Immune Complexes Enhance Uptake of Blood Coagulation Factor VIII by Antigen Presenting Cells

Abstract 37

The X-chromosome-linked bleeding disorder hemophilia A is caused by the absence or dysfunction of blood coagulation factor VIII (FVIII) which can be corrected by regular intravenous administration of FVIII. Development of antibodies directed against FVIII (referred to as FVIII inhibitors) is common complication in hemophilia care. Immune tolerance induction (ITI) comprising frequent administration of high doses of FVIII is the treatment of choice for hemophilia A patients with inhibitors. Thus far, the molecular mechanisms contributing to tolerance induction have not been defined. Here we sought to study the role of immune complex (IC) formation in endocytosis and functional presentation of FVIII by antigen presenting cells. Bone marrow-derived dendritic cells (DC) were used as model antigen-presenting cells, as DCs are known for their ability to take up and process immune complexes via FcγR. Purified recombinant FVIII was labeled using the Microscale Alexa Fluor 488 protein-labeling kit. For T-cell proliferation assays, FVIII^−/− mice were injected intravenously 5× weekly with 1 μg of recombinant FVIII. Subsequently, spleens collected after weekly injections were processed into single-cell suspensions and used as a source of FVIII-specific T cells. Bone marrow-derived murine DCs were able to efficiently take up FVIII pre-complexed with anti-FVIII antibodies (FVIII-IC) in a dose-dependent manner. Endocytosis of FVIII-IC was 3–6 fold more efficient when compared to equimolar concentrations of soluble FVIII. Uptake of FVIII-IC, but not FVIII alone, could be inhibited with 2.4G2 antibody indicating functional involvement of FcγRII/III in this process. These results were confirmed using murine DCs isolated from FcγR-deficient mice. Moreover, antibodies used for IC formation were analysed based on isotype – IgG2a antibodies were most efficient in mediating IC endocytosis. Experiments performed with FVIII-specific T cells showed that in the low concentration range (<1 nM), FVIII-IC induced stronger T cell proliferation when compared to soluble FVIII. Collectively, these data provide further insight and understanding of modulation of anti-FVIII immune response in hemophilia A patients with pre-existing FVIII inhibitors.