• Immune responses to FVIII sequence variants encoded by ns-SNPs do not contribute appreciably to inhibitor development in African Americans.• African American HA subjects with an intron-22 inversion had a 2-to 3-times-higher inhibitor incidence than whites with the same mutation.African American hemophilia A (HA) patients experience a higher incidence of neutralizing anti-factor VIII (FVIII) antibodies ("inhibitors") vis-à-vis white patients. Nonsynonymous single-nucleotide polymorphisms (ns-SNPs) in the F8 gene encoding FVIII-H484, FVIII-E1241, and FVIII-V2238 are more prevalent in African Americans. This study tested the hypothesis that immune responses to these sites provoke inhibitors. Blood samples were obtained from 174 African American and 198 white HA subjects and their F8 gene sequences determined. Major histocompatibility complex class II binding and T-cell recognition of polymorphic sequences were evaluated using quantitative binding assays and HLA-DRB1 tetramers. Peptides corresponding to 4 common ns-SNPs showed limited binding to 11 HLA-DRB1 proteins. CD4 T cells from 22 subjects treated with FVIII products having sequences at residues FVIII-484, 1241, and 2238 differing from those of putative proteins encoded by their F8 genes did not show high-avidity tetramer binding, whereas positive-control staining of tetanus-specific CD4 T cells was routinely successful. African Americans with an intron-22 inversion mutation showed a 2-3 times-higher inhibitor incidence than whites with the same mutation (odds ratio 5 2.3 [1.1-5.0, P 5 .04]), but this did not correlate with any of the ns-SNPs. We conclude that immune responses to "sequence-mismatched" FVIII products are unlikely to contribute appreciably to the inhibitor incidence in African Americans. (Blood. 2015;126(7):895-904)
• An HA subject with a multiexon F8 deletion showed a highly clonal response to 1 FVIII epitope via an immunodominant TCR.• The same HLA-DRA*01-DRB1*01:01-restricted FVIII epitope was recognized by T cells from 3 HA subjects.Factor VIII (FVIII)-neutralizing antibodies ("inhibitors") are a serious problem in hemophilia A (HA). The aim of this study was to characterize HLA-restricted T-cell responses from a severe HA subject with a persistent inhibitor and from 2 previously studied mild HA inhibitor subjects. Major histocompatibility complex II tetramers corresponding to both of the severe HA subject's HLA-DRA-DRB1 alleles were loaded with peptides spanning FVIII-A2, C1, and C2 domains. Interestingly, only 1 epitope was identified, in peptide FVIII [2194][2195][2196][2197][2198][2199][2200][2201][2202][2203][2204][2205][2206][2207][2208][2209][2210][2211][2212][2213] , and it was identical to the HLA-DRA*01-DRB1*01:01-restricted epitope recognized by the mild HA subjects. Multiple T-cell clones and polyclonal lines having different avidities for the peptide-loaded tetramer were isolated from all subjects. Only high-and medium-avidity T cells proliferated and secreted cytokines when stimulated with FVIII 2194-2213 . T-cell receptor b (TCRB) gene sequencing of 15 T-cell clones from the severe HA subject revealed that all high-avidity clones expressed the same TCRB gene. High-throughput immunosequencing of high-, medium-, and low-avidity cells sorted from a severe HA polyclonal line revealed that 94% of the high-avidity cells expressed the same TCRB gene as the high-avidity clones. TCRB sequencing of clones and lines from the mild HA subjects also identified a limited TCRB gene repertoire. These results suggest a limited number of epitopes in FVIII drive inhibitor responses and that the T-cell repertoires of FVIII-responsive T cells can be quite narrow. The limited diversity of both epitopes and TCRB gene usage suggests that targeting of specific epitopes and/or T-cell clones may be a promising approach to achieve tolerance to FVIII. (Blood. 2016; 128(16):2043-2054
Key Points Less immunogenic FVIII muteins were designed by defining and replacing MHCII anchor residues with amino acids that reduced MHCII binding. Patient-derived T-cell clones show lower proliferation in response to FVIII-F2196K, which had normal FVIII activity and expression level.
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