pDCs robustly produce IFNα in response to DENV infected cells.

<p>(<b>A</b>) Quantification of IFNα in the supernatants of PBMCs, pDC-depleted PBMCs and isolated pDCs (Responders) co-cultured with DENV infected Huh7.5.1 cells (DENV cells) or treated with 100 µl of supernatants from the latter (DENV SN), as indicated (Inducers). Viral titers of DENV SN ≈2.5×10⁶ foci forming units (ffu)/ml. pDC depletion/enrichment was performed using an anti-BDCA-4 antibody. Cell #; number of co-cultured responder cells. Cont cells; uninfected Huh-7.5.1 cells. Arrows indicate results below the detection threshold of the IFNα ELISA (<i>i.e.</i>, 12.5 pg/ml). Results are representative of 4 independent experiments. Error bars represent the means ± SD. (<b>B</b>) Representative FACS analysis of pDC depletion and isolation from PBMCs using the pDC selective markers, CD123 and BDCA-2. (<b>C</b>) Quantification of IL6 in the supernatants of PBMCs and pDC-depleted PBMCs triggered by LPS (10 µg/mL for 20 hours). Results are expressed as percentage relative to LPS-treated PBMCs. 3 independent experiments in triplicate (error bars, means ± SD), paired Student's t test, ***p<0.0001, NS p>0.1, Δ; indicates separated group by ANOVA. (<b>D</b>) Total PBMC population containing a number of pDCs equivalent to the purified pDCs as determined by FACS analysis as described in (B) were co-cultured with DENV infected cells. IFNα productions were thus compared for equal numbers of pDCs. PBMCs and pDC-depleted PBMCs were compared using equal cell numbers. The results are expressed as IFNα levels relative to the co-culture with PBMCs, set at 100, 3 independent experiments in triplicate, paired Student's t test, ***p<0.0001, NS p>0.05, Δ; indicates separated group by ANOVA (error bars, means ± SD). (<b>E</b>) Quantification of IFNα secretion by pDCs isolated from the blood of a healthy donor cohort (n = 20) co-cultured with infected cells or treated with their supernatant. IFNα levels in the supernatants of pDCs co-cultured with uninfected (cont) cells or with DENV SN were all below the detection threshold (<i>i.e.</i>, 12.5 pg/ml).