Unexpected Enhancement of FVIII Immunogenicity by Endothelial Expression in Lentivirus-transduced and Transgenic Mice
ISTH Academy. Shi Q. Jul 10, 2019; 273938; OC 76.1 Topic: Coagulation Factors & Inhibitors
Dr. Qizhen Shi
Dr. Qizhen Shi
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OC 76.1

Unexpected Enhancement of FVIII Immunogenicity by Endothelial Expression in Lentivirus-transduced and Transgenic Mice

Q. Shi1, C. Carman2, Y. Chen1, F. Xue1, X. Liang3, G. Gilbert3
1Medical College of Wisconsin, Blood Research Institute, Children's Research Institute, Milwaukee, United States, 2Harvard School of Public Health, Boston, United States, 3VA Boston Healthcare System and Harvard Medical School, Boston, United States

Main Topic: Coagulation and Anticoagulation
Category: Coagulation Factors & Inhibitors

Background: Endothelial cells (ECs) provide the physiologic site for FVIII production, However, the potential for therapeutic expression of FVIII from ECs remains uncertainty and the effect on development of inhibitory antibodies (inhibitors) remains unknown.
Aims: To explore the potential role of endothelial cells for in situ engineered FVIII expression and the impact on FVIII immunogenicity.
Methods: FVIII expression was introduced by lentivirus-mediated in situ transduction or embryonic stem cell-mediated transgenesis. FVIII activity and inhibitor titers were determined by standard techniques. A T cell proliferation assay was used to examine the antigen presenting cell (APC) functions of ECs. Confocal microscopy was used to examine FVIII uptake by ECs.
Results: All HA mice unexpectedly developed inhibitors after in situ T2F8 lentivirus-mediated EC transduction. Inhibitors, but not FVIII activity, were detected in plasma. In contrast lentivirus-mediated bone marrow transduction, producing FVIII in platelets led to tolerization. T2F8 transgenic (T2F8Tg) mice, in which FVIII expression is driven by the EC-specific Tie 2 promoter normalized plasma FVIII levels but contributed to an unexpectedly robust immune response. A single FVIII - incomplete Freund's adjuvant injection led high titers of inhibitors, reducing plasma-FVIII to undetectable levels. Titers were significantly higher than in wild-type C57BL/6 controls and in platelet-targeted FVIII transgenic mice. Because EC's are putative non-hematopoietic, semi-professional-APCs we asked whether EC's might directly influence the FVIII immune response. T cell proliferation assays confirmed that microvascular ECs that were conditioned with inflammatory cytokines could take up exogenous FVIII and stimulate T follicular helper (Tfh) cells to FVIII-specific proliferation. Imaging studies confirmed that these ECs indeed bind and take up FVIII protein.
Conclusions: Our results demonstrate unanticipated antigenicity of endothelial-expressed FVIII and a possible pathway for immune enhancement of FVIII immunogenicity through EC function as auxiliary APCs for Tfh cells.

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