FIG 2.

HBsAg-induced DC maturation is dependent on CD14 and TLR4. (A and B) mDC were preincubated with αCD14 or isotype control and subsequently incubated with medium or 100 ng/ml pHBsAg for 20 h. Means ± SEM of percent CD83⁺ (%CD83⁺) DC and CD40 MFI (A) and IL-6 or IL-12p40 (B) for 4 or 5 experiments with different donors. n.d., not detected. (C) PBMCs depleted for CD14⁺ cells were preincubated with medium (−), αCD14, or isotype control and subsequently exposed to 1 μg/ml FL-HBsAg for 2 h at 37°C. mDC exposed to medium and FL-HBsAg at 4°C were used as controls. The percentage of HBsAg⁺ cells of BDCA1⁺CD19-DC was determined by flow cytometry. Representative FACS histograms and summary of means ± SD from duplicate assays showing the percentage of HBsAg⁺ DC. Data are representative of three experiments with different donors. (D and E) mDC were preincubated with medium (−), αCD14, αTLR4, a combination of αCD14 and αTLR4 (combi), or isotype control and subsequently exposed to medium or 100 ng/ml pHBsAg (D) or recombinant HBsAg (rHBsAg) (E) for 20 h. Mean ± SEM %CD83⁺ DC from four (D) or three (E) independent experiments with different donors. Paired Student's t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (F) Example of flow cytometry analysis of CD14 and BDCA1 expression on PBMC. Indicated percentage represents the percentage of CD14⁺ BDCA1⁺ cells. The plot is representative of five experiments with different donors. (G) mDC, isolated either by standard procedure (white bars) or with additional CD14 depletion (black bars), were preincubated with αCD14 or isotype control and subsequently exposed to medium (−) or pHBsAg. Data represent mean ± SEM %CD83⁺ mDC for three experiments with different donors.