Il 21

Monocyte-derived dendritic cells (DCs) were generated from PBMCs. Monocytes were isolated from PBMCs by adherence to a plastic tissue culture dish (Becton Dickinson) and cultured in the presence of 1000 IU/mL of granulocyte-macrophage colony-stimulating factor (R&D System) and 1000 IU/mL of interleukin (IL)−4 (R&D System) in AIM-V medium (Invitrogen) supplemented with 2% human AB serum (SIGMA) for 7 days. On day 5, OK-432 (Chugai Pharmaceutical) was added in the culture medium to induce the maturation of DCs (final concentration: 0.1 KE/mL). Autologous CD8⁺ T cells (3.0 × 10⁵ cells) purified from PBMCs with CD8-Positive Isolation Kit (Dynal) were cultured with DCs (1.5 × 10⁴ cells) with 20 μg/mL of each peptide in AIM-V medium containing 2% human AB serum, 10 ng/mL of IL-7 (R&D System), and 30 ng/mL of IL-21 (Cell Genix). After 3 days of the culture, CD8⁺ T cells were restimulated with DCs and 20 μg/mL of each peptide. DCs were prepared by the same procedure described above. At day 7 of the culture, CD8⁺ T cells were further stimulated with 48 IU/mL of IL-2 (Novartis), 5 ng/mL of IL-7 (Novoprotein), and 5 ng/mL of IL-15 (Novoprotein) in AIM-V medium supplemented with 2% human AB serum. At day 11 of the culture, CD8⁺ T cell responses were examined by an IFN-γ ELISPOT assay.

Matured DCs and pre-thawed CD8⁺ cells were cocultured at a 1:4 ~ 10 ratio in AIM-V medium with 2% autologous serum and IL21 (30 ng/ml, CellGenix) in flask (T75 or T175) for two or three days. IL2 (40 ~ 50IU/ml, CellGenix), IL-7 (5 ~ 10 ng/ml, PrimeGene), and IL15 (1 ~ 2 ng/ml, PrimeGene) were added into the medium. Then cells were incubated for 8–10 days. These T cells were re-stimulated again, and incubated for the next 8–10 days. T cells were collected and transferred into infusion bags for treatment.