cell division provides an opportunity for inter-molecular pairing between the duplicated Tlox on sister chromatids. Upon pairing, Cre recombinase can convert Tlox to a single loxP and consequently express the fluorescent marker permanently. We showed that, in vitro and in vivo, reporter activation is indeed dependent on both active cell cycles and Cre expression. By combining Ox40-Cre with the Tlox reporter, we separated Tregs based on their proliferative history in naive animals. Functional and transcriptomic analysis indicted that, within the Treg pool, proliferated Tregs form a distinct population and are highly enriched with effector phenotypes. Thus, the newly established lineage tracking system provides a novel tool for assessing dynamitic changes among Treg cells during their tolerance response in live animals.