Suppressive property of CD4⁺CD25⁺ cells is not dependent on suppressive cytokines or other soluble factors and cannot be explained by IL-2 consumption by these cells.

(A) 5000 CTLL-2 cells (▪) were cultured with various concentrations (Conc) of rh–IL-2 in the presence or absence of either CD4⁺CD25⁺ (⋄) or CD4⁺CD25⁻ (○) cells (2 × 10⁴cells/well) for 36 hours with ³H-thymidine added during the last 12 hours of culture. The proliferation of CD4⁺CD25⁺ or CD4⁺CD25⁻cells to IL-2 alone is negligible (mean cpm = 69 and 95, respectively) compared with that of the CTLL-2 cells in the presence of IL-2. (B) Supernatants from cultures of either CD4⁺CD25⁺or CD4⁺CD25⁻ (1 × 10⁴) cells with ACs (2 × 10⁴) and PHA (2 μg/mL) were measured for the production of IL-10 and TGF-β in 3 separate experiments. (C,D) CD4⁺CD25⁻ cells (1.5 × 10⁵cells/well) were cultured in 24-well plates in the presence of ACs (1.5 × 10⁵) and PHA (2 μg/mL). CD4⁺CD25⁺ or CD4⁺CD25⁻cells (1.5 × 10⁵) cells were added to the upper or lower chamber as indicated (ACs were present in both the lower and upper chambers). The proliferative responses were measured separately from the cells harvested from the lower wells (C) or upper wells (D) after 3 days culture. (E) CD4⁺CD25⁺ and CD4⁺CD25⁻ (1 × 10⁴) cells were cultured in various combinations with ACs (1 × 10⁴) plus PHA (2 μg/mL) and the indicated antibody or IL-2.