(A) Rag^–/– mice were reconstituted with 2.5 × 10⁶ Tregs or naive conventional T cells at a dose of 1.25 × 10⁶ or 2.5 × 10⁶ (n = 4 per group). Naive conventional T cells were flow sorted as CD3⁺CD4⁺CD25^–CD38^–CD44^–. Expression of CD80 (left panel) and CD86 (right panel) by migratory DCs in pLN is shown. Data are representative of 2 independent experiments. (B) 1 × 10⁶ CFSE-labeled polyclonal CD4⁺ T cells were transferred into unreconstituted, Treg-reconstituted, or conventional T cell–reconstituted Rag^–/– mice. Second daily IL-2/JES6-1 treatment was continued for all groups until lymphoid organ harvest 7 days after CFSE-labeled cell transfer. Adoptively transferred CD4⁺ WT T cells were identified based on differential expression of CD45 alleles. Left panel: Representative CFSE division profiles from the pLN day 7 after transfer. Right panel: Ratio of CFSE^– T cells (>7 divisions) to CFSE⁺ T cells (0–3 divisions) within adoptively transferred T cells was calculated (n = 6 per group). Data are pooled from 2 independent experiments. (C) Proliferation of adoptively transferred 5C.C7 T cells in response to 1 μg i.v. peptide in Rag^–/– hosts, either unreconstituted or reconstituted with 2.5 × 10⁶ Tregs or conventional T cells (n = 4 per group). Left panel: Representative CFSE division profiles in pLN of Rag^–/– hosts (unfilled histograms) compared with WT controls (filled histograms) 3 days after adoptive transfer of 5C.C7 T cells. Right panel: MFI of CFSE expression by adoptively transferred 5C.C7 T cells. Data are representative of 2 independent experiments. Statistical analysis of A–C was performed using one-way ANOVA with a Newman-Keuls post-test. Bars represent mean ± SEM with individual values indicated by the open circles. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.