Mononuclear cells were isolated from 50 mL of bone marrow under sterile condition and induced into DCs in vitro in the serum-free culture medium (AIMV) containing granulocyte-macrophage colony-stimulating factor (GM-CSF) 1000 U/mL, inter- leukin -4 (IL-4) 1000 U/mL, stem cell factor (SCF) 10 ng/mL, TNF-a 1000 U/mL, IL-6 10 ng/mL, IL-3 10 ng/mL.
Ten days later, the peripheral blood mononuclear cells (PBMCs) were collected with Cobe Spectra Apheresis System (COBE Spectra) and adjusted to a density of 1×106/mL. DCs and PBMCs were co- cultured in the culture medium (AIMV) supplemented with IFN-y 2000 U/mL, IL-2 1000 U/mL, anti-CD3 monoclonal antibody 50 μg/L. The culture medium was changed every two to three days, and the cells were cultured in Cellstar cell culture flasks (650 mL) with 5% CO2 at 37°C. The expansion period was maintained for 10–15 days till the cell number was around 5×109 for every patient.
After centrifugation, DC-CIK cells were washed once with 0.9% sodium chloride including 2% albumin and then suspended in 300–400 mL 0.9% sodium chloride with 5% albumin and IL-2 1000 U/mL in the 500 mL medical infusion bags.
DC-CIK cells were divided into two parts and infused to the patients themselves at an interval of 24 hours. Usually, the final DC-CIK products were infused into the patients in 30 min. After the second infusion of DC-CIK cells, all patients were injected with IL-2 1×106 U/day subcutaneously every other day for a total of 10 times.
Before infusion, the tumor killing activity of DC-CIKs was confirmed. The results of lymphocytes subsets in 15 cases of AML before and 3 months after DC-CIKs therapy, it was found that the ratio of CD3+/CD8+ cells was increased dramatically after the therapy, suggesting the significant increase of cytotoxic CIKs in the patients.
One patient received three DC-CIK therapies (interval=3 months), 2 patients received two (interval=3 months), and the remaining patients received one therapy. All patients reached CR before the DC-CIK therapy. The median follow-up duration from DC-CIK infusion was 71 months, ranging from 30 to 108 months. Twenty patients were in consistent CR (90.9%); two cases (9.1%) relapsed (one relapsed 19 months after DC-CIK therapy and the other relapsed 28 months after the treatment). Five cases were MRD positive before treatment but became negative during re-check, 3 months after the therapy. After the treatment, the genes of AML1/ETO-positive samples became negative.
There were no side effects in patients before treatments. For the infusion of DC-CIK, 7 patients showed side effects (31.28%). Among them, 6 (27.27%) patients suffered from high fever and chills which were easily treated with fever drugs and Phenergan. Hives were found on 3 (13.64%) patients and 2 (9.09%) patients had low fever after infusion and the symptom disappeared without special medical care. No obvious side effects were observed during the period of IL-2 treatment. No damages were found in heart, liver and kidney. Blood routine test showed blood cells were in normal range before and after treatment.
DC-CIK therapy is relatively safe with few side effects. The therapy can eliminate the leukemia cells and eradicate MRD in children with AML in a short time of follow-up.
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Scientific article publishing date: 10/22/2015
Immucura identifier BSC21_327EN