Human CD3⁺CD4⁺CD25⁺ T cells from DKO-hu HSC mice are functional Treg cells. Human Treg cells (huCD45⁺CD3⁺CD4⁺CD25⁺) from DKO-hu HSC mice were isolated by FACS (> 95% purity), and cocultured with naive CD4⁺CD25⁻ T cells from human PBMCs labeled with CFSE. Cells were activated either with antihuman CD3/CD28 mAb (A-C) or with mDCs pulsed with SEB (10 pg/mL; D-F). At 4 to 5 days after activation, cells were analyzed for CFSE levels by flow cytometry. Numbers indicate percentage of CFSE^low (proliferated) responder cells. (A) CFSE-labeled responder cells without CD3/CD28 stimulation. (B) CFSE-labeled responder cells with CD3/CD28 stimulation.(C) Treg cells plus CFSE-labeled responder cells (at a 1:2 Treg/responder ratio) with CD3/CD28 stimulation. (D) CFSE-labeled responder cells with SEB-pulsed mDCs. (E) CFSE-labeled responder cells and SEB-pulsed mDCs plus CD4⁺CD25⁻ naive T cells isolated from DKO-hu mice. (F) CFSE-labeled responder cells and SEB-pulsed mDCs plus CD4⁺CD25⁺ Treg cells. Both CD4⁺CD25⁻ control and CD4⁺CD25⁺ Treg cells were used at 1:1 ratio relative to responder cells. Data shown are representative of 2 independent experiments.