Generating MAGE-A3-specific T cells for Immunotherapy

T cells obtained from healthy donors were purified using magnetic beads (Miltenyi Biotec) and then transduced with TCR genes specific for the DP4/MAGE-A3_243–258 complex^{33 (link)}. IFN-γ ELISPOT assays were performed as previously described^{32 (link),34 (link),35 (link)}. Briefly, PVDF plates (Millipore, Bedford, MA) were incubated with capture mAb (1D1K; MABTECH, Mariemont, OH) at a 1:200 ratio overnight at 4 °C. After thorough washing with 2%FCS/PBS, DP4-MAGE-A3_243–258-specific T cells were then incubated with 2 × 10⁴ of the indicated stimulator cells for 20–24 hours at 37 °C. After washing with PBS, plates were incubated with biotin-conjugated detection mAb (7-B6–1; MABTECH) at a 1:2000 ratio overnight at 4 °C. After washing with PBS, plates were incubated with HRP-conjugated Streptavidin (DAKO, Carpenteria, CA) at 1:5000, and IFN-γ spots were subsequently developed. The peptides used as controls were: MAGE-A3_243–258 (₂₄₃KKLLTQHFVQENYLEY₂₅₈) (Genway, San Diego, CA) and tetanus toxin_947–967 (₉₄₇FNNFTVSFWLRVPKVSASHLE₉₆₇) (Genway, San Diego, CA).