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

Summary. 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) IC₅₀ 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.