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

Here, we report on experiments in double-transgenic mice, in which 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. CD4+CD25+ RFP+GFP- and CD4+CD25+ RFP+GFP+ T cells from pooled lymph nodes and pooled spleens of 20 mice were FACS sorted for RNA extraction and hybridization on Affymetrix microarrays in duplicates.