DMF5 and gp100(154) TCR retroviral constructs conferred greater antitumor reactivity to donor PBLs than the original DMF4 receptor. (A) Donor PBLs were stimulated with anti-CD3 mAb OKT-3 and separated into CD4 and CD8 populations before retroviral transduction with DMF4, DMF5, or gp100(154) TCR constructs. TCR expression was analyzed 7 days later by tetramer staining and flow cytometry. (B) Donor PBLs transduced with retroviral TCR constructs were cocultured with T2 cells pulsed with MART-1:27-35 or gp100:154-162 peptide, and IFN-γ secretion was measured by ELISA. (C) Transduced PBLs were cocultured with mel624⁺ melanomas, and tumor target lysis was evaluated by ⁵¹Cr-release assay. Cells did not lyse HLA-mismatched tumors (data not shown).