LAUSR

Despite over 80 years of clinical experience with coagulation factor (F)VIII inhibitors, surprisingly little is known about the in vivo mechanism of this most serious complication of replacement therapy for hemophilia A. These neutralizing anti-drug alloantibodies arise in ~30% of patients. Inhibitor formation is T cell-dependent, but events leading up to helper T cell activation have been elusive due in part to the complex anatomy and cellular makeup of the spleen. Here, we show that FVIII antigen presentation to CD4+ T cells critically depends on a select set of several anatomically distinct antigen presenting cells, whereby marginal zone B cells, marginal zone and marginal metallophilic but not red pulp macrophages (RPMFs) participate in shuttling FVIII to the white pulp where conventional dendritic cells (DCs) prime helper T cells, which then differentiate into follicular helper T (Tfh) cells. Toll-like receptor 9 (TLR9) stimulation accelerated Tfh responses, Germinal Center and inhibitor formation, while systemic administration of FVIII alone in hemophilia A mice increased frequencies of monocyte-derived and plasmacytoid DCs. Moreover, FVIII enhanced T cell proliferation to another protein antigen (ovalbumin), and inflammatory signaling-deficient mice were less likely to develop inhibitors, indicating that FVIII may have intrinsic immunostimulatory properties. Ovalbumin, which, unlike FVIII, is absorbed into the RPMF compartment, fails to elicit T cell proliferative and antibody responses when administered at the same dose as FVIII. Altogether, we propose that an antigen trafficking pattern that results in efficient in vivo delivery to DCs and inflammatory signaling, shape the immunogenicity of FVIII.