Administration of Dexamethasone during Initial Exposure to Factor VIII Prevents the Development of Anti-Factor VIII Antibodies and Increases the Proportion of Thymic but Not Splenic Regulatory T Cells in the Exon 16 Knockout C57Bl6 Hemophilia Α Mouse Model

Abstract Background: The most severe complication of hemophilia A (HA) treatment is the development of inhibitory antibodies (inhibitors) to factor VIII (FVIII). The presence of immunological ‘danger signals’ during initial exposure to FVIII may increase the risk of developing inhibitors. Promoting an anti-inflammatory environment during initial exposure to FVIII may therefore prevent inhibitor development in HA patients. Glucocorticoids are used clinically for their immunosuppressive and anti-inflammatory properties. There has also been evidence to suggest that these agents have the ability to induce regulatory T cells (Tregs): a CD4+CD25+FoxP3+T cell subset involved in the maintenance of tolerance to self-antigens. Previous work in our laboratory showed that administration of the glucocorticoid dexamethasone (Dex) during initial FVIII exposure renders tolerance to FVIII in exon 17 knockout (E17KO) HLADRB1*1501 C57Bl6/S129 HA mice. When treated with recombinant human FVIII (rhFVIII) this model has an anti-FVIII antibody incidence of 30%. In contrast, the E16KO C57Bl6 HA mouse model has 100% antibody incidence when treated with rhFVIII. Using this second model provides a method for validating our treatment protocol in an animal model with no inherent tolerance to FVIII and facilitates future mechanistic studies due to its homogeneous genetic background. Aims: To assess the ability of Dex administration during initial FVIII exposure to reduce the FVIII immune response in a HA mouse model with high propensity for inhibitor development, and enumerate lymphocyte subsets in the spleen and thymus to examine possible mechanisms of this treatment. Methods: E16KO C57Bl6 HA mice received Dex (75 μg, intraperitoneally) and rhFVIII (6 IU, intravenously) (rhFVIII+Dex Group) or rhFVIII alone (rhFVIII Group) for five consecutive days. Five weeks later, blood was collected via cardiac puncture. Plasma anti-FVIII antibody titres and FVIII inhibitory activity were determined using an anti-FVIII immunoglobulin G (IgG) ELISA and Bethesda assay respectively. Statistical comparisons were calculated using the Fisher’s exact and Mann-Whitney U tests. To elucidate early effects of the treatment, E16KO C57Bl6 mice received rhFVIII, Dex, rhFVIII+Dex, or HBSS for five consecutive days. Three days later the spleen and thymus were harvested. The percentages of splenic B cells (CD19+) and Tregs (CD4+CD25+FoxP3+) as well as thymic Tregs (CD4+CD8-CD25+FoxP3+) were quantified by flow cytometry. Statistical comparisons were calculated using a 2-tailed Student’s t-test. Results: Five weeks after treatment, 77% of mice in the rhFVIII+Dex Group vs 100% of mice in the rhFVIII Group developed detectable anti-FVIII IgG (p=0.0485). Furthermore, mice in the rhFVIII+Dex Group had overall lower anti-FVIII IgG titres (p=0.0063) and lower inhibitory activity (p=0.0783) than mice in the rhFVIII Group. Examination of the spleen three days after Dex treatment showed a decrease in the percentage of CD19+ cells in comparison to HBSS (41% vs 54%, p=0.0152). The same effect was observed with Dex+rhFVIII in comparison to rhFVIII (48% vs 54%, p=0.0489) or HBSS (48% vs 54%, p=0.0212). No significant changes were detected in the percentage of CD4+CD25+FoxP3+splenocytes across treatment groups. In the thymus, Dex caused a decrease in the percentage of CD4+CD8+ cells (42% vs 76%, p=0.0184) and an increase in the percentage of CD4-CD8+ cells (24% vs 7%, p=0.0107) in comparison to HBSS. The same effects were observed with Dex+rhFVIII in comparison to rhFVIII (40% vs 72%, p=0.0204; 20% vs 8%, p=0.0368) or HBSS (40% vs 76%, p=0.0137; 20% vs 7%, p=0.0266). The percentage of CD4+CD8- cells remained unchanged across treatment conditions. Dex caused an increase in the percentage of thymic CD4+CD8-CD25+FoxP3+ cells when compared to HBSS (11% vs 4%, p=0.0188). The same effect was observed with Dex+rhFVIII in comparison to rhFVIII (12% vs 5%, p=0.0006) or HBSS (12% vs 4%, p=0.0001). Conclusions: Administration of Dex during initial FVIII exposure reduces the anti-FVIII immune response in a HA mouse model with high propensity for inhibitor development. The effect is accompanied by a decrease in the percentage of splenic B cells and thymic CD4+CD8+ cells, and an increase in the percentage of thymic CD4-CD8+ cells and Tregs. These findings suggest potential mechanisms whereby glucocorticoid administration results in tolerance to FVIII in HA mice. Disclosures Moorehead: Baxter: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria. Reipert:Baxter Innovation GmbH: Employment. Hough:Bayer: Research Funding. Lillicrap:Bayer: Research Funding; Baxter: Research Funding; Biogen-Idec.: Research Funding; CSL-Behring: Research Funding.

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