Adaptation to replating of dendritic cells synergizes with Toll-like receptor stimuli and enhances the pro-inflammatory cytokine profile

As initiators of the adaptive immune response, dendritic cells (DCs) can be used for anti-cancer immunotherapy. On addition of proper maturation stimuli DCs mature and produce pro-inflammatory cytokines that skew T cells in the direction needed for anti-cancer therapy. Further optimization of DC maturation might improve the efficacy of DCs for clinical application. We describe that replating and a subsequent resting period enhance the inflammatory properties of the DCs. Cultured immature monocyte-derived DCs were harvested and, after replating, were stimulated immediately or after 2 h of rest. Cytokine production was assessed using enzyme-linked immunosorbent assay (ELISA). Dynamics of mitogen-activated protein kinase (MAPK) and nuclear factor kappa b (NFκB) activation in DCs was analyzed using flow cytometry and imaging flow cytometry. Resting immature DCs after replating, before addition of Toll-like receptor (TLR) ligands, increased the production of pro-inflammatory but not anti-inflammatory cytokines. In addition, the speed of MAPK phosphorylation and nuclear translocation of NFκB was increased when DCs were allowed to rest before TLR stimulation. The effect was imprinted, transient and did not reflect a temporary loss of responsiveness, indicating that signaling induced by culture adaptation of DCs synergizes with TLR signals to increase cytokine production. DCs rested before TLR stimulation induced more interferon (IFN)-γ production in CD4-positive and CD8-positive T cells. Introduction of a resting step in the DC maturation method, which is cheap and easy to implement, will improve the generation of pro-inflammatory DCs for cancer immunotherapy. These DCs enhanced Th1 polarization and IFN-γ production by CD8 T cells, both important hallmarks for the induction of efficient anti-cancer immunity