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SummaryDevelopment of inhibitory antibodies against factor VIII (FVIII) is a severe complication of replacement therapy in haemophilia A. Patients with inhibitors are treated with high FVIII doses in the context of immune tolerance therapy (ITT). Data from haemophilia A mouse model suggest that high FVIII concentrations prevent the formation of antibody secreting cells (ASCs) from memory B cells (MBCs) by inducing apoptosis. Fc gamma receptor 2B (CD32) is an important regulator of B cell function, mediating inhibitory signals after cross-linking with the B cell receptor. Here, the role of CD32 in the regulation of FVIII-specific MBCs was investigated using F8-/- and F8-/-CD32-/- knockout mice and monoclonal antibodies (mAbs). The initial immune response was similar between F8-/- and F8-/-CD32-/- mice, including concentration of anti-FVIII antibodies and number of FVIII-specific ASCs in spleen and bone marrow. In contrast, formation of ASCs from MBCs upon rhFVIII re-stimulation in vitro was abolished in F8-/-CD32-/- mice, whereas FVIII/anti-FVIII immune complexes significantly enhanced ASC formation in F8-/- mice. Inhibition of CD32 by mAbs or F(ab)2 fragments prevented ASC formation in a dose-dependent manner. Transfer of B cell-depleted splenocytes using CD45R (B220) depletion from CD32-competent mice did not restore ASC formation in F8-/-CD32-/- cells confirming that CD32 is required on B cells. We conclude that CD32 is a crucial regulator of FVIII-specific B cells and is required for the differentiation of MBCs into ASCs. Inhibition of CD32 could potentially improve the efficacy of FVIII in the context of ITT.

Abstract 204 The formation of inhibitory antibodies to factor VIII (FVIII) is the foremost complication of replacement therapy in hemophilia A. Patients with inhibitors are treated with very high doses of FVIII, over prolonged periods of time, to induce immune tolerance. Studies in a hemophilia A mouse model demonstrated that very high doses of FVIII can induce apoptosis in FVIII-specific memory B cells and prevent their differentiation into antibody-secreting cells. The Fc gamma receptor IIb (FcgRIIb) is expressed on B cells and mediates inhibitory signals after crosslinking with the B cell receptor. Here, we studied the potential role of this receptor in the regulation of memory B cell response to FVIII. FVIII knockout mice (B6;129S4-F8tm2Kaz/J) were crossed with FcgRIIb knockout mice (B6;129S4-Fcgr2btm1Ttk/J). Comparing F8−/− mice and F8−/−/FcgR2b−/− double knockout mice, the initial anti-FVIII antibody formation was similar after intravenous exposure to 4 weekly doses of 80 or 400 IU/kg. Similar numbers of FVIII-specific antibody-secreting cells were detected in the spleen and bone marrow by ELISPOT. As previously shown, in vitro re-stimulation of memory B cells from spleens of immunized F8−/− mice at doses of 1 to 200 ng/ml induced their differentiation into antibody-secreting cells. Higher doses of 400 to 800 ng/ml prevented differentiation. In F8−/−/FcgR2b−/− double knockout mice, however, formation of antibody-secreting cells was completely inhibited across all FVIII doses tested. Addition of B220-depleted splenocytes from F8−/− mice did not restore memory B cell function in F8−/−/FcgR2b−/− double knockout mice, indicating that the observed effect was not due to dysfunction of follicular dendritic cells or other antigen-presenting cells. Inhibition of FcgRIIb using a monoclonal antibody prevented the FVIII-specific memory B cell response in splenocytes from immunized F8−/− mice. Staining with propidium iodide, annexin V, or anti-caspase 3 indicated increased rates of apoptosis when FcgRIIb was blocked during re-stimulation. In summary, FcgRIIb plays a crucial role for the differentiation of FVIII-specific splenic memory B cells into antibody-secreting cells. Inhibition of FcgRIIb appears to sensitize B cells for apoptosis during re-stimulation with FVIII. This mechanism could potentially facilitate the eradication of FVIII-specific memory B cells during ITI. Disclosures: No relevant conflicts of interest to declare.