Lymphopenic Rag^–/– mice were reconstituted with purified Tregs by adoptively transferring either 0.25 × 10⁶, 0.5 × 10⁶, 1.25 × 10⁶, or 2.5 × 10⁶ bead-selected CD4⁺CD25⁺ Tregs. Mice were treated with IL-2/JES6-1 complexes second daily to support Treg expansion. (A) Donor cells were distinguished from host cells by a CD45 allelic difference. Shown is expression of CD25 and Foxp3 on donor CD4⁺ T cells in pLN (n = 4 per group). (B) The total number of CD4⁺CD25⁺Foxp3⁺ Tregs was calculated for each Treg dose in pLN, mLN, and spleen of Treg-reconstituted mice, and for WT controls (n = 4 per group). Data in A and B are from a single experiment. The comparison of WT, Rag^–/– mice, and Rag^–/– mice reconstituted with 2.5 × 10⁶ CD4⁺CD25⁺ Tregs is representative of more than 10 independent experiments. (C) At day 7 after Treg transfer, 1 × 10⁶ CFSE-labeled CD4⁺CD25^– conventional T cells were adoptively transferred into unreconstituted or Treg-reconstituted Rag^–/– mice, as described for A. All mice were treated with second daily IL-2/JES6-1 complexes throughout the experiment. Representative CFSE division profiles of conventional CD4⁺ T cells are shown at 7 days after transfer (left panels). (D) The ratio of CFSE^– cells (>7 rounds of division) to CFSE⁺ T cells (0–3 rounds of division) was calculated. Data in C and D are from one experiment with n = 3–4 per group. (E) Comparison of the ratio of divided to undivided cells in the pLN, mLN, and spleen of unreconstituted or Treg-reconstituted Rag^–/– mice. Data are pooled from 6 independent experiments (n = 9–19 per group). Data in B and D were analyzed using one-way ANOVA with a Newman-Keuls post-test, while data in E were analyzed using a 2-tailed unpaired t test. Bars represent mean ± SEM with individual values indicated by the open circles. *P < 0.05, **P < 0.01, and ****P < 0.0001.