6 to 6.0 [23]. Hu et al. reported that CD8+ depleted PBMCs from four non-inhibitor haemophilic subjects proliferated when stimulated by FVIII or by several A2 domain peptides [25]. Reding et al. did not consistently observe proliferation of T cells from non-inhibitor haemophilic subjects in response to A3 domain peptides [26], while C2 domain peptides spanning FVIII residues 2190–2210 and 2291–2330 elicited proliferation in some subjects with and without haemophilia or inhibitors [27]. We cloned T cells from an individual with haemophilia A but no clinically
significant inhibitor (IV-2); these clones retained their antigen specificity for FVIII2194–2213 presented click here by DR0101, as demonstrated by both tetramer binding and proliferation assays. The SI values for proliferation assays utilizing six of these T-cell clones were highly significant, ranging between 62 and 248 at a low peptide concentration of 0.1 μm. To our knowledge, this study is the first to demonstrate conclusively an HLA-DR-restricted
T-cell response, validated by isolation of relevant epitope-specific T-cell clones, to a FVIII epitope in a haemophilia A subject without a clinically significant inhibitor. We isolated FVIII-responsive T cells from two haemophilia A subjects who did not have a clinically significant inhibitor, one of whom NVP-LDE225 cell line had received infusions of wild-type FVIII to achieve haemostasis. T cells from these subjects may well have different immune characteristics compared to those
from subjects with an established antibody response to FVIII. A recent study demonstrated that cytokine production by FVIII-stimulated polyclonal CD4+ T cells differs between healthy subjects, haemophilia A subjects without inhibitors, and haemophilia A subjects with inhibitors [39]. T cells from haemophilia A subjects with inhibitors produced higher levels of IFN-γ and IL-4, whereas T cells from controls and haemophilia A subjects without inhibitors secreted higher levels of TGF-β Clomifene but did not produce IL-4. We are utilizing the T-cell clones isolated from the A2201P missense substitution subjects, which are restricted by the same HLA-DR-FVIII peptide complex, to investigate additional features of T-cell immune responses to FVIII, including cytokine secretion and T-cell receptor variations. As noted above, T-cell responses to the haemophilic peptide differed in these individuals. Their T cells may also have different thresholds of responsiveness to haemophilic and/or wild-type peptides. DRB1 proteins corresponding to the second allele (DRB1*1503) of inhibitor subject IV-1 were not available, but his brother’s second allele was DRB1*0401. This subject, IV-2, did not have DR0401-restricted T cells that recognized FVIII C2 domain peptides, although he did have a pronounced DR0101-restricted T-cell response to one of these FVIII peptides.