Removal of Mannose-Ending Glycan at Asn2118 Abrogates FVIII Presentation by Human Monocyte-Derived Dendritic Cells.

Sandrine Delignat,Bagirath Gangadharan,Olivier D Christophe,Cécile V Denis,Srinivas V Kaveri,Julie Rayes,Sébastien Lacroix-Desmazes,Jagadeesh Bayry,Suryasarathi Dasgupta

doi:10.3389/fimmu.2020.00393

Abstract

The development of an immune response against therapeutic factor VIII is the major complication in hemophilia A patients. Oligomannose carbohydrates at N239 and/or N2118 on factor VIII allow its binding to the macrophage mannose receptor expressed on human dendritic cells, thereby leading to factor VIII endocytosis and presentation to CD4+ T lymphocytes. Here, we investigated whether altering the interaction of factor VIII with mannose-sensitive receptors on antigen-presenting cells may be a strategy to reduce factor VIII immunogenicity. Gene transfer experiments in factor VIII-deficient mice indicated that N239Q and/or N2118Q factor VIII mutants have similar specific activities as compared to non-mutated factor VIII; N239Q/N2118Q mutant corrected blood loss upon tail clip. Production of the corresponding recombinant FVIII mutants or light chains indicated that removal of the N-linked glycosylation site at N2118 is sufficient to abrogate in vitro the activation of FVIII-specific CD4+ T cells by human monocyte-derived dendritic cells. However, removal of mannose-ending glycans at N2118 did not alter factor VIII endocytosis and presentation to CD4+ T cells by mouse antigen-presenting cells. In agreement with this, the N2118Q mutation did not reduce factor VIII immunogenicity in factor VIII-deficient mice. Our results highlight differences in the endocytic pathways between human and mouse dendritic cell subsets, and dissimilarities in tissue distribution and function of endocytic receptors such as CD206 in both species. Further investigations in preclinical models of hemophilia A closer to humans are needed to decipher the exact role of mannose-ending glycans in factor VIII immunogenicity.

Highlights

Five to thirty percent of patients with hemophilia A develop inhibitory anti-factor VIII (FVIII) antibodies following replacement therapy with therapeutic FVIII [1]
Missense mutations in 580 (42%) of the 1384 amino acids that encompass activated FVIII lead to moderate, mild or severe hemophilia A (HAMSTeRS database November 2016)1. This illustrates the critical relationship between the structure and function of proteins in general, and of FVIII in particular, which prompted us to estimate the repercussion of removing N-glycosylation sites of FVIII on its pro-coagulant activity
2FVIII239Q/2118Q protected mice from experimentally induced blood loss, and FVIII239Q/2118Q bound normally to von Willebrand factor (VWF) and phosphatidylserine. These observations indicate that removal of oligomannose carbohydrates at N239 and N2118 does not alter the pro-coagulant activity of FVIII

Summary

Introduction

Five to thirty percent of patients with hemophilia A develop inhibitory anti-factor VIII (FVIII) antibodies following replacement therapy with therapeutic FVIII [1]. A mandatory step for developing an anti-FVIII immune response is the endocytosis of the exogenously administered FVIII by APCs, such as dendritic cells (DCs) and macrophages, and the presentation of FVIIIderived peptides on major histocompatibility complex class II (MHC II) molecules by APCs to CD4+ T lymphocytes [8, 9] These last decades, we and others have explored the mechanisms by which FVIII is endocytosed by APCs either by targeting endocytic receptors [10, 11] or by masking and mutating specific amino acid residues in FVIII [11,12,13]. Both plasma-derived FVIII, recombinant fulllength (FL) and B domain-deleted FVIII (BDD-FVIII) have been reported to contain mannose-ending glycans at positions N239 and N2118 of the A1 and C1 domain, respectively [16, 17]

Methods

Results

Conclusion