ALBERT

Description: Key Points 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.

Description: Key Points Generation and function of specific human Tregs. Specific regulation of FVIII responses by engineered human Tregs.

Description: Key Points 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.

Description: The development of inhibitory anti-FVIII antibodies in hemophilia A patients, referred to clinically as “inhibitors”, results from stimulation of T cells when the infused FVIII is recognized as foreign. T-cell stimulation follows the binding of specific epitopes within FVIII-derived peptides to HLA-DR molecules on the surface of antigen-presenting cells. We are investigating the HLA dependence of immune responses to FVIII and characterizing T-cell epitopes to better understand the process of T-cell stimulation leading to anti-FVIII antibody production. Inhibitors are less common in mild/moderately severe patients than in severe hemophilia A, but an elevated risk of inhibitor formation has been noted for patients with missense mutation R593C in the FVIII A2 domain. CD4+ T cells were isolated from two unrelated hemophilic subjects with this missense genotype, one Dutch and one American. Both had a DRB1*1101 haplotype and longstanding low- to moderate-titer inhibitors. Antigen-specific T cells from both subjects were identified by staining with fluorescent, peptide-loaded MHC Class II tetramers. The tetramer staining was carried out after stimulating the cells with synthetic peptides corresponding to the FVIII A2 domain sequence. Both hemophilic subjects, but not an HLA-matched healthy control, had a DRB1*1101-restricted response to peptide FVIII589-608, which contained the wild-type R593 sequence. Single-cell sorting and expansion with PHA facilitated the isolation of A2-specific T-cell clones from both subjects. A polyclonal population of A2-specific T cells was also isolated from the American subject’s CD4+ T cells. All T-cell clones and the polyclonal T-cell line proliferated in response to the FVIII589-608 peptide and to FVIII. However, the cytokine profiles were somewhat different for peptide-stimulated cells from the two subjects. The single clone obtained from the Dutch subject produced modest levels (10,000 pg/mL of IFN-g. Somewhat surprisingly, all of the cloned and polyclonal A2-specific cultured cells proliferated in response to the hemophilic peptide FVIII589-608,593C, despite the fact that this peptide elicited no response from primary T-cell cultures isolated from the American subject. FVIII589-608 bound to recombinant, monomeric DRB1*1101 protein with high affinity, while the affinity of the hemophilic peptide FVIII589-608,593C was threefold lower. FVIII residues 594-602 (FLPNPAGVQ) comprise a predicted high-affinity DRB1*1101-binding motif. Proliferation assays carried out with truncated peptides (FVIII592-608, FVIII593-608, and FVIII594-608) confirmed the responsiveness of the clones and polyclonal T-cell line to peptides containing this predicted motif, but proliferation decreased markedly when residue R593 was removed. The immunogenicity of this FVIII region with respect to other HLA types was evaluated by binding assays utilizing several recombinant DR molecules, in order to determine the relative promiscuity of this T-cell epitope. The effects of modifying amino acid residues within this epitope are also under investigation, as substitutions at “anchor” positions should decrease the immunogenicity of the corresponding peptides. Similar substitutions in recombinant FVIII may allow development of less immunogenic variants of human FVIII proteins.

