Generation of MP1-specific T cells by coculture with T-APCs. (A) Expansion of MP1-tetramer⁺ T cells by coculture with autologous CD8⁺HyMP1⁺ T-APCs. PBMCs from a homozygous HLA-A2⁺ donor were cocultured for 21 days with 5 U/mL rhIL-2 in the presence of (top row) CM alone or (middle row) a 5:1 (responder/stimulator) ratio of autologous γ-irradiated hygromycin B–resistant CD8⁺Hy⁺MP1^– T-APCs (transfected with the plasmid pMGP̂ac), or (bottom row) autologous γ-irradiated CD8⁺HyMP1⁺ T-APCs (transfected with the plasmid HyMP1-pEK). Cultures were supplemented with irradiated T-APCs every 7 days. Responding cells were analyzed at the indicated time points by flow cytometry using FITC-conjugated anti-CD8 and PE-conjugated MP1-HLA-A2*0201 tetramer. Propidium iodine⁺ cells were excluded from analysis. T-APCs themselves are MP1-tetramer^neg (data not shown). The percentage of tetramer⁺ T-cells is shown after electronic gating on CD8⁺ T cells. (B) Expansion of MP1-tetramer⁺ T cells by coculture with autologous CD4⁺ HyMP1⁺ T-APCs. PBMCs from a heterozygous HLA-A2⁺ donor were cocultured for 7 days with 5 U/mL rhIL-2 in the presence of (top row) CM alone, (middle row) a 5:1 (responder/stimulator) ratio of autologous γ-irradiated hygromycin B–resistant CD4⁺Hy⁺MP1^– T-APCs (transfected with the plasmid pMGP̂ac), or (bottom row) autologous γ-irradiated CD4⁺HyMP1⁺ T-APCs (transfected with the plasmid HyMP1-pEK). Responding cells were analyzed at the indicated time points by flow cytometry using anti–CD8-FITC and PE-conjugated MP1-HLA-A2*201 tetramer. The percentage of tetramer⁺ T cells is shown after electronic gating on CD8⁺ T cells. (C) Proliferation of CD8⁺MP1-tetramer⁺ T cells on T-APCs. 5 × 10⁴ HLA A2⁺ CD8⁺ MP1-tetramer⁺ T-cells were cocultured at a 1:1 (responder/stimulator) ratio with media or with thawed γ-irradiated autologous CD8⁺Hy⁺ and CD8⁺HyMP1⁺ T-APCs. The analysis was performed in quadruplicate, and the mean incorporated ³H-thymidine is shown along with SD. (D) Numerical expansion of CD8⁺MP1-tetramer⁺ T cells by T-APCs. 25 × 10⁶ HLA A2⁺ PBMCs were cocultured for 21 days at a 5:1 (responder/stimulator) ratio in low-dose rhIL-2 with thawed γ-irradiated autologous CD8⁺HyMP1⁺ T-APCs. Cultures were supplemented with irradiated T-APCs every 7 days. Viable cells were counted based on the trypan blue dye exclusion method.