Charactering the immune signature of antigen presenting cells in the immune response to factor VIII in a murine model of hemophilia A using single-cell RNA sequencing

Abstract

Abstract Deficiency of the procoagulant protein factor VIII (FVIII) is the defining characteristic for individuals with the inherited X-linked bleeding disorder hemophilia A. These individuals require intravenous infusions of FVIII to treat bleeding events, however ~30% develop neutralizing alloantibodies (i.e., inhibitors) against exogenous FVIII. Transcriptome analysis of FVIII-stimulated peripheral blood mononuclear cells in patients with hemophilia A and active inhibitors showed increased expression of innate immune modulators and proinflammatory pathways. Murine models of hemophilia A (FVIII KO) similarly develop anti-FVIII antibodies with FVIII exposure. Although the antibody response to FVIII is CD4-T cell dependent, the key antigen presenting cells (APCs) that mediate inhibitor formation is unclear. Bone marrow chimeras of transgenic mice with depleted dendritic cell (DC) subsets in FVIII KO suggest that type 2 classical DCs (cDC2), but not cDC1 or plasmacytoid DCs, mediate FVIII immunity. We then evaluated the evolution of splenic APCs and their immune cell signatures in FVIII KO mice injected with 0, 1, 3, or 5 doses of FVIII using flow cytometry and single-cell RNA sequencing (scRNAseq). FVIII KO injected with 5 doses of FVIII had similar frequencies of pDC, cDC1, and cDC2 subsets compared to mice injected with normal saline as a control. However, preliminary scRNAseq analysis demonstrated increased expression of phagocytic and interferon-related genes among DC and macrophage clusters, particularly CD8a −CD4 +Itgax −DCs. Further scRNAseq analyses of APC subclusters and differentially expressed genes will be assessed. Overall, these findings suggest that cDCs, specifically cDC2, are critical in FVIII immunity. Supported by NIH K99HL150595 and Hemophilia of Georgia Clinical Scientist Development Grant.