Humanized E17 Hemophilic Mice Are a Major Breakthrough in the Design of New Preclinical Models for Developing Factor VIII Products with Reduced Immunogenicity.
Authors: Reipert Birgit M. , Hausl Christina , Sasgary Maria , Schuster Maria , Ahmad Rafi U. , Baumgartner Bernhard , Ilas Josenato , Bauer Natalie , Ehrlich Hartmut , Schwarz Hans Peter 
Published: October 2019

MHC class II molecules are crucial for regulating adaptive immune responses against self and foreign protein antigens. They determine the antigenic peptides that are presented to CD4+ T cells and are essential for shaping the CD4+ T-cell repertoire in the thymus. Thus, the structure of MHC class II molecules is a major determinant for protein antigen immunogenicity. Structural differences between murine and human MHC class II complexes fundamentally limit the use of conventional murine hemophilia A models for dissecting immune responses to human factor VIII and developing new factor VIII products with reduced immunogenicity. To overcome this limitation, we humanized the murine E17 model of hemophilia A by introducing the human MHC class II haplotype HLA-DRB1*1501 on the background of a complete knockout of all murine MHC class II genes. Any anti-FVIII antibody response in this new humanized hemophilia A model is driven by CD4+ T cells that recognize FVIII-derived peptides that are presented by human HLA-DRB1*1501. The MHC class II haplotype HLA-DRB1*1501 is particularly relevant for the situation in hemophilia A patients because it is found in about 25% of Caucasians and 32% of Africans and has been shown to be associated with an increased risk that patients with severe hemophilia A have for developing FVIII inhibitors. We validated the relevance of this new model by asking the question whether HLA-DRB1*1501 hemophilic E17 mice develop FVIII inhibitors that are similar to those observed in patients with hemophilia A. Furthermore, we wanted to show that anti-FVIII antibody responses in these mice depend on the expression of the human DRB1*1501 molecule. Mice were treated with 8 intravenous doses of human FVIII and tested for anti-FVIII antibodies, anti-FVIII antibody-producing plasma cells and FVIII-specific T cells. About 90% of all humanized hemophilic E17 mice tested developed anti-FVIII antibodies that were similar to FVIII inhibitors found in patients. These antibodies were not restricted isotypically and contained mainly IgG1, IgG2a and IgG2b antibodies. Detection of antibodies in the circulation correlated with the presence of anti-FVIII antibody-producing plasma cells in the spleen. Development of anti-FVIII antibodies depended on the activation of FVIII-specific T cells and strictly depended on the expression of the HLA-DRB1*1501 molecule. Mice that did not express any MHC class II molecules did not develop anti-FVIII antibodies. We conclude that this new humanized E17 model for hemophilia A is a major advance towards developing suitable animal models needed to design future immunomodulatory strategies for patients with FVIII inhibitors and develop new FVIII products with reduced immunogenicity. Furthermore, it provides a tool for identifying T-cell epitopes of human FVIII restricted by MHC class II molecules that can be used for monitoring FVIII-specific T cells in patients who receive replacement therapy with FVIII products.