Immunodominant T-cell epitopes in the factor VIII C2 domain are located within an inhibitory antibody binding site

Abstract

Formation of inhibitor antibodies to factor VIII (FVIII) is a major complication of FVIII replacement therapy for hemophilia A patients, and it occurs through a T-cell dependent process. The C2 domain of FVIII contains epitopes that are recognized by antibody inhibitors. We have examined regions of the C2 domain that form epitopes for T cells in mice congenitally deficient in FVIII. We obtained CD4(+)T cells from mice immunized by intravenous infusion of therapeutic doses of recombinant human FVIII (rFVIII), or by subcutaneous injections of rFVIII or recombinant human C2 domain in adjuvant. In all cases, the T cells recognized most strongly and consistently two overlapping peptides that spanned residues 2191 to 2220 of the C2 domain. Analysis of the crystal structure of human factor VIII C2 bound to a human monoclonal antibody, BO2C11, showed these residues also constitute part of a human alloimmune B-cell epitope (Spiegel et al., Blood 2001; 98: 13-19). This region includes one of the "hydrophobic spike" protrusions, consisting of M2199 and F2200, as well as the basic residues R2215 and R2220. These residues contribute to membrane binding and to association with von Willebrand factor (vWF). These findings suggest that a major T-cell epitope in the C2 domain recognized by hemophilic mice is located within the same region that binds to inhibitors, vWF, and activated membranes.

HLA-DR-presented Peptide Repertoires Derived From Human Monocyte-derived Dendritic Cells Pulsed With Blood Coagulation Factor VIII

“…Both donors that present this peptide in our study are DRB1*0101 as well. Two other C2 peptides identified in this study were found previously to induce T-cell proliferation (35,37). The fact that most peptides found were donor-specific suggests that these peptides are presented in a HLA-specific context.…”

“…Proliferation of this clone has been used by another group as a general readout for FVIII endocytosis by dendritic cells (22). Region 2196 -2204 was identified as a region against which CD4ϩ T cells are directed both in a study with hemophilic mice (35) and in a study with a hemophilia A patient (36). This sequence was identified using DRB1*0101 tetramers.…”

Lower Inhibitor Development in Hemophilia A Mice following Administration of Recombinant Factor VIII-O-Phospho-L-serine Complex

“…The immunoprecipitation and inhibitor neutralization assays of the inhibitor plasma from hemophilia A patients clearly indicated that the anti-light chain antibody titer was the highest (12). More recently, Reding et al (13) and Pratt et al (14) have identified several universal epitopes for CD4ϩ T-cells in the 2291-2330 region of the C2 domain using proliferation assays with CD4ϩ cells from normal humans, hemophilia A patients (13), and mice (14). Three-dimensional models proposed based on crystallographic studies and mutational analysis show that the C2 domain also contains 2-4 hydrophobic loops and other charged residues that promote lipid binding ( Fig.…”

Induction of tolerance to factor VIII inhibitors by gene therapy with immunodominant A2 and C2 domains presented by B cells as Ig fusion proteins

“…[10][11][12] We have shown that recipients of B-cell blasts, transduced with an Ig fusion of a variety of model antigens or autoimmune targets constructs, are tolerant to the protein epitopes of the expressed genes and that this therapy can lead to striking modulation of clinical disease. 8,[13][14][15][16] Importantly, tolerance is more effective and of longer duration in the presence of the IgG backbone 9 (T.C.L., Yan Su, and D.W.S., manuscript in preparation).It is known that most inhibitory antibodies are reactive with conformational epitopes on the exposed surfaces of C2 and A2 domains of fVIII, 1,3,4,17,18 and that immunodominant T-cell epitopes can be found in the C2 region. 17 In this study, we inserted the coding sequences for residues S2173-Y2332 (C2 domain) and S373-R740 (A2 domain) in frame with the IgG heavy chain backbone in a retroviral vector to induce tolerance in hemophilic mice.…”