No DR1104-restricted T cells were detected in total CD4+ T-cell cultures (Fig. 3a).

However, tetramer-positive responses to peptide pool 2 were observed for 0.7% of the cells when the total CD4+ fraction was depleted of CD4+CD25+ T cells (Fig. 3b). This small but above-background T-cell response to peptide pool 2 was seen on analysis of two separate blood samples collected three months apart. Decoding identified FVIII2202–2221 (peptide sequence: TWSPSKARLHLQGRSNAWRP), BKM120 purchase which is adjacent to the A2201P missense substitution site, as the immunodominant DR1104-restricted T-cell epitope (Fig. 3c). The possibility of additional minor T-cell epitopes being present cannot be dismissed because of the above-background staining by tetramers loaded with FVIII2186–2205 and FVIII2194–2213. No DR0901-restricted T-cells were detected in total CD4+ or CD4+CD25+-depleted T-cell cultures (data not shown). Subject II-3, the great-uncle of IV-1 and IV-2 and great-grandfather of IV-3, has HLA alleles DRB1*0404 and DRB1*1501; Cisplatin cost despite prior FVIII infusions, he has shown no evidence of an inhibitor. A blood sample was obtained from him several months after his last FVIII exposure and TGEM was carried out as above

using his total CD4+ and CD4+CD25+-depleted T-cell fractions to screen for DR0404- and DR1501-restricted T-cell epitopes. Staining above background (0.9% tetramer-stained CD4 cells) was seen for total CD4 cells cultured with DR0404 tetramers loaded with pool 4 peptides. However, when tetramer binding was plotted versus CD25 staining the learn more tetramer-positive cells did not form a distinct cell

population, suggesting that this signal included significant non-specific binding, so we concluded that this staining did not indicate a legitimate epitope. Similar results were observed for the CD4+CD25+-depleted cultures (data not shown). No DR1501-restricted T cells were detected in total CD4+ or in CD4+CD25+-depleted T-cell cultures (data not shown). Subject III-2, the obligate carrier mother of IV-1 and IV-2, is HLA-DRB1*0101, 0901, and subject III-4, the obligate carrier mother of subject IV-3, is HLA-DRB1*1104, 1501. Blood samples from each were screened for FVIII T-cell epitopes using TGEM with pooled peptides as above. Neither total CD4+ cells or CD4+CD25+-depleted CD4+ cells from the carrier mothers showed T-cell responses to the pooled peptides restricted by their respective HLA-DR allelic proteins (data not shown). T cells from the first blood draw of haemophilic subject IV-2 that were stimulated with peptide pool 2 were next stained using DR0101 tetramers carrying FVIII2194–2213 and then were single-cell sorted into 96-well plates as described above. Cells in 21 wells expanded sufficiently to be tested for tetramer binding, and 20/21 wells contained expanded T cells that were 99–100% tetramer-positive (data not shown). Six of these clones were cryo-preserved.