Anti cd3 mab clone okt3

Peptide-specific CD8⁺ T cells were expanded using an aAPC as described previously (31 (link), 32 (link), 40 (link)–42 (link)). PBMCs were isolated from healthy volunteers and stimulated with 50 ng/ml anti-CD3 mAb (clone OKT3) in the presence of 100 IU/ml human IL-2 (Novartis) 3 days before transduction. Activated T cells were retrovirally transduced with TCR genes by centrifuging 1 hour at 1,000 g at 32°C. Following transduction, CD8⁺ T cells were purified and plated at 2 × 10⁶ cells/well in RPMI 1640 supplemented with 10% human AB serum. The stimulator wt-aAPC or mut-aAPC was pulsed with 10 μg/ml A2-restricted wild-type MART1_27–35 or heteroclitic NY-ESO-1_157–165 peptide for 6 hours at room temperature. The aAPC was then irradiated at 200 Gy, washed, and added to the responder T cells at a responder to stimulator ratio of 20:1. Starting the next day, 10 IU/ml IL-2 (Novartis) and 10 ng/ml IL-15 (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. A2/HIV pol_476–484 peptide was used as a control.

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.