Maturation markers CCR7, CD80 and CD83. However, several differences were observed in the phenotype of mDC derived from monocytes of the same patients but matured either conventionally or by IRX-2 as shown inexpression of APM components in DC matured conventionally or with IRX-2 was compared. Both the conventional cytokine cocktail and IRX-2 up-regulated 25033180 the expression levels of the APM components LMP2, TAP1, TAP2, Tapasin and Calreticulin as compared to iDC from the same donors (the data for iDC are shown in Figure S1). However, as shown in Figure 2, IRX-2 induced higher levels of LMP2, TAP1, TAP2 and Tapasin (p,0.05 for all) in 
mDC than did conventional cytokines. No significant differences in the expression of Calreticulin and surface MHC Class I molecules were evident between mDC matured with IRX-2 and the conventional mix.IRX-2-matured DC Induce TA-specific CTL in vitroThe induction of TA-specific T cells (CTL) is the final and critical endpoint of antigen presentation by mDC. In IVS cultures, we generated CTL from PBMC of HLA-A2+ HNSCC patients using mDC which were GNF-7 biological activity cultured in the presence of IRXIRX-2 Up-Regulates DC MaturationFigure 1. Phenotype and migration of DC matured in IRX-2 or conventional cytokines. (A) DC obtained from HNSCC patients were matured for 48 h either with IRX-2 or the conventional maturation cocktail. While conventionally matured DC (white bars) expressed higher levels of CD80, CD83 and CD86 (*, p,0.05), IRX-2 matured DC (black bars) showed higher expression of CD11c, CD40 and CCR7 (*, p,0.05). The data are mean x-fold of MFI 6 SEM for cells obtained from 12 different HNSCC patients. (B) Representative histograms showing expression of DC markers after maturation with IRX-2 or the conventional cytokine cocktail in DC generated from monocytes of one HNSCC patient. The shaded peaks represent isotype controls. (C) Migration of mDC in vitro: Migration assays were performed as described in Materials Methods using DC generated from peripheral blood monocytes of HNSCC patients. While iDC showed very little migration in response to CCL21, both 
conventional- and IRX-2-matured DC migrated significantly better. Results are shown as the mean absolute numbers of migrated cells 6 SEM obtained from 5 different HNSCC patients. doi:10.1371/journal.pone.0047234.g2 or conventional cytokines. Lysates of the HLA-A2+ HNSCC cell line PCI-13 served as an antigen source in the IVS culture. As shown in Figure 3, both conventional and IRX-2 matured DC induced CTL which were able to kill PCI-13 target cells. Anti-HLA class I blocking Abs inhibited cytotoxicity and CTL showed only low cytotoxicity against the irrelevant target MCF-7 (data not shown). However, CTL generated in the presence of IRX-2-matured DC showed higher cytotoxicity as compared to CTL generated with conventional mDC. Taken together, IRX2-matured DC were more effective in inducing tumor cellspecific CTL in vitro as compared to conventional mDC.IRX-2 Matured DC Cross-present Antigen more Efficiently than Conventionally-matured DCKnowing that both conventionally- and IRX-2-matured DC are able to cross-prime PCI-13 specific CTL populations, we decided to use these in vitro generated CTL to HIF-2��-IN-1 web explore the ability of mDC to cross-present tumor antigens. As summarized in Figure 4A, CTL were generated by IVS using mDC, which were either matured by conventional cytokines (Conv CTL) or IRX-2 (IRX-2 CTL). PCI-13 HNSCC cells were used as an antigen source for both types of CTL. DC.Maturation markers CCR7, CD80 and CD83. However, several differences were observed in the phenotype of mDC derived from monocytes of the same patients but matured either conventionally or by IRX-2 as shown inexpression of APM components in DC matured conventionally or with IRX-2 was compared. Both the conventional cytokine cocktail and IRX-2 up-regulated 25033180 the expression levels of the APM components LMP2, TAP1, TAP2, Tapasin and Calreticulin as compared to iDC from the same donors (the data for iDC are shown in Figure S1). However, as shown in Figure 2, IRX-2 induced higher levels of LMP2, TAP1, TAP2 and Tapasin (p,0.05 for all) in mDC than did conventional cytokines. No significant differences in the expression of Calreticulin and surface MHC Class I molecules were evident between mDC matured with IRX-2 and the conventional mix.IRX-2-matured DC Induce TA-specific CTL in vitroThe induction of TA-specific T cells (CTL) is the final and critical endpoint of antigen presentation by mDC. In IVS cultures, we generated CTL from PBMC of HLA-A2+ HNSCC patients using mDC which were cultured in the presence of IRXIRX-2 Up-Regulates DC MaturationFigure 1. Phenotype and migration of DC matured in IRX-2 or conventional cytokines. (A) DC obtained from HNSCC patients were matured for 48 h either with IRX-2 or the conventional maturation cocktail. While conventionally matured DC (white bars) expressed higher levels of CD80, CD83 and CD86 (*, p,0.05), IRX-2 matured DC (black bars) showed higher expression of CD11c, CD40 and CCR7 (*, p,0.05). The data are mean x-fold of MFI 6 SEM for cells obtained from 12 different HNSCC patients. (B) Representative histograms showing expression of DC markers after maturation with IRX-2 or the conventional cytokine cocktail in DC generated from monocytes of one HNSCC patient. The shaded peaks represent isotype controls. (C) Migration of mDC in vitro: Migration assays were performed as described in Materials Methods using DC generated from peripheral blood monocytes of HNSCC patients. While iDC showed very little migration in response to CCL21, both conventional- and IRX-2-matured DC migrated significantly better. Results are shown as the mean absolute numbers of migrated cells 6 SEM obtained from 5 different HNSCC patients. doi:10.1371/journal.pone.0047234.g2 or conventional cytokines. Lysates of the HLA-A2+ HNSCC cell line PCI-13 served as an antigen source in the IVS culture. As shown in Figure 3, both conventional and IRX-2 matured DC induced CTL which were able to kill PCI-13 target cells. Anti-HLA class I blocking Abs inhibited cytotoxicity and CTL showed only low cytotoxicity against the irrelevant target MCF-7 (data not shown). However, CTL generated in the presence of IRX-2-matured DC showed higher cytotoxicity as compared to CTL generated with conventional mDC. Taken together, IRX2-matured DC were more effective in inducing tumor cellspecific CTL in vitro as compared to conventional mDC.IRX-2 Matured DC Cross-present Antigen more Efficiently than Conventionally-matured DCKnowing that both conventionally- and IRX-2-matured DC are able to cross-prime PCI-13 specific CTL populations, we decided to use these in vitro generated CTL to explore the ability of mDC to cross-present tumor antigens. As summarized in Figure 4A, CTL were generated by IVS using mDC, which were either matured by conventional cytokines (Conv CTL) or IRX-2 (IRX-2 CTL). PCI-13 HNSCC cells were used as an antigen source for both types of CTL. DC.
