TCR-transduction does not alter the ability of Tregs to function in a non-Ag–specific suppressive activity. (A) Primary human Tregs or CD4 T cells isolated from cord blood samples were stimulated with anti-CD3/28–coated beads for 3 or 1 day(s), respectively, and then either mock transduced or transduced with lentiviral vectors encoding the A2-SL9 TCR awt/b6. After 7-10 days of additional culture, transduction efficiency was determined by staining for the A2-SL9 Vβ-specific chain (Vβ5a), and the percentage of TCR transduced cells was determined by flow cytometry. (B) awt/b6 A2-SL9 TCR-transduced Tregs from panel A were cocultured with 1 × 105 CFSE-stained PBMCs at ratios ranging from 1:2 (1 Treg: 1 PBMC) to 1:32. Cells were stimulated with anti-CD3 Ab-coated beads at a ratio of 1 PBMC to 1 bead for 5 days, and then FACS analysis was performed to measure CFSE dilution of CD8 T cells. The percent suppression was calculated as described in the “In vitro suppression assay.” (C) Data were compiled from 3 independent experiments showing the percent suppression achieved at a 1:8 Treg:Teff ratio. Each symbol represents 1 experiment.
