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

• DOI: 10.1160/th14-06-0535
• Publication Date: 2015-08-06T10:58:52Z
• Authors: Nadine Vollack; Marcus von Hornung; Katy Kalippke; Julia Kutzschbach; Arne Trummer; Arnold Ganser; Andreas Tiede; Sonja Werwitzke
• Keywords: Mice, Knockout; 0301 basic medicine; Factor VIII; Receptors, IgG; B-Lymphocyte Subsets; Dose-Response Relationship, Immunologic; Antibodies, Monoclonal; Antibodies, Heterophile; Apoptosis; Antigen-Antibody Complex; Hemophilia A; Lymphocyte Activation; Adoptive Transfer; Antibodies, Neutralizing; 3. Good health; Disease Models, Animal; Mice; 03 medical and health sciences; Bone Marrow; Animals; Leukocyte Common Antigens; Immunologic Memory; Cells, Cultured

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.