Abstract
BackgroundDesign of tumour specific immunotherapies using the patients' own dendritic cells (DC) is a fast advancing scientific field. The functional qualities of the DC generated in vitro are critical, and today's gold standard for maturation is a cytokine cocktail consisting of IL-1β, IL-6, TNF-α and PGE2 generating cells lacking IL-12p70 production. OK432 is an immunotherapeutic agent derived from killed Streptococcus pyogenes that has been used clinically to treat malignant and benign neoplasms for decades.MethodsIn this study, we analysed the effects of OK432 on DC maturation, DC migration, cytokine and chemokine secretion as well as T-cell stimulatory capacity, and compared it to the cytokine cocktail alone and combinations of OK432 with the cytokine cocktail.ResultsOK432 induced a marked up-regulation of CD40 on the cell surface as well as a strong inflammatory response from the DC with significantly more secretion of 19 different cytokines and chemokines compared to the cytokine cocktail. Interestingly, secretion of IL-15 and IL-12p70 was detected at high concentrations after maturation of DC with OK432. However, the OK432 treated DC did not migrate as well as DC treated with cytokine cocktail in a transwell migration assay. During allogeneic T-cell stimulation OK432 treated DC induced proliferation of over 50 percent of CD4 and 30 percent of CD8 T-cells for more than two cell divisions, whereas cytokine cocktail treated DC induced proliferation of 12 and 11 percent of CD4 and CD8 T-cells, respectively.ConclusionsThe clinically approved compound OK432 has interesting properties that warrants its use in DC immunotherapy and should be considered as a potential immunomodulating agent in cancer immunotherapy.
Highlights
Design of tumour specific immunotherapies using the patients’ own dendritic cells (DC) is a fast advancing scientific field
After a 24-hour maturation stimulus with either 0.1 KE OK432 or cytokine cocktail, both populations had a clear (p < 0.05) up-regulation on the level of HLA-DR and CD86 compared to the immature DC (Figure 1)
Chemotaxis assay After maturation, the DC were harvested and washed extensively. 50 000 live cells were added to the upper chamber of an 8 μm transwell 96-well plate (Corning Lifescience, Lowell, MA) and left to migrate towards CCL19 (100 ng/ml, Immunotools, Friesoythe; Germany) for 16 hours at 37°C, 5% CO2 humidified atmosphere
Summary
Design of tumour specific immunotherapies using the patients’ own dendritic cells (DC) is a fast advancing scientific field. The functional qualities of the DC generated in vitro are critical, and today’s gold standard for maturation is a cytokine cocktail consisting of IL-1b, IL-6, TNF-a and PGE2 generating cells lacking IL12p70 production. Dendritic cells (DC) are a pivotal part of the immune system, bridging the innate and adaptive immune response. After receiving maturation stimuli such as inflammatory cytokines, direct T-cell stimulation or recognition of pathogen-associated molecular patterns (PAMP), the DC up-regulate the surface expression of major histocompatibility complex (MHC) class II as well as a number of co-stimulatory markers [1]. Immunotherapies with the aid of DC have been shown to be a safe and non-reactogenic way to improve the immune response towards cancer [4,5,6,7,8].
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