Investigating persistent factor VIII-specific IgM in the humoral immune response to factor VIII

Abstract

Abstract Hemophilia A is an X-linked bleeding disorder resulting in bleeding events due to insufficient levels of factor VIII (FVIII). The most significant complication in the management of hemophilia A is the formation of polyclonal neutralizing IgG (i.e., inhibitors) which impede FVIII procoagulant activity. In a hemophilia A (FVIII KO) murine model injected with multiple doses of FVIII, FVIII-specific IgM were found to persist despite an established IgG response. In attempt to characterize the role of IgM in FVIII immunity, 18 IgMs were purified from immortal FVIII-specific hybridomas and FVIII binding was confirmed via ELISA. IgMs demonstrated weak binding to FVIII in a static ELISA model with mean optical densities (OD) at 405 nm of 0.33 ± 0.43 compared to anti-FVIII IgG with a mean OD of 1.85 ± 0.02. A novel fluid-phase ELISA, developed in our lab, utilizing N-hydroxysuccinimide (NHS)-activated magnetic beads indirectly conjugated to FVIII increased IgM-FVIII binding with mean OD of 1.15 ± 0.60. IgM binding also demonstrated porcine FVIII cross-reactivity. To evaluate the effect of IgM on antibody titers in vivo, FVIII KO mice received 5 weekly injections of either FVIII or 4 separate immune complexes (IC) of IgM-FVIII. Mice injected with IgM-FVIII IC showed slightly higher median IgG ELISA titers than FVIII alone. These findings suggest a potential role for persistent anti-FVIII IgM in propagating FVIII inhibitor development. Further studies determining the effect of IgM-FVIII IC on complement activation, as well as anti-FVIII IgM epitope specificity and clonality utilizing computational methods of RNA-sequencing, may provide additional mechanistic insight into the role of persistent IgM in the humoral immune response to FVIII. Hemostasis and Thrombosis Research Society- Student Research Award (EY), NIH K99HL150595 (GB), and Hemophilia of Georgia Clinical Scientist Development Grant (GB).