CD4^highCD25⁺ alloantigen-specific Treg have no cytotoxic capacity, and their suppressor function is dependent on cell-cell contact and partially relies on CTLA-4 expression. CD4^highCD25⁺ Treg or CD4^mediumCD25⁻ T cells were sorted after 7 days of allostimulation as shown in Figure 3B. (A) Cytotoxic capacity of induced CD4^highCD25⁺ Treg. (B) The alloantigen-specific suppressor function of CD4^highCD25⁺ Treg is cell-cell contact dependent. (C) Neutralizing anti–CTLA-4 mAb partially reverses the alloantigen-specific suppression mediated by CD4^highCD25⁺ Treg, but neutralizing mAbs to IL-4, IL-10, TGF-β, and GITR fails to reverse that suppression. Responder (R) CD4⁺CD25⁻ and gamma-irradiated stimulator PBMC (S) were cocultured with or without sorted CD4^highCD25⁺ Treg or CD4^mediumCD25⁻ T cells. The cytotoxic activities (A) of human IL-2–activated NK cells against K562 cells were set as positive controls (PC). Stimulator (S) or responder (R) cells alone were set as controls. For transwell experiments (B), the same amount of responder (R) and stimulator (S) cells were plated in the bottom wells of a transwell system. The top well insert was inoculated with same amount of sorted CD4^highCD25⁺ Treg. For the blocking experiments (C), the neutralization mAbs (□) and their relevant isotype controls (■) were added in the coculture system. Proliferation (y-axis) is shown for day 3 of cultures. Data for 4 different experiments are shown (n = 4). The 2-tailed unpaired Student t tests were used for comparison. *P < .01.