Description: Some, but not all, hemophilia A patients who receive factor VIII (FVIII) replacement therapy develop antibodies that interfere with FVIII coagulation activity (“inhibitors”), and their development is T-cell dependent. Immune tolerance induction protocols induce longterm tolerance to FVIII in a number of these patients, and although this clinical success is remarkable, its multifactorial molecular basis is still poorly understood. Our laboratory is characterizing protein-protein interactions and subsequent cytokine signaling that occurs when antigen-presenting cells present FVIII-derived peptides to T-cell receptors (TCRs). We previously mapped HLA-DR-restricted T-cell epitopes recognized by T cells from several related individuals with mild hemophilia A due to the missense substitution FVIIIA2201P (James EA, et al. J Thromb Haemost5:2399, 2007; Ettinger et al., submitted). Two brothers had unambiguous HLA-DRA-DRB1*0101-restricted T-cell responses to the same epitope in the FVIII C2 domain, which included the missense substitution site at FVIII residue 2201. Both had received FVIII infusions but only the proband developed a high-titer inhibitor. Although a standard Bethesda assay indicated the brother did not have an inhibitor, his concentrated IgG showed detectable FVIII inhibition. The present study addresses the question of why a high-titer inhibitor developed in only one of two hemophilic brothers, despite strong recognition of the same HLA-restricted epitope by T cells from both. In other words, why was tolerance to wild-type FVIII broken in the proband? Several antigen-specific T-cell clones were isolated from each subject’s CD4+ T cells using MHC Class II tetramers: PE-labeled, tetrameric DR0101 proteins complexed with synthetic peptides containing the relevant FVIII epitope were used to label and then single-cell sort antigen-specific T cells, which were then expanded in culture following standard protocols. The clones from the proband were isolated 19 weeks after his peak inhibitor response, and clones were isolated from a blood sample obtained from his brother two years after his last FVIII exposure. Cloned T cells from both proband and brother showed highly similar tetramer staining and dose-dependent proliferation upon stimulation with the peptide FVIII2194–2213 presented by irradiated HLA-DR-matched peripheral blood mononuclear cells. Cytokine ELISA assays of supernatants following stimulation of these clones with 1 mM FVIII2194–2213 showed the following patterns for the proband: three clones secreted IL-17 (20–600 pg/ml), relatively low levels of IFN-g and TNF-a (0–200 pg/ml) and no IL-4 or IL-10; two clones secreted IL-4 and IL-10 (100–500 pg/ml), relatively low IFN-g and TNF-a (0–200 pg/ml) and no IL-17. In contrast, all six clones from the brother had a similar profile, secreting relatively high levels of IFN-g (500–1500 pg/ml), moderate levels of TNF-a, IL-4, and IL-10 (50–300 pg/ml) and no IL-17. These profiles indicate that FVIII-specific clones from the proband are of the Th17 and Th2 lineage, whereas the brother had a Th1high/Th2low T-cell response. We next sequenced the TCRBV-D-J genes to investigate whether differences in the TCRs correlated with T-cell responses. Four distinct TCRBV-D-J sequences were identified among the 11 clones that correlated with the observed differences in cytokine secretion, suggesting a structural basis for these functional differences. Expression patterns of chemokine receptors considered to be markers of Th1, Th2, and Th17 cells provided additional evidence for the Th lineage designations of the clones. Eight additional clones were isolated from the proband 21 months following his peak inhibitor response, when his inhibitor titer was 5 Bethesda units/mL, and none of them secreted IL-17, suggesting Th17 cells may play a significant role in early stages of anti-FVIII antibody responses. The importance of Th17 cells in various inflammatory as well as autoimmune responses has been recognized only recently. This is the first report of Th17 involvement in epitope-specific T-cell responses to FVIII.

Description: 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 (TH)17/TH1- or TH1/TH2-polarized, whereas all 8 clones isolated 21 months postinhibitor development were TH2-polarized cells. In contrast, all 6 clones from the brother who did not develop an inhibitor were TH1-polarized, indicating that tolerance to FVIII can be maintained even with circulating TH1-polarized cells that respond vigorously to in vitro FVIII stimulation. This is the first evidence that TH17/TH1-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 TH17/TH1 clones using standard culture conditions.

