Long-term in vitro culture and vector comparison. (A) PBMCs transduced with the 1D3 TCR (■) and nontransduced (□) PBMCs were restimulated every 14 days (indicated by arrows) by addition of irradiated feeder cells and phytohemagglutinin. At the time points indicated, total cell numbers were determined. (B) TCR expression in 1D3-transduced cells was determined by staining with anti-CD8, anti-CD4, and A2.1-Mart-1_{(26-35, 27 A>L)} tetramer. The numbers indicate the percentage of tetramer⁺ CD8⁺ (■) and tetramer⁺ CD4⁺ (□) cells. (C) Four weeks after transduction, cytolytic activity of 1D3 TCR–transduced cells against different HLA-A2.1⁺, Mart⁺ cell lines—AKR (●), GDO (■), and 526 (♦)—was determined. The HLA-A2.1⁻, Mart⁺ cell line 938 (▴) was used as a control. Cytolytic activity of nontransduced cells is indicated by open symbols: AKR (○), GDO (□), 526 (◇), and 938 (▵). Error bars represent standard deviations (n = 3) (D,E) Transient viral supernatant of the indicated vectors all encoding the 1D3 TCR was used to transduce Jurkat/MA cells (D) or PBMCs (E). Four days after transduction, TCR expression in Jurkat/MA cells was determined by staining with anti-TCRαβ. Expression in PBMCs was determined by staining with anti-CD8 and A2.1-Mart-1_{(26-35,27 A>L)} tetramer. The numbers indicate the percentage of tetramer⁺ CD8⁺ cells.