Binding of HCV-LPs to monocyte-derived DCs. (A) Immunophenotyping of immature (imDC) and mature DCs (mDC) used for HCV-LP binding. DC phenotypes were characterized by flow cytometry of HLA-DR, CD86, CD80, and CD83 surface expression (LIN indicates a combination of anti-CD3, -CD14, -CD16, -CD19, -CD20, and -CD56 antibodies). Histograms corresponding to cell surface expression of the respective cell surface molecules (shaded curves) are overlaid with histograms of cells incubated with the appropriate isotype control antibody (open curves). The absence of CD3, CD14, CD16, CD19, and CD56 expression excluded the presence of B cells, T cells, natural killer (NK) cells, or macrophage/monocytes in the DC preparation. (B) Binding of HCV-LPs to monocytes, immature, and mature DCs. Flow cytometry histograms of HCV-LP binding to target cells (open curves) are shown. Background fluorescence (gray shaded curves) was measured using cells incubated with insect cell control preparations. Cellular binding of HCV-LPs was analyzed using mouse anti-E2 mAb (16A6) and PE-conjugated anti–mouse IgG. (C) Dose-dependent binding of HCV-LPs to target cells. Immature (▪) and mature DCs (□; left graph), monocytes (▴), T (○), and B cells (⋄; right graph) were incubated with increasing concentrations of HCV-LP or insect cell control preparations. On the y-axis, net mean fluorescence intensity (Δ MFI) values for each HCV-LP E2 concentration were calculated by subtracting the MFI of insect cell control preparations from that obtained with the respective HCV-LP E2 concentration (x-axis). Concentration of HCV-LP E2 was quantified by an ELISA as described earlier.³³