Hemophilia A subjects with an intron-22 gene inversion mutation show CD4+ T-effector responses to multiple epitopes in FVIII.

Background: Almost half of severe hemophilia A (HA) is caused by an intron 22 inversion mutation (Int22Inv), which disrupts the 26-exon F8 gene. Inverted F8 mRNA exons 1-22 are transcribed, while F8B mRNA, containing F8 exons 23-26, is transcribed from a promoter within intron 22. Neither FVIII activity nor FVIII antigen (cross-reacting material, CRM) are detectable in plasma of patients with an intron-22 inversion. Objectives: To test the hypothesis that (putative) intracellular synthesis of FVIII proteins encoded by inverted F8 and F8B mRNAs confers T-cell tolerance to almost the entire FVIII sequence, and to evaluate the immunogenicity of the region encoded by the F8 exon 22-23 junction sequence. Patients/Methods: Peripheral blood mononuclear cells (PBMCs) from 30 severe or moderate HA subjects (17 with an Int22Inv mutation) were tested by ELISPOT assays to detect cytokine secretion in response to FVIII proteins and peptides and to map immunodominant T-cell epitopes. Potential immunogenicity of FVIII sequences encoded by the F8 exon 22-23 junction region was also tested using peptide-MHCII binding assays. Results: Eight of the Int22Inv subjects showed robust cytokine secretion from PBMCs stimulated with FVIII proteins and/or peptides, consistent with earlier publications from the Conti-Fine group. Peptide ELISPOT assays identified immunogenic regions of FVIII. Specificity for sequences encoded within F8 mRNA exons 1-22 and F8B mRNA was confirmed by staining Int22Inv CD4+ T cells with peptide-loaded HLA-Class II tetramers. FVIII peptides spanning the F8 exon 22-23 junction (encoding M2124-V2125) showed limited binding to MHCII proteins and low immunogenicity, with cytokine secretion from only one Int22Inv subject. Conclusions: PBMCs from multiple subjects with an Int22Inv mutation, with and without a current FVIII inhibitor, responded to FVIII epitopes. Furthermore, the FVIII region encoded by the exon 22-23 junction sequence was not remarkably immunoreactive and is therefore unlikely to contain an immunodominant, promiscuous CD4+ T-cell epitope. Our results indicate that putative intracellular expression of partial FVIII proteins does not confer T-cell tolerance to FVIII regions encoded by inverted F8 mRNA or F8B mRNA.

Anti-FVIII antibodies in Black and White hemophilia A subjects: do F8 haplotypes play a role?

The most common complication in hemophilia A (HA) treatment, affecting 25% to 30% of patients with severe HA, is the development of alloimmune inhibitors that foreclose the ability of infused factor VIII (FVIII) to participate in coagulation. Inhibitors confer significant pathology on affected individuals and present major complexities in their management. Inhibitors are more common in African American patients, and it has been hypothesized that this is a consequence of haplotype (H)-treatment product mismatch. F8 haplotypes H1 to H5 are defined by nonsynonymous single-nucleotide polymorphisms encoding sequence variations at FVIII residues 1241, 2238, and 484. Haplotypes H2 to H5 are more prevalent in individuals with Black African ancestry, whereas 80% to 90% of the White population has the H1 haplotype. This study used an established multiplex fluorescence immunoassay to determine anti-FVIII antibody titers in plasma from 394 individuals with HA (188 Black, 206 White), measuring their binding to recombinant full-length H1 and H2 and B-domain-deleted (BDD) H1/H2, H3/H5, and H4 FVIII proteins. Inhibitor titers were determined using a chromogenic assay and linear B-cell epitopes characterized using peptide microarrays. FVIII-reactive antibodies were readily detected in most individuals with HA, with higher titers in those with a current inhibitor, as expected. Neither total nor inhibitory antibody titers correlated with F8 haplotype mismatches, and peptides with D1241E and M2238V polymorphisms did not comprise linear B-cell epitopes. Interestingly, compared with the full-length FVIII products, the BDD-FVIII proteins were markedly more reactive with plasma antibodies. The stronger immunoreactivity of BDD-FVIII suggests that B-domain removal might expose novel B-cell epitopes, perhaps through conformational rearrangements of FVIII domains.

The humanized DRAGA mouse (HLA-A2. HLA-DR4. RAG1 KO. IL-2R g c KO. NOD) establishes inducible and transmissible models for influenza type A infections.

