Defining the Innate Immune Cell Profile of Factor VIII Immunity - PROJECT SUMMARY The development of inhibitors (i.e., neutralizing antibodies) against infused coagulation protein factor VIII (FVIII) is the most significant treatment-related complication in modern hemophilia A care. Administration of frequent and high doses of FVIII over months-years, referred to as immune tolerance induction, remains the primary strategy for inhibitor eradication for the last 40 years. There is a critical need for novel, innovative, and less burdensome approaches to immune tolerance induction to restore FVIII hemostatic efficacy in the management of acute bleeds or surgeries in persons with hemophilia A (PwHA) and inhibitors. Despite numerous novel treatments recently approved or in the pipeline for hemophilia A treatment, the lack of therapeutic strategies for inhibitor prevention or universally successful inhibitor eradication methods are persistent barriers within the field that warrant continued research focus. Moreover, a poor understanding of the underlying immunologic processes that mediate FVIII immunity further contribute to the limited advancements in therapies for peripheral tolerance induction. The long-term goal of this work is to define the key cellular components and early immunologic events that mediate FVIII immunity to ultimately develop and test targeted immunomodulatory therapies that prevent inhibitor formation and induce peripheral FVIII tolerance. The rationale for this R00 proposal stems from preliminary data demonstrating that type 2 conventional dendritic cells (cDC2) are key mediators in anti-FVIII antibody development in a murine model of hemophilia A. Additionally, single cell RNA sequencing (scRNAseq) analysis show upregulation of phagocytic and interferon genes by cDC2 following FVIII injections in hemophilia A mice signifying their contribution in FVIII recognition and presentation. The central hypothesis is that with early FVIII exposure conventional DC (cDC), specifically cDC2, mediate CD4+ T cell activation in a proinflammatory cytokine microenvironment that induces B cell activation and production of inhibitory anti-FVIII antibodies. The central hypothesis will be tested through the following 2 aims: 1) Define the cytokine microenvironment of cDC2 activation and CD4+ T cell differentiation in FVIII inhibitor development, and 2) Determine the innate immune profile across the lifespan of PwHA with and without inhibitors. Aim 1 will examine the cDC2-activated cytokine microenvironment, focusing of interferon gamma (IFN-γ) and IL-12 production and cellular source, that dictates antibody production in FVIII knockout mice with and without cDC2 depletion. Aim 2 will investigate and compare the innate immune transcriptomic profile of peripheral blood mononuclear cells isolated from PwHA based on age groupings, inhibitor status, and treatment regimen utilizing 10X Genomics scRNAseq and flow cytometry. This research is significant because it will advance understanding of the innate immune response to FVIII and help illuminate therapeutic strategies that can be administered during early FVIII exposure to prevent inhibitor development and induce peripheral immune tolerance.
