Rag^–/– hosts were reconstituted with flow-sorted WT or Ctla-4^–/– Tregs. Ctla-4^–/– and WT Tregs were purified from mixed BM chimeras generated with an 80:20 ratio of Ctla-4^–/–/WT BM. Fully reconstituted chimeras (>3 months after irradiation) were treated with IL-2/JES6-1 at –5, –4, and –3 days to expand Treg numbers before harvest at day 0. Treg reconstitution was performed as described in Figure 1. (A) MFI of CD80 (left) and CD86 (right) on migratory DCs from pLN (n = 4 per group). Bars represent mean ± SEM with individual values indicated by the open circles. Data are from 1 of 2 independent experiments. (B) 1 × 10⁶ CFSE-labeled WT polyclonal CD4⁺ T cells were transferred into Rag^–/– mice, either unreconstituted or reconstituted with WT or CTLA-4–deficient Tregs (n = 3 per group). All groups were treated with second daily IL-2/JES6-1 throughout the course of the experiment. One group of Rag^–/– mice reconstituted with WT Tregs received water supplemented with 5 mg/ml 1-MT from 3 days before CFSE-labeled cell transfer until organ harvest. Shown are representative CFSE division profiles from the pLN and spleen at day 7 after transfer. (C) The ratio of fully divided CFSE^– T cells (>7 divisions) to CFSE⁺ T cells (0–3 divisions) within adoptively transferred CD4⁺ T cells was calculated for pLN and spleen, and summarized for all LIP experiments performed, totaling 6 independent experiments for Rag^–/– and WT Treg groups, 3 experiments with Ctla-4^–/– Tregs, and 2 experiments for 1-MT treatment (n = 6–19 per group). Statistical analysis was performed using one-way ANOVA with a Newman-Keuls post-test. **P < 0.01, ***P < 0.001, ****P < 0.0001.