T-cell responses in two unrelated hemophilia A inhibitor subjects include an epitope at the factor VIII R593C missense site

J Thromb Haemost. 2011 Apr;9(4):689-99. doi: 10.1111/j.1538-7836.2011.04202.x.

Abstract

Background: Development of neutralizing anti-factor (F)VIII antibodies ('inhibitors') is a serious clinical problem in hemophilia A. Increased inhibitor risk has been associated with certain FVIII missense substitutions, including R593C in the A2 domain.

Objectives: The aim of the present study was to identify T-cell epitopes in FVIII and characterize T-cell responses in two unrelated hemophilia A subjects sharing F8-R593C and HLA-DRB1*1101 genotypes. We hypothesized that the hemophilic substitution site coincides with an important T-cell epitope.

Patients/methods: The binding affinities of peptides for recombinant HLA-DR proteins were measured and compared with epitope prediction results. CD4+ T cells were stimulated using peptides and stained with fluorescent, peptide-loaded tetramers.

Results: The inhibitor subjects, but not HLA-matched controls, had high-avidity HLA-DRB1*1101-restricted T-cell responses against FVIII(589-608), which contains the hemophilic missense site. Antigen-specific T cells secreted Th1 and Th2 cytokines and proliferated in response to FVIII and FVIII(592-603). FVIII(589-608) bound with physiologically relevant (micromolar) IC(50) values to recombinant DR0101, DR1101 and DR1501 proteins.

Conclusions: Hemophilia A patients with R593C missense substitutions and these HLA haplotypes had an increased incidence of inhibitors in our cohorts, supporting a paradigm in which presentation of FVIII epitopes containing the wild-type R593 influences inhibitor risk in this hemophilia A sub-population.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cell Proliferation
  • Enzyme-Linked Immunosorbent Assay
  • Epitopes / immunology*
  • Factor VIII / genetics*
  • HLA-DR Antigens / genetics
  • HLA-DRB1 Chains
  • Hemophilia A / immunology*
  • Humans
  • Molecular Sequence Data
  • Mutation, Missense*
  • T-Lymphocytes / cytology
  • T-Lymphocytes / immunology*

Substances

  • Epitopes
  • HLA-DR Antigens
  • HLA-DRB1 Chains
  • Factor VIII