Description: Antibody responses (inhibitors) against human factor VIII (FVIII) can obviate successful therapeutic efficacy of FVIII replacement therapy in hemophilia A patients. Novel efforts are necessary to overcome this problem: we propose that specific T regulatory cells (Tregs) can be used to prevent and reverse inhibitor formation. Tregs are the most important T subset for the maintenance of self-tolerance and homeostasis of our immune system. Clinical applications using Tregs are now considered a next-generation cellular therapy in a variety of autoimmune and inflammatory immune disorders such as type 1 diabetes, GVHD, and transplantation, and potentially as a treatment for antibody-mediated diseases such as hemophilia inhibitor formation. However, therapy with polyclonal Tregs is non-specific and a potential drawback is that it could be generally immunosuppressive. Therapy with antigen-specific Tregs (CARTs) would be highly preferable, as these have proved more potent in animal models of autoimmune diseases. We demonstrate herein the generation and functional properties of human T regulatory cells that specifically recognize FVIII. Tregs were generated that expressed the same T cell receptor (TCR) as that found on an HLA-DRB1*01:01-restricted T cell clone isolated from a hemophilia subject with a high-titer inhibitor. The T effector clone was isolated by MHC (DR0101) tetramer staining of this subject’s CD4 cells followed by single-cell sorting and expansion in cell culture. Nucleotide/amino acid sequences were identified to design retroviral FVIII-specific TCR constructs. To produce FVIII-specific human Foxp3+ Tregs, we transduced both expanded T effector cells and Tregs with a TCR expressed on an effector CD4 clone isolated from an inhibitor subject; these T cells recognize the same HLA-DRB1*01:01 restricted epitope as the “parent” effector T cell clone. Specific reactivity of the transduced cells bearing the designed TCR was validated by staining of the transduced T effector and Treg cells by DR0101 tetramers loaded with the same peptide as that recognized by the original effector T cell clone. Transduced effector T cells were expanded by stimulation with the expected FVIII peptide in vitro. Moreover, activation by this peptide in the presence of oligonucleotides mediated functional expansion and stabilization of transduced Tregs in ex vivo culture. Importantly, the expanded transduced Tregs were immunosuppressive only when stimulated by FVIII, even when FVIII-specific effector T cells were present in large excess. (Supported by NIH grant RO1 HL061883-15 to DWS and unrestricted research funding from Bayer, Pfizer and CSL Behring to KPP, and the intramural program of the NIAID to EMS). Disclosures: Pratt: Bayer: Research Funding; Pfizer: Research Funding; CSL Behring: Research Funding.

Description: Abstract 39 Neutralizing anti-factor VIII (FVIII) antibodies, or “inhibitors”, interfere with FVIII pro-coagulant activity, and persistent inhibitors can result in significant morbidity and mortality in hemophilia A (HA) patients and in individuals who develop autoantibodies to their endogenous FVIII. Inhibitor production follows stimulation of helper T cells by linear amino acid sequences in FVIII corresponding to HLA-restricted T-cell epitopes. An immunodominant HLA-DRB1*01:01-restricted T-cell epitope within a peptide corresponding to FVIII residues 2194–2213 was identified previously using blood samples from two mild HA subjects with hemophilic mutation A2201P. This same immunodominant epitope was found recently in inhibitor subjects with (a) a large F8 gene deletion and b) an F8 nonsense mutation in exon 12. The present study aims to identify amino acid substitutions in FVIII that will neutralize this T-cell epitope while preserving the pro-coagulant activity of the modified FVIII protein. MHC class II - peptide binding assays were carried out using truncated FVIII peptides to determine the shortest sequence with full binding affinity for a recombinant HLA-DR0101 protein, and subsequently using peptides having systematic arginine substitutions at each position to identify the specific residues that confer this binding affinity. The results indicated that FVIII2194–2205 is the minimal binding epitope and that residues F2196, M2199, A2201 and S2204 interact with the peptide-binding groove of HLA-DR0101. Next, four T-cell clones that all proliferate in response to this epitope but have different T-cell receptors were stimulated with 12 peptides having systematic alanine substitutions at each position of FVIII2194–2205. The F2196A substitution abrogated proliferation of all four clones. The M2199A-substituted peptide stimulated three of the clones more weakly than the wild-type peptide. Peptide binding and T-cell assays were next carried out with FVIII2194–2205 peptides in which the 19 common non-phenylalanine amino acids were substituted at position 2196. These results identified 12 different amino acid substitutions that decreased both MHC binding and T-cell proliferation more than 10-fold. The binding of FVIII2194–2205 and FVIII2194–2205, F2196A to 10 common HLA-DRB1 proteins was measured to determine the potential promiscuity of this epitope. Moderate or low affinity binding of FVIII2194–2205 (IC50 〈 50 mM) to DR0401, DR0404, DR0901, DR1001, and DR1501 was observed. FVIII2194–2205, 2196A did not bind to any of the HLA-DRB1 proteins, suggesting that this substitution would not introduce a neo-epitope recognized by these other common MHC class II receptors. A recombinant FVIII-C2 domain protein with substitution F2196A was generated in E. coli and purified to homogeneity following a procedure that removes endotoxin. This FVIII-C2 mutein failed to stimulate the same four T-cell clones, all of which showed a strong, dose-dependent response to wild-type FVIII-C2. Recombinant B-domain-deleted FVIII (BDD-FVIII) proteins with substitutions F2196A, F2196L, F2196K, M2199A, M2199W and M2199R were expressed in BHK-M cell lines. Multiple cell lines were generated to express wild-type BDD-FVIII and each of these mutant proteins. Expression levels of the muteins were similar to that of wild-type BDD-FVIII except for the M2199W and F2196A variants, which had expression levels ∼30 and 10% that of wild-type BDD-FVIII, respectively. Specific activities of the muteins, measured using chromogenic and clotting assays, were similar to that of wild-type BDD-FVIII. Binding of these muteins to plasma-derived von Willebrand factor was evaluated by ELISAs, as a surrogate assay to indicate possible effects of specific mutations on FVIII half-life in the circulation. Their affinities for VWF ranged from ∼40–100% that of wild-type BDD-FVIII. Our results suggest that FVIII muteins with amino acid substitutions that abolish binding to DR0101 and retain reasonable FVIII functionality could be developed as less immunogenic therapeutic proteins, in order to avoid HLA-DRB1*01:01-restricted immune responses in HA patients with this common allele. The immunogenicity of this T-cell epitope and of the sequence-modified peptides and proteins in HA subjects with other HLA-DRB1 alleles is currently under investigation. Disclosures: Pratt: Puget Sound Blood Center: Employment, patent describing design of novel factor VIII proteins Other.

