Abstract
There are numerous different DC maturation protocols but many contain non-GMP approved reagents and are thus not suitable for the production of vaccines to be used in patients. We aimed to generate an optimized DC product using GMP-grade reagents.
Monocyte-derived DC were matured using a two-step maturation protocol. Monocytes were purified and cultured in CellGro® GMP DC medium for 48 hours in presence of IL-4 and GM-CSF plus 18 h with different combinations of TNF-α, IFN-γ and Toll-like receptor (TLR) agonists for TLR-3, -4 and -7/8. Following maturation, production of IL-12 was evaluated.
Variations between individuals in the ability to respond to each maturation stimulus as well as in the overall IL-12 producing capacity was observed. The presence of both IFN-γgand TLR-7/8 agonist was essential for DC to produce high levels of IL-12. Furthermore, the highest induction of IL-12 production was consistently achieved by the combination of IFN-γ, TLR-3, -4 and -7/8 agonist.
For TLR-4 triggering, synthetic monophosphoryl lipid A (MPLA) was used. Addition of non-GMP lipopolysaccharide (LPS) resulted in increased activation of DC. However, addition of MPLA did not result in any measurable activation of DC. Therefore, TLR-4 triggering was excluded. In absence of TLR-4 triggering, the combination including IFN-γ, TLR-3 and TLR-7/8 agonists induced the highest IL-12 levels. For TLR-7/8 triggering GMP-grade synthetic compound R848 was used throughout the study. For TLR-3 triggering poly I:C was used.
Notably, poly I:C from different companies had high variability in the ability to activate DC. This was likely to a large extent due to contamination with LPS and possibly also other substances in non-GMP grade products. However, heterogeneity in the dsRNA molecule could also potentially affect the ability to trigger TLR-3 and cytosolic RNA-sensors. In the end we decided to use the GMP-grade, long poly I:C product Hiltonol®.
The ability of the DC to activate allogeneic T cells to IFN-γ production was analyzed and correlated well to the production of IL-12 from the DC. This was also the case for tyrosinase-peptide loaded DC activation of tyrosinase-specific TCR transduced T cells. We are now investigating the ability of tumor-lysate loaded DC to trigger activation of tumor-specific T cells.
In summary, we have developed a maturation protocol to generate GMP-grade DC with excellent ability to produce IL-12 and activate T cells.
Citation Format: Lars Adamson, Dhifaf Sarhan, Bhavesh Choudhary, Jeroen Melief, Maria Nyström, Ulrika Edbäck, Renee Vermeij, Gina Scurti, Michael Nishimura, Andreas Lundqvist, Rolf Kiessling, Tanja Lövgren. Enhanced IL-12 production and T cell stimulation ability by dendritic cells matured in presence of GMP-grade Toll-like receptor ligands and IFN-γ. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B071.
