Figure 8. Sustained Foxp3 expression is required for maintenance of a suppressive phenotype.

A. Marilyn CD4⁺ T cells were transduced with cFoxp3 and either treated or not treated with 4HT to induce nuclear localisation of GFP-Foxp3-ERT. After 48 h, transduced cells were sorted based on CD4 and GFP expression (sorted cells indicated by rectangles). Data are representative of three experiments.

B. Splenic CD4⁺ cells from Marilyn mice were transduced with cFoxp3 and half received in vitro 4HT treatment. 2×10⁴ or 1×10⁶ cells were sorted for expression of GFP and transferred into B6.Rag^−/− mice. These mice received daily tamoxifen (1mg i.p.). As controls, some recipients did not receive in vivo tamoxifen treatment, but received vehicle alone. Graft rejection was monitored daily. On day 28, tamoxifen treatment was ceased and graft rejection followed until day 56. Data are representative of two separate experiments.

C. MoFlo FACS profile of sorted GFP⁺ cTreg used in (4D).

D. 5×10⁵ MoFlo sorted in vitro 4HT-treated cFoxp3-transduced cells were transferred into Marilyn.Foxp3^hCD2 mice (n=4) followed the next day by transplantation of a male CBA.Rag^−/− graft. Daily tamoxifen (1mg) was administered. Control recipients (n=4) did not receive cells, but were treated with daily tamoxifen. Data are representative of two separate experiments.