A MHC class I–restricted TCR that is unable to redirect CD4 T effector responses is capable of conferring robust Ag-specific suppression to Tregs. RNA encoding either the A2-SC9 specific TCR (C-D) or the A2-SL9 TCR (B-D) was transfected into an equal mixture of resting and purified CD4 and CD8 T cells and, after overnight culture, the ability to bind SC9 tetramer (A-C), introduced Vβ13 (B,D), SL9 tetramer (E,G), or introduced Vβ5 (F,H) was measured by flow cytometry using nontransfected cells stained with the same reagent to set the respective gates. (I) An equal mixture of CD4 and CD8 T cells were transfected with A2-SC9–specific TCR and incubated with the aAPCs described in Figure 1 or phorbol 12-myristate 13-acetate and ionomycin for 5 hours. The ability of these stimulated cells to produce IFNγ and IL-2 was measured by intracellular cytokine staining. Freshly isolated cord blood Tregs were isolated, and RNA transfected with nothing (polyclonal; J), A2-SL9 (K), or A2-SC9 (L). These Tregs were mixed at various ratios with CFSE-labeled CD8 T cells that had been transfected with awt/b6 A2-SL9–specific TCR. These T-cell mixtures were stimulated with aAPC as 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.”