Purified cultured CD4⁺CD25⁺ cells markedly suppress MLRs. The MLR cultures contain various test-cell populations at a 1:2 suppressor-responder cell ratio. Shown are kinetic curves of proliferation over a 1-week MLR. Cultures were pulsed daily with ³H-thymidine for last 16 hours of culture. (A) Representative cell lines derived from CD4⁺CD25⁺ cells (double-column lineage-depletion purification) are good suppressors (•). In contrast, cell lines derived from CD25^- cells (▵) augment the MLR versus control MLR cultures (□). Results are representative of 22 experiments. (B) Fresh standard MACS-purified CD4⁺CD25⁺ cells (♦), added to the MLR, compared with a representative weakly suppressive CD4⁺CD25⁺ cell line (•) versus control MLR (□). Results are representative of 4 experiments. (C) The MLR was nearly completely blocked by addition of potently suppressive cultured CD4⁺CD25⁺ cells (•) versus control MLR (□). Representative of 7 potent suppressor cell lines, tested in 14 MLRs. Error bars represent 1 SD above and below the mean.