We have engineered a Human Immune System (HIS)-reconstituted mouse strain (DRAGA mouse: HLA-A2. HLA-DR4. Rag1 KO. IL-2Rγc KO. NOD) in which the murine immune system has been replaced by a long-term, functional HIS via infusion of CD34+ hematopoietic stem cells (HSC) from cord blood. Herein, we report that the DRAGA mice can sustain inducible and transmissible H1N1 and H3N2 influenza A viral (IAV) infections. DRAGA female mice were significantly more resilient than the males to the H3N2/Aichi infection, but not to H3N2/Hong Kong, H3N2/Victoria, or H1N1/PR8 sub-lethal infections. Consistently associated with large pulmonary hemorrhagic areas, both human and murine Factor 8 mRNA transcripts were undetectable in the damaged lung tissues but not in livers of DRAGA mice advancing to severe H1N1/PR8 infection. Infected DRAGA mice mounted a neutralizing anti-viral antibody response and developed lung-resident CD103 T cells. These results indicate that the DRAGA mouse model for IAV infections can more closely approximate the human lung pathology and anti-viral immune responses compared to non-HIS mice. This mouse model may also allow further investigations into gender-based resilience to IAV infections, and may potentially be used to evaluate the efficacy of IAV vaccine regimens for humans.

Transfusion-related immunomodulation: gamma irradiation alters the effects of leukoreduction on alloimmunization.

BACKGROUND: Adverse events following blood transfusion include allosensitization and generalized immunosuppression, collectively referred to as transfusion-related immune modulation. We evaluated the immunological effects of red blood cell (RBC) and platelet transfusions on alloantibody responses and on immunoregulatory cells in nonimmunosuppressed patients undergoing cardiovascular surgery. STUDY DESIGN AND METHODS: Patients were randomized to receive standard unmodified (STD), leukoreduced (LR), or leukoreduced and γ-irradiated (LRγ) RBCs. Patients received only apheresis platelets that were in-process LR and were γ-irradiated for the third arm. Nontransfused patients served as controls for the effects of surgery itself on immunologic changes. Antibodies to HLA were assessed with use of solid-phase assays. The effects of transfusion on adaptive and innate immunity were evaluated by assessing T regulatory cells (Tregs) and invariant natural killer T (iNKT) cells. RESULTS: LR of blood products reduced the development of human leukocyte antigen (HLA) alloantibodies, but only in patients without preexisting HLA antibodies. However, if LR blood products were γ-irradiated, HLA antibody production was not reduced. Compared to nontransfused patients, recipients of STD or LR transfusions showed a significant increase in CD4+CD25hi T cells expressing FoxP3 or CTLA4 and also of iNKT cells producing interleukin-4. In contrast, recipients of LRγ blood products showed markedly lower increases in all three cellular assays. CONCLUSION: LR decreased HLA alloantibody production in naïve recipients, but did not reduce the immunosuppressive effects of transfusion. LRγ reduced immunosuppression and was not associated with decreased HLA alloantibody production.

FVIII proteins with a modified immunodominant T-cell epitope exhibit reduced immunogenicity and normal FVIII activity.

Factor VIII (FVIII)-neutralizing antibodies (inhibitors) are a serious complication in hemophilia A (HA). The peptide FVIII2194-2213 contains an immunodominant HLA-DRA*01-DRB1*01:01 (DRB1*01:01)-restricted epitope recognized by CD4+ T-effector cells from HA subjects. The aim of this study was to identify amino acid substitutions to deimmunize this epitope while retaining procoagulant function and expression levels comparable to those of wild-type (WT) FVIII proteins. The shortest DRB1*01:01-binding peptide was FVIII2194-2205, and residues important for affinity were identified as F2196, M2199, A2201, and S2204. T-cell proliferation experiments with Ala-substituted FVIII2194-2205 peptides identified F2196A as a substitution that abrogated proliferation of clones specific for the WT sequence. T-cell clones that were stimulated by recombinant WT-FVIII-C2 (rWT-FVIII-C2) protein did not proliferate when cultured with rFVIII-C2-F2196A, indicating the immunogenic peptide includes a naturally processed T-cell epitope. Additional amino acid substitutions at F2196 and M2199 were evaluated by peptide-MHC class II (MHCII)-binding assays, T-cell proliferation assays, epitope prediction algorithms, and sequence homologies. Six B-domain-deleted (BDD)-FVIII proteins with substitutions F2196A, F2196L, F2196K, M2199A, M2199W, or M2199R were produced. Proliferation of T-cell clones and polyclonal lines in response to rBDD-FVIII-F2196K and rBDD-FVIII-M2199A was reduced compared with responses to WT-BDD-FVIII. The BDD-FVIII-F2196K sequence modification appears to be the most promising sequence variant tested here, due to its effectiveness at eliminating DRB1*01:01-restricted immunogenicity, low potential immunogenicity in the context of other MHCII alleles, expression level comparable to WT-BDD-FVIII, and retained procoagulant activity. These results provide proof of principle for the design of less immunogenic FVIII proteins targeted to specific subsets of HA patients.

