Lineages of human T-cell clones, including T helper 17/T helper 1 cells, isolated at different stages of anti–factor VIII immune responses

Fletcher

et al. 2015

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• 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)

Section: Discussionmentioning

confidence: 99%

Factor VIII gene variants and inhibitor risk in African American hemophilia A patients

Gunasekera

Fletcher

et al. 2015

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“…FVIII antigen was undetectable in his plasma (supplemental Data, available on the Blood Web site). Mild HA subjects GS1-17A 28,29,36 and GS1-32A 29,36 with missense substitution FVIII-A2201P and T-cell clones isolated from these subjects 36 were described previously. T-cell lines were isolated from a subsequent blood sample collected from GS1-17A ;5 years after his initial 250 BU/mL inhibitor was detected, at which time the titer had decreased to 2 to 13 BU/mL.…”

Section: Subjects and Blood Samplesmentioning

confidence: 99%

“…TCRB genes in clones from mild HA subjects GS1-17A 28,36 and GS1-32A, 29,36 which had the same epitope specificity as the clones and For personal use only. on May 12, 2018. by guest www.bloodjournal.org From line from the severe HA subject, were sequenced (Table 3 52 ).…”

Section: Org Frommentioning

confidence: 99%

“…29 Secretion of interferon-g (IFN-g), interleukin-4 (IL-4), IL-17A, and IL-21 in cell supernatants was measured by sandwich enzyme-linked immunosorbent assays (ELISAs). 30,36 RNA was isolated from T-cell clones using an RNeasy mini kit (Qiagen, Valencia, CA). For 56A clones, complementary DNA (cDNA) was generated from 250 ng total RNA using a QuantiTect reverse transcription kit (Qiagen).…”

mentioning

confidence: 99%

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T cells from hemophilia A subjects recognize the same HLA-restricted FVIII epitope with a narrow TCR repertoire

Paz

James³

et al. 2016

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• 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

“…This experimental approach may be used to target functional B-cell epitopes, including critical residues in antigenic loops in the FVIII A2 domain and in other regions of FVIII, [46][47][48][49][50][51][52] in designing novel FVIII muteins that could provide useful bypass therapy options for inhibitor patients. Because their sequences would be closer to that of the FVIII used to treat the original bleeding disorder, the risk of provoking new T-cell responses and subsequent new inhibitors [53][54][55][56][57][58] to such rationally designed therapeutic FVIII muteins might also be lowered, in comparison with porcine FVIII used as bypass therapy. We expect that sequence modifications to neutralize immunodominant B-cell and T-cell epitopes will eventually be a feature of therapeutic FVIII proteins targeted to patients with refractory inhibitor responses, as well as to patients with poor prognostic factors such as high-risk F8 gene mutations or a family history of inhibitors.…”

Section: Blood 24 April 2014 X Volume 123 Number 17 B-cell Epitopesmentioning

confidence: 99%

High-resolution mapping of epitopes on the C2 domain of factor VIII by analysis of point mutants using surface plasmon resonance

Nguyen

Lewis

et al. 2014

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• Amino acid residues comprising B-cell epitopes recognized by neutralizing antifactor VIII antibodies (inhibitors) have been identified. • Amino acids contributing significant antigen-antibody binding avidity are candidates for mutagenesis in the design of less antigenic proteins.Neutralizing anti-factor VIII (FVIII) antibodies that develop in patients with hemophilia A and in murine hemophilia A models, clinically termed "inhibitors," bind to several distinct surfaces on the FVIII-C2 domain. To map these epitopes at high resolution, 60 recombinant FVIII-C2 proteins were generated, each having a single surface-exposed residue mutated to alanine or a conservative substitution. The binding kinetics of these muteins to 11 monoclonal, inhibitory anti-FVIII-C2 antibodies were evaluated by surface plasmon resonance and the results compared with those obtained for wild-type FVIII-C2. Clusters of residues with significantly altered binding kinetics identified "functional" B-cell epitopes, defined as those residues contributing appreciable antigen-antibody avidity. These antibodies were previously shown to neutralize FVIII activity by interfering with proteolytic activation of FVIII by thrombin or factor Xa, or with its binding to phospholipid surfaces, von Willebrand factor, or other components of the intrinsic tenase complex. Fine mapping of epitopes by surface plasmon resonance also indicated surfaces through which FVIII interacts with proteins and phospholipids as it participates in coagulation. Mutations that significantly altered the dissociation times/half-lives identified functionally important interactions within antigenantibody interfaces and suggested specific sequence modifications to generate novel, less antigenic FVIII proteins with possible therapeutic potential for treatment of inhibitor patients. (Blood. 2014;123(17):2732-2739 IntroductionThe development of neutralizing anti-factor VIII (FVIII) antibodies is a serious complication that may be encountered when FVIII replacement therapy is administered to patients with hemophilia A (HA). It affects 25% to 30% of the treated HA population, with a peak occurrence after ;14 FVIII infusions.1-3 Autoimmune responses to FVIII can also occur, 4 and although this happens only rarely, the resulting bleeding phenotype can be severe. Inhibitors can be difficult and extremely expensive to manage clinically. Interestingly, porcine FVIII has been used effectively in the clinic as a "bypass" therapy; that is, a therapeutic protein that can evade neutralization by anti-FVIII antibodies in many allo-and autoimmune inhibitor patients. [5][6][7] However, some patients have or could develop antibodies that neutralize porcine FVIII as well, 8 because of antigenic cross-reactivity 9 or because regions in which the porcine sequence differs from the human FVIII sequence stimulate effector T cells, leading to antibody production. Identification of the binding sites (B-cell epitopes) on FVIII that are recognized by inhibitors would allow rational design of novel therapeutic FVIII...