Expansion of CD4⁺CD25⁺ suppressor cell lines. Proliferation of CD4⁺CD25⁺ cells in short-term assays and accumulation in long-term cultures. (A) Proliferation of highly purified CD4⁺CD25^- cells (▵) and double-column lineage-negative CD4⁺CD25⁺ cells (▪), in short-term 96-well ³H-thymidine incorporation assays. CD4⁺CD25^- cells markedly proliferate, whereas CD4⁺CD25⁺ cells minimally and transiently proliferate. (B) Augmentation of proliferation of highly purified double-column lineage-negative CD4⁺CD25⁺ cells (▪), in short-term 96-well ³H-thymidine incorporation assays. IL-2 at 100 IU/mL augments expansion (♦). However, irradiated CD4⁺CD25^- feeder cell supplementation (1:1 ratio; •) provides for increased expansion, which is more sustained. Representative of 4 experiments. (C) Long-term culture accumulation of CD4⁺CD25⁺ cell lines. Cell lines stimulated once with anti-CD3/anti-CD28 mAb-coated beads (□), or with immobilized anti-CD3 (▪), both supplemented with feeder cells. Cells were split and fed IL-2 every 3 to 4 days as needed. Data are reported as fold expansion of cell number and are representative of 22 cultures for anti-CD3/CD28 mAb-coated beads and 3 cultures for plastic-immobilized anti-CD3 mAbs. Error bars represent 1 SD above and below the mean.