PMC

The high-titer inhibitor subject previously described [] is depicted here as the first member of generation IV, or IV-1. Mild haemophilia A is indicated by shaded squares, and the half-shaded circles indicate the obligate carrier mothers.

A. T-cell clones #15, 19, 11, and 12 were stimulated with HLA-mismatched PBMCs and PHA. Thirteen days later, the clones were incubated with PE-labeled DR1104 tetramers loaded with peptide FVIII_2202-2221 and APC-labeled anti-human CD4 IgG. B. Two of the clones were incubated with DR1104 tetramers loaded with an irrelevant peptide (FVIII_2186-2205) as a negative control.

A. T-cell clones #5, 14, 15, 16, 18, and 21 were stimulated with HLA-mismatched PBMCs and PHA. Fourteen days later, the clones were incubated with PE-labeled DR0101 tetramers loaded with peptide FVIII_2194-2213 and FITC-labeled anti-human CD4 IgG. B. Two of the clones were incubated with DR0101 tetramers loaded with an irrelevant peptide (FVIII_2218-2237) as a negative control.

Resting T-cell clones #5, 14, 15, 16, 18, and 21 were stimulated with PBMCs from a healthy DRB1*0101 donor plus wild-type peptide FVIII_2194-2213 (triangle symbols), haemophilic peptide FVIII_{2194-2213 2201P} (square symbols), or irrelevant peptide FVIII_519-538 (circle symbols) at 0, 0.1, 1.0, and 10 μM final concentration. [³H]thymidine uptake was measured. Data show mean ± SD of triplicate determinations.

Total CD4+ cells (A) and CD4+ cells depleted of CD4+CD25+ cells (B) were stimulated with pooled peptides spanning the FVIII C2 domain sequence. Fifteen days later, the cells were incubated with PE-labeled DR1104 tetramers loaded with FVIII pooled peptides and antibodies as described in Methods. Decoding of DR1104-restricted responses to peptide pool 2 using tetramers loaded with individual peptides comprising the pool was carried out 22 days after stimulation of CD4+CD25+-depleted CD4+ cells with peptide pool 2 (C).

CD4+ T cells were stimulated with pooled peptides spanning the FVIII C2 domain sequence. Eighteen days later, the cells were incubated with PE-labeled DR0101 tetramers loaded with FVIII C2 peptide pools (A) or with DR0401 tetramers loaded with FVIII C2 peptide pools (B) and antibodies as described in Methods. Decoding of positive CD4+ responses to DR0101 tetramers loaded with peptide pools 1 and 2 was carried out 22 days after stimulation of total CD4+ cells (top row) or CD4+CD25+-depleted CD4+ cells (bottom row), respectively (C). Decoding of DR0101-restricted responses to peptide pool 1 using tetramers loaded with individual peptides comprising the pool is shown in the top row. Decoding of DR0101-restricted responses to peptide pool 2 is shown in the bottom row.