Cultured CD4⁺CD25⁺ cells consistently and markedly suppress MLR proliferation and cytokine secretion. Potent CD25⁺ suppressor cell lines were tested in multiple MLRs from various unrelated donors. (A) Shown are 8 separate MLRs displaying variance of control and suppressed proliferation. Most all donor combinations were markedly impaired. Control MLR cultures (▦) and suppressed MLR (▪). Results are representative of more than 20 experiments with 7 different potent suppressor cell lines. (B) Graded numbers of potent cultured suppressor cells were added to the MLR to determine the minimum number needed for inhibition. Up to a 1:16 dilution (roughly 3125 suppressors) still would markedly impair the MLR when using the most potent suppressor cell lines. Three plots are shown representative of 6 potent suppressor cell lines. Error bars represent 1 SD above and below the mean. (C) Daily assessment of IL-2 levels in culture supernatant reveals profound block in IL-2 accumulation in suppressed MLR cultures (•) versus control MLR cultures (□). Representative of 4 MLRs. (D) Assessment of other cytokines produced by activated T cells reveals profound impairment of accumulation. Significant levels of TNF-α, IFN-γ, GM-CSF, IL-6, or IL-10 are not produced. Shown are levels on day 6, peak of accumulation in control MLR cultures (▦) versus suppressed MLR cultures (▪). Representative of 4 MLRs.