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

Abstract

Fc gamma receptors (FcγRs) for IgG regulate adaptive immune responses by modulating activating and inhibitory signalling pathways within immune cells. Data from a haemophilia A mouse model demonstrate that genetic deletion or blockade of the inhibitory FcγR (CD32) suppresses the formation of antibody-secreting cells (ASCs) in vitro. Mechanisms preventing the FVIII-specific recall response, however, remain unclear. Here, the potential role of CD32 inhibition was studied by differentially modulating receptor activity with selected anti-CD32 monoclonal antibodies (mAbs). Splenocytes from immunized FVIII mice were restimulated with FVIII in the absence or presence of different anti-CD32 mAbs over 6 days. At day 6, cytokine release was quantified from cell culture supernatant and the formation of FVIII-specific ASCs assessed. Binding of FVIII-containing immune complexes (F8-ICs) to bone marrow-derived dendritic cells (BMdDCs) was also investigated. The antagonistic CD32 mAb AT128 suppressed the formation of FVIII-specific ASCs and reduced secretion of IFN-γ and IL-10. In contrast, the agonistic mAbs AT130-2 and AT130-5, and their F(ab') fragments, allowed the formation of FVIII-specific ASCs, even though the full IgG of AT130-2 reduced binding of F8-ICs to CD32. Data suggest that an inhibitory signal is transmitted when F8-ICs bind to CD32 and that this signal is required during memory B cell (MBC) activation to support formation of FVIII-specific ASCs. If the inhibitory signal is lacking due to CD32 deletion or blockade with antagonistic anti-CD32 mAbs, FVIII-specific T cell stimulation and ASC formation are suppressed, whereas agonistic stimulation of CD32 restores T cell stimulation and ASC formation.

New revelations from an old receptor: Immunoregulatory functions of the inhibitory Fc gamma receptor, FcγRIIB (CD32B)

“…In agreement with this, in FcγRIIB‐deficient mice, mouse IgG2a ICs enhanced primary Ab responses, development of germinal centers and immunological memory . Inhibition of mouse FcγRIIB with an antagonistic mAb (AT128) has also been shown to suppress recall responses to human FVIII ex vivo, presumably by inhibiting binding of FVIII ICs to FcγRIIB expressed by splenic memory B cells and/or effector cells …”

Factor VIII antibody immune complexes modulate the humoral response to factor VIII in an epitope-dependent manner

“…The Fcγ receptor, specifically the inhibitory FcγRIIb (CD32), has been described as the primary mediator of endocytosis of FVIII-IC by DC and recall of FVIII-specific memory B-cell responses ( 19 , 26 , 36 ). In this study, FcγRIIb/FcγRIII blockade significantly reduced the uptake of FVIII-IC by BMDC derived from FVIII -/- mice.…”

“…Moreover, antigen reexposure in mice primed with immune complexes efficiently induces plasma cell memory responses ( 25 ). The Fcγ receptor (FcγR) has been implicated as the mediator of enhanced FVIII internalization when complexed with polyclonal anti-FVIII IgG ( 19 , 26 ). DCs in mice express activating FcγRI and FcγRIII and the inhibitory FcγRIIb, which have different binding affinities for IgG isotypes versus IgG-containing immune complexes ( 27 ).…”

Recombinant Factor VIII Fc Inhibits B Cell Activation via Engagement of the FcγRIIB Receptor

“…Case reports and retrospective studies of hemophilia A patients undergoing ITI with rFVIIIFc have suggested a quicker time to tolerization when compared to ITI using conventional FVIII concentrates (17,18). Finally, antibodies targeted to FcγRIIB have been shown to modulate the FVIII immune response (19). Based on this evidence we hypothesize that rFVIIIFc may inhibit FVIII-specific B cells more efficiently than FVIII due to its ability to cross-link the BCR of these cells with FcγRIIB.…”

“…Whole splenocytes from naïve or FVIII-exposed mice as well as 413 cells were incubated with varying doses of BDD FVIII (0, 0.1, 0.2, and 0.4 µg/test) or APC-conjugated anti-FcγRIIB (APC anti-FcγRIIB: 0, 0.1, 0.2, and 0.4 µg/test) for 30 min at 4 • C in order to block potential binding sites of rFVIIIFc on these cells. Anti-FcγRIIB antibody clone AT130-2 was used because it has previously been shown to have agonistic effects against its target (24) and prevent binding of FVIII immune complexes to FcγRIIB (19). YFP rFVIIIFc was then added at 0.3 µg/test for 30 min at 4 • C. The amount of YFP rFVIIIFc binding was then measured via flow cytometry (SH800S, Sony).…”