Figure 2
Figure 2. Impairment of DC functions in HPS2 patients. DCs were generated from freshly isolated monocytes by adding IL-4 and GM-CSF for 6 days. Final maturation has been induced after the addition of LPS for another 24 hours. Analysis of MIP1-β/CCL4, MIG/CXCL9, IL-12, TNF-α, and IL-23 production (A, all panels) by immature DCs (gray bars) and mature DCs (black bars) derived from HPS2 patients or from a healthy subject. Statistical analysis by non-parametric test shows a significant difference (*P < .05). (B) mDCs (○) and pDCs (♢) were evaluated by FACS analysis in peripheral blood from the HPS2 patients (● and ♦) as compared with the percentages detected in healthy subjects of comparable age (○ and ♢). Statistical analysis of DC counts shows that mDCs and pDCs were significantly lower in HPS2 patients after 2 independent measurements as compared with cell counts in 24 normal control subjects (**P < .005; ***P < .001). (C) IFN-α production, measured in triplicate, by freshly isolated PBMCs after stimulation for 24 hours with 6 μg/mL CpG, HSV-1 (copies/200 μL), or medium alone in HPS2 and 1 control subject. Statistical analysis by non-parametric test shows a significant difference (*P < .05). This experiment is representative of 2 experiments performed. (D) PBMCs were stimulated with 6 μg/mL CpG for 5 hours in the presence of brefeldin A for the last 3 hours. At the end of stimulation, PBMCs were stained with anti–BDCA2-FITC Ab and anti–CD123-Vioblue Ab to identify pDCs, and with an anti-IFNα–antigen-presenting cell Ab. The graph shows the percentage (gray bars) and (MFI, black bars) of IFN-α–positive cells. (E) PBMCs were stimulated with 6 μg/mL CpG for 5 hours. At the end of incubation, PBMCs were collected and IFN-α levels were evaluated by real-time polymerase chain reaction. Levels of HPRT messenger RNA were used for normalization. (F) MLR was performed by evaluation of the percentage of proliferating CD3+ cells after incubation with irradiated PBMCs from HPS2 patients or a control subject for 7 days. Statistical analysis by non-parametric test shows a significant difference (*P < .05). Ab, antibody; CTR, control; HPRT, hypoxanthine guanine phosphoribosyltransferase; MFI, mean fluorescence intensity; Pt, patient.

Impairment of DC functions in HPS2 patients. DCs were generated from freshly isolated monocytes by adding IL-4 and GM-CSF for 6 days. Final maturation has been induced after the addition of LPS for another 24 hours. Analysis of MIP1-β/CCL4, MIG/CXCL9, IL-12, TNF-α, and IL-23 production (A, all panels) by immature DCs (gray bars) and mature DCs (black bars) derived from HPS2 patients or from a healthy subject. Statistical analysis by non-parametric test shows a significant difference (*P < .05). (B) mDCs (○) and pDCs (♢) were evaluated by FACS analysis in peripheral blood from the HPS2 patients (● and ♦) as compared with the percentages detected in healthy subjects of comparable age (○ and ♢). Statistical analysis of DC counts shows that mDCs and pDCs were significantly lower in HPS2 patients after 2 independent measurements as compared with cell counts in 24 normal control subjects (**P < .005; ***P < .001). (C) IFN-α production, measured in triplicate, by freshly isolated PBMCs after stimulation for 24 hours with 6 μg/mL CpG, HSV-1 (copies/200 μL), or medium alone in HPS2 and 1 control subject. Statistical analysis by non-parametric test shows a significant difference (*P < .05). This experiment is representative of 2 experiments performed. (D) PBMCs were stimulated with 6 μg/mL CpG for 5 hours in the presence of brefeldin A for the last 3 hours. At the end of stimulation, PBMCs were stained with anti–BDCA2-FITC Ab and anti–CD123-Vioblue Ab to identify pDCs, and with an anti-IFNα–antigen-presenting cell Ab. The graph shows the percentage (gray bars) and (MFI, black bars) of IFN-α–positive cells. (E) PBMCs were stimulated with 6 μg/mL CpG for 5 hours. At the end of incubation, PBMCs were collected and IFN-α levels were evaluated by real-time polymerase chain reaction. Levels of HPRT messenger RNA were used for normalization. (F) MLR was performed by evaluation of the percentage of proliferating CD3+ cells after incubation with irradiated PBMCs from HPS2 patients or a control subject for 7 days. Statistical analysis by non-parametric test shows a significant difference (*P < .05). Ab, antibody; CTR, control; HPRT, hypoxanthine guanine phosphoribosyltransferase; MFI, mean fluorescence intensity; Pt, patient.

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