PBMC were expanded over four rounds of peptide-specific stimulation, with cells being analysed by flow cytometry at the end of rounds one and four. Briefly, 3×106 PBMC were initially resuspended in NGM at 1.5×106 cells/mL in a 24-well plate, in the presence of rhIL-2 (20 U/mL, Roche, Basel, Switzerland), rhIL-7 (2 ng/ml, R&D Systems, Abingdon, Oxfordshire, UK), rhIL-15 (5 ng/ml, R&D Systems) and rmIL-21 (0.5 ng/ml, R&D Systems). Peptide-specific stimulation was performed by adding pCEA691 or CMV pp65 directly into specific wells, at a final concentration of 10μM. After a first round of 7–9 days duration, cells were moved to re-stimulation. Three rounds of re-stimulation were performed as described below, each of which were 7–9 days duration.
Re-stimulation was conducted by re-suspending 5×105 cells in 2 ml of cytokine-supplemented NGM in a new 24-well plate, and stimulating them with irradiated (70 Gy) T2 cells (2×105) pulsed with the appropriate peptide, at a final concentration of 10μM. Irradiated (35 Gy) autologous PBMCs or PBMC from healthy HLA-A2+ donors obtained from the National Blood Service (Colindale, UK) were used as feeder cells (2×106).
Re-stimulation was conducted by re-suspending 5×105 cells in 2 ml of cytokine-supplemented NGM in a new 24-well plate, and stimulating them with irradiated (70 Gy) T2 cells (2×105) pulsed with the appropriate peptide, at a final concentration of 10μM. Irradiated (35 Gy) autologous PBMCs or PBMC from healthy HLA-A2+ donors obtained from the National Blood Service (Colindale, UK) were used as feeder cells (2×106).