Fluorochrome-based definition of naturally occurring Foxp3+ regulatory T cells of intra- and extrathymic origin

Under physiological conditions, studies on the biology of naturally induced Foxp3(+) Treg cells of intra- and extrathymic origin have been hampered by the lack of unambiguous markers to discriminate the mature progeny of such developmental Treg- cell sublineages. Here, we report on experiments in double- transgenic mice, in which red fluorescent protein (RFP) is expressed in all Foxp3(+) Treg cells, whereas Foxp3- dependent GFP expression is exclusively confined to intrathymically induced Foxp3(+) Treg cells. This novel molecular genetic tool enabled us to faithfully track and characterize naturally induced Treg cells of intrathymic (RFP+ GFP(+)) and extrathymic (RFP+ GFP(-)) origin in otherwise unmanipulated mice. These experiments directly demonstrate that extrathymically induced Treg cells substantially contribute to the overall pool of mature Foxp3(+) Treg cells residing in peripheral lymphoid tissues of steady- state mice. Furthermore, we provide evidence that intra- and extrathymically induced Foxp3(+) Treg cells represent distinct phenotypic and functional sublineages.