Maturation by Toll Like Receptor Ligand R848 Improves the Uptake of Apoptotic Leukemic Cells by Monocyte Derived Dendritic Cells.

Therapeutic vaccination with dendritic cells (DC) is currently considered as an investigational therapy in acute myeloid leukemia (AML) for eradication of minimal residual disease (MRD). Various strategies are being explored in manufacturing DC vaccines ex vivo, e.g. monocyte derived DC (MoDC) loaded with leukemia associated antigens (LAA). Many sources of LAA, whether or not combined with various adjuvants and different methods of loading of antigen onto DC, have been used in an attempt to optimize anti-tumor responses. However, the optimal source of tumor antigen combined with adjuvants remains unclear. Leukemic cells harboring all unknown and known potential LAA are an attractive antigen source. Here we explore the use of leukemic blast derived apoptotic cells and lysates loaded onto MoDC combined with various maturation stimuli, such as the clinically applicable toll like receptor 7/8 ligand (TLR-L) R848 combined with conventional cytokine cocktail consisting of IL1β, IL6, TNFα and PGE₂.

CD14+ monocytes isolated from peripheral blood of healthy donors were differentiated into MoDC with GM-CSF and IL-4 in serum free medium. Leukemic blasts were labeled with CFSE, followed by induction of apoptosis in serum deprived medium with heat shock (2h 42°C) or preparation of lysate by 3 freeze/thaw cycles. Apoptosis was confirmed by flow cytometry using Syto(62) and 7AAD. Subsequently, the CFSE-labeled apoptotic blasts and lysates were incubated in various ratios with immunofluorescently labeled MoDC in the presence of TLR-L (Poly-(I:C), LPS, PGN, Flagellin or R848) and/or the cytokine cocktail in different time frames. Uptake was defined by flow cytometry as percentage of CFSE positive MoDC. MoDC were analyzed for expression of co-stimulatory molecules, the chemokine receptor CCR7 and maturation-associated antigens. Migratory capacity towards CCL19 was measured in a transwell system. Mixed leukocyte reaction was performed to study T cell stimulation and IL12 secretion was quantified after CD40 ligation.

Apoptotic cells or lysates were taken up by MoDC in a dose dependent manner, up to 26% (12-49%) and 17% (median; range: 3-47%), respectively. Of all tested TLR-L, R848 most effectively enhanced the uptake of apoptotic cells, but not of lysates. Addition of the cytokine cocktail resulted in a decreased uptake by MoDC of both apoptotic cells and lysate (n=7), but most effectively induced phenotypic DC maturation as determined by up-regulation of co-stimulatory molecules, enhanced migratory capacity and enhanced ability to stimulate T cells. Both R848 and cytokine cocktail stimulated MoDC were able to produce IL-12 (n=6). To combine the enhanced uptake induced by R848 and the optimal maturation of MoDC by the cytokine cocktail, we co-incubated blasts and MoDC in the presence of R848 for 24h followed by a 48hr incubation with cytokine cocktail (n=9). The resulting uptake was superior to the cytokine cocktail alone, and migratory and co-stimulatory capacity was improved as compared to R848 alone.

The TLR-L R848 significantly improved the uptake of leukemic blasts by MoDC. Combination of R848 and cytokines resulted in sufficient maturation induction and subsequent T cell priming. These data may add significantly to the development of new strategies for optimization of vaccine preparation in AML.

No relevant conflicts of interest to declare.