Maturation of monocyte-derived dendritic cells with Toll-like receptor 3 and 7/8 ligands combined with prostaglandin E2 results in high interleukin-12 production and cell migration

Abstract

Dendritic cells (DC) are professional antigen-presenting cells of the immune system that play a key role in regulating T cell-based immunity. In vivo, the capacity of DC to activate T cells depends on their ability to migrate to the T cell areas of lymph nodes as well as on their maturation state. Depending on their cytokine-secreting profile, DC are able to skew the immune response in a specific direction. In particular, IL-12p70 producing DC drive T cells towards a T helper 1 type response. A serious disadvantage of current clinical grade ex vivo generated monocyte-derived DC is the poor IL-12p70 production. We have investigated the effects of Toll-like receptor (TLR)-mediated maturation on ex vivo generated human monocyte-derived DC. We demonstrate that in contrast to cytokine-matured DC, DC matured with poly(I:C) (TLR3 ligand) and/or R848 (TLR7/8 ligand) are able to produce vast amounts of IL-12p70, but exhibit a reduced migratory capacity. The addition of prostaglandin E2 (PGE2) improved the migratory capacity of TLR-ligand matured DC while maintaining their IL-12p70 production upon T cell encounter. We propose a novel clinical grade maturation protocol in which TLR ligands poly(I:C) and R848 are combined with PGE2 to generate DC with both high migratory capacity and IL-12p70 production upon T cell encounter.

Fig. 1

Phenotype and IL-12p70 production after DC maturation with different cocktails. Immature DC were matured for 48 h with different maturation cocktails, maturation markers were evaluated by flow cytometry and IL-12p70 secreted was measured in the supernatant by ELISA. a DC from three different donors matured with the conventional cytokine cocktail (TNFα, IL-6, IL-1α and PGE₂), TLR3 ligand poly(I:C), TLR7/8 ligand (R848) or both. Upper panels show the different maturation cocktails used. Horizontally the expression of CD80, CD83, CD86, CCR7 and MHC class II from one representative donor measured by flow cytometry is shown (bold line). The thin line represents the isotype control. The lower panel shows the IL-12p70 production in the supernatant measured by ELISA. b DC from different donors were matured with the conventional cytokine cocktail (TNFα, IL-6, IL-1β and PGE₂), or poly(I:C) or R848 or poly(I:C)/R848 in the combination with TNFα, IFNα, IFNγ with and without PGE₂. IL-12p70 production was measured after 48 h. Per condition each symbol represents one donor. Means are shown for each maturation cocktail and were compared to cDC (*  p < 0.05), comparison of the different cocktails with TLR-DC were significant with all the different cocktails. There is no significant difference between PGE₂-TLR-DC and the TNFα/IFNα/IFNγ-PGE₂-TLR-DC

Fig. 2

Migration of the TLR-ligand DC is poor and can be restored upon addition of PGE₂. Differently matured DC from three to six different donors were added to a fibronectin-coated plate and individual cells were monitored for 60 min for their motility. The migrated distance (in μm) (a) and the activity (the percentage of time the cells were migrating) (b) is plotted for the different maturation cocktails and compared to cDC. Per condition, each symbol represents the mean for each donor (individual donors indicated by different symbols). Means are shown for each maturation cocktail and were compared to cDC (* = p < 0.005)

Fig. 3

CCR7 expression and CCR7-mediated chemotaxis of cDC, TLR-DC and PGE₂-TLR-DC. DC from three different donors were matured with the conventional cytokine-cocktail or with poly(I:C), R848 with or without PGE₂. a CCR7 expression of the different matured DC and CD80, CD83, CD86 and MHC class II expression of PGE₂-TLR-DC measured by flowcytometry is shown. b Chemotaxis was determined by the number of cells that had migrated into the lower compartment of a transwell system containing increasing concentrations of CCL21, counted in 1 minute by flow cytometry. Kinesis was determined by the number of cells that had migrated into the lower compartment when CCL21 is present in both compartments in the same concentration. The graph shown here is representative for three different donors. The error bars show the standard deviation

Fig. 4

IL-12p70 production upon secondary stimulation. DC were matured with the conventional cytokine cocktail (TNFα, IL-6, IL-1β and PGE₂), or TLR ligands (poly(I:C)/R848) or TLR ligands in combination with PGE₂ for 48 h and then cultured for another 24 h with or without PBL or CD40L-trimers. IL-12p70 production was measured in the supernatant by ELISA. Per condition, each symbol represents the mean for each donor. Means are shown for each maturation cocktail (open symbols cDC, gray symbols TLR-DC, black symbols PGE₂-TLR-DC, NS non significant)

Fig. 5

Cytokine production of T cells in contact with cDC, TLR-DC and PGE₂-TLR-DC. a The profile of cytokines secreted by PBL upon contact with cDC, TLR-DC and PGE₂-TLR-DC was measured by cytokine bead array. The graph shows the fold change in the cytokine production of TLR-DC and PGE₂-TLR-DC relative to cDC of five different donors. The table presents the mean ± SEM concentration (pg/ml) of each cytokine in absolute numbers for all conditions. b KLH-specific proliferation of PBL from patients vaccinated with KLH-loaded DC. Four patients were tested, one representative graph is shown. PBL were cocultured with autologous DC matured with the cytokine cocktail, TLR-ligands or TLR-ligands with PGE₂ with or without KLH. Proliferation was measured by incorporation of tritiated thymidine. Filled symbols represent DC loaded with KLH. Open symbols represent DC without KLH. Error bars show the standard deviation within the experiment