TCR affinity does not modulate Treg suppressive activity. (A-B) Freshly isolated cord blood Tregs were transduced with an A2-SL9–specific (awt/b6) TCR or mock-transduced and expanded for 12-18 days. Effector CD8 T cells derived from adult PBMCs were also transduced with the same A2-SL9–specific (awt/b6) TCR and cultured until they stopped expanding. Transduced or nontransduced Tregs were mixed with A2-SL9–specific CD8 T cells at the indicated ratios, along with K.A2.SL9.CD86.4-1BBL aAPCs. The ability of the polyclonal Tregs (▴) or A2-SL9–specific Tregs (■) to suppress the proliferation of the A2-SL9–specific CD8 T cells was measured using the bead-count assay described in supplemental Figure 1. Representative data are shown in panel A and composite data from 4 independent experiments are shown in panel B. (C) Suppressive activity of the A2-SL9 TCR-transduced Tregs was determined by mixing Tregs at the indicated ratios with CFSE-labeled CD8 T cells that had been transfected with RNA encoding the a2/b1 or the a11/b6 A2-SL9–specific TCR. These T-cell mixtures were stimulated with the aAPCs described in Figure 1B, and CFSE dilution was measured 5 days later by flow cytometry. The percent suppression was calculated as described in the “In vitro suppression assay.” A negative value indicates that more cell divisions were measured in these conditions relative to the control without Tregs.