OC 37.5 - Liver sinusoidal endothelial cells as a novel target for tolerance induction to FVIII

Background: A major complication of current treatment of hemophilia A is the development of FVIII inhibitory antibodies. Currently, immune tolerance induction (ITI) is used to eradicate FVIII inhibitors. Since ITI fails in 30% of patients within the EDUC8 consortium we are exploring novel ways to induce tolerance to FVIII.

Aims: Explore the potential of targeting FVIII or FVIII derived peptides to liver sinusoidal endothelial cells (LSEC) since these cells have been shown to promote tolerance instead of immunity towards the antigens presented on their surface.

Methods: Human hepatic sinusoidal endothelial cells were cultured and characterized. We studied their phenotype by FACS analysis and confocal imaging using specific antibodies against FVIII, stabilin-2 and other markers of LSEC and endothelial cells. In a parallel approach we also employed stabilin-2 transfected HEK293 cells as a model system for stabilin-2 dependent targeting of LSEC.

Results: Our results show that primary human hepatic endothelial cells express von Willebrand factor. Primary human liver endothelial cells also express VE-Cadherin and evidence was obtained for expression of stabilin-2 on the cell membrane employing confocal microscopy. FACS analysis confirmed these findings and provided evidence for the expression of CD32, previously shown to be expressed on LSEC. In parallel we established a transient system for expressing stabilin-2 in HEK293 cells which will be used to study the interaction of FVIII and VWF with stabilin-2 and explore whether the internalization is linked to activation of tolerogenic pathways in LSEC.

Conclusion(s): Our findings indicate that LSECs are a potential target for the induction of tolerance. The primary human hepatic endothelial cells used in this study express several LSEC specific markers. The presence of stabilin-2 on the surface of these cells will allow us to target this receptor employing recombinant proteins containing FVIII immunogenic domains or peptides with the ultimate goal of inducing tolerance to FVIII.