Gene expression profiles of IFNα1, α2, α8, α21 and IFNβ-DC were analyzed on HG-U133 Plus 2.0 microarrays. A. Hierarchical clustering of IFN-DC. B. Genes differentially expressed between IFNα-DC and IFNβ-DC. Results are expressed as median ± SD. In color, genes identified as differentially expressed by 2 or more probe sets. In dark grey genes subsequently analyzed by RT-qPCR in DC differentiated from monocytes isolated from several blood donors.

A. IFNα1, α2, α8, α21 and ω-DC were generated from monocytes from three independent donors and gene expression level of CXCL11, VSIG4, Clec5A and IDO1 analyzed by RT-qPCR. Expression level is relative to GAPDH. One experiment representative of the three is shown. B. IFNα2, ω and β-DC were generated from monocytes from 5 to 11 independent donors and 9 genes were analyzed by RT-qPCR for their expression. Results are expressed as a ratio: (gene expression in IFNβ-DC/GAPDH)/(gene expression in IFNα-DC or IFNω-DC/GAPDH). Closed symbols are for IFNα2-DC and open symbols for IFNω-DC.

After 3 days of differentiation, IFN-DC supernatants were collected and amount of chemokines produced quantified by luminex. Error bars represent the SEM. P value was determined using a Wilcoxon matched pairs test. Experiments were done on cells from nine independent blood donors for CXCL11, CXCL10, CCL4, CCL3, CCL7, CXCL9, CCL2, CCL5 and IL8, eight independent blood donors for CCL8 and six independent blood donors for CCL11. *** indicates P<0.005, ** P<0.01 and * P<0.05. NS: not significant.

A. Mixed lymphocyte reaction assay in which IFN-DC were used as stimulators. Proliferation of allogenic CD4⁺ T cells was determined after 6 days of co-culture by measuring thymidine incorporation. Data are from three independent experiments performed on cells from three independent blood donors and error bars indicate SEM. B–C. Naïve allogenic CD4⁺ T cells were cocultured with IFNα2-DC or IFNβ-DC for 6 days and differentiated T cells were analyzed by FACS for the expression of CXCR3 (B), CCR4 and CCR6 (C). In B, experiments were performed on cells from five independent blood donors. Error bars indicate SEM. In C, one experiment carried out on cells from a single blood donor and representative of five experiments done on five independent donors is shown. D. RT-qPCR analysis of T-bet, GATA3, RORγt and FoxP3 mRNA levels in differentiated T cells after coculture of naïve CD4⁺ T cells with IFN-DC for 6 days. Results are expressed as a ratio: gene expression level in naïve CD4⁺ T cells cultured with DC relative to gene expression level in naïve CD4⁺ T cells alone. Data are from samples from one of the five independent experiments reported in panel B.

IFN-DC were pulsed or not (control) with Flu-HA for 24 h and were then cocultured with autologous CFSE-labelled CD4⁺ (A) or CD8⁺ (B) T cells at a ratio of 1∶5 in AIM-V medium. T cells were analysed by FACS 5 days later for CFSE dilution after gating on viable (7-AAD⁻) CD4⁺ or CD8⁺ T cells. One experiment representative of six, carried out on five independent donor samples and of four experiments carried out on four independent donors are shown for CD4 and CD8 T cells, respectively.

Chemotaxis assay was performed on IFN-DC in presence of 500 ng/ml CCL4 or 100 ng/ml CCL19. After 2 hours, cells that have migrated through the membrane were counted under the microscope. Error bars represent SEM. Data are from three independent blood donors. The migration index was calculated as the number of cells that migrated toward the chemokine gradient divided by the number of cells that migrated toward the medium alone.

A.B. Dendritic cells were cocultured with apoptotic CellTracker®Green-labeled (A) or necrotic PKH26-labeled (B) LY28 cells for 4 h and then analyzed by FACS. The percentage of phagocytosis was calculated as described in . In left panels, one representative experiment is shown. Results in right panel A, are from nine experiments carried out on samples from seven independent blood donors. Results in right panel B are from six experiments carried out on five independent blood donors. Error bars indicate SEM. p value was calculated using Student's t-test.

A.B. IFN-DC were cultured with yellow-green fluorescent latex bead for 4 h (A) or with dextran-FITC for 1 h (B) before being analyzed by FACS. The percentage of internalization was defined as the percentage of green-labeled-DC at 37°C minus the percentage of green-labeled-DC at 4°C. Experiments were performed on three (B) or four (A) independent donor samples. Error bars indicate SEM.