Uptake of blood coagulation factor VIII by dendritic cells is mediated via its C1 domain

Abstract

The uptake and processing of blood coagulation factor VIII (FVIII) by antigen-presenting cells and the subsequent presentation of FVIII-derived peptides to CD4(+) Tcells direct the immune response to FVIII in patients with hemophilia A. Multiple receptors including mannose receptor and low-density lipoprotein receptor-related protein-1 (LRP1) have been implicated in FVIII uptake. This work studies the involvement of receptor candidates in FVIII uptake by dendritic cells (DCs). Furthermore, we explore FVIII residues that mediate endocytosis. FVIII uptake was performed with human monocyte-derived and murine bone marrow-derived DCs. To investigate FVIII endocytosis, competition assays with soluble receptor ligands, binding studies with recombinant receptor fragments, and small-interfering RNA-induced gene silencing were performed. In addition, FVIII-targeting monoclonal antibodies KM33 and VK34 were used. To confirm invitro results, hemophilic E17 knockout mice were pretreated with antibodies prior to FVIII injections and anti-FVIII titers were determined. Upon treatment of DCs with mannan or LRP ligand α2-macroglobulin, we observed only a minor decrease in FVIII internalization. In addition, small interfering RNA-mediated knockdown of LRP, mannose receptor, or DC-SIGN expression in monocyte-derived dendritic cells did not prevent FVIII uptake. Binding studies using Fc chimeras revealed that LRP, DC-SIGN, and mannose receptor can bind to FVIII; however, we did not observe a critical role for these receptors in FVIII uptake. Previous studies have shown that human antibodies targeting the C1 (KM33) and A2 (VK34) domains of FVIII interfere with binding to endocytic receptors. Preincubation of FVIII with VK34 did not influence FVIII uptake; however, KM33 completely inhibited FVIII endocytosis by both monocyte-derived dendritic cells and bone marrow-derived dendritic cells. Accordingly, anti-FVIII antibody titers were greatly reduced following the preadministration of KM33 invivo. Together, our observations emphasize the physiological significance of KM33-targeted residues within theC1 domain in the uptake of FVIII by DCs invitro and invivo.