Cultured Treg cells impair the activation of responding T cells and can suppress MLRs driven by mature DCs. MLRs were evaluated for expression of activation antigens after 1 day of culture. Shown are control MLRs stained for CD69 (A), CD25 (B), and OX40 (CD134; C). In suppressed MLRs responding T cells were first gated on HLA-A2 to distinguish them from the HLA-A2^- suppressor cells. Shown are responder cells from suppressed MLRs stained for CD69 (D), CD25 (E), and OX40 (CD134; F). Results are representative of 3 experiments. (G) Maturation of DCs prior to MLR by LPS or TNF/poly (I:C) combination, or inclusion of these stimulating factors in MLRs fails to bypass suppression. Control MLR cultures (▦) and suppressed MLR (▪).