Cultured cord blood–derived CD4⁺CD25⁺ cells markedly suppress MLRs. Analysis of suppressor cell function in MLR assays by proliferative inhibition. (A) Kinetic curves of proliferation over a 1-week MLR. Control MLR (▴) cord blood–derived cells essentially block MLRs (•); directly MACS-selected adult cell lines had weak suppressor function (□); and stringently selected adult cells (CD25⁺⁺lineage^-) had moderate potency (▪). CD25^- cells are not suppressive (▵). Results representative of 10 experiments. Cultures are pulsed daily with ³H-thymidine for the last 16 hours of culture. (B) Scatter plot showing the consistency of suppression (at day 6 of MLR) of individual cell lines; cord blood–derived (•) versus direct MACS adult cell lines (□) or stringently purified adult lines (CD25⁺⁺lineage^-) (▪). (C) Graded numbers of cultured T_reg cells were added to the MLR reaction to determine the minimum number needed for potent inhibition. Up to a 1:32 dilution (roughly 1560 suppressors per 50 000 responders) still would markedly impair MLRs when using cord blood–derived suppressor cell lines (•), versus 1:16 for a selected potent subset of stringently purified adult lines (pCD25⁺⁺lin^-) (▵). Two lines each are shown, representative of 6 adult- and cord blood–derived suppressor cell lines. (D) Maturation of DCs prior to MLR, by LPS or TNF–poly I:C combination, or inclusion of these stimulating factors in the MLR fails to bypass suppression. Representative of 3 experiments.