4a). When the DR0101 Talazoparib in vivo tetramer was loaded with FVIII2218–2237, staining of the T-cell clones was at background levels, demonstrating the specificity of the clones for the DR0101-FVIII2194–2213 peptide complex (Fig. 4b). The antigen specificity of the T-cell clones
was further tested by proliferation assays. Each T-cell clone proliferated in response to FVIII2194–2213 presented by DR0101 (Fig. 5). The amount of proliferation was dose-dependent over the range of peptide concentrations tested (0.1–10 μm). The stimulation indices (ratios of measured proliferation/background proliferation) were highly significant, ranging from 62 to 248 at 0.1 μm peptide concentration for the six clones. The T-cell clones also proliferated in response to a peptide with the haemophilic sequence, FVIII2194–2213, 2201P, but at significantly reduced levels. However, this proliferation was above the background levels established using an irrelevant peptide, FVIII519–538. This stimulation by the haemophilic (missense) sequence was in contrast to the behaviour of clones isolated from the clinical inhibitor subject IV-1, which did not proliferate in response to the haemophilic peptide [33]. The T cells from haemophilic subject IV-3 that were stimulated with peptide pool 2 were next stained using DR1104 tetramers carrying FVIII2202–2221 and then
single-cell sorted into 96-well plates as described above. Cells in 13 wells expanded sufficiently to be tested for tetramer binding, and cells in four of these wells were tetramer-positive (Fig. 6). The binding avidity of these T-cell clones for the DR1104 tetramer Ceritinib molecular weight loaded with FVIII2202–2221 was low (Fig. 6a) compared with that of the clones shown in Fig. 4a. These T-cell clones did not bind to the DR1104 tetramer loaded with FVIII2186–2205 (Fig. 6b), verifying the specificity
of these T-cell clones for a peptide adjacent to the missense substitution A2201P. None of these T-cell clones expanded well under conditions routinely used for expansion of human 3-oxoacyl-(acyl-carrier-protein) reductase T-cell clones, so they could not be further analysed or cryo-preserved. The presence of FVIII-responsive T cells in subject IV-2, who did not have an inhibitor but whose T-cell response was highly similar to that of his brother, raised the possibility that he might have circulating inhibitory antibodies, albeit at a sub-clinical level. To address this possibility, IgG was Protein G-purified from his plasma and Bethesda assays were carried out for serial dilutions of the concentrated IgG samples. At 10 mg mL−1, his IgG sample had an inhibitor titre of 1 BU mL−1, demonstrating a trace level of circulating inhibitory antibody. Our previous study of T-cell responses to FVIII peptides in a mild haemophilia A inhibitor subject with missense substitution FVIII-A2201P established a powerful approach to identify HLA-DR-restricted T-cell epitopes in FVIII [33].