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<p>Abstract</p> <p>Background</p> <p>Optimization of the current dendritic cell (DC) culture protocol in order to promote the therapeutic efficacy of DC-based immunotherapy is warranted. Alternative differentiation of monocyte-derived DCs using granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-15 has been propagated as an attractive strategy in that regard. The applicability of these so-called IL-15 DCs has not yet been firmly established. We therefore developed a novel pre-clinical approach for the generation of IL-15 DCs with potent immunostimulatory properties.</p> <p>Methods</p> <p>Human CD14⁺monocytes were differentiated with GM-CSF and IL-15 into immature DCs. Monocyte-derived DCs, conventionally differentiated in the presence of GM-CSF and IL-4, served as control. Subsequent maturation of IL-15 DCs was induced using two clinical grade maturation protocols: <it>(i) </it>a classic combination of pro-inflammatory cytokines (tumor necrosis factor-α, IL-1β, IL-6, prostaglandin E₂) and <it>(ii) </it>a Toll-like receptor (TLR)7/8 agonist-based cocktail (R-848, interferon-γ, TNF-α and prostaglandin E₂). In addition, both short-term (2-3 days) and long-term (6-7 days) DC culture protocols were compared. The different DC populations were characterized with respect to their phenotypic profile, migratory properties, cytokine production and T cell stimulation capacity.</p> <p>Results</p> <p>The use of a TLR7/8 agonist-based cocktail resulted in a more optimal maturation of IL-15 DCs, as reflected by the higher phenotypic expression of CD83 and costimulatory molecules (CD70, CD80, CD86). The functional superiority of TLR7/8-activated IL-15 DCs over conventionally matured IL-15 DCs was evidenced by their <it>(i) </it>higher migratory potential, <it>(ii) </it>advantageous cytokine secretion profile (interferon-γ, IL-12p70) and <it>(iii) </it>superior capacity to stimulate autologous, antigen-specific T cell responses after passive peptide pulsing. Aside from a less pronounced production of bioactive IL-12p70, short-term <it>versus </it>long-term culture of TLR7/8-activated IL-15 DCs resulted in a migratory profile and T cell stimulation capacity that was in favour of short-term DC culture. In addition, we demonstrate that mRNA electroporation serves as an efficient antigen loading strategy of IL-15 DCs.</p> <p>Conclusions</p> <p>Here we show that short-term cultured and TLR7/8-activated IL-15 DCs fulfill all pre-clinical prerequisites of immunostimulatory DCs. The results of the present study might pave the way for the implementation of IL-15 DCs in immunotherapy protocols.</p>