Interleukin 7 (il 7)

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.

Peripheral blood was obtained from healthy volunteers. Ethical permission was granted by the School of Medicine, Zhejiang University, and all of the blood samples were handled following the required ethical and safety procedures. PBMCs were isolated by Ficoll density gradient centrifugation (Dakewe). Then T lymphocytes were purified using the Pan T Cell Isolation Kit (Miltenyi Biotec) and activated with CD3/CD28 T cell Activator Dynabeads (Thermo Fisher Scientific) and cultured in X‐VIVO15 Serum‐free Hematopoietic Cell Medium (Lonza) supplemented with 10% FBS, 1% penicillin/streptomycin, 5 ng ml⁻¹ interleukin‐7 (IL7) and 5 ng ml⁻¹ interleukin‐15 (IL15) (Novoprotein). The medium was changed every 2 days, and cells were plated at 10⁶ cells ml⁻¹.

Human blood was obtained from healthy donors with written approval. PBMC was isolated using human lymphocyte isolation kit (Dakewe, Shenzhen, China). T cells were further purified using Pan T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and cultured with X-VIVO 15 medium (Lonza) supplemented with 10% FBS, 1% P/S, 5 ng/ml IL-7 (Novoprotein, Shanghai, China) and 5 ng/ml IL-15 (Novoprotein). Immediately after T cell isolation, they were stimulated with CD3/CD28 T cell Activator Dynabeads (Invitrogen, Carlsbad, United States) at a ratio of 1:1. T cells transduction was performed after 48 h. Retrovirus was produced from 293T cell lines. T cells were transduced with retrovirus supernatants in retronectin (Takara, Otsu, Japan)-coated plates. To enhance transduction efficiency, the plates were centrifuged with 3000 rpm for 90 min.