The Relevance of the Inhibitory Receptor FcγIIB for the Regulation of Anti-Factor VIII Antibody Responses in Murine Models.
Authors: Hausl Christina , van Helden Pauline M. , Ahmad Rafi U. , Muchitsch Eva M. , Schwarz Hans Peter , Ehrlich Hartmut , Voorberg Jan , Reipert Birgit M. 
Published: October 2019

Abstract
The prevention of neutralizing anti-factor VIII (FVIII) antibodies remains the major challenge in the treatment of hemophilia A patients with FVIII products. Therefore, it is important to understand how B cells differentiate into antibody-producing plasma cells, how this process is regulated and how it can be inhibited. A number of inhibitory receptors have been identified that are expressed on B cells and might provide targets for the prevention of antibody development against FVIII. The inhibitory receptor FcγIIB has emerged as a key regulator of B cell responses in the later phase of antibody responses. It has been described as a potent inhibitor of B-cell-receptor (BCR) signaling when co-ligated to the BCR by engagement of antigen-containing immune complexes. Based on these findings we asked whether the inhibitory receptor FcγIIB is involved in the natural regulation of anti-FVIII antibody responses in murine models. If this receptor were an important negative regulator of anti-FVIII antibody responses, mice that do not express this receptor should develop higher titers of anti-FVIII antibodies than mice that express the receptor. We treated wildtype mice and FcγRIIB knockout mice, both on a C57BL/6J background, with four intravenous doses of 200ng of human FVIII (80U/kg) and analyzed titers of anti-FVIII antibodies after the second, the third and the fourth dose. Surprisingly, we did not see any significant difference in antibody titers between both strains of mice. We extended our study and asked under which conditions FcγRIIB knockout mice would develop higher titers of anti-FVIII antibodies than wildtype mice. We tested different application routes (i.v. and i.p.) of FVIII, compared low doses (200ng) and high doses (80μg) and combined the i.p. application with or without Freund’s adjuvant. Furthermore, we included KLH-TNP (i.p. application of 100μg with Freund’s adjuvant) as a positive control that was previously shown to induce different levels of antibodies in wildtype mice compared with FcγRIIB knockout mice. We found significant differences in the development of specific antibodies between both strains of mice when we used the positive control (i.p. KLH-TNP with adjuvants) and when we applied high dose FVIII (80μg) with Freund’s adjuvant i.p. We did not see any differences in antibody titers between both strains of mice with any of the other treatment schedules. Our results indicate that the inhibitory receptor FcγIIB is only active as a negative regulator for antibody responses against foreign proteins in vivo when a certain threshold level of antibodies is reached in the circulation. This critical threshold level is obviously not reached when mice are treated with clinically relevant doses of FVIII. Based on our results we conclude that the inhibitory receptor FcγIIB is not involved in the regulation of antibody responses against therapeutically relevant doses of human FVIII in murine models. Whether the same situation is true for patients with hemophilia A remains to be shown. Furthermore, the relevance of the inhibitory receptor FcγIIB might differ between patients who react to the FVIII product as a foreign protein and patients who react to FVIII as an altered self protein or self protein.