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


OBJECTIVE
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.


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.
Benaroya Research Institute, Seattle, WA, USA.
E A
EA
James
S D
SD
van Haren
R A
RA
Ettinger
K
K
Fijnvandraat
J A
JA
Liberman
W W
WW
Kwok
J
J
Voorberg
K P
KP
Pratt
D000595:Amino Acid Sequence; D049109:Cell Proliferation; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D005169:Factor VIII / Q000235:genetics*; D006684:HLA-DR Antigens / Q000235:genetics; D059811:HLA-DRB1 Chains; D006467:Hemophilia A / Q000276:immunology*; D006801:Humans; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology*
D000939:Epitopes; D006684:HLA-DR Antigens; D059811:HLA-DRB1 Chains; D005169:Factor VIII
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