Target Ags must be expressed on the same cell to observe bystander immunosuppression. (A) RNA encoding the A2-SC9 TCRs was transfected into Tregs, and the ability of these cells to bind A2-SC9 tetramer was measured the following day by flow cytometry. (B) Same as panel A except TCR encoding RNA was not added before the transfection (mock control). (C) RNA encoding the A2-SL9 TCRs was transfected into a mixture of CD4 and CD8 Teffs, and the ability of these cells to bind A2-SL9 tetramer was measured the following day by flow cytometry. (D) Same as panel C except TCR encoding RNA was not added before the transfection (mock control). (E) A2-SL9 Teffs were mixed with the indicated ratios of mock-transfected, polyclonal Tregs. These T-cell mixtures were then incubated with an equal number of K.A2.SL9 and K.A2.NY-ESO aAPCs. The final ratio of T cells to aAPCs was 1. (F) A2-SL9 Teffs were mixed with the indicated ratios of A2-SC9–transfected Tregs. These T-cell mixtures were then incubated with an equal number of K.A2.SL9 and K.A2.NY-ESO aAPCs. The final ratio of T cells to aAPCs was 1. (G) A2-SL9 Teffs were mixed with the indicated ratios of mock-transfected, polyclonal Tregs. These T-cell mixtures were then incubated with an equal number of K.A2.SL9.NY-ESO aAPCs. The final ratio of T cells to aAPCs was 1. (H) A2-SL9 effectors were mixed with the indicated ratios of A2-SC9–transfected Tregs. These T-cell mixtures were then incubated with an equal number of K.A2.SL9.NY-ESO aAPCs. The final ratio of T cells to aAPCs was 1. A cartoon denoting the cell mixtures is displayed below each data panel. The percent suppression was calculated as described in “In vitro suppression assay.” A negative value indicates that more cell divisions were measured in these conditions relative to the control without Tregs.