MHC class I–restricted TCR-transduced Tregs can be expanded and enriched by aAPCs expressing cognate pMHC. (A) awt/b6 A2-SL9 TCR transduced and nontransduced Tregs were allowed to expand and after resting (on day 12), transduced Tregs were restimulated with K64.A2.SL9.41BBL and nontransduced Tregs were restimulated with K.64.A2.41BBL coated with anti-CD3 (OKT3) Ab. Population doublings were determined at the indicated time points. (B) Transduced Tregs (22% TCR positive) were restimulated with either Ag-specific stimulation provided by K.64.A2.SL9.41BBL or polyclonal stimulation provided by K.64.A2.41BBL coated with OKT3 Ab. The frequency of TCR-transduced Tregs was reevaluated by SL9 tetramer staining 7 days after restimulation. (C) A2-SL9 TCR-transduced Tregs or nontransduced Tregs from panel B were again restimulated using K.64.A2.SL9.41BBL or K.64.A2.41BBL coated with OKT3 Ab, respectively. After another 7-10 days of culture, transduced Tregs (black line) and nontransduced Tregs (gray shading) were stained for Foxp3, CD27, and CD62L. Isotype control staining is shown as a thin black line. (D) TCR-transduced Tregs, nontransduced Tregs, and CD4 T cells were expanded after CD3/28 bead stimulation. After resting, cells were treated with phorbol 12-myristate 13-acetate plus ionomycin for 6 hours and intracellular IL-2 was measured by flow cytometry. These data are representative of 3 independent experiments.