Expression of the APM components and on TA presentation to T cells. It is well documented that only adequately matured DC are able to cross-prime T cells and that only mDC migrate to lymph nodes where they can prime T-cell responses [17,18]. Further, mDC produce higher levels of IL12p70, the cytokine necessary for Th1 and CTL responses, than iDC [15,17,19]. In addition, maturation greatly enhances antigen presentation and APM component expression as well as the expression of co-stimulatory molecules on the surface of DC [10,17,20]. Therefore, optimal maturation is essential DC function. Various combinations of cytokines and/or toll-like-receptor (TLR) ligands have been used to mature human monocyte-derived DC [17,21]. Usually, these cocktails contain variable levels of multiple cytokines, e.g., 50 ng/ml TNF-a, 25 ng/ml IL-1b and 10 ng/ml IL-6 in the most widely used conventional cocktail (“the conv. mix”). In contrast, IRX-2 used in this study Pentagastrin site contained 15?0 times lower concentrations of these cytokines (Table S2) than the conv. mix. In direct comparisons, while the conv. mix induced higher expression levels of CD80, CD83 and CD86 on moDC, IRX-2 induced considerably higher expression levels of CCR7, CD11c and CD40. Importantly, IRX-2-matured DC not only expressed higher surface levels of CCR7 but also migrated better in response to the CCR7 ligand, CCL21. CCR7 is essential forFigure 4. Cross-presentation of lysed tumor cells by mDC. CTL were generated using either IRX-2- or conventionally-matured mDC (IRX-2 CTL or conv CTL) as described in Materials and Methods. ELISPOT assays were performed using either IRX-2- or conventionally-matured mDC loaded with lysed PCI13 and co-incubated for 24 h with IRX-2 CTL or conv CTL. The highest number of spots was observed when IRX-2 mDC were co-incubated with IRX-2 CTL from the same donor (p,0.05 as compared to the other possible combinations of CTL and mDC). Co-incubation of IRX-2 mDC with conv CTL or conv mDC with IRX-2 CTL led to a similar number of spots, while co-incubation of conv CTL with conv mDC led to a significantly lower number of spots. IRX-2 and conv CTL showed similar levels of IFN-c release in the absence of mDC (white bars). The mean number of spots per 105 CD8+ T cells 6 SEM from 4 independent experiments is 
shown. doi:10.1371/journal.pone.0047234.gIRX-2 Up-Regulates DC MaturationDC migration to lymph nodes, where interaction of DC and T cells takes place [22], suggesting that IRX-2-matured DC might also migrate more efficiently in vivo. CD40, a member of the TNFR superfamily, serves as a receptor on DC which interacts with CD154 on CD4+ T cells, leading to further DC maturation, and it induces IL-12p70 production [23]. It has been suggested that DC expressing low levels of CD40 might induce tolerance instead of CASIN web immunity [24]. The induction of high levels of CD40 on DC matured with IRX-2 indicates that these cells are unlikely to induce tolerance, a clearly undesirable event in cancer patients receiving immunotherapy. The function of CD11c, also known as complement rector 4, 16574785 remains less clear, but studies indicate its role in cell adhesion but also in antigen presentation by DC [25]. A higher CD11c expression on IRX-2-matured DC than on conventionally-matured DC could thereby contribute to their superior antigen presentation and induction of CTL. The APM expression in DC is necessary not only for the presentation of antigens derived from self-proteins via the classical cytosolic.Expression of the APM components and on TA presentation to T cells. It is well documented that only adequately matured DC are able to cross-prime T cells and that only mDC migrate to lymph nodes where they can prime T-cell responses [17,18]. Further, mDC produce higher levels of IL12p70, the cytokine necessary for Th1 and CTL responses, than iDC [15,17,19]. In addition, maturation greatly enhances antigen presentation and APM component expression as well as the expression of co-stimulatory molecules on the surface of DC [10,17,20]. Therefore, optimal maturation is essential DC function. Various combinations of cytokines and/or toll-like-receptor (TLR) ligands have been used to mature human monocyte-derived DC [17,21]. Usually, these cocktails contain variable levels of multiple cytokines, e.g., 50 ng/ml TNF-a, 25 ng/ml IL-1b and 10 ng/ml IL-6 in the most widely used conventional cocktail (“the conv. mix”). In contrast, IRX-2 used in this study contained 15?0 times lower concentrations of these cytokines (Table S2) than the conv. mix. In direct comparisons, while the conv. mix induced higher expression levels of CD80, CD83 and CD86 on moDC, IRX-2 induced considerably higher expression levels of CCR7, CD11c and CD40. Importantly, IRX-2-matured DC not only expressed higher surface levels of CCR7 but also migrated better in response to the CCR7 ligand, CCL21. CCR7 is essential forFigure 4. Cross-presentation of lysed tumor cells by mDC. CTL were generated using either IRX-2- or conventionally-matured mDC (IRX-2 CTL or conv CTL) as described in Materials and Methods. ELISPOT assays were performed using either IRX-2- or conventionally-matured mDC loaded with lysed PCI13 and co-incubated for 24 h with IRX-2 CTL or conv CTL. The highest number of spots was observed when IRX-2 mDC were co-incubated with IRX-2 CTL from the same donor (p,0.05 as compared to the other possible combinations of CTL 
and mDC). Co-incubation of IRX-2 mDC with conv CTL or conv mDC with IRX-2 CTL led to a similar number of spots, while co-incubation of conv CTL with conv mDC led to a significantly lower number of spots. IRX-2 and conv CTL showed similar levels of IFN-c release in the absence of mDC (white bars). The mean number of spots per 105 CD8+ T cells 6 SEM from 4 independent experiments is shown. doi:10.1371/journal.pone.0047234.gIRX-2 Up-Regulates DC MaturationDC migration to lymph nodes, where interaction of DC and T cells takes place [22], suggesting that IRX-2-matured DC might also migrate more efficiently in vivo. CD40, a member of the TNFR superfamily, serves as a receptor on DC which interacts with CD154 on CD4+ T cells, leading to further DC maturation, and it induces IL-12p70 production [23]. It has been suggested that DC expressing low levels of CD40 might induce tolerance instead of immunity [24]. The induction of high levels of CD40 on DC matured with IRX-2 indicates that these cells are unlikely to induce tolerance, a clearly undesirable event in cancer patients receiving immunotherapy. The function of CD11c, also known as complement rector 4, 16574785 remains less clear, but studies indicate its role in cell adhesion but also in antigen presentation by DC [25]. A higher CD11c expression on IRX-2-matured DC than on conventionally-matured DC could thereby contribute to their superior antigen presentation and induction of CTL. The APM expression in DC is necessary not only for the presentation of antigens derived from self-proteins via the classical cytosolic.
