Figure 6.

Sialic acid does not alter FVIII binding to, and maturation of splenocytes and dendritic cells in vitro, and does not modulate the FVIII immune response in vivo. Desialylated and control rFVIII were incubated with FVIII naïve splenocytes for 1 hr at 4°C and assessed for binding by flow cytometry. Cells were gated as total (A) splenocytes or (B) CD11c⁺ dendritic cells (DCs). rFVIII association with DCs was quantified as the proportion of FVIII+CD11c⁺ out of all CD11c⁺ cells. Flow cytometry characterization of the surface co-stimulatory molecules, CD80 and CD86, were performed on (C) splenocytes and (D) CD11c⁺ DCs incubated for 24 hr under the indicated conditions (1 μg/mL LPS or rFVIII; n=4). HA-R593C mice were administered 1 μg of rFVIII and its desialylated glycoform subcutaneously biweekly for 2 weeks. Plasma samples 28 days after the initial infusion were assessed for (E) the incidence of FVIII-specific IgG and (F) the titre of the anti-FVIII IgG response. Line and error bars represent mean and SEM respectively. Data representative of at least 3 independent deglycosylation reactions.^**P<0.01; ^***P<0.001. MFI: mean fluorescence intensity; rFVIII: recombinant factor VIII; Ig: immunoglobulin; n.s. not significant.