Identification of Specific Residues in a Human FVIII T-Cell Epitope That Are Essential for An HLA-DRA-DRB1*0101-Restricted T-Cell Response Associated with Inhibitor Development.

Abstract The development of antibodies that interfere with the pro-coagulant activity of factor VIII (FVIII), clinically referred to as “inhibitors”, can complicate the treatment of individuals with hemophilia A, and inhibitors are associated with significantly higher morbidity and mortality. The production of both allo- and auto-immune inhibitors depends on the involvement of helper T cells. In order to better understand T-cell signaling events, it is necessary to identify and characterize the relevant T-cell epitopes in FVIII, i.e. the linear amino acid sequences that bind to MHC Class II (HLA-DR) molecules on the surface of antigen-presenting cells (APCs). These peptide-Class II complexes are recognized in turn by T-cell receptors, and sustained interaction between T cells and APCs through these complexes results in T-cell activation. We previously identified an HLA-DRA-DRB1*0101 restricted T-cell epitope recognized by CD4+ T cells from a mild hemophilia A subject with missense mutation A2201P in the FVIII C2 domain. This individual developed a high-titer inhibitor after receiving FVIII infusions for hemostatic support during surgery (James et al., J. Thrombos. Haemostas. 5:2399–2407, 2007). The epitope was identified using tetramer-guided epitope mapping, in which biotinylated DR0101 protein was incubated with synthetic 20-mer peptides having overlapping sequences spanning the FVIII C2 domain. The DR0101 proteins were converted to tetramers by addition of PE-labeled streptavidin, and T cells with receptors that recognized particular peptide-loaded tetramers were detected by flow cytometry. Peptides with sequences corresponding to FVIII residues 2186–2205, 2187–2205 and 2194–2213 were shown to contain a DRB1*0101 restricted epitope, suggesting that the epitope resided within the overlapping sequence S2194–P2205. Competition binding assays indicated peptide FVIII2194-2213 bound to DR0101 with ~20-fold higher affinity than a similar peptide with the hemophilic substitution A2201P. In the present study, we precisely defined the minimal epitope within this region by determining the affinities of a series of modified peptides for monomeric DR0101 protein. Truncated peptides were used to identify the minimal epitope, and their affinities as well as those of of peptides with single arginine substitutions indicated putative “anchor” residues that fit into pockets within the DR0101 peptide-binding groove. Briefly, DR0101 was incubated with biotinylated influenza virus haemagglutinin 306–318 (bHA) in the presence of several concentrations of a truncated and/or sequence-modified peptide. DR0101-bound peptide was separated from free peptide by immunoprecipitation of DR0101, and bHA peptide bound to DR0101 was detected using europium-labeled streptavidin. In this assay effective competition is thus indicated by a reduced fluorescent signal. N- and C-terminal truncated peptides corresponding to FVIII residues 2195-2206, 2196–2206, 2197–2206, 2194–2211, 2194–2207, 2194–2206, 2194–2205, 2194–2204 and 2194–2203 were tested, and the results indicated the minimal epitope consisted of S2194–P2205, with F2196 and S2204 serving as anchors at positions 1 and 9, respectively. The affinities of a series of peptides corresponding to residues 2194–2205, with a single residue at each position substituted to arginine, were also evaluated. Reduced binding affinities of several peptides indicated that the DR0101 anchor residues are F2196, M2199, A2201 and S2204. Our identification of anchor residues that contribute affinity necessary for productive DR0101-peptide complex formation indicates sites in FVIII that could be modified by amino acid substitutions to modulate DRB1*0101-restricted T-cell responses. The binding of these peptides to other prevalent DR molecules is currently being evaluated to indicate whether this epitope is immunodominant. Modification of immunodominant epitopes in next-generation recombinant FVIII proteins may eventually provide therapeutic benefits, especially in conjunction with clinical tolerance-inducing regimes, as reduced T-cell signaling could lead to lower antibody titres.