Expansion of Tumor-Specific CD8+ T Cells

Peripheral T cells were stimulated with 50 ng/mL anti-CD3 mAb (clone OKT3) and 100 IU/mL human IL2 (Novartis) for 3 days and retrovirally transduced with TAK1α or β gene fused with ΔNGFR or with ΔNGFR alone (control). CD8⁺ T cells were purified using the CD8⁺ T Cell Isolation Kit (Miltenyi Biotec) and subsequently expanded using aAPCs as described previously (38 (link), 39 (link), 41 (link)). Briefly, CD8⁺ T cells were plated at 2×10⁶ cells/well in RPMI 1640 supplemented with 10% human AB serum. Where indicated, A24-aAPCs was pulsed with 1 μg/ml A24-restricted WT1_235–243 peptide (GenWay Biotech) for 6 hrs at room temperature and irradiated at 200 Gy before use. B57-aAPCs were always used without any peptide pulse. Starting the next day, 10 IU/ml IL2 (Novartis) and 10 ng/ml IL15 (Peprotech) were added to the cultures every three days. T cells were harvested, counted, and restimulated every week. T-cell analysis was performed one day prior to or on the day of restimulation.