Recombinant human granulocyte macrophage colony stimulating factor

Blood samples were donated by healthy volunteers who had undertaken informed consent in accordance with local Research Ethics Committee approval. Peripheral blood mononuclear cells were isolated from citrated peripheral blood samples by density gradient separation using Lympholyte (Cedarlane Labs), and subsequent CD14+ positive selection using the MACS Miltenyi Biotec Human CD14 microbead protocol (Miltenyi Biotec). CD14+ cells (3 × 10⁵ cells per well) were differentiated into macrophages using recombinant human granulocyte-macrophage colony-stimulating factor (200 ng/mL GM-CSF) and recombinant human interferon gamma (50 ng/ml IFNg) (Peprotech) in standard tissue culture DMEM media containing 10% fetal calf serum.
The human primary monocytes were extracted from the blood from donors who gave their samples voluntarily without any compensation. They gave informed consent via a Good Clinical Practice registered member of staff from Royal Papworth Hospital NHS Foundation Trust Cambridge under local ethics committee approval (REC No. 12/WA/0148). No human-derived cells were stored after the experiments had been completed.

Heparinised whole blood was obtained by venepuncture from the antecubital fossa of the arm of healthy volunteers after written informed consent (Ethics from University of Nottingham Ethics committee, ref 161–1711). Peripheral blood mononuclear cells (PBMC) were immediately separated by density centrifugation over Ficoll Histopaque 1077 (Sigma) followed by washes in endotoxin-free phosphate-buffered saline (PBS, Sigma). PBMC were washed in MACS buffer (PBS + 1% foetal calf serum (FCS, Sigma) + 2 µM EDTA (Sigma)) then incubated with CD14 microbeads (Miltenyi Biotech) and monocytes isolated by magnetic separation (purity routinely >95%). Purified CD14 + monocytes were differentiated into macrophages at 37 °C/5%CO₂ for 7 days at 1 × 10⁶/well in low-attachment 24-well plates (Corning Costar) in macrophage medium (RPMI 1640 (Sigma) supplemented with 10% FCS and 1% sodium pyruvate (Sigma)) and 20U/mL recombinant human granulocyte-macrophage colony-stimulating factor (Peprotech) with medium plus additives supplemented at day 4.

Dendritic cells (DCs) were generated from CD14⁺ cells isolated from PBMCs with CD14 MicroBeads (Miltenyi Biotec) and subsequent culturing with 1000 IU/mL recombinant human granulocyte-macrophage colony-stimulating factor (Peprotech) and 500 IU/mL human recombinant interleukin 4 (Peprotech), as described previously.^{17 (link),18 (link)} Epstein-Barr virus lymphoblastoid cell lines (EBV-LCLs) were generated from patient PBMCs using the viral supernatant from the B95-8 producer cell line (ATCC) using standard procedures and cultured in Iscove’s Modified Dulbecco’s Medium (Life Technologies) supplemented with 10% fetal calf serum (HyClone) and standard additives.

Human peripheral blood mononuclear cells (PBMNCs) were obtained from healthy volunteers (provided by Zhejiang Blood Center, Zhejiang, China), and SFs were isolated from non-tumoral gastric walls of the patients who underwent surgery in our hospital. Informed consent was obtained from all volunteers and patients. PBMNCs were isolated using a human Lymphoprep solution (Axis-shield PoC AS, Oslo, Norway), and cultured in a 10-cm Petri dish and incubated for 24 h to allow them to adhere to the dish’s surface. Adherent cells were induced to form immature DCs, supplemented with 120 ng/mL recombinant human granulocyte macrophage colony-stimulating factor (PeproTech, Offenbach, Germany), and 60 ng/mL recombinant human interleukin-4 (PeproTech) to decrease contamination by macrophages for 5 days. Nonadherent cells were harvested and used as human lymphocytes. SFs were prepared by transferring the gastric tissue to a T25 flask and cutting into 1mm³ pieces. Incubate the chopped material with 5 ml of trypsin EDTA for 5 min at 37 °C, and then the trypsin was Inactivated by adding 1 ml FBS. The cell pellet was obtained by centrifuging of suspension at 200 g speed for 10 min, which was transferred to a T25 flask containing DMEM medium with 20% FBS and 2 × Penicillin/Streptomycin) after resuspend using DMEM.

Monocytes were purified, cultured, and matured to monocyte-derived dendritic cells (MD-DCs) as described previously (2 (link)). Briefly, CD14⁺ cells were selected using magnetic beads (Miltenyi Biotec) and cultured for 7 days in the presence of 2 ng/ml rhIL-4 and 10 ng/ml recombinant human granulocyte-macrophage colony-stimulating factor (both from Peprotech, Rocky Hill, NJ). MD-DCs were matured by addition of 10 ng/ml recombinant human TNF (eBioscience) on day 6. Matured MD-DCs were pulsed with a 10 μM concentration of the relevant peptide. In parallel, autologous CD8⁺ T cells were purified using magnetic beads (Miltenyi Biotec) and labeled with Pacific Blue succinimidyl ester (PBSE; Life Technologies) as described previously (2 (link)). Subsequently, MD-DCs and CD8⁺ T cells were cocultured in the presence of anti-CD28 MAb (0.5 μg/ml; BD Biosciences) at a ratio of 1:30 (MD-DCs/CD8⁺ T cells). rhIL-2 (20 U/ml) was added on days 4 and 8. On day 12, cells were used for phenotypic and functional analyses.

The experimental procedures were approved by the Ethics Committee of Dalian Medical University and written consent forms were obtained from each volunteer participant. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation from freshly drawn peripheral blood (50 mL). Cells were allowed to adhere in plastic cell culture flask for 2 h at 37 °C in the incubator. Adherent cells were induced into DCs using 500 U/mL of recombinant human interleukin-4 (PeproTech) and 1000 U/mL of recombinant human granulocyte-macrophage colony stimulating factor (PeproTech) for 4 days, before treated with 1000 U/mL of tumor necrosis factor alpha (PeproTech) to generate mature DCs. Cells were then treated with DMSO (control), curcumin (25 μM), or apigenin (30 μM). Non-adherent cells were collected for cytokine-induced killer (CIK) cells using serum-free medium (Lonza, X-VIVO 15) supplemented with 1000 U/mL IFN-γ (Chemo Wanbang, China) for 24 h. Then cells were treated with 100 ng/mL of anti-CD3 antibody and 1000 U/mL of recombinant human IL-2 (Sanyao, China). The medium was replenished every 3 days. At day 7, CIK cells were harvested and separated into equal parts for co-culture with previously treated DCs at a ratio of 3:1 for another 7 days. CIK and DC-CIK cells were used in co-culture assays with A375 cells according to methods described above.