Deletion or inhibition of Fc gamma receptor 2B (CD32) prevents FVIII-specific activation of memory B cells in vitro

Summary

Development 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.

Keywords

Haemophilia A mouse model - CD32 - inhibitor - memory B cells - recall response

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