HLA-DR-restricted T-cell responses to factor VIII epitopes in a mild haemophilia A family with missense substitution A2201P

Abstract

An HLA-DRA-DRB1*0101-restricted T-cell epitope in the factor VIII (FVIII) C2 domain occurred in a mild haemophilia A patient with missense substitution FVIII-A2201P. His T cells responded to synthetic peptides FVIII(2186-2205) and FVIII(2194-2213) (J Thromb Haemost 2007; 5: 2399). T cells from family members with genotype FVIII-A2201P were analysed to determine if FVIII-specific T cells occur in individuals with a haemophilic mutation but no clinically significant inhibitor response. Fluorescent MHC class II tetramers corresponding to subjects'HLA-DRB1 types were loaded with 20-mer peptides and utilized to label antigen-specific CD4+ T cells. T-cell responses to peptides spanning the FVIII-C2 sequence were evaluated. T cells recognizing specific peptides were cloned, and antigen specificity was verified by proliferation assays. Plasma and/or purified IgG samples were tested for FVIII inhibitory activity. CD4+ T cells and T-cell clones from two brothers who shared the DRB1*0101 allele responded to FVIII(2194-2213). A haemophilic cousin's HLA-DRA-DRB1*1104-restricted response to FVIII(2202-2221) was detected only when CD4+CD25+ cells were depleted. A great uncle and two obligate carriers had no detectable FVIII-C2-specific T cells. Concentrated IgG from the brother without a clinical inhibitor response showed a low-titre FVIII inhibitor. FVIII-specific T cells and inhibitory IgG were found in a previously infused, haemophilic subject who had a sub-clinical FVIII inhibitor. CD4+CD25+ depleted T cells from a non-infused haemophilic cousin recognized an overlapping FVIII epitope, indicating a latent HLA-DRA-DRB1*1104-restricted T-cell response to FVIII. Specific T-cell responses to FVIII can occur without clinically significant inhibitors.

Figure 1. The A2201P mild haemophilia A family pedigree

The high-titer inhibitor subject previously described [33] 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.

Figure 2. T-cell epitope mapping for haemophilic subject IV-2

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.

Figure 3. T-cell epitope mapping for haemophilic subject IV-3

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).

Figure 4. Tetramer staining of T-cell clones isolated from haemophilic subject IV-2

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.

Figure 5. Antigen-specific proliferation of T-cell clones from haemophilic subject IV-2

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.

Figure 6. Tetramer staining of T-cell clones isolated from haemophilic subject IV-3

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.