Early mechanisms of factor VIII immunity in hemophilia A
The most serious complication in treating hemophilia A (HA) is the development of an immune response to the therapeutically-administered factor VIII (FVIII). A better understanding of initiating events leading to FVIII immunity may highlight alternative treatment interventions. We have explored this synapse at the level of the FVIII protein, the cellular interactions, and the surrounding microenvironment. At the protein level, we have confirmed epidemiological findings suggesting that recombinant FVIII produced in a baby hamster kidney cell line is more immunogenic than FVIII produced in Chinese hamster ovary cell line. We have attributed this, in part, to differences in protein N-linked glycosylation, where incomplete occupancy of N-linked consensus sequences reveals epitopes for naturally circulating IgM antibodies. To elucidate cellular immune responses, we observed that human FVIII interacts initially with MARCO+ macrophages in the marginal zone of the mouse spleen. Functional enrichment analysis of the upregulated splenic transcriptome 3 hours after intravenous FVIII administration suggests a modulation of STAT1 and STAT3 signaling, while a similar analysis in the liver implicate Wnt signaling. Interestingly, an endocytic receptor has never been implicated in FVIII immunity in vivo. A GWAS study recently implicated a role of the endothelial cell scavenger receptor, stabilin-2, on circulating FVIII levels. Here, we showed that stabilin-2 knockout mice exhibit an attenuated immune response against FVIII compared to wildtype mice in a human von Willebrand factor-dependent manner. Lastly, we assessed the role of an inflammatory microenvironment on FVIII immunogenicity. The proximity of pediatric vaccination and the initiation of FVIII replacement therapy has prompted clinical concerns that concurrent exposure to inflammatory viral constituents may increase the risk of FVIII immune responses. In a transgenic HA mouse model, we have shown that intramuscular immunization against influenza decreases FVIII immune responses through a mechanism of antigenic competition leading to CD4+ T cell diversion towards the site of immunization. Collectively, these studies highlight potential areas for therapeutic intervention, and also provide clinicians with mechanistic evidence that may aid in the treatment of HA.