Artikel
Serum-free generation of mature dendritic cells from malignant glioma patients
Serumfreie Gewinnung reifer dendritischer Zellen von Gliompatienten
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Autoren
Veröffentlicht: | 4. Mai 2005 |
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Gliederung
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Objective
Immunotherapy with tumor-antigen loaded dendritic cells (DC) is a promising approach, which may allow the specific killing of tumor cells without excessive damage to normal tissues. However, it relies on ex-vivo generation of sufficient numbers of functionally competent DC, which may be impaired in patients. Here, mature monocyte-derived DC were generated from malignant glioma patients in a two-step culture protocol using either plasma-supplemented or serum-free medium.
Methods
DC generation in glioma patients and controls was compared, using GMP-quality, serum-free CellGro DC medium and plasma-supplemented X-Vivo 15 medium. Monocytes were enriched immunomagnetically to over 96% CD14+ purity and cultured in the presence of GM-CSF and IL-4 for the first 6 days, followed by an additional 3-day culture period in the presence of GM-CSF, IL-4 and TNFα, to induce DC maturation. Resulting DC were characterized by flow cytometry.
Results
In patients (n=14), frequencies of lymphocytes and CD3+ T-cells were significantly reduced, whereas the frequency of neutrophils was increased. Immunophenotypic characterization of T-cells revealed a decreased frequency of CD25+ cells in patients. Functionally, there was a slightly better mitogen-responsiveness of T-cells in patients as compared to controls. In the monocyte compartment, the only phenotypic difference observed was a lower frequency of CD80+ cells in patients. DC differentiation of monocytes revealed more homogenous generation of mature CD83+/CD14- DC for patients compared to controls in the presence of X-vivo 15 medium supplemented with autologous plasma. Residual CD14 expression was lower and expression of CD83, CD80 and CD25 was higher for patients. When serum-free CellGro DC medium was used instead, all of these differences disappeared, besides the higher frequency of CD25+ DC in patients.
Conclusions
Overall, the serum-free medium was superior to plasma-supplemented X-vivo 15 medium. Thus, only minor differences between glioma patients and controls were observed, which probably will not affect DC-based immunotherapy. In addition, a serum-free culture system is defined, which allows efficient and homogenous ex vivo generation of mature DC from glioma patients for clinical application.