Relatively enhanced activity of new (Rag-GFP⁺) tTreg cell generation along with reduced thymic-reentered (Rag-GFP^-neg) Treg cells were related to the severity of thymic atrophy.

<p><b>(A)</b> A representative flow cytometric gate strategy from young mice of FF (normal thymus, top panels) or FC (atrophied thymus, bottom panels) with a Rag-GFP reporter shows gates of CD4^SP (left panels), CD4^SP-GFP⁺ (middle panels), and tTcon cells and tTreg cells in the CD4^SP-GFP⁺ population from the same thymus (right panels). <b>(B)</b> Summarized absolute cell numbers per thymus from panel-A of newly generated (Rag-GFP⁺) CD4^SP, tTreg cells, and tTcon cells from FF-Rag-GFP and FC-Rag-GFP mice treated with TM x3 (for induction of acute <i>FoxN1</i>^fx/fx gene deletion), respectively. A Student <i>t</i> test was used to determine statistical significance between groups. <b>(C)</b> <i>FoxN1</i>^fx/fx time-course deletion (due to the CreER^T autoleakage over time) induced dynamic changes in ratio (left panel) and absolute cell numbers per thymus (right panel) of newly generated (Rag-GFP⁺) tTreg cells and tTcon cells in the same thymus. <b>(D)</b> Potential peripheral thymic-reentered (Rag-GFP^-neg) Treg cells and Tcon cells in the same thymus. Two curves in left panels of C and D show linear regression (green lines) and nonlinear one-phase decay (red lines), respectively. Lines in right panels of C and D are linear regression, while data presented by nonlinear one-phase decay are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003352#pbio.2003352.s002" target="_blank">S2A Fig</a>. GraphPad-5 statistic software was used for analysis. Each symbol represents an individual animal sample. Underlying data used in the generation of this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003352#pbio.2003352.s007" target="_blank">S1 Data</a>. CreER^T, ubiquitous promoter-driven Cre-recombinase and estrogen-receptor fusion protein; FC, <i>FoxN1</i>^fx/fx/CreER^T; FF, <i>FoxN1</i>^fx/fx without CreER^T for controls; <i>Foxn1</i>, Forkhead box protein N1; <i>FoxN1</i>^fx/fx, <i>loxp</i>-flanked <i>FoxN1</i> gene; GFP, green fluorescent protein; Tcon, conventional T cell; TM, tamoxifen; tTcon, thymic conventional T cell; tTreg, thymic regulatory T cell.