Administered Treg wane away with time. (A) DEREG splenocytes were analyzed by flow cytometry. CD25 vs Foxp3-eGFP expression on electronically gated CD4⁺ cells. (B) Expression of CD25 vs Foxp3-eGFP by purified DEREG Treg was analyzed before (left) and after (right) in vitro culture with donor-type irradiated splenocytes as stimulators. Dot plots are representative of 4 experiments. Numbers indicate percentages within indicated electronic gates. Sublethally irradiated B6 WT mice were grafted with CBA BM under cover of 2.10⁶  DEREG Treg in vitro cultured with (CBA × B6)F1 antigen-presenting cells. (C) Hematopoietic chimerism in PBMC and percentage of Foxp3-eGFP⁺ cells among CD4⁺ T lymphocytes in (D-E) blood and (F) spleen, lymph node (LN), and BM was assessed at the indicated time points. Indicated are mean values ± SD (n ≥ 3, 4 independent experiments). (G) Four weeks after BM grafting, chimeric mice were left untreated (white bars) or received (black bars) 2 consecutive injections of diphtheria toxin (DTx). Percentage of Foxp3-eGFP⁺ cells among CD4⁺ T lymphocytes was analyzed in spleen, lymph node (LN), BM, and PBMC. Indicated are mean values ± SD (n = 5, 2 independent experiments).