A unique major histocompatibility complex Class II-binding register correlates with HLA-DR11-associated immunogenicity of the major K blood group antigen.

BACKGROUND: Kell is a glycoprotein expressed on red blood cells (RBCs). Its K and k variants contain either Met (K antigen) or Thr (k antigen) at Position 193, respectively. Development of anti-K after K-mismatched antigen exposure via blood transfusions or pregnancy can destroy RBCs, leading to hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. The immunogenicity of overlapping 15-mer Kell peptides with M193 or T193 at every possible position was investigated previously. Interestingly, Peptide W179 to M193, with the polymorphic M193T residue at the peptide's C-terminus, was the most effective at stimulating CD4 T cells from a series of K-immunized women. STUDY DESIGN AND METHODS: This study investigates the basis for HLA restriction of anti-K immune responses. Major histocompatibility complex Class II (MHCII)-binding prediction algorithms and quantitative peptide-MHCII-binding assays were employed to determine the binding registers; anchor residues; and affinities of wild-type, truncated, and sequence-modified K and k peptides. Predictions were generated using Immune Epitope Database and ProPred algorithms. Competitive peptide-MHCII-binding assays utilized 12 recombinant HLA-DR proteins, K and k peptides, and high-affinity MHCII-restricted reference peptides. RESULTS: The peptide-MHCII-binding assays identified a unique K peptide-binding register (W179-S187) restricted to HLA-DRB1*11:01, in addition to partially overlapping binding registers that included the K/k M193T polymorphic site and that bound promiscuously to multiple HLA-DR proteins. CONCLUSION: Three partially overlapping MHCII-binding motifs for HLA-DRB1*11:01 result in high-avidity K-peptide binding, which may contribute to HLA-DR11-restricted immunogenicity associated with the K allele.

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

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 FVIII2194-2213, 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 FVIII2194-2213 T-cell receptor ß (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.

Accurate, simple, and inexpensive assays to diagnose F8 gene inversion mutations in hemophilia A patients and carriers.

The most frequent mutations resulting in hemophilia A are an intron 22 or intron 1 gene inversion, which together cause ∼50% of severe hemophilia A cases. We report a simple and accurate RNA-based assay to detect these mutations in patients and heterozygous carriers. The assays do not require specialized equipment or expensive reagents; therefore, they may provide useful and economic protocols that could be standardized for central laboratory testing. RNA is purified from a blood sample, and reverse transcription nested polymerase chain reaction (RT-NPCR) reactions amplify DNA fragments with the F8 sequence spanning the exon 22 to 23 splice site (intron 22 inversion test) or the exon 1 to 2 splice site (intron 1 inversion test). These sequences will be amplified only from F8 RNA without an intron 22 or intron 1 inversion mutation, respectively. Additional RT-NPCR reactions are then carried out to amplify the inverted sequences extending from F8 exon 19 to the first in-frame stop codon within intron 22 or a chimeric transcript containing F8 exon 1 and the VBP1 gene. These latter 2 products are produced only by individuals with an intron 22 or intron 1 inversion mutation, respectively. The intron 22 inversion mutations may be further classified (eg, as type 1 or type 2, reflecting the specific homologous recombination sites) by the standard DNA-based "inverse-shifting" PCR assay if desired. Efficient Bcl I and T4 DNA ligase enzymes that cleave and ligate DNA in minutes were used, which is a substantial improvement over previous protocols that required overnight incubations. These protocols can accurately detect F8 inversion mutations via same-day testing of patient samples.

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

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 = 2.3 [1.1-5.0, P = .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.