American Society of Hematology

Figure 5

$Figure 5. IFN-α production by CD141+ cDCs in response to TLR3 ligand. (A) Human cDCs produce IFN-α in response to polyICLC. PBMCs of 3 donors were separated into a cDC and a non-cDC fraction by magnetic-activated cell sorting (MACS) separation. Then, 2 × 105 cells were plated and stimulated for 14 hours with 25 μg/mL polyICLC, 5 μg/mL GLA, or 4 μg/mL R848. Pan IFN-α levels were determined in the cell supernatants after 14 hours by the use of ELISA. (B) IFN-α production by DC subsets after TLR stimulation. PBMCs were sequentially separated into CD141+ cDCs, CD304+ pDCs, and CD1c+ cDCs by MACS separation. A total of 0.25 × 105 cells of the positive fractions as well as the final negative fraction were plated, stimulated, and IFN-α levels were determined as in panel A. (C) Same as in panel B but showing composite data from 3 donors. (D) Intracellular staining for IFN-α in CD141+ cDCs stimulated with polyICLC. cDCs were isolated from PBMCs by MACS separation and stimulated with polyICLC for 9 hours with 10 μg/mL brefeldin A. Cells were stained for surface markers followed by intracellular cytokine staining for IFN-α. Gating was performed as described in Figure 1A, and plots display the CD141+ cDCs. Numbers indicate the percentage of IFN-α producing cells with (right) and without (left) polyICLC stimulation. (E) Fold change of intracellular levels of IFN-α in 4 donors. Same as in panel D but shown for the CD1c+ and CD141+ cDCs fractions. Statistical analysis was performed with a paired t test. (F) IFN-α secretion by DC subsets. Cellular supernatants shown in panel C were run on pan-IFN-α−specific and IFN-α2−specific ELISAs in parallel. The plot shows composite data for the percentage of IFN-α2 of the pan IFN-α amount for 3 donors. Error bars indicate SD. (G) Transcriptional profiles for IFN-α subtypes in CD141+ cDCs. CD141+ cDCs were isolated from 3 donors by MACS and stimulated with polyICLC for 8 hours before RNA isolation. Sybr-green based Q-PCR was performed for the different IFN-α subtypes and transcript numbers were related to 1000 GAPDH copies. Results are displayed as percentage of the indicated IFN-α subtype transcript number in relation to the total IFN-α count. Error bars indicate SD. (H) Same as in panel H but for CD304+ pDCs stimulated with R848 for 1 hour.$

IFN-α production by CD141⁺ cDCs in response to TLR3 ligand. (A) Human cDCs produce IFN-α in response to polyICLC. PBMCs of 3 donors were separated into a cDC and a non-cDC fraction by magnetic-activated cell sorting (MACS) separation. Then, 2 × 10⁵ cells were plated and stimulated for 14 hours with 25 μg/mL polyICLC, 5 μg/mL GLA, or 4 μg/mL R848. Pan IFN-α levels were determined in the cell supernatants after 14 hours by the use of ELISA. (B) IFN-α production by DC subsets after TLR stimulation. PBMCs were sequentially separated into CD141⁺ cDCs, CD304⁺ pDCs, and CD1c⁺ cDCs by MACS separation. A total of 0.25 × 10⁵ cells of the positive fractions as well as the final negative fraction were plated, stimulated, and IFN-α levels were determined as in panel A. (C) Same as in panel B but showing composite data from 3 donors. (D) Intracellular staining for IFN-α in CD141⁺ cDCs stimulated with polyICLC. cDCs were isolated from PBMCs by MACS separation and stimulated with polyICLC for 9 hours with 10 μg/mL brefeldin A. Cells were stained for surface markers followed by intracellular cytokine staining for IFN-α. Gating was performed as described in Figure 1A, and plots display the CD141⁺ cDCs. Numbers indicate the percentage of IFN-α producing cells with (right) and without (left) polyICLC stimulation. (E) Fold change of intracellular levels of IFN-α in 4 donors. Same as in panel D but shown for the CD1c⁺ and CD141⁺ cDCs fractions. Statistical analysis was performed with a paired t test. (F) IFN-α secretion by DC subsets. Cellular supernatants shown in panel C were run on pan-IFN-α−specific and IFN-α2−specific ELISAs in parallel. The plot shows composite data for the percentage of IFN-α2 of the pan IFN-α amount for 3 donors. Error bars indicate SD. (G) Transcriptional profiles for IFN-α subtypes in CD141⁺ cDCs. CD141⁺ cDCs were isolated from 3 donors by MACS and stimulated with polyICLC for 8 hours before RNA isolation. Sybr-green based Q-PCR was performed for the different IFN-α subtypes and transcript numbers were related to 1000 GAPDH copies. Results are displayed as percentage of the indicated IFN-α subtype transcript number in relation to the total IFN-α count. Error bars indicate SD. (H) Same as in panel H but for CD304⁺ pDCs stimulated with R848 for 1 hour.

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