Enhanced uptake of blood coagulation factor VIII containing immune complexes by antigen presenting cells

Abstract

Background A major complication in the treatment of hemophilia A is the development of inhibitory antibodies targeting coagulation factor VIII (FVIII). Eradication of these inhibitors can be established by immune tolerance induction (ITI), which consists of daily administration of high dosages of FVIII. FVIII immune complexes (FVIII-IC) could be formed following FVIII infusion in patients with pre-existing anti-FVIII antibodies. Objectives Here we studied endocytosis of FVIII-IC by bone marrow-derived dendritic cells (BMDCs). Methods BMDCs were pulsed with FVIII/FVIII-IC and uptake was assessed by flow cytometry and confocal imaging. Results BMDCs were able to efficiently internalize FVIII-IC in a dose-dependent manner, 3-4-fold more efficiently when compared with equimolar concentrations of non-complexed FVIII. Uptake of FVIII-IC, but not FVIII alone, could be inhibited with anti-Fcγ receptor (FcγR) antibody 2.4G2, indicating functional involvement of FcγR. No internalization of FVIII-IC was observed in BMDCs lacking FcγRI, FcγRIIb, FcγRIII and FcγRIV. Genetic ablation of FcγRIIb, FcγRIII or FcγRIV individually did not affect the ability of anti-FVIII IgG to promote the uptake of FVIII. BMDCs lacking FcγRI showed lower FVIII-IC uptake levels when compared with other single FcγR null BMDCs. Expression of the inhibitory FcγRIIb alone was sufficient to internalize FVIII-IC more efficiently than FVIII. Conclusions FcγR are critical in the internalization of FVIII-IC by BMDCs and multiple FcγR can contribute independently to this process. Our findings provide a basis for future studies to address whether the outcome of ITI is dependent on the interplay between FVIII-IC and inhibitory and activating FcγR.

N-linked glycosylation modulates the immunogenicity of recombinant human factor VIII in hemophilia A mice

“…These rFVIII immune complexes may provide an explanation for the enhanced immunogenicity documented with BHK-rFVIII through fragment crystallizable (Fc)-mediated uptake by DCs. 49 The binding of IgM to antigens further facilitates the binding of mannose-binding lectin, and both can trigger the deposition of complement that greatly increases the binding potential of FVIII to endocytic receptors. 50 These data therefore support the observation of increased clearance and immunogenicity of BHK-rFVIII.…”

Anti‐FcγRIIB (CD32) Antibodies Differentially Modulate Murine FVIII‐Specific Recall Response in vitro

“…Interestingly, recent data showed that murine BMdDCs were able to internalize FVIII complexed to anti‐FVIII IgG antibodies more efficiently than pure FVIII . This internalization was Fc γ R‐dependent.…”

“…Haemophilia A patients are treated with human FVIII to control their condition, but unfortunately, one of three patients develop neutralizing antibodies against FVIII . The FVIII‐specific immune response can be established and maintained by circulating FVIII‐containing immune complexes (F8‐ICs) suggesting an interaction of inhibitory and/or activating Fc γ Rs with the Fc portion of the anti‐FVIII antibodies . Consistently, CD32 deficiency or blockade with the dual anti‐CD16/32‐specific mAb suppresses the differentiation of murine FVIII‐specific memory B cells (MBCs) into antibody‐secreting cells (ASCs) in vitro .…”

Advances in knowledge of inhibitor formation in severe haemophilia A

“…Upon additional T‐cell‐dependent or ‐independent mechanisms, these pre‐existing B cells could undergo rapid affinity maturation, potentially giving rise to the reported 100‐fold increase in affinity towards FVIII, thus enabling the affinity‐selected antibodies to develop FVIII‐neutralising capabilities (Hofbauer et al , ). These non‐neutralising antibodies, at least in part, could be contributing to inhibitor production through mediating uptake by APCs via Fcγ receptors, as Ig‐opsonised FVIII leads to a more efficient and immunogenic response to the FVIII protein (Hartholt et al , ).…”