Description: The development of inhibitory anti-FVIII antibodies is a major clinical problem in hemophilia A. While less common in mild/moderately severe patients, the relative risk of inhibitor formation is elevated in patients with missense mutations in the FVIII A2 domain, especially those with an R593C genotype. In this study, T-cell responses to 14 FVIII A2 domain peptides with predicted DRB1*1101 MHC binding motifs were investigated using MHC class II tetramer reagents. CD4+ T cells were isolated from a hemophilic subject (DRB1*1101, 1302) with FVIII missense genotype R593C and stimulated with pooled peptides. This subject had developed a longstanding low titer inhibitor after receiving multiple FVIII infusions. Staining with fluorescent, peptide-loaded tetramers revealed that the hemophilic subject, but not an HLA-matched healthy control, had a DRB1*1101-restricted response to peptide A2589–608, which contained the wild-type R593 sequence. MHC class II tetramers with bound A2589–608 were used to sort antigen-specific T cells and then generate a T-cell clone recognizing this high avidity DRB1*1101-restricted epitope. FVIII residues 594–602 (FLPNPAGVQ) comprise a predicted high-affinity binding motif. Peptide binding assays confirmed that A2589–608 bound to recombinant, monomeric DRB1*1101 protein with high affinity. A peptide with the corresponding hemophilic sequence (A2589–608, 593C) bound with affinity that was threefold lower, but in a range that should be sufficient for presentation to T cells. The other 12 A2 peptides had a wide spectrum of binding affinities for the DRB1*1101 protein. Interestingly, prediction algorithms suggest that the peptides containing residue 593 occupy the MHC Class II peptide-binding groove in a register that places this residue just outside the p1 anchor position. Thus we anticipate that residue 593 modulates T-cell recognition of peptide A2589–608. This provides a potential explanation for inhibitor development in mild hemophilia A patients with a FVIII R593C genotype who are HLA-DRB1*1101. Immunogenicity of this FVIII region with respect to other HLA types is under investigation.