Design of tumour-specific immunotherapies using dendritic cells – analyses of IL15-DC

Abstract

Immunotherapy of malignancies aims at activating the patient’s own immune system to fight the tumour affecting the patient. Even though the use of dendritic cells (DC) has shown promising results, the DC vaccination strategy needs improvement, as only few relevant clinical responses could be documented so far. Aim: In this study, the standard protocol to generate monocyte derived DC using GM-CSF and IL-4 was compared to the use of GM-CSF and IL-15. Methods: Monocytes were isolated by plastic adherence from peripheral blood mononuclear cells and cultured for 6 days with GM-CSF and either IL-4 (IL4-DC) or IL-15 (IL15-DC). A fraction of the IL4-DC was stimulated with a cytokine cocktail, while a fraction of the IL15-DC was stimulated by adding TNF-α 24 hours before harvesting. The phenotypes of the four DC populations were determined using flow cytometry. Intracellular signalling pathways were investigated using phospho-specific antibodies in a Western blot. IL-12 production was analysed in an ELISA. Migratory capacity was determined in a chemotaxis assay. Results: Monocytes cultured with GM-CSF and IL-15 developed a DC-like morphology. Phenotypic analyses revealed that both IL4-DC and IL15-DC had down-regulated CD14 expression and up-regulated CD1a expression, although IL15-DC to a lesser extent. IL15-DC showed an enhanced surface expression of co-stimulatory molecules CD80 and CD86 whereas no difference was observed in surface expression of MHC class II and CD40. Upon stimulation, an up-regulation of the maturation marker CD83 and CD86 were observed on IL4-DC only. The IL15-DC had more phosphorylated JNK and ERK than IL4-DC whereas the phosphorylation level of p38 in both IL4-DC and IL15-DC were approximately the same. None of the cell populations produced IL-12 or showed chemotaxis towards CCL19. Conclusions: The generation of IL15-DC turned out to be more problematic compared to the generation of IL4-DC. The stimulatory activity of IL-15 on T-cell proliferation resulted in a high degree of contamination with T -cells in the IL15-DC cultures as I did not have a pure monocyte population to start with. This problem might be overcome by using either alternative monocyte isolation protocols or by reducing the culture period from 5-6 to 3-4 days. Because of the variation in the results of the experiments the data needs to be confirmed with this approach.