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Short-term cultured, interleukin-15 differentiated dendritic cells have potent immunostimulatory properties.

ABSTRACT: BACKGROUND: 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. METHODS: 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: (i) a classic combination of pro-inflammatory cytokines (tumor necrosis factor-alpha, IL-1beta, IL-6, prostaglandin E2) and (ii) a Toll-like receptor (TLR)7/8 agonist-based cocktail (R-848, interferon-gamma, TNF-alpha 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. RESULTS: 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 (i) higher migratory potential, (ii) advantageous cytokine secretion profile (interferon-gamma, IL-12p70) and (iii) 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 versus 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. CONCLUSIONS: 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.

SUBMITTER: Anguille S

PROVIDER: S-EPMC2807857 | biostudies-other | 2009

REPOSITORIES: biostudies-other

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Short-term cultured, interleukin-15 differentiated dendritic cells have potent immunostimulatory properties.

Anguille Sébastien S Smits Evelien L J M EL Cools Nathalie N Goossens Herman H Berneman Zwi N ZN Van Tendeloo Vigor F I VF

Journal of translational medicine 20091218

<h4>Background</h4>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 a ...[more]

PMID: 20021667

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Project description:Dendritic cells (DCs) are important innate and adaptive immune effectors, and have a key role in antigen presentation and T-cell activation. Different lineages of DCs can be developed from hematopoietic progenitors following cytokine signaling, and the various lineages of DCs display distinct morphology, phenotype and functions. There has been limited information on differential cytokine-mediated molecular signaling in DCs. Analyses of surface molecules by flow cytometry and quantitative RNA profiling revealed differences between DCs derived from interleukin-4 (IL-4) versus IL-15 signaling, yet both lineages of DCs exhibited similar levels of surface molecules key to immune activation. Functional assays confirmed that IL-15-derived DCs elicited greater antigen-specific, primary and secondary CD8 and CD4 T-cell responses than did IL-4-derived DCs. Importantly, IL-15 DCs secreted substantial amounts of proinflammatory cytokines, including IL-6, interferon-? (IFN-?) and tumor necrosis factor-? (TNF?), which helped polarize a strong T-cell response. Assessment of signaling pathways revealed that IL-15 DCs exhibited a lower levels of activated signal transducer and activator of transcription 5 (STAT5), STAT6 and extracellular signal-regulated kinase 1/2 than IL-4 DCs, but after lipopolysaccharide (LPS)/TNF? treatment, the STAT3 and p38 mitogen-activated protein kinase (MAPK) activities were significantly enhanced in the IL-15 DCs. Surprisingly, contrary to the canonical IL-15-mediated STAT5 signaling pathway in lymphoid cells, IL-15 did not mediate a strong STAT5 or STAT3 activation in DCs. Further analysis using specific inhibitors to STAT3 and p38 MAPK pathways revealed that the STAT3 signaling, but not p38 MAPK signaling, contributed to IFN-? production in DCs. Therefore, while IL-15 does not promote the STAT signaling in DCs, the increased STAT3 activity after LPS/TNF? treatment of the IL-15 DCs has a key role in their high IFN-? effector activities.

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Short-term cultured, interleukin-15 differentiated dendritic cells have potent immunostimulatory properties.

Publications

Short-term cultured, interleukin-15 differentiated dendritic cells have potent immunostimulatory properties.

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