DEREG or C57BL/6 mice were treated with 12 ng/g Diphtheria toxin (DT) i.p. for 3 consecutive days, following which blood and splenocytes were collected and stained for the presence of FoxP3⁺ T cells. A and B are gated on CD3⁺CD4⁺ cells and show representative plots (n = 3). DT administration did not alter the percentage of FoxP3⁺ cells in the blood (A) or spleen (B) of C57BL/6 mice. In contrast, CD3⁺CD4⁺ T cells expressing the GFP reporter under the control of FoxP3 were almost completely absent from DT-treated DEREG mice, while a small residual population of FoxP3⁺GFP^– cells remained following depletion. C (gated on CD3⁺CD4⁺ cells) summarizes the proportion of cells expressing FoxP3 in all DT-treated mice. In DEREG mice, FoxP3 cells declined from 14.4% ± 1.7% to 0.96% ± 0.19% of CD4⁺ T cells in blood (P = 0.015) and from 23.1% ± 1.8% to 5.7% ± 0.8% of CD4⁺ T cells in spleen (P = 0.010), upon DT administration. (D) Absolute numbers of CD4⁺FoxP3⁺ T cells were reduced from 42,067 ± 3,982 to 971 ± 192 in the peripheral blood of DT-treated DEREG mice (P = 0.0005) and from 1.5 × 10⁶ ± 0.5 × 10⁶ to 1.08 × 10⁵ ± 0.63 × 10⁵ in spleen (P = 0.0038). Boxes in C and D show minimum to maximum with a line at the mean. Data described as mean ± SEM, statistical analyses were performed using an unpaired t test; n = 3/group. (E and F) Survival of B6.Kd skin grafts to DEREG mice transduced with AAV-K^d was not influenced by DT treatment. Conversely, B6.Kd skin graft survival in DEREG mice inoculated with AAV-K^d-D227K was reduced from 27 to 18 days (P = 0.0046) when DT was coadministered, abrogating the survival prolongation due to expression of the K^d-D227K transgene. n = 6/group; survival analysis was performed using the log-rank test.