Figure 9.

Foxp3-Tocky identifies newly generated Treg cells. (A) Use of Foxp3-Tocky mice to investigate in vivo dynamics of Foxp3 transcription. (B) Construct for the generation of Foxp3-Tocky BAC transgenic mice. (C) Splenic T cells were sored into blue⁻red+, blue⁺red⁺, and blue⁻red⁻ and analyzed for intracellular Foxp3 proteins. (D) Foxp3-Tocky mice were crossed with Foxp3-IRES-GFP, and analyzed for the coexpression of GFP and Timer. (E) Timer-negative naive T cells and splenic Timer-positive Treg cells from Foxp3-Tocky mice were isolated and stimulated by anti-CD3 and anti-CD28 for 0, 22, or 50 h in the presence of IL-2 (and TGF-β for iTreg cells). Flow cytometry plots display raw blue versus red expression during the cultures. (F and G) Timer Angle versus Timer Intensity (F) or Timer Angle versus time in the data from E (F).