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 Haemost 5:2399, 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:

  1. 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;

  2. 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.

Topics:
clone cells, factor ix, interleukin-17, th17 cells, t-lymphocytes, epitopes, human leukocyte antigens, cytokine, interferon type ii, interleukin-10

Disclosures: No relevant conflicts of interest to declare.

Author notes

Corresponding author

2008, The American Society of Hematology
2008
Sign in via your Institution