HLA‐DR‐restricted T‐cell responses to factor VIII epitopes in a mild haemophilia A family with missense substitution A2201P

Fletcher

et al. 2015

• 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

“…In a previous study, 16 the proband and his (half-)brother were designated IV-1 and IV-2, reflecting their assigned designations in the family pedigree. They are referred to in this study as subjects 17A (the proband) and 32A (his brother), respectively, because these clinical study identifiers were also used to label T-cell clones isolated from their blood.…”

Section: Human Samples and Inhibitor Titersmentioning

confidence: 99%

“…11 T H 17-polarized cells are associated with chronic inflammation and with various autoimmune disorders, 12,13 but there has as yet been no report of their possible role in hemophilia A immune responses. T-cell clones that respond to distinct epitopes in the FVIII A2, C1, and C2 domains have been isolated from several hemophilic subjects, [14][15][16][17] and these monoclonal CD4 ϩ cell lines are proving useful in mechanistic investigations of inhibitor development.…”

Section: Introductionmentioning

confidence: 99%

“…15,16 The objective of the present study was to investigate the phenotypes of these clones by characterizing cytokine production, chemokine receptor expression, and transcription factor usage, as phenotypic changes over time and differences between these subjects should offer clues as to why only one of them developed a clinically significant inhibitor. Thirteen T-cell clones were isolated from the proband's blood at 2 time points, and 6 clones were obtained from a single sample obtained from his brother.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

James²,

Kwok

et al. 2009

The development of neutralizing antibodies (inhibitors) after factor VIII (FVIII) infusions is a serious complication that affects approximately one-quarter of hemophilia A patients who have access to replacement therapy. To investigate the differentiation of naive T cells into FVIII-specific helper T cells that promote B-cell activation and antibody secretion, HLA-DRA-DRB1*0101-restricted T-cell clones that respond to a specific epitope in FVIII were isolated from a mild hemophilia A subject (the proband) 19 weeks and 21 months after his development of a high-titer inhibitor. Clones responding to the same epitope were also isolated from his multiply infused brother, who has not developed a clinically significant inhibitor. The 19-week proband clones were T helper (T H )17/T H 1-or T H 1/T H 2-polarized, whereas all 8 clones isolated 21 months postinhibitor development were T H 2-polarized cells. In contrast, all 6 clones from the brother who did not develop an inhibitor were T H 1-polarized, indicating that tolerance to FVIII can be maintained even with circulating T H 1-polarized cells that respond vigorously to in vitro FVIII stimulation. This is the first evidence that T H 17/ T H 1-polarized cells play a role in hemophilic immune responses to FVIII. Furthermore, this is the first report of successful isolation and expansion of antigen-specific human T H 17/T H 1 clones using standard culture conditions. (Blood.

“…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

• 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...