THROMBOSIS AND HEMOSTASIS Plant-based oral tolerance to hemophilia therapy employs a complex immune regulatory response including LAP 1 CD 4 1 T cells

• Coadministering FIX orally and systemically induces tolerance via complex immune regulation, involving tolerogenic dendritic and T-cell subsets. • Induced CD4CD25LAP regulatory T cells with increased IL-10 and TGF-b expression and CD4CD25 regulatory T cells suppress antibody formation against FIX. Coagulation factor replacement therapy for the X-linked bleeding disorder hemophilia is severely complicated by antibody (“inhibitor”) formation. We previously found that oral delivery to hemophilic mice of cholera toxin B subunit-coagulation factor fusion proteins expressed in chloroplasts of transgenic plants suppressed inhibitor formation directed against factors VIII and IX and anaphylaxis against factor IX (FIX). This observation and the relatively high concentration of antigen in the chloroplasts prompted us to evaluate the underlying tolerance mechanisms. The combination of oral delivery of bioencapsulated FIX and intravenous replacement therapy induced a complex, interleukin-10 (IL-10)–dependent, antigen-specific systemic immune suppression of pathogenic antibody formation (immunoglobulin [Ig] 1/inhibitors, IgE) in hemophilia B mice. Tolerance induction was also successful in preimmune mice but required prolonged oral delivery once replacement therapy was resumed. Orally delivered antigen, initially targeted to epithelial cells, was taken up by dendritic cells throughout the small intestine and additionally by F4/80 cells in the duodenum. Consistent with the immunomodulatory responses, frequencies of tolerogenic CD103 and plasmacytoid dendritic cells were increased. Ultimately, latency-associated peptide expressing CD4 regulatory T cells (CD4CD25LAP cells with upregulated IL-10 and transforming growth factor-b (TGF-b) expression) aswell asconventionalCD4CD25 regulatoryTcells systemicallysuppressedanti-FIX responses. (Blood. 2015;125(15):2